Obstacles for the diagnosis of Zika virus

Jan Felix Drexler

Institute of Virology, University of Bonn Medical Centre

German Centre for Infection Research

Obstacle 1Areas most affected by arboviruses

are often resource-limited

Cleton et al. PNTD, 2015

In house testing: Often the only affordable resource

Some challenges, Brazilian example:

• Up to 6 weeks delays for receipt of reagents(primers << LNA / MGB probes)

• 100% import tax – unaffordable pricing

• Difficult maintenance (Power cuts, costly repairs…)

Drexler et al. PMED, 2009

Transfer of essential reagents allowsrobust work in resource-limited settings

Zika virus:universal control RNA for assay

validation and quantification

Corman et al., Bull WHO, 2016©Asuragen

Armored RNA:Thermostable, quantified, non-infectious

Obstacle 2Nucletide mismatches between viral genomes and

primers/probes may affect assay sensitivity

Drexler et al., JCM, 2007

10,000-100,000x deviation

Zika virus: Up to 10 potential mismatches with existing assays already

Corman et al., Bull WHO, 2016

Need forconstantmonitoring

Obstacle 3 - Specificity

Many arboviruses co-circulate

Need for thoroughassessments

Need for virus panels(example EVA)

Corman et al., Bull WHO, 2016

Obstacle 4 - Sensitivity

Zika virus: Most around 5 copies/reaction,1000 in one assay

Need for controls

Need for EQACorman et al., Bull WHO, 2016

Obstacle 5 Viral loads are low for some arboviruses

ZIKV loads about 10000 copies/mL: close to technical limit of qPCR limited capacity of PCR to exclude infection Need for serology

Corman et al., Bull WHO, 2016

Even very good assayswill miss ca. 5%

Related issue: Blood safety

• WNV: similarly lowviral loads

• Fatal transfusion-associated cases due to false-negative NAT in minipool (6x)

• First cases in Brazil

Paired specimensfrom 6 outbreakpatients, same day

Obstacle 6Selection of feasible specimens

Corman et al., Bull WHO, 2016

Florida: Urine positive > than blood

MMWR, May 2016; Pessôa et al. Eurosurveillance, 2016

Brazil: Blood positive > than urine

Semen: Prolonged ZIKV shedding at high loads?

62 days postsymptomonset

EID, May 2016

Lancet ID, April 2016

100,000x higherthan in blood & urine

Whole blood positive for longerperiod?

• Has been suggestedbefore for WNV, Chikungunya…

• New difficulties in extraction methods, PCR inhibitors

• Validation neededon large patientcollective

Murray et al., EID, 2017

• Necessity to co-detect Zika, Dengue andChikungunya viruses

• Low ZIKV concentrations may challengemultiplex approaches

US CDC’s “Trioplex”: May miss 40% of Zika infections

28. September, 2016

Obstacle 7Multiplexing ususally decreases PCR

sensitivityDENV-1,-2,-3forward CAGATCTCTGATGAACAACCAACG

DENV-2forwardC→T CAGATCTCTGATGAATAACCAACG

DENV-3forwardC→T CAGATTTCTGATGAACAACCAACG

DENV-4forward GATCTCTGGAAAAATGAAC

DENV-1,-3reverse TTTGAGAATCTCTTCGCCAAC

DENV-2reverse AGTTGACACGCGGTTTCTCT

DENV-2reverseA→G AGTCGACACGCGGTTTCTCT

DENV-4reverse AGAATCTCTTCACCAACC

DENVprobeA FAM CTCGCGCGTTTCAGCATAT BHQplus

DENVprobeB FAM CTCTCGCGTTTCAGCATAT BHQplus

DENVprobeC FAM CTCTCACGTTTCAGCATATTG BHQplus

DENVprobeD FAM CTCACGCGTTTCAGCATAT BHQplus

Dengue Waggoner et al. JCM 2013

Chikungunya Panning et al. EID 2008ChikSI TGATCCCGACTCAACCATCCT

ChikSII CCGACTCAACCATCCTGGAT

ChikAsI GGCAAACGCAGTGGTACTTCCT

ChikAsII GGCAGACGCAGTGGTACTTCCT

ChikP FAM TCCGACATCATCCTCCTTGCTGGC BHQ-1

Zika Waggoner et al. EID 2016

ZCD Probe CYGTTGTGGATGGAATAGTGG

ZCD Forward CAGCTGGCATCATGAAGAAYC

ZCD Reverse 2 CACCTGTCCCATCTTTTTCTCC

ZCD Reverse 1 CACTTGTCCCATCTTCTTCTCC

This combination:15 primers6 probes3 if internal control – 24 oligos!

Unspecific bindingDimer formationCompetition…

Example from our lab:a Dengue virus qPCR in 1-/2-/3-plex

Single

Double

Triple7 cycles= ca. 100 fold deviationbetween 1- & 3-plex

Risk of missing> 50%!

Current efforts: Universal quantifiedcontrol for CHIKV, DENV & ZIKV

Panning et al. EID 2008Waggoner et al. JCV 2016

Waggoner et al. JCM 2013Leparc-Goffart et al. JCV 2009Drosten et al. JCM 2003Alm et al. PNTD 2014

Corman et al. Bull WHO 2016Lanciotti et al. EID 2008Waggoner & Pinsky JCM 2016Faye et al. Virol J. 2013Pyke et al. PLOS 2014Tappe et al. EID 2015Waggoner et al. EID 2016

Free of chargesupply foracademicresearch

Serology

• Complicated in endemicsettings by co-circulationof antigenically relatedviruses: DENV 1-4, YFV, Ilheus, Rocio…

• Less reliable IgMcompared to previouslyunexposed individuals

• IgA? IgG subclass assays(despite shorter half-life)?

CDC.Gov for DENV diagnostics

Available methods

• Euroimmun ELISA: NS1-based, 100% specific??? No! But still OK.

• Brazil bought several million tests for LACENs

• MAC-ELISA: good for IgM, but time-consuming, robustness?

• Do we need IgG for cohort studies?

• Even for NTs: cross-reactivity for DENV and ZIKV possible (what is the gold standard? need forrepeated sera for titer changes, seroconversion)

Virology, BonnVictor Max CormanAndrea RascheMonika Eschbach-BludauSebastian BrüninkTobias BleickerChristian Drosten

AMU, FranceCecile BarontiXavier de Lamballerie

AMC, NetherlandsMartin Grobusch

EMC, RotterdamChantal ReuskenMarion KoopmansSuzan Pas

Bernhard Nocht Institut, HHJonas Schmidt-ChanasitDaniel Cadar