Complicated viral infections

Transcription

1 Complicated viral infections Clinical case discussion Diagnostic dilemmas NSW State Reference Laboratory for HIV St Vincent s Hospital Sydney

2 Diagnostic dilemmas Indeterminate or discordant serology (western blot and immunoassays) Acute HIV infection - (pre-seroconversion) Genetic variants of HIV Infant diagnosis in the setting of a HIV seropositive mother

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4 Discordant, indeterminate serology Detection of acute / primary HIV infection DILEMMA

5 Serological diagnosis of HIV infection Serological eclipse

6 Serology of primary HIV-1 infection Antibody tests may be negative or low level (indeterminate) (Western Blot) Antibodies to viral proteins are incrementally detected increasing in intensity over time (diagnostic)

7 gp160 gp120 p68 p55 p53 gp41-45 p40 p34 p24 p18 p12 acute established late DNA PCR RNA PCR p24 Ag 3rd gen ELISA 1st gen ELISA Incidence ELISA 1wk 2wk 3wk 6wk 2mo 6mo 1yr 2yr 3yr +8yr

8 Case 1 - BA,RI 30 y/o African male Ghana Immigration detention centre NSW Heterosexual Exposure FSW 1 year ago Currently 1 syphilis Recurrent episodes malaria treated as outpatient several times over last few years

9 BA,RI HIV serology HIV-1/2 Ab/Ag 4 th gen EIA reactive HIV-1/2 3 rd generation EIA - reactive HIV-1/2 rapid test (X2) reactive HIV-1 western blot POSITIVE N P gp160 gp120 p68 p55/51 gp41 p40 HIV direct detection HIV-1 p24 antigen not detected HIV proviral DNA PCR not detected HIV RNA (gag) PCR not detected p34 p24 p18 α-huigg

10 What next? Follow up sample clinical history HIV serology testing strategies Supplementary EIAs CD4 lymphocyte count 480 (normal) CD4:8 ratio not inverted HIV-2 serology Negative to specific gp 36 antigens HIV DNA (gag) - not detected HIV RNA (pol) - Not detected by bdna RNA test HIV culture - virus isolation CD8 depleted, IL2 enriched, PHA stimulated PBMC co-culture gp140 gp105 p68 p56 gp36 p34 p26 p16

11 HIV Testing Direct Detection of Virus HIV-1 p24 antigen - serology Simple serology no additional sample required LLD 10pg p24 only assays qualitative and quantitative mode p24 in combination with antibody Virus isolation PBMC co-culture Insensitive Requires containment laboratory (PC3) ~ 3-6 weeks culture Detect with p24 antigen or RT activity in supernate Nucleic acid detection - (NAT) Qualitative tests more sensitive and specific blood/tissue screening Most products available are viral load tests not designed for diagnosis Plasma RNA (virus load) or cellular DNA Dedicated samples required Nucleic acid laboratories

12 NAT tests for HIV DNA RNA RNA Qualitative Quantitative (viral load) Qualitative <10 copies <50 copies <10 copies Highly specific HIV supplemental diagnostic test Resolve indeterminate serology Acute infection diagnosis (pre-seroconversion) Early infant diagnosis Integrated and unintegrated cellular DNA Whole blood Dried Blood Spot (DBS) Specificity issues at low range HIV monitoring test Treatment response Prognostic marker Clinical decision point Extracellular RNA (free virion) Plasma DBS Highly specific Blood and tissue donor screening Detection of HIV in pooled or single blood/tissue donors Extracellular RNA (free virion) Plasma Cadaveric blood

13 UG273 DJ258 DJ263 US1 US2 US3 US4 CM237 BK132 BZ167 ZB18 US278 P3100 SE364 SM145 SE365 UG270 UG274 CM235 CM238 CM240 CM243 POC30506 RA12 RA17 NP1465 BZ126 BZ162 BZ163 MIKA G A B C D E F G Isolate and Subtype HIV RNA copies/ml Not detected Qualitative vs quantitative assays

14 Geographic distribution of HIV Subtype B: represents ~10% of HIV-1 infections worldwide

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16 HIV subtype distribution in USA

17 Target two regions within the HIV-1 genome Simultaneous Amplification and Detection LTR Primers FAM labeled LTR probe GAG Primers FAM labeled GAG probe HIV Viral Load Method Technology Assay Target Drug target Previous Roche HIV-1 Methods PCR gag no CAP/CTM HIV-1 Test v2.0 PCR gag and LTR no VERSANT HIV-1 RNA 3.0 bdna Polymerase (pol) yes Abbott RealTime HIV-1 PCR Integrase (int) yes

18 Case 2 - Patient History 37 year old male Born in Ghana, Africa Malarial infection as an infant Blood transfusion in ~1974 Migrated to UK as an infant Multiple partners in UK and Australia Asymptomatic and good health at diagnosis

19 Results ASSAY Initial sample Followup sample Abbott Architect HIV1/2 Ag/Ab Combo EIA BioRad Genscreen HIV 1/2 EIA BioRad HIV-1 p24 Ag EIA Reactive Reactive Non Reactive Reactive Reactive Non Reactive

