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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 Interpretation only possible if this information is provided! ESCMID eLibrary 23 © by author Rapid test (RDT) • 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 22 April,ESCMID 2017 eLibrary 26 © by author Diagnostic opportunities in urine • 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 22 April,ESCMID 2017 eLibrary 27 © by author Arbovirus detecion in semen: Zika
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