Differential Arbovirus Diagnostics Dr

Differential Arbovirus Diagnostics Dr. Isabella Eckerle Institute of Virology University of Bonn Medical Centre Bonn, Germany

ECCMID ESCMID2017, Vienna PREPARE eLibrary is funded by the European Commission under grant number 602525 © by author Arboviruses – global relevance

• Emerging group of viruses • Complex ecology: Spread and emergence depending on a large variety of factors (reservoir, vector, climate, environment, modification of landscapes, human behaviour, globalization, virus evolution) • Vector control is complex; eradication of vectors or disease not possible at this stage  Arboviruses will almost certainly become an increasing health concern in many countries, including Europe! ESCMID eLibrary 2 © by author Clinical presentation

Febrile syndrome Hemorrhagic syndrome • Fever & malaise • Low platelet count, liver enlargement • Headache & retro-orbital pain • Petechiae • Myalgia • Spontaneous or persistent bleeding • Shock Neurological syndrome • Meningitis, encephalitis, myelitis Arthralgia, arthritis, rash • Convulsion & coma • Exanthema or maculopapular rash • Paralysis • Polyarthralgia & polyarthritis

DD: Malaria, other viruses (Influenza, Enterovirus, Herpesvirus, Hantavirus, Ebola, Rabies...), typhoid fever, bacterial meningitis, other bacterial infections, non-communicable etiologies ESCMID eLibrary 3 © by author Clinically important arboviruses

Virus family Virus genus Species examples Flaviviridae Flavivirus Dengue 1-4 (DENV) West Nile virus (WNV) Yellow fever virus (YFV) Zika virus (ZIKV) Tick-borne encephalitis virus (TBEV) Togaviridae Alphavirus Chikungunya virus (CHIKV) O‘nyong Nyong virus (ONNV) Mayaro virus (MAYV) Sindbis virus (SINV) Ross River virus (RRV) Bunyaviridae Nairovirus, Phlebovirus, Crimean Congo hemorrhagic virus (CCHFV) Orthobunyavirus Sandfly fever virus (SFV, TOSV) Rift Valley fever virus (RVFV) Oropouche virus (OROV) Reoviridae Coltivirus, Seadornavirus Colorado tick borne fever (CTFV) ESCMIDBanna eLibrary virus (BANV) 4 © by author Distribution and syndrome diversity

Arboviruses are not exclusively tropical diseases! Cleton et al., Plos NTD 2015 ESCMID eLibrary 5 © by author ?

22 April,ESCMID 2017 eLibrary 6 © by author Arboviruse diagnostics - important aspects

• Travel history: • Where? Geographical range! • How long? Likelihood of exposure! • Date of return? Incubation period! • Exposure, activities, reason for travel Likelihood of exposure! • Onset of symptoms Incubation period! • Clinical presentation Hints towards a certain syndrome • Other risk factors (medical conditions, pregnancy, immunosuppression, ....) Interpretation of results (serology) • Vaccination history Cross-reactivity in serology

ESCMID eLibrary 7 © by author Now it‘s your turn!

How many samples received in a diagnostic laboratory are accompanied by the information on „days post onset“?

A) 75% B) 42% C) 15% D) 5.5% E) 0.5%

22 April,ESCMID 2017 eLibrary 8 © by author Now it‘s your turn!

How many samples received in a diagnostic laboratory are accompanied by the information on „days post onset“?

Price VA et al. General A) 75% physicians do not take adequate travel histories. B) 42% J Travel Med. 2011 C) 15% Travel history in less than 20 % of patients D) 5.5% presenting with fever, rash, diarrhea, vomiting, E) 0.5% jaundice

Source: Cleton N et al., Travel Med Infect Dis. 2014 22 April,ESCMID 2017 eLibrary 9 © by author Which patient materials are useful for arbovirus diagnostics?

