Molecular Surveillance of recent HIV-Infections in Germany - PowerPoint PPT Presentation

Molecular Surveillance of recent HIV-Infections in Germany (2013-2015) Andrea Hauser 30.04.2016 Köln

Molecular Surveillance of recent HIV-Infections in Germany (2013-2015) Andrea Hauser 30.04.2016 Köln

Estimation of new HIV-infections/new HIV-diagnosis Part of routine HIV-surveillance since 2011 Sampling strategy is based on HIV-reporting system: ~ 60% of all HIV- Anonymus new DSS RKI diagnosis Diagnostic labs (n=~82) HIV-Report (DriedSerumSpots) (IfSG) 30.04.16 AREVIR 2016 1

Representativeness of InzSurvHIV (2011-2015) DSS total 30.04.16 AREVIR 2016 2

Workflow in the Lab Sample receipt Serological “Recency Test” RNA extraction (magnetic beads) Viral load and RT-PCR Population sequencing + HIV-genotyping A) HIV-1 subtype B) HIV-resistance 30.04.16 AREVIR 2016 2

Workflow in the Lab Sample receipt Serological “Recency Test” RNA extraction (magnetic beads) Viral load and RT-PCR Population sequencing + HIV-genotyping A) HIV-1 subtype B) HIV-resistance 30.04.16 AREVIR 2016 4

Course of HIV-infection  recent infection long-standing infection (> 6 months) (< 6 months) Evolution of laboratory markers during HIV infection HIV-specific IgG: Increase of Titer + Avidity 1-2 years 30.04.16 AREVIR 2016 5

Serological recency assays Based on increase of HIV-spec. IgG i) TITER or ii) AVIDITY HIV-1 specific IgG i) BED IgG-Capture ELISA (BED-CEIA) total IgG - proportion of HIV-1 specific ELISA-plate reader among total IgG  applied in Germany OD n < 0.8 = recent ii) Avidity assays 2-well assay: Addition of mild denat. reagent in one well -> dissotiation of low binding antibodies AI < 40% = recent 30.04.16 AREVIR 2016 6

Limitations Immune response differs between individuals • Immune response differs between HIV-subtypes • delay of antibody maturation for HIV-subtype A+D infections Low antibody concentration in long term infected persons • in late state disease • on ART • ⇒ High false classifications ⇒ i.p. false recent => over estimation of incidence 30.04.16 AREVIR 2016 7

Workflow in the Lab Sample receipt BED IgG capture ELISA RNA extraction False recent rate: 14% (magnetic beads) (Loschen et al . 2008) Viral load and RT-PCR Population sequencing + HIV-genotyping A) HIV-1 subtype B) HIV-resistance 30.04.16 AREVIR 2016 8

Workflow in the Lab Sample receipt BED IgG capture ELISA 4x 100µl Recent infections: RNA isolation Viral load and RT-PCR Population sequencing + HIV-genotyping A) HIV-1 subtype B) HIV-resistance 30.04.16 AREVIR 2016 9

Workflow in the Lab Sample receipt BED IgG capture ELISA Recent infections: RNA isolation Viral load and RT-PCR Population sequencing + HIV-genotyping A) HIV-1 subtype B) HIV-resistance 30.04.16 AREVIR 2016 10

Workflow in the Lab Sample receipt BED IgG capture ELISA Recent infections: RNA isolation Viral load and RT-PCR Sanger/Next generation sequencing A) HIV-1 subtype B) HIV-resistance 30.04.16 AREVIR 2016 11

Workflow in the Lab Sample receipt BED IgG capture ELISA Recent infections: RNA isolation Viral load and RT-PCR Sanger/Next generation sequencing A) HIV-resistance (SDRM list) B) HIV-1 Subtyp (REGA) + socio demographic & clinical data from HIV-report 30.04.16 AREVIR 2016 12

Results (1) HIV-1 genotypes (PR/RT) 2013-2015/I: n= 1197 Characteristics of patients with recent infection Study Population % Gender Male 88.2 Female 11.1 Not reported 0.7 Transmission group Men who have sex with men (MSM) 57.7 Persons with heterosexual contacts (HET) 9.8 Persons with intravenous drug use (PWID) 2.9 Not reported/other 29.6 Country of origin [CI: 9,0; 12,5] Germany 57.7 Other 20.2 Not reported 22.1 Country of infection Germany 61.7 Other 12.3 Not reported 26.1 30.04.16 AREVIR 2016 13

Results (2) HIV-1 genotypes (PR/RT) 2013-2015/I: n= 1197 Transmitted drug resistance in recent infections P tend TDR > 0.5 Proportion (%) [CI: 9,0; 12,5]  stable proportion 30.04.16 AREVIR 2016 14

