pcr vs. serology example mers how to interpret … · jan felix drexler souhaib aldabbagh sebastian...

Copyright: Marcel A. Müller

Dr. Marcel A. Müller

Institute of Virology

University of Bonn Medical Center Bonn, Germany

[email protected]

www.virology-bonn.de

January 20th, 2016

CONSISE Meeting Paris

PCR vs. Serology – example MERS

How to interpret results in

seroepidemiologic studies

mailto:[email protected]

http://www.virology-bonn.de/


1. Reliable diagnostic tools available?

2. What kind of samples/ parameters should be

tested?

3. Optimal time points for testing?

Challenges in diagnostics


Incre

ase

Vir

al

load

/ an

tib

od

yti

ter

Time post infection (days)

Incu

bati

on

tim

e

Virus

IgM

Antibody: IgG

10-143-10 180

Limit of

detection

Neutralizing IgG

(long time detection

and protection)

General virus infection

PCRSerology

Overview of MERS-CoV real-time PCR assays

Screening with upELimit: 3.4 RNA copies/reaction

Confirmation 1ALimit: 4.1 RNA copies/reaction

Amplification & Sequencing

Amplification & Sequencing

Features:

No cross-reactivity with clinical samples containing other respiratory viruses

No cross-reactivity with other hCoV isolates

Suitable for all known MERS-CoV isolates/sequences as of Dec 2015

Basis for development of commercial kits

Corman VM; Euro Surveill. 2012 Sep 27; Corman VM, Müller MA; Euro Surveill. 2012 Dec 6


Incre

ase

Vir

al

load

/ an

tib

od

yti

ter


Incu

bati

on

tim

e

Virus

IgM

Antibody: IgG

10-143-10 180

Limit of

detection

Neutralizing IgG

(long time detection

and protection)

Serology

PCRSerology


Serodiagnostic assays

Recombinant IFA (rIFA)

Antigen: complete MERS Spike

Plaque Reduction Neutralization Test

Virus or Virus-like particle-based

ELISA

Antigens:

Recombinant S1 Spike Subdomain

Recombinant N protein

Inactivated virus

Virus immunofluorescence assay (vIFA)

Antigen: MERS-CoV


Challenge: Vast diversity of coronaviruses

circulating in animals and human

Drexler et al., AVR 2014

MERS

SARS


Human Coronaviruses

Name Genus Clinical picture

HCoV-229E Alpha

10% of all “Common Colds”

>90% of humans have IgG

HCoV-NL63 Alpha

HCoV-OC43 Beta

HCoV-HKU1 Beta

SARS-CoV Beta Severe acute respiratory syndrome

Diagnostic issue:

Cross-reactive antibodies in low serum dilutions


Cross-reactivity CoV

Non-structural proteins (NSP)

CONSERVEDStructural proteins

VARIABLE

MERS-CoV

HCoV-OC43

HCoV-HKU1

HCoV-NL63


Example: Non-specific reactivity in CoV

serodiagnostics (serum dilution 1:100)

Negative

serum

Cross-reactive

serum

MERS patient

serum

Fu

llv

iru

s

IFA


Differential serology

Recombinant spike-based IFA (rIFA)

VeroB4 +

Spike encoding plasmid

24h

fix (4% PFA)

permeabilize (acetone/methanol; 1:1)

stain (heat-inactivated patient serum)

spotting,

drying

6h

229E NL63 OC43 HKU1 SARS MERS

Transfection


Non-specific reactivity in CoV serodiagnostics

Negative

serum

Cross-reactive

serum

MERS patient

serum

Fu

llv

iru

s

IFA

Reco

mb

inan

t

IFA

Buchholz/Müller et al., Eurosurveillance 2013

rIFA more specific!


DifferentialrIFAassaysinMERS-S1ELISAreactivesera vIFA ELISA(Ratio) ELISAResult rIFA

SubjectID MERS MERSS1 MERSS1 229E HKU1 MERS NL63 OC43 SARS

3 neg. 0,278 reactive 320 neg. neg. 320 320 neg.

44 neg. 0,561 reactive neg. 320 neg. 320 320 neg.

94 neg. 0,207 reactive 320 320 neg. 320 >1000 neg.

161 neg. 0,269 reactive 320 320 neg. neg. >1000 neg.


185 10 0,182 reactive >1000 320 neg. 320 >1000 neg.


211 neg. 0,748 reactive 320 320 neg. 320 320 neg.

217 neg. 0,424 reactive 320 320 neg. 320 320 neg.


229 neg. 0,278 reactive neg. 320 neg. 320 320 neg.


