HIV viral load testing in the era of ART. Christian Noah Labor Lademannbogen, Hamburg
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1 HIV viral load testing in the era of ART Christian Noah Labor Lademannbogen, Hamburg 1
2 Life expectancy of patients on ART Data from the UK Collaborative HIV Cohort (UK CHIC) Requirements: Early diagnosis Timely initiation of ART Adherence Periodic monitoring Successfully treated HIV patients have the chance of a normal life expectancy May et al. (2014), AIDS, 28,
3 HIV monitoring CD4 cell count Lymphocyte differentiation Marker of overall immune function Predictor of disease progession Urgency to initiate ART Indication for prophylaxis against opportunistic infection Viral load PCR Marker of treatment success and adherence Monitoring of the efficacy of ART Detection of virologic failure Selection of antivirals Substances/combinations not recommended > copies/ml: Rilpivirin Abacavir/Lamivudine + Efavirenz or Atazanavir/r Darunavir/r+Raltegravir Risk of transmission 3
4 Monitoring intervals DAIG/DÖAG 2015 EACS 2015 Viral load pre ART: 2-3 months at ART initiation/switch: more frequent if suppressed: 2-4 months 3-6 months at ART initiation/switch: more frequent CD4 cells same as viral load 3-6 months every 12 months if stable on ART CD4 >350; VL <LOD IAS 2014 at least every 3 months 4 weeks after ART initiation at least every 3 months at least every 6 months if stable on ART CD4 >350; VL <LOD >1 year optional if stable on ART CD4 >500; VL <LOD >2 years DHHS 2016 every 3-4 months 2-8 weeks after ART start/switch every 6 months (adherence, immunologically stable, <LOD >2 years) every 3-6 months first 2 years on ART, CD4 <300, viremia every 12 months after 2 years on ART, VL <LOD, CD4 >300 optional CD4 >500 4
5 Monitoring intervals DAIG/DÖAG 2015 EACS 2015 IAS 2014 DHHS 2016 Viral load pre ART: 2-3 months at ART initiation/switch: more frequent if suppressed: 2-4 months every 3 months 3-6 months at ART initiation/switch: more frequent baseline before ART every 1-2 months after at least every 3 months 4 ART weeks initiation after ART until initiation suppression consider lower frequency (every 6 months) if <LOD >2 years every 3-4 months 2-8 weeks >350 after CD4 ART cells start/switch every adherence 6 months (adherence, immunologically stable, <LOD >2 years) CD4 cells same as viral load 3-6 months every 12 months if stable on ART CD4 >350; VL <LOD at least every 3 months at least every 6 months if stable on ART CD4 >350; VL <LOD >1 year optional if stable on ART CD4 >500; VL <LOD >2 years every 3-6 months first 2 years on ART, CD4 <300, viremia every 12 months after 2 years on ART, VL <LOD, CD4 >300 optional CD4 >500 5
6 Future role of CD4 cell count in HIV monitoring Metaanalysis (13 studies) virologically suppressed patients Proportion of an unexplained, confirmed CD4 decline: 0,4 % (95 % CI 0,2-0,6) No adverse events among patients experiencing CD4 declines Ford et al. 2015; JIAS 18:
7 Definition of treatment response Viral load [copies/ml] Limit of detection (LOD) Months after ART initiation 7
8 Definition of treatment response Cobas Amplicor (Roche Diagnostics) defined by a technical cutoff: 50 copies/ml 8
9 Limits of detection: the Roche history Cobas Amplicor Cobas Ampliprep/Taqman Cobas (1995) ultrasensitive 50 (1998) Version 1 40 (2005) Version 2 20 (2009) 20 copies/ml (2015) 9
10 Platforms for viral load measurement Roche COBAS Ampliprep/Taqman Siemens VERSANT kpcr Molecular System Abbott m2000rt Real Time PCR System 20 Kopien/ml Roche Cobas Kopien/ml Qiagen QIAsymphony RGQ 40 Kopien/ml 20 Kopien/ml 34 Kopien/ml 10
11 Definition of optimal treatment response Viral load [copies/ml] DAIG/DÖAG 2015 EACS 2015 <50 after 3-4 months after 6 months if viral load was high at baseline <50 after 6 months Limit of detection (LOD) IAS 2014 <LOD (<20-75) after 6 months Months after ART initiation DHHS 2016 <LOD (<20-75) after 6 months 11
12 Treatment failure? Viral load [copies/ml] Months after ART initiation 12
13 Treatment failure? No, just a blip... Viral load [copies/ml] Blip = transient viremia Months after ART initiation 13
14 Biological causes of blips Infections Vaccination Syphilis: 27,6 % viremic during active infection Palacios R (2007), J Acquir Immune Defic Syndr 44(3), Tuberculosis: fold increase of viral load during active infection Goletti D (1996), J Immunol 157(3), Influenza: 20,6 % viremic 2-4 weeks after vaccination Kolber MA (2002), AIDS 16(4), Tetanus 100 % viremic after vaccination Stanley SK (1996) N Engl J Med 334(19), Pneumococi Vigano A (1998), AIDS Res Human Retroviruses 14(9),
15 Technical causes of blips 1. Pre-analytical errors proviral DNA avoid hemolysis Plasma should be separated from cells within 24 h after blood withdrawal 2. Assay variation 15