• Serum / plasma  Virus detection / Antibodies • CSF  Virus detection / Antibodies • Urine (WNV, DENV, ZIKV)  Virus detection • Semen  Virus detection • Saliva  IgA • Tissues (post mortem), amniotic fluid (Zika) etc.  Virus detection

ESCMID eLibrary 10 © by author Virological methods– Overview

• Virus isolation (cell culture)

• Detection of viral nucleic acid (RT-PCR, real-time RT-PCR)

• Detection of virus antigen (ELISA)

• Detection of antibodies (Immunofluorescence, ELISA, NT)

ESCMID eLibrary 11 © by author Confidence Accessability

Virus Genome Antigen Serology isolation detection detection IgM/IgG Neutra- lization assay

Costs & lab requirements! ESCMID eLibrary © by author Virus isolation

• BSL-2/3/4 facilities needed depending on virus species • Most arboviruses are category 3 (Zika: BSL-2; DENV, CHIKV, WNV, TBEV: BSL-3; CCHF: BSL- 4) • Time consuming, expensive, experienced staff needed • Role in routine diagnostic negligible (in most countries) BUT important for emerging virus surveillance/research/pandemic preparedness! • Same laboratory requirements/considerations also necessary for virus neutralization assay (serological test method)

ESCMID eLibrary 13 © by author Detection of the viral genome I

• Methods: One-step RT-PCR, nested RT-PCR, real-time RT-PCR, LAMP • Detection of viral genome (serum, CSF, urine, tissue) = confirmative of infection • Multiplex-PCR assays: Assessment of several viruses at the same time • PCR: rather expensive methods • More ‘exotic/rare’ arboviruses: PCR diagnostic usually limited to a small number of (reference/specialized) laboratories • Primers need to cover the local strains/diversity of circulating variants

ESCMID eLibrary 14 © by author Detection of the viral genome II

• Dengue: Differentiation of serotypes possible by real-time RT-PCR • Possibility to sequence PCR products (interesting from an epidemiological point of view/surveillance) • Additional options: Detection of RNA in autopsy tissue specimens by immunofluorescence or immunohistochemical methods (role in routine diagnostics rather low)

Eckerle et al., EID 2015 ESCMID eLibrary 15 © by author Limitations & pitfalls - direct virus detection • Viremic period of most arboviruses is short! • Viral load is low (often close to detection limit) • Humans are dead-end hosts: WNV, JEV, TBEV, SFV • Peak for viraemia before symptoms / in early (unspecific) symptomatic period • In travellers: late presentation after return travel (day 5-7)

ESCMID eLibrary 16 © by author Diagnostic opportunities in arboviruses

Serology

Virus isolation RNA detection IgM IgG

Window for virus detection short! Antibody response Viraemia

....-2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 ... Onset of symptoms (days) ESCMID22 April, 2017 eLibrary © by author Virus kinetics in dengue virus infection Dengue: Prolonged detection of NS-1 compared to viral RNA

Guzman et al. Nat Rev Microbiol 2010 22 April,ESCMID 2017 eLibrary 18 © by author Virus kinetics in CCHF 

22 April, 2017 19 ESCMID eLibraryErgonul et al. Lancet Inf Dis 2006 © by author Serology • Antibody detection: IgG, IgM, IgA • Methods: ELISA, IFT, Immunochromatographie • Seroconversion • IgG/IgM 4x titer increase • 2nd sample needed with 14 day interval • IgM in liquor in case of neurological disease  confirmative • Mosaic-chips (immunofluorescence): Detection of antibodies to multiple flaviviruses at the same time • Neutralization assay (requirements similar to virus isolation, also cross-neutralization possible) • IgA / IgG in saliva (experimental) ESCMID eLibrary 20 © by author Now it‘s your turn!

For how many initial samples is a second sample provided?

A) 75% B) 45% C) 25% D) 20% E) < 10%

22 April,ESCMID 2017 eLibrary 21 © by author Now it‘s your turn!

For how many initial samples is a second sample provided?

A) 75% B) 45% C) 25% D) 20% E) < 10%

Source: Cleton N et al., Travel Med Infect Dis. 2014 22 April,ESCMID 2017 eLibrary 22 © by author Pitfalls in serology

Cross-reactivity with vaccine antibodies •Yellow fever virus (South Americas, Africa) •Japanese encephalitis virus (Asia) •Tick-borne encephalitis virus (Central and Eastern Europe) •Dengue virus (Central and South Americas) Suboptimal antibody response •Young, old, pregnant, immunosuppression (“YOPI”): low/no immune response •Prolonged viremia & IgM detection possible Previous infections (Antigenic sin) •Stimulation of memory B cells from a previous infection