From Drosten, NEJM 2014 (supplementary data)

Non-specific sera show high titers against

other HCoVs


Goldstandard: Plaque Reduction-Neutralisation-Test

(PRNT)

1:160

1:20

1:20 1:40 1:80

Serum

1:2 1:2 1:2

1:20 1:40 1:80 1:160

50 PFU

Y

Y

Y

Y

No Infection No plaques

Infection Plaques

Y

Y

Y

Y

Neutralized

Y

Y

Y

Y

Not neutralized

1h


Screening

Stage 1

Stage 2

Established staged serological testing

algorithm for camels and humans

S1 ELISA

S1 ELISA rIFA full S

S1 ELISA rIFA full S PRNT

Drosten et al. 2014, NEJM & Müller et al. 2015, Lancet ID

Testing dilution ELISA, vIFA

1:100

Starting dilution PRNT:

1:10

Cutoff PRNT50: 1:20

ELISA and vIFA commercial

rIFA and PRNT: in-house

vIFA

Testing dilution rIFA:

1:40



2. What kind of samples/ parameters should

be tested?




PCR: Which samples?

Corman et al., CID 2015

BAL, Sputum optimal for MERS detection

Lower resp. tract Upper resp. tract

VIR

AL

LO

AD

(lo

g c

op

ies

per

ml)

NO

. S

AM

PL

ES

93% pos. 47.6% pos.

Mean:

5.0x106

Mean

2.0x104

Low viral loadHigh viral load


Other body compartments?

14.6% pos.

2.4% pos.

30.6% pos.

1.6x104

1.3x102

2.5x103



Antibodies: IgM or IgG?

IgM

IgG


N=37 MERS patients

IgM and IgG increase is comparable

IgM higher risk of cross-reactivity

IgM indicates recent infection



2. What kind of samples should be tested?




Time point for PCR testing


High viral loads up to 15 days post diagnosis

VIR

AL

LO

AD

(lo

g c

op

ies

per

ml)

NO

. S

AM

PL

ES

93% pos.

Mean:

5.0x106

High viral load


3-6 years

Incre

ase

Vir

al

load

/ an

tib

od

yti

ter


Incu

bati

on

tim

e

Virus

105 180

Limit of

detection

Summary: MERS-CoV infection and

diagnostics

PCRSerology

PCR: up to day 15 post diagnosis high viral loads

(>10e6 copies per ml) approx. 25 days post infection

20 90


3-6 years

Incre

ase

Vir

al

load

/ an

tib

od

yti

ter


Incu

bati

on

tim

e

IgM

IgG

105 180

Limit of

detection

Neutralizing IgG

Summary: MERS-CoV infection and

diagnostics

PCRSerology

Reinfection

possible?

Serology: IgM, IgG increase simultaneously approx 9

days post onset of symptoms

20 90


Application & Interpretation

1. Identification of the source of infection

2. Clinical/ Outbreak investigations

3. Cross-sectional studies

4. Case-contact studies


1. Source: Livestock screening

MERS-CoV antibodies in dromedaries

Reusken/Haagmans/Müller et al. Lancet ID, 2013

Mohamed Abdiwahab/AFP/Getty Images

SE

RO

LO

GY


Young camels as source of infection

Wernery et al. EID 2015

SerumantibodyELISA

RNA detection RT-PCR (%)

Virus isolation

Adults, >4 yrs 298/310 (96.1) 0/250 (0) 0/12 (0)

Subadults, 2–4 yrs 328/340 (96.5) 10/344 (2.9) 1/14 (7.1)

Calves, <1 yr 92/108 (85.2) 24/68 (35.3) 6/44 (13.6)

Young camels < 1 year virus-positive!

SE

RO

LO

GY

+P

CR


Long-term and widespread circulation of

MERS-CoV in dromedaries

1983/84 Sudan/Somalia

Müller et al. EID 2014

1992-96Saudi Arabia

Alagaili et al.mBio 2014

2009/10Saudi Arabia

Alagaili et al.mBio 2014

1997Egypt

Müller et al.EID 2014

2003UAE

Meyer et al. EID 2014

2013UAE/Oman/

Egypt/ Jordan

Meyer et al. EID 2014Reusken TLID 2013Chu et al. EID 2013Reusken et al.Euro Surv 2013Perera et al.Euro Surv 2013

1992Kenya

Corman et al EID 2014

2010/11Ethiopia/ Nigeria

Reusken et al.EID 2014

2009Tunisia

Reusken et al.EID 2014

Red=Africa

Black=Arabian Peninsula

SE

RO

LO

GY

+P

CR


MERS-CoV originates from Africa

Nigeria

Egypt

Arabian

Peninsula

Korea

First human isolate

Egypt

PC

R


2. Outbreak: Jeddah April 2014

PCR diagnostic laboratory artifact? NO CONTAMINATION

Increase of zoonotic transmission?

Mutant Virus?

Drosten et al. 2014, CID

2014

PC

R


Monophyly of Jeddah outbreak viruses

Jed

dah

Single introduction of virus in Jeddah outbreak

7 full-length genomes

Jeddah viruses monophyletic


PC

R


Diagnostic laboratory artifact? NO CONTAMINATION

Increase of zoonotic transmission? NO

Mutant Virus?