20 Results HIV-1 Western Blot gp160 + gp160 + gp120 +/- gp120 - p68 ++ p68 ++ p55 +/- p55 +/- p53 - p53 - gp /- gp p40 - p40 - p p p24 +/- p24 - p18 +/- p18 +/-

21 Supplemental tests ASSAY Initial sample Followup sample Roche Amplicor HIV-1 DNA Test v. 1.5 BioRad Multispot HIV-1/HIV-2 Rapid Test (neat) BioRad Multispot HIV-1/HIV-2 Rapid Test (1:100 dilution) Roche Amplicor HIV-1 RNA Test v. 1.5 Drug Resistance Genotype Borderline Positive HIV-1 Ab detected HIV-2 Ab detected HIV-1 Ab negative HIV-2 Ab detected N/A N/A Borderline positive HIV -1 Ab detected HIV-2 Ab detected HIV -1 Ab negative HIV-2 Ab detected 2900 cpy/ml Unable to sequence CD4% N/A 3% (R.R %)

22 HIV-2 western blot gp140 gp105 p68 p56 p36 p34 p All glycoproteins and viral specific bands present at high intensity Indicates presence of HIV-2 antibodies in patient sample Confirms Multispot Rapid Test result of HIV-2 p16 +

23 40 y/o gay male admitted to emergency with suspected pneumonia First presentation (1980 s) Case 3 - LE,LA Laboratory Weak reactive EIA (brdln negative) Indeterminate 4 WB p24 antigen EIA reactive (failed to neutralize) HIV proviral DNA detectable HIV RNA >750,000 cpy/ml CD4+ lymphocyte <20 cells/mm 3

24 Diagnosis of neonatal HIV infection DILEMMA

25 Case 4 - VI,MI 34 y/o Australian born female (HIV+) 21 weeks pregnant Unremarkable 1 st pregnancy 40 y/o partner HIV+ (HIV acquired sub Saharan Africa)

26 Testing Where to? HIV antibodies in the neonate HIV direct detection HIV DNA PCR HIV proviral DNA v1.0 negative in the mother Version 1.5 (modified primers) detectable HIV viral load (RNA >5000) HIV drug resistance sequencing Treatment optimisation Subtype classification (A/D)

27 Screening during Pregnancy Universal counseling and voluntary HIV testing (incl. opt-out testing) in early pregnancy (AII) 3rd-trimester repeat testing recommended if there is a high risk of infection because of behavior or residence in a highprevalence area (AII) September 2010 AETC National Resource Center,

28 Screening in Labor and Delivery Rapid testing during labor for women without documented HIV status and if positive, initiation of prophylaxis (AII) If acute HIV infection is suspected, a virologic test (e.g., plasma HIV RNA assay) should be performed (AII)

29 Screening during Postnatal Period If no previous HIV results available, offer immediate postpartum maternal test or newborn HIV test and if positive, initiate neonatal ARV prophylaxis and advise mother not to breastfeed pending results of confirmatory testing (AII)

30 Diagnostic Testing in Infants In infants <18 mos, use virologic assays that directly detect HIV: HIV DNA PCR or HIV RNA (AII) Persistent maternal antibody may give false positive antibody result Virologic testing of HIV-exposed infant (AII) days of age 1-2 months 4-6 months Virologic testing at birth should be considered for infants at high risk of infection (BIII) HIV antibody test months to document seroreversion in HIV-uninfected infants (BIII) Diagnostic test for children 18 months (AII)

31 Criteria for HIV Diagnosis 2 positive HIV virologic tests on separate blood samples (regardless of age) (AIl) Positive HIV antibody test with confirmatory Western blot (or IFA) at age 18 months (AII)

32 Choice of Diagnostic Test HIV DNA PCR and HIV RNA assays are preferred virologic assay (AII) HIV DNA PCR Sensitivity <40% at <48 hours of age Sensitivity increases to >90% by 2-4 weeks HIV RNA assays As sensitive/specific as DNA PCR for early diagnosis (results of <5,000 copies/ml may be false positives and should repeated) Unclear whether sensitivity affected by maternal or infant ARV prophylaxis HIV qualitative RNA assay is an alternative diagnostic test that can be used with infants

33 Choice of Diagnostic Test (2) HIV culture: not used for routine HIV diagnostic testing Sensitivity similar to HIV DNA PCR Complex, expensive, results in 2-4 weeks

34 14d 48h HIV infection reasonably excluded in non-breast fed infant if negative in 2 or more ( 1month and 4months) 1-2mo 3-6mo >2 Negative HIV Ab tests (<1month apart) Loss HIV Ab/ Neg DNA = uninfected 6-12mo Infant still Ab Pos at 12mo retest 15-18mo >15-18m Positive 48h (likely intrauterine Infection - early) Positive 14d (likely intrapartum Infection - late) Months post partum? consider stop prophylaxis Anti-HIV Positive >18m = HIV infection HIV DNA testing in the infant blood

35 Cse 5 - SU-BI gp160 gp120 6 week old baby girl HIV seropositive mother undetectable HIV RNA, unremarkable pregnancy C-section delivery, non-breast feed p68 p55 p53 gp41-45 p40 p34 p24 Laboratory (infant blood) p18 HIV antibody positive Direct detection NAT hu-igg HIV proviral DNA not detected Sero-reversion in the infant 12-Oct Jan Jul-2010