16 Definition of the limit of detection (LOD) Viral load detected with a probability of 95 % Example: COBAS AmpliPrep/COBAS Taqman HIV-1-Test, v. 2,0 LOD 20 copies/ml Concentration [copies/ml] Replicates Positive Detection (WHO standard) rate [%] PROBIT 95 % analysis: 16,5 copies/ml 95 %.confidence interval: 14,3-19,8 16
17 Target not detected negative 130 patients 3TC/d4T + LPV/r ornfv mit 3TC und d4t als Backbone <50 copies/ml within 24 weeks <50 copies/ml after 60 weeks After 2 years: No significant drop of the viral load After 60 weeks: 83 % of the patients viremic Single Copy PCR LOD 1 copy/ml After 7 years: 77 % of the patients viremic Median 3,34 copies/ml After treatment intensification (Raltegravir): No significant drop of the viral load Median 3,1 copies/ml Maldarelli F et al. (2007), PLoS Pathogens, 3(4), 46 Palmer S et al. (2008) PNAS, 105 (10), 3879 Gandhi RT et al. (2010). PLoS Medicine 7(8), e
18 Precision of viral load assays (Roche) 15 runs 3 lots 3 replicates Viral load log10/ml Charge 1 Total SD (log) Charge 2 Total SD (log) Charge 3 Total SD (log) Charge 1-3 Total -SD (log) Total VC der Log- Normalverteilung (%) ,19 0,07 0,07 0,04 0,10 0,11 0,16 0,09 0,07 0,05 0,09 0,12 0,17 0,07 0,06 0,07 0,10 0,14 0,17 0,08 0,07 0,06 0,10 0, Example: Viral load 10 2 /ml = 100 copies/ml 10 2,17 /ml = 148 copies/ml 10 1,83 /ml = 68 copies/ml 18
19 Precision of viral load assays (Siemens) 19
20 Precision of viral load assays (Abbott) 20
21 Comparison of viral load assays Results from over 4000 paired plasma samples Roche Taqman Version 1+2 Roche Amplicor Abbott RealTime J Clin Microbiol (2014). 52(2), Overall good correlation (0,90-0,97) Low level viremia <200: 0,45-0,85 21
22 Assay variation Residual viremia <LOD Ability of commercial assays to detect HIV RNA <LOD Inaccurate quantification at low levels Overquantification of residual viremia = Blip 22
23 Treatment failure? Viral load [copies/ml] Months after ART initiation 23
24 Treatment failure? Maybe yes, maybe no... Viral load [copies/ml] Resistance development persistent viremia low-level very-low-level high-level Months after ART initiation 24
25 Risk factors for persistent viremia ADHERENCE Interactions Pharmacogenomics Stage of infection high baseline VL low CD4 cell count CDC state Very low level viremia overall conflicting data most cases multifactorial no one factor is determinative Regimen PI>NNRTI Duration of suppression <50 Ryscavage R. et al. (2014), AAC, 58(7),
26 How to manage viremia? Which viral load is predictive for treatment failure? Viral load level Persistence 26
27 Significance of persistent low-level viremia Data from 18 cohorts including patients No LLV 93,8 % LLV ,5 % LLV ,7 % Virologic failure (VL 500 copies/ml): 1903 patients (10.6 %) No LLV 1745 (10,4%) LLV (7,9 %) LLV (22,6 %) 91,7 % of patients with VF without any previous LLV LLV strongly associated with VF (adjusted HR 3,97) LLV weakly associated with VF No association with VF: type of regimen duration of LLV Vandenhende 2015, CROI, Poster
28 Significance of low-level viremia (LLV) LLV is common: blips: % persistent LLV: % high level viremia: 6-9 % Persistent LLV associated with an increased risk of resistance 19 % resistance mutations at first LLV ( ) Risk correlated with viral load level Median VL in patients evolving resistance: 472 copies/ml vs. 369 copies/ml in patients not evolving resistance (p=0,067) Ryscavage R. et al. (2014), AAC, 58(7),
29 Definition of treatment failure DAIG/DÖAG 2015 EACS 2015 IAS 2014 Detectable viral load >50 copies/ml (confirmed) Drop of <2 log10 after 4 weeks Detectable viral load 6 months after ART start Detectable viral load >50 copies/ml (confirmed) Depending on assay limit could be higher or lower Detectable viral load 6 months after ART start Detectable viral load >50 copies/ml (confirmed) Detectable viral load >200 copies/ml DHHS 2016 Detectable viral load >200 copies/ml (confirmed) 29
30 Management of viremia Confirmation Assessment of factors leading to suboptimal response Adherence (TDM?) Interactions (TDM?) Infections Vaccinations Pre-analytic issues (incl. sample confusion)... 30
31 Management of viremia VL (=low-level viremia) Confirmation within 4 weeks VL <50 (=very low-level viremia): No clinical relevance Regular follow-up 3 months later VL >500: Virologic failure Resistance test Switch regimen as soon as possible No resistance mutations: assess adherence (again?) a) Not confirmed: Blip No clinical relevance Regimen with low genetic resistance barrier: consider shorter follow-up interval b) Confirmed: Weak association with virologic failure Regular Follow-up 3 months later Consider resistance test (optional) Predictive for virologic failure Resistance test Switch of regimen No resistance mutations: assess adherence (again?) 31
32 32
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