2014

PC

R

Copyright: Marcel A. MüllerDrosten, Muth, Corman, Hussain et al., CID 2014

No hints toward

changes in shedding

from patients

No relevant / unique

changes in spike

ALIGNMENT OF SEQUENCES

PC

R


Nosocomial outbreak in Jeddah

Nosocomial infections starting at

King Fahd Hospital



Tested >10,000 serum samples from all 13 provinces

3. Cross-sectional serosurvey in

Saudi Arabia

Müller et al. 2015, Lancet ID

SE

RO

LO

GY

AGE RANGE


Screening 152 (1.5%)

Stage 1 17 (0.2%)

Stage 2 15 (0.2%)

Results cross-sectional study

S1 ELISA

S1 ELISA rIFA full S

S1 ELISA rIFA full S PRNT

Total N=10,009


SE

RO

LO

GY



-15/10,009 (0.15%) in 6/13

provinces had MERS

antibodies (asymptomatic!)

-up to 23-fold increase in

camel-exposed individuals

Seroprevalence in Saudi Arabia


Saudi-Arabia

26 Index MERS Patients

–

280 Household Contacts

Drosten et al. 2014, NEJM

Case-contact studyS

ER

OL

OG

Y+

PC

R

Copyright: Marcel A. MüllerDrosten, New England Journal of Medicine 2014

26 MERS Patients

RT-PCR

7 positive

280 Contacts

Serology

5 positive

12 transmissions in

26 households, max 1 transmission per

householdR < 0.5


Overview/ Summary

Study PCR Serology

1. Source -Identify virus and putative mutations-Phylogenetic analyses-Monitor acute infection in animals

-Largescale screening of livestock-Retrospective detection-Infection dynamics in livestock

2. Outbreak -Identify virus and mutations-Phylogenetic analyses-Monitor patients and contacts-Quarantine decision making-Prognostic value (e.g. viral load andoutcome of disease)

-Identify asymptomatic cases, contacts (e.g. HCW)-Retrospective control ofquarantine success-Prognostic value (antibody kineticsand outcome of disease)

3. Cross-sectionalserosurvey

Not applicable -Largescale screening of human sera for „background“ seroprevalence-Seasonality

4. Case-contact -Identify acutely infected contacts-Quarantine decision making-R0 value estimates

-Identify unrecognized(asymptomatic) contacts-R0 value estimates


Open questions

R0<1

Young camels

as amplifiers

Hospital

outbreaks„Silent“

transmissions

MERS-CoV a common cold virus for

camels! Why?

Has camel MERS-CoV evolved towards

better transmissibility? Look at diversity of

camel-associated MERS-CoV in Africa, old

respiratory samples needed!

Influence of husbandry, farming?

Seasonality? Parturition differences

between African countries and Arabian

Peninsula!

Are there human cases in Africa? Lack of

testing, predispositions, co-morbidities?

MERS: Why 2012? Only on the Arabian Peninsula?


Intervention?

Hygiene

In hospitals, ER

Avoid contact with (young)

camels

Vaccination

MVA-based vaccine:

Phase 1 clinical trial

planned

Proof of principle

vaccination in camels

showed protection

(Haagmans et al Science

2015)Source: CDC und REUTERS/Faisal Al Nasser


Capacity building/ workshops

First WHO-MERS diagnostic workshop in Dubai in 2015 organized

by WHO-EMRO, University of Bonn, MOH Dubai, CVRL and

University of Hong Kong

Increase awareness, improve quality


a n t i gone

Bonn:

Victor Corman

Doreen Muth

Isabella Eckerle

Jan Felix Drexler

Souhaib Aldabbagh

Sebastian Brünink

Acknowledgments

Funding: BMBF (SARS), DFG

Africa, DFG SPP1596, EU grants

ANTIGONE, EVA, EMPERIE,

COMPARE, PREPARE, ZAPI

ERASMUS Rotterdam, NL

Bart Haagmans

Ron Fouchier

Ab Osterhaus

V. Raj Stalin

Chantal Reusken

Marion Koopmans

EUROIMMUN AG Lübeck

Erik Lattwein

Kai Fechner

Katja Steinhagen

Global Centre for Mass

Gatherings Medicine (GCMGM),

Ministry of Health, Riyadh, KSA

Malak Al-Masri

Abdulhafeez Turkestani

Raafat F Alhakeem

Abdullah M Assiri

Ziad A. Memish

Utrecht University

Berend-Jan Bosch

Huihui Mou

ILRI, Kenya

Jörg Jores

Mario Younan

National Vet Institute, Sweden

Set Bornstein

CVRL, UAE

Ulrich Wernery

All volunteers and

assistants for sampling

Müller lab:

Benjamin Meyer

Andrea Sieberg

Daniel Ritz

Daniela Niemeyer

Jan Papies

Eva-Maria Klein

Robert Wollny

Tasnim Suliman

Stephan Kallies

Ute Winke

John Hopkins Aramco Healthcare,

Dhahran, KSA

Jaffar A. Al-Tawfiq

Saudi Center for Disease

Control, Riyadh, KSA

Ali M. Albarrak

University of Edinburgh, UK

Paul WikramaratnaKing Abdulaziz Medical City

&Advisor Royal court,

Riyadh, KSA

A A Alrabeeah

National Health Laboratory,

Riyadh, KSA

Ali M. Al-Shangiti

Head:

Christian Drosten

pcr vs. serology example mers how to interpret … · jan felix drexler souhaib aldabbagh sebastian...

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