Zika virus: laboratory diagnosis
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1 Zika virus: laboratory diagnosis Dr Linda Hueston Principal Scientist Arbovirus Emerging Diseases Unit CIDMLS-ICPMR Westmead Hospital
2 Laboratory Diagnosis Flaviviruses >70 members of the family All cross react with each other but don t cross protect Antigen hall of mirrors Exhibit antibody dependent enhancement Test interpretation is critical Often requires testing against panels of flaviviruses Need clinical, travel and vaccination history
3 Laboratory Diagnosis Virus isolation Sine que non Difficult to obtain with most arboviruses as viraemia is low and short lived in humans Antigen detection ELISA based using monoclonal antibodies PCR Serology Majority of diagnoses are achieved serologically
4 Laboratory Diagnosis Serology Neutralisation (quantitative) Most sensitive & specific Requires use of live virus Haemagglutination inhibition (quantitative) Cross reactive Need to be able to make own antigen CF (quantitative) Short lived, more specific than HI but still some cross reactivity Need to make own antigen
5 Laboratory Diagnosis Serology IFA (semi quantitative or quantitative) In-house need to make slides Commercial slides available but not validated ELISA In- house ELISA (semi quantitative or quantitative) IgG IgM Defined epitope blocking ELISA Commercial ELISA (semi quantitative) IgG IgM
6 Virus isolation for Zika Established cultures of ZIKV grow well in a variety of mammalian and insect cell lines The virus has been very difficult to isolate from primary specimens Partly due to the almost universally low to very low viral loads in clinical specimens. Culture methods are reserved for specialised reference laboratories, Always worth attempting as isolates are extremely valuable
7 Key points laboratory testing Nucleic acid test Real-time rtpcr assay for detection of viral RNA Serum - detectable 3 to 7 days (Israeli study suggests RNA detectable in whole blood for up to 58 days) Urine - detectable up to a month Semen - detectable up to 2 weeks (occ cases >92 days) Shorter in secondary flavi infections NEGATIVE PCR DOES NOT EXCLUDE INFECTION
8 Key points laboratory testing Nucleic acid test Longest known period between infection & sexual transmission to another days post onset Variability across sample type Don t know if presence of RNA indicates ability to transmit the virus NEGATIVE PCR DOES NOT EXCLUDE INFECTION 80% of serologic diagnoses were PCR negative
9 Serologic testing for Zika Neutralisation (use 90% endpoint, titres 20) Detects total antibody, Appears within 3 to 5 days Detectable for decades Most specific particularly in primary infections Some cross reactivity in non primary infections
10 Serologic testing for Zika IFA ELISA Detects IgG and IgM IgM detectable within 5 to 7 days for 8 to 10 weeks IgG detectable at 7 to 10 days for decades Some commercial slides available but not validated CROSS REACTIVE Commercial Euroimmun IgM detectable 8 to 12 days for 6 to 8 weeks IgG detectable 18 to 21 days NOT SPECIFIC
11 Laboratory Diagnosis Serology testing for Zika virus ELISA Commercial Euroimmun Cross reactions in IgM with YF, Den, JE, Kunjin/West Nile, Kokobera Cross reactions in IgG with YF, Den, JE, Kunjin/WNV, MVE, Kokobera, Stratford and Edge Hill Also missed early infections suggest repeat sample 21 days post onset Best used as a screen for negatives (if two samples are tested) Positives should be sent for confirmation
12 Key points laboratory testing Serology Cross reactivity presents laboratory diagnostic challenges Study released 2016 suggests that presence of preexisting dengue antibody may lead to an antibody dependent enhancement of Zika infection May explain the severity of disease seen in the Americas where dengue and YFV are endemic This has implications for vaccination options
13 Key points laboratory testing Serology In Australia antibody testing is being used as A routine antenatal screen A routine STD screen Regardless of travel and clinical history Given the issues with flavivirus serology this could lead to problems if patients return +ve results
14 Key information for laboratory testing Clinical history Onset date of symptoms Travel history dates of travel, countries visited, length of stay, type of trip. Vaccination history YFV, JE, TBE Past medical history any previous history of flavivirus infection
15 Limitations Infection may not be excluded on one sample alone Negative PCR does not exclude infection and should only be performed in symptomatic patients 80% of PCR negative symptomatic patients were IgM positive Follow-up any pos PCR with serology Follow up positive IgM s Titrate positive IgG and IgM (titres valuable) Test against a panel of flaviviruses to investigate cross reactivity These results suggest past infection with Zika virus or another closely related flavivirus or flavivirus vaccination. Further differentiation would require neutralisation testing
16 Conclusions Rapid and accurate diagnosis of ZIKV infection is an international priority. NAT has limited utility in ZIKV diagnosis because most patients are either asymptomatic or present for testing after the brief period of likely viral shedding. The greatest need and most difficult challenge is the development of commercially available antibody tests for the specific and sensitive diagnosis of recent ZIKV infection.
17 Conclusions Identifying ZIKV epitopes that do not cross-react with other flavivirus antigens could lead to virus specific tests As ZIKV becomes endemic in new areas it adds another layer to the diagnostic dilemma of flavivirus diagnosis
18 Cases Visited relatives in Caribbean for Xmas Returned to Aust febrile illness upon return requested Zika serology Zika IgG IFA: Pos (titre 640) Zika IgM IFA: Neg Den specific total antibody: Pos (titre 2560) Den IgM: Negative Neut testing: Zika: 80 Den 1and 3 Neg Den 2: 320 Den 4: 320 PCR negative
19 Acute and Convalescent testing Acute Zika IgG IFA: Pos (titre 640) Zika IgM IFA: Neg Den specific total antibody: Pos (titre 2560) Den IgM: Negative Neut testing: Zika: 80 Den 1: <10 Den 2: 320 Den 3: <10 Den 4: 320 PCR negative Convalescent Zika IgG IFA: Pos (titre 5120) Zika IgM IFA: Equvocal Den specific total antibody: Pos 5120 Den IgM: Negative Neut testing: Zika: 10,240 Den 1: <10 Den 2: 320 Den 3: <10 Den 4: 320 PCR negative Interpretation: Recent Zika virus infection, secondary flavivirus
20 Cases Visited Caribbean Returned to Aust febrile illness upon return requested Zika serology Zika IgG IFA: <10 Zika IgM IFA: <10 Den specific total antibody: Negative Den IgM: Negative PCR negative
21 Acute and Convalescent testing Acute Zika IgG IFA: <10 Zika IgM IFA: Neg Den specific total antibody: Neg Den IgM: Negative Neut testing: Zika: <10 PCR negative Convalescent Zika IgG IFA: Pos (titre 320) Zika IgM IFA: Pos Den specific total antibody: Neg Den IgM: Negative Neut testing: Zika: 1,280 PCR negative Interpretation: Recent Zika virus infection
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