• Also: lateral flow test, bedside test, dipstick test • Easy to use, quick results (15-30 mins) • Sensitivity and specifity varies between manufacturers • Cross-reactivation positive: e.g. reports of positive DENV- RDT results in acute Zika infection (under debate)* • Most commonly used test: Dengue NS-1 or Dengue IgG/IgM + NS-1 • Useful in outbreak situations & low resource countries • Many new formats in development *Gyurech et al. False positive dengue NS1 antigen test in a traveller with an acute Zika virus infection imported into Switzerland. Swiss Med Wkly. 2016; Matheus S et al. Specificity of Dengue NS1 Antigen in Differential Diagnosis of Dengue and Zika Virus Infection. Emerg Infect Dis. 2016 ESCMID eLibrary 24 © by author Arbovirus diagnostics – future perspectives

Chan et al. Rapid, Affordable and Portable Priye et al. A smartphone-based diagnostic Medium-Throughput Molecular Device for platform for rapid detection of Zika, Zika Virus. Sci Rep. 2016 Dec 9;6:38223 chikungunya, and dengue viruses. Sci Rep. 2017 Mar 20;7:44778 ESCMID eLibrary 25 © by author Beyond blood testing: Arboviruses in urine and semen

• WNV, DENV, ZIKV: Detection in urine was shown in several studies • Potential to extent period of direct virus detection • Zika: Prolonged virus shedding in urine compared to serum; CDC: NAT testing recommended of urine samples (<14 days after symptom onset). • TBEV: Reports of prolonged PCR positivity in urine

• 23 men in UK with proven symptomatic ZIKA • PCR semen for ZIK RNA • 13/23 (56.5%) strong positive • 10/16 (62.5%) in those diagnosed by PCR Emerg Infect Dis. 2017 • 2/7 (28.6%) of those diagnosed by serology alone

• less virus (higher Ct value) with time in all patients • Some positive to about 60 days • longest positive sample 91 days • one patient near threshold for positivity 167 days • Infectivity unknown

No recommendation at this time Possible scenarios: • PCR negative but Zika later shedded in semen • IgM negative, but testing performed before onset of IgM response • IgM negativ, but testing performed after IgM response has waned Testing of semen or vaginal fluid? No recommendation at this time Possible scenarios: • Interpretation of PCR-positive findings (RNA=infectivity?) • Intermittend shedding possible (how many samples needed to exclude infection?) • Evaluation of sensitivity/specifity 22 April,ESCMID 2017 eLibrary 29 © by author Patient advice regarding pregnancy

US: Women and Men with Possible Zika Virus Exposure Who Desire Pregnancy •Female exposure: wait at least 8 weeks after symptoms or last possible exposure •Male exposure: wait at least 6 months after symptoms or last possible exposure •Waiting period: condoms for vaginal, anal, and oral sex, in addition to their chosen method of birth control, or to not have sex. Source: https://www.cdc.gov/zika/hc-providers/reproductive-age/desire-pregnancy.htmlMale with or without symptoms of ZIKV

UK: Precautions for possible sexual risk •Female with or without symptoms of ZIKV: Barrier methods should be used for 8 weeks following return from an area with high or moderate ZIKV risk or after last exposure •Male with or without symptoms of ZIKV: Barrier methods should be used for 6 months following return from an area with high or moderate ZIKV risk or after last possible ZIKV exposure

Source: http://travelhealthpro.org.uk/news/4/zika-virus--update-and-advice-for-travellers-including-pregnant-women-and-those- planning-pregnancy 22 April,ESCMID 2017 eLibrary 30 © by author Now it‘s your turn!

What will be the next emerging arbovirus keeping us busy?

A) Mayaro virus B) O‘nyong nyong C) Ross River Fever Virus D) Tahyna Virus E) Yellow Fever virus in Asia

What will be the next emerging arbovirus keeping us busy?

A) Mayaro virus B) O‘nyong nyong C) Ross River Fever Virus D) Tahyna Virus E) Yellow Fever virus in Asia

22 April,ESCMID 2017 eLibrary 32 © by author Thanks to: Natalie Cleton, Ramona Mögling, Chantal Reusken, Erasmus Medical Centre; Nick Beeching, Liverpool School of Tropical Medicine; PREPARE team; ESCMID Thank you for your attention!