Results (2) HIV-1 genotypes (PR/RT) 2013-2015/I: n= 1197 Transmitted drug resistance in recent infections P trend > 0.05 Proportion (%)  stable proportion 30.04.16 AREVIR 2016 14

Results (3) HIV-1 genotypes (PR/RT) 2013-2015/I: n= 1197 HIV-1 B and non-B subtypes in recent infections P trend nonB = 0.002 Proportion (%) [CI: 23,7; 28,8]  significant increase of non B-infections 30.04.16 AREVIR 2016 15

Results (4) HIV-1 genotypes (PR/RT) 2013-2015/I: n= 1197 HIV-1 B and non-B subtypes in recent infections  A (East europe + Asia + Africa)  Rare recombinants (CRF + URF) Proportion (%) P trend subtype A = 0.01 P trend CRF/URF > 0.05  significant increase of subtype A infections 30.04.16 AREVIR 2016 16

Results (5) HIV-1 genotypes (PR/RT) 2013-2015/I: n= 1197 In which populations / transmission groups subtype A is circulating? Origin Transmission route Proportion (%) Proportion (%)  Subtype A in MSM (53%)  Subtype A in Germans (50%)  + by heterosexuel contacts (38%)  + East europeans (30%) 30.04.16 AREVIR 2016 17

Results (6) HIV-1 genotypes (PR/RT) 2013-2015/I: n= 1197 Subtype A infections according origin + transmission route P trend D/MSM = 0.05 30.04.16 AREVIR 2016 18

Results (6) HIV-1 genotypes (PR/RT) 2013-2015/I: n= 1197 Which recombinant forms were identified? Jan 2015 CRF (n=28) CRF06_cpx (9) CRF07_BC (1) CRF12_BF (5) CRF18_cpx (3) CRF19_cpx (2) CRF22_01A1 (1) CRF31_BC (2) CRF34_01B (1) Aug 2015 CRF35_AD (2) CRF44_BF (2) URF ( n=26) 30.04.16 AREVIR 2016 19

Results (6) HIV-1 genotypes (PR/RT) 2013-2015/I: n= 1197 In which groups rare recombinants (CRF/URF) are circulating? Origin Transmission route n= 14 n= 20 n= 21 2 1 2 5 3 2  55% in Germans  69% in MSM  35% by heterosexuel contacts 30.04.16 AREVIR 2016 20

Summary & Conclusion (1)  Proportion of TDR is stably high >10% in recent infections => maintain resistance testing  Proportion of non-B infections in Germany is high (22%) and increasing - in particular for subtype A => further analysis by Kirsten Hanke - rare recombinants => diversification of HIV 30.04.16 AREVIR 2016 21

Testing for recency of infection => new approach: Multi Assay Algorithm (MAA) Rated successively: Cut offs (*Laeyendecker et al 2012) 30.04.16 AREVIR 2016 22

Evaluation of a `German MAA´ ~ 𝟖𝟖𝟖 specimen of “German Seroconverter Cohorte” (known duration of infection) MAA: ≥1.0 ≥85 <100 30.04.16 AREVIR 2016 13

Conclusion (2)  `Multi Assay Algorithm´ (MAA) to distinguish between recent and long term infection significantly improves recency testing compared to the BED-CEIA alone.  The MAA is currently applied in the incidence surveillance as a pilot study. 30.04.16 AREVIR 2016 14

Diagnostic labs Thank you for your attention! 30.04.16 AREVIR 2016 15

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Patients & Methods  Trainings Panel (n=136) reflecting the present German situation in newly diagnosed HIV cases (2013/14) Proportion (1:2) Distribution of HIV-1 subtypes 33% recent ( ≤155 days) 67% long-term (>220 days)  plasma specimens from German Seroconverter Cohort with well defined duration of HIV-infection (recent / long-term) 06.04.16 26th Annual Meeting of the Society for Virology 30

Estimation of DAA cut-offs 1. Trainings panel: Dual Assay Algorithm BED-CEIA (ODn) + BioRad Avidity *(AI%) 91 2. Test results rated successively 3. Cut-offs resulting in the highest accuracy applied to validation panel (n=475) (194 recent, 281 long term, 409 B , 66 non-B) 4. Calculation of FRR + FLTR from the validation panel (*BioRad HIV-1/2 screening EIA and modified protocol from Masciotra 2010) 06.04.16 26th Annual Meeting of the Society for Virology 31

Results for DAA cut offs from the trainings panel  highest accuracy 93.4% (*BioRad HIV-1/2 screening EIA and modified protocol from Masciotra 2010) 06.04.16 26th Annual Meeting of the Society for Virology 32

Evaluation of “German approche” 500 specimen of “German Seroconverter Cohorte” (known duration of infection) MAA 1: MAA 1: MAA 2: MAA 2: 30.04.16 AREVIR 2016 33