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

30.04.16 AREVIR 2016 1

Estimation of new HIV-infections/new HIV-diagnosis

Diagnostic labs (n=~82) DSS

(DriedSerumSpots)

Anonymus HIV-Report (IfSG) RKI

~ 60%

• f all HIV-

new diagnosis Part of routine HIV-surveillance since 2011 Sampling strategy is based on HIV-reporting system:

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Representativeness of InzSurvHIV

(2011-2015) total DSS

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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

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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

Evolution of laboratory markers during HIV infection

recent infection  long-standing infection

(< 6 months) (> 6 months)

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Course of HIV-infection

HIV-specific IgG: Increase of Titer + Avidity 1-2 years

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Serological recency assays

i) BED IgG-Capture ELISA (BED-CEIA)

• proportion of HIV-1 specific

among total IgG

• applied in Germany

HIV-1 specific IgG total IgG

ELISA-plate reader

OD n < 0.8 = recent

Based on increase of HIV-spec. IgG i) TITER or ii) AVIDITY

ii) Avidity assays 2-well assay: Addition of mild denat. reagent in one well

• > dissotiation of low binding antibodies

AI < 40% = recent

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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
• n ART

⇒ High false classifications ⇒ i.p. false recent => over estimation of incidence

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Workflow in the Lab

Sample receipt BED IgG capture ELISA RNA extraction (magnetic beads) Viral load and RT-PCR Population sequencing + HIV-genotyping A) HIV-1 subtype B) HIV-resistance

False recent rate: 14%

(Loschen et al. 2008)

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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

4x 100µl

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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

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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

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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

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Results (1)

HIV-1 genotypes (PR/RT) 2013-2015/I: n= 1197

[CI: 9,0; 12,5]

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 Germany 57.7 Other 20.2 Not reported 22.1 Country of infection Germany 61.7 Other 12.3 Not reported 26.1

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Results (2)

HIV-1 genotypes (PR/RT) 2013-2015/I: n= 1197

Transmitted drug resistance in recent infections

• stable proportion

Ptend TDR > 0.5

Proportion (%) [CI: 9,0; 12,5]

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Results (2)

HIV-1 genotypes (PR/RT) 2013-2015/I: n= 1197

Transmitted drug resistance in recent infections

Ptrend > 0.05

• stable proportion

Proportion (%)

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Results (3)

HIV-1 genotypes (PR/RT) 2013-2015/I: n= 1197

HIV-1 B and non-B subtypes in recent infections

Ptrend nonB = 0.002

• significant increase of non B-infections

Proportion (%)

[CI: 23,7; 28,8]

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Results (4)

HIV-1 genotypes (PR/RT) 2013-2015/I: n= 1197

HIV-1 B and non-B subtypes in recent infections

Ptrend subtype A = 0.01 Ptrend CRF/URF > 0.05

• significant increase of subtype A infections

Proportion (%)

• A (East europe + Asia + Africa)
• Rare recombinants (CRF + URF)

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Results (5)

HIV-1 genotypes (PR/RT) 2013-2015/I: n= 1197

In which populations / transmission groups subtype A is circulating?

• Subtype A in Germans (50%)
• + East europeans (30%)

Origin

Proportion (%)

Transmission route

• Subtype A in MSM (53%)
• + by heterosexuel contacts (38%)

Proportion (%)

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Results (6)

HIV-1 genotypes (PR/RT) 2013-2015/I: n= 1197

Subtype A infections according origin + transmission route

Ptrend D/MSM = 0.05

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Results (6)

HIV-1 genotypes (PR/RT) 2013-2015/I: n= 1197

Which recombinant forms were identified? 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) CRF35_AD (2) CRF44_BF (2)

URF ( n=26)

Jan 2015 Aug 2015

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Results (6)

HIV-1 genotypes (PR/RT) 2013-2015/I: n= 1197

Transmission route

• 69% in MSM
• 35% by heterosexuel contacts

n= 20 n= 14

• 55% in Germans

Origin

n= 21 2 1 2 5 3 2

In which groups rare recombinants (CRF/URF) are circulating?

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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

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Testing for recency of infection => new approach: Multi Assay Algorithm (MAA)

(*Laeyendecker et al 2012)

Cut offs

Rated successively:

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Evaluation of a `German MAA´

~𝟖𝟖𝟖 specimen of “German Seroconverter Cohorte” (known duration of infection) MAA:

≥1.0 ≥85 <100

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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.

Thank you for your attention!

Diagnostic labs

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Patients & Methods

• Trainings Panel (n=136)

reflecting the present German situation in newly diagnosed HIV cases (2013/14) Distribution of HIV-1 subtypes Proportion (1:2)

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)

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Estimation of DAA cut-offs

Dual Assay Algorithm

1. Trainings panel: BED-CEIA (ODn) + BioRad Avidity *(AI%) 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)

91

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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)

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Evaluation of “German approche”

500 specimen of “German Seroconverter Cohorte” (known duration of infection) MAA 1: MAA 1: MAA 2: MAA 2: