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Zika virus   2018-2                                                         

Nucleic Acid Based Diagnostics

An External Quality Control Study (2017) covering 15 laboratories of seven Brazilian Member States for the comparative assessment of sensitivity and specificity of nucleic acid based molecular diagnostic tests for the laboratory detection of Zika infection (ZIKV).

Fischer C. et al. (+26) (2018): External Quality Assessment for Zika Virus Molecular Diagnostic Testing, Brazil     Emerg Infect Dis. 24: 888–892.  PMID: 29470164
The geographic location (approx. along 2,500 km) of the laboratories involved in the study provided opportunity for the comparative analysis of ZIKV diagnostics in areas less affected and also in areas heavily affected by the epidemic (the northeastern region).
Participants in the Study: laboratories at universities and public hospitals, federal public health institutions and a diagnostic company.
Study panels: 12 lyophilized samples/panel/locale > heat-inactivated virus inoculated into arbovirus-negative human plasma (supernatant of virus-infected in vitro cell culture, see below) + control sample.


4 samples for testing virus load and sensitivity of detection: 103–106 ZIKV-RNA copies/ml. Mixed population of virus strains: Zika-Asia + Zika-America (strain MRS_OPY_Martinique_PaRi_2015) + Zika-Africa (strain MR766).
» 7 samples of
heterologous virus content and negative for ZIKV, for assessing test specificity. Heterologous virus content: Dengue 2 and 4 serotypes (DENV 2,4), Japanese encephalitis virus (JEV), St.Louis enecephalitis virus (SLEV), West Nile virus (WNV), Yellow fever virus (YFV), Chikungunya virus (CHIKV)  ≈105 virus /ml  (a dose of 50% infection capacity in cells of culture).

» 1 plasma sample negative for viruses.

» WHO standard* to quantify ZIKV in infection.

*Instead of WHO standard: 
to avoid biohazard risks and limitations, further, to avoid logistics and cost problems, a synthetic approach was invented. Zika RNA (pseudovirus) based on RT-PCR sequence experience was synthesized and packaged in bacteriophage protein. The advantages of this synthetic approach are: product stability, non-infectious character, test control at hand and the cost-effective nature.
When quantitat
ive determination of ZIKV load, no significant difference was obtained between results with WHO Zika standard and results with synthetic Zika RNA control.

» Practical basis for the interlaboratory comparative assessment of ZIKV tests: in 14 of the 15 laboratories participating in the study, the same real-time RT-PCR (rRT-PCR) protocol was in use.
Lanciotti R.S. et al. (2008): 
Genetic and Serologic Properties of Zika Virus Associated with an Epidemic, Yap State, Micronesia, 2007  
Emerg Infect Dis. 14: 1232–1239.

  •  In 4/15 laboratories (27%), correct results were obtained with all the samples.
  • In 5/15 laboratories (33%), false negative results were reported with samples of low ZIKV concentration.
  • Only two samples at the two highest ZIKV concentrations (8.1 × 105 and 7.0 × 103 ZIKV-RNA copies/ml) were able to lead to uniformly correct results in all laboratories.  
  • Results above raise the issue of sensitivity in rRT-PCR tests since in acute infection the virus concentration of 103 - 104 copies/ml in body fluids (blood, urine, saliva) is common.
  • In 6/15 laboratories, false positive results were reported with ZIKV-negative samples(False positive test results may culminate in induced abortion since the correlation between ZIKV infection and detrimental neurogenesis of the embryo is proven). However, it should be emphasized that the 6/15 false positive results were not associated with other, heterologous Flavivirus infection (» the nonspecific oligonucleotide binding is to be excluded). False positive results: suspected sequelae of laboratory contamination?
  • In 7/15 laboratories virus RNA was subjected to manual extraction; this efficiency was surmounted in further 8/15 laboratories where virus RNA was subjected to mechanical extraction. 
  • In summary: considering deficiencies already known, the combined use of genetic and serological tests in ZIKV diagnostics remains indispensable.
    ?? In question ??
    With regard to the global spread and the epidemic potential of ZIKV,  the aim of inventions is a unified and reliable Zika diagnostics. Obstacles: lack of resources in areas heavily affected by ZIKV epidemics (e.g. regions in Latin America, Africa, Asia) where purchase of costly (duty, distributor margin) rRT-PCR reagents is hampered. Along with this, the published study suggests the need for regular external quality assessment in Zika diagnostics and the extension of it to all continents.



      Development goal is ZIKV differential diagnosis separating other members of the Flaviviridae family.
      As is known, in infected hosts the target to be attacked by IgM and neutralizing antibodies generated by diverse flaviviruses (such as ZIKV, DENV...), is the virion surface 'E' ('env') glycoprotein, a Flaviviridae member antigen with high similarities in protein sequences and spatial conformations. These similarities result in flavivirus serological cross-reactions rendering current ZIKV serodiagnostics be only effective in geographic areas devoid of other flavivirus' spread. However, this latter is an unrealistic surmise since ZIKV biogeographic spread is becoming more common in zones gained by other flaviviruses (e.g. Dengue, Tick-borne encephalitis, Yellow Fever ...).

      In line with those above, Gao X. et al. elaborated a differential diagnostic test for ZIKV based on the antigenicity of virus non-structural protein (non-structural protein 1 = NS1)  published in 2017. Further in this approach, kinetic aspects of ZIKV-NS1 diagnostics unknown so far, were also underlined.

      Gao X. et al. (2018):  Delayed and highly specific antibody response to nonstructural protein 1 (NS1) revealed during natural human ZIKV infection by NS1-based capture ELISA
      BMC Infectious Diseases  18: 275

      In their IgG capture ELISA procedure, recombinant ZIKV-NS1 (full protein chain) antigen expressed in mammalian 293T cell line (HEK293T - transformed cell line from human embryonal kidney) was used in follow-up tests of two Chinese travelers having returned from Latin America with ZIKV infection.
      *  ZIKV-NS1 and Dengue(DENV1-4)-NS1 full protein chain produced in mammalian 293T cells,
      *  blood samples taken regularly from travelers after onset of ZIKV infection symptoms,
      *  checking serological cross reactions with all four serotypes of Dengue (DENV1-4)
      *   isolation of ZIKV-NS1 specific monoclonal antigens from blood samples
      * tests for ZIKV-NS1 binding in sera of DENV-1 infected population.


      • Eight samples of sera taken from Traveler-1 (4-7-15-32-65-106-188-322 days after onset of symptoms) and three samples of sera taken from Traveler-2 (6-12-66 days after onset of symptoms) in dilutions 1:100 and 1:1000 proved the significant time delays in anti-NS1 release and reach of peak concentration, when compared to anti-E antibodies.
      • Traveler-1 and Traveler-2 memory B cells in vitro for isolation and characterization of five monoclonal antibodies (mAbs): strong binding of 4/5 mAbs to ZIKV-NS1 antigen and no reaction with Dengue[DENV(1-4)]-NS1 antigens was found. 1/5 mAb cross-reacted with DENV2-NS1 and DENV4-NS1 antigens, though in slighter reaction compared to that with ZIKV-NS1 antigen (partial similarities in sequence and structure)
      • Sera samples taken from DENV1-infected 108 test subjects in their convalescence phases (35 persons in primary infection, 20 persons in secondary infection, 53 persons unknown ). Sera in dilution 1:100 were taken for ELISA tests of ZIKV-NS1 and DENV(1-4)NS1 binding. In 106/108 test subjects no cross-reaction with ZIKV-NS1 was observed. In only 2/108 test subjects DENV1 immune serum reacted with DENV(2-3-4) antigens as well and also with ZIKV-NS1 antigen. 
      • In summary: Regarding serological cross reactions when targeting DENV 'E' and ZIKV 'E' antigens, a more advantageous strategy seems to be the targeting NS1 antigen when performing ZIKV differential diagnostics.
      • ??  In question ?? 
      • The regional shortages in financial resources when purchasing ZIKV diagnostic reagents, further, the need for medical laboratory when running current diagnostic procedures (nucleic acid based + serodiagnostics), all make the implementation of urgent at site diagnostics (POC / Point of Care) less accessible and/or less economical.   
      • Bridging the shortcomings above, Draz M.S. et al. prepared a thrifty cellulose microchip with embedded microelectrodes in it to follow changes in electric conductivity of ZIKV-infected samples in the presence of platina-nanoparticles. Draz M.S. et al. (2018): Nanoparticle-enhanced electrical detection of Zika virus on paper microchips  Nanoscale. 10(25):11841-11849. doi: 10.1039/c8nr01646a
      • Essence of the method: isolation of viruses ("fishing") from biosamples with the use of magnetic beads conjugated to virus specific antibodies, labelling of the isolate with platina-nanoparticles (PtNP) capable for giving electric signals, transfer of the labelled virus complex to surface of cellulose-plastic microchip (0,18mm-and-0,1mm in thickness) with embedded micoelectrodes in it, rinsing of the labelled virus complex with low-conductivity solution and with detergent, the latter making the charged molecules and PtNP particles free from the complex. In result, electric conductivity of the solution will change and can be measured by impedance spectroscopy. 
      • Results obtained so far point to the highly specific detection of ZIKV with no interference with related flaviviruses (DENV-1 and DENV-2)
      • Limit of detection (LOD) = 101 virus particle/μl.
      For consideration: the diagnostic effectivity of ZIKV-NS1 was also suggested in an earlier paper presenting the serodiagnostic follow-up of Belgian (9 samples), Czech (38 samples), German (6 samples), Italian (13 samples), Chilean (6 samples), Israeli (33 samples) travelers returning from ZIKV-endemic areas. In the kinetic studies after onset of symptoms, significant difference in IgM and IgG diagnostic sensitivities were observed along with time. Consequently, the relevance of combined (multiplex) demonstration of ZIKV-NS1 specific IgM and IgG antibodies is emphasized. 
      for History:  Actuality 2017/Zika virus/...
                                  ... Zika vaccine constructs ...)

      Purified inactivated Zika virus: conventional active immunization

      1. NCT02963909 / situation on 12-06-2018: Active  
      A Phase 1, First-in-human, Double-blinded, Randomized, Placebo-controlled Trial of a Zika Virus Purified Inactivated Vaccine (ZPIV) With Alum Adjuvant in Healthy Flavivirus-naive and Flavivirus-Primed Subjects
      Study Start Date: November 1, 2016
      Estimated Primary Completion Date: February 1, 2019

      Preliminary Publication > Modjarrad K. et al (+36) Lancet 391: 563-571. (2018)

      2. NCT03008122 / situation on 12-06-2018: Recruiting
      Phase I, Randomized, Double-blinded, Placebo-Controlled Dose De-escalation Study to Evaluate Safety and Immunogenicity of Alum Adjuvanted Zika Virus Purified Inactivated Vaccine (ZPIV) in Adults in a Flavivirus Endemic Area
      Study Start Date: February 24, 2017
      Estimated Primary Completion Date: July 18, 2019
      Estimated Study Completion Date: January 15, 2020

      NCT02952833 situation on 12-06-2018: Active
      ZIKA Vaccine in Naive Subjects
      (Phase 1, Double-blinded, Placebo-Controlled Study of the Safety and Immunogenicity of Alum Adjuvanted Zika Virus Purified Inactivated Vaccine (ZPIV) Administered by the Intramuscular Route in Flavivirus Naïve Adult Subjects)

      Study Start Date: October 14, 2016
      Estimated Primary Completion Date: December 18, 2018
      Estimated Study Completion Date: June 25, 2019
      Preliminary Publication > Modjarrad K. et al (+36) Lancet 391: 563-571. (2018)

      Plasmid DNA vaccine coding for virus surface structural proteins 'prM-E' or 'M-E'
      1. NCT02809443situation on 12-06-2018: Active
      Study of GLS-5700 in Healthy Volunteers
      Study Start Date: July, 2016
      Estimated Primary Completion Date: November, 2017
      Estimated Study Completion Date: December, 2017  
      Preliminary Report: Tebas P. et al (2017): Safety and Immunogenicity of an
      Anti–Zika Virus
      DNA Vaccine
      - Preliminary Report
      DOI: 10.1056/NEJMoa1708120

      2. NCT02887482 situation on 12-06-2018: Active
      Study of GLS-5700 in Dengue Virus Seropositive Adults
      Study Start Date: August, 2016
      Estimated Primary Completion Date: October, 2017
      Estimated Study Completion Date: June, 2018
                                          No further results available at present.

      NCT02840487 situation on 12-06-2018: Active
      Safety and Immunogenicity of a Zika Virus DNA Vaccine, VRC-ZKADNA085-00-VP, in Healthy Adults
      Study Start Date: August 2, 2016
      Estimated Primary Completion Date: December 28, 2018
      Estimated Study Completion Date: December 28, 2018
      Preliminary Publication > Gaudinski M.R. et al (+35) Lancet 391: 552-562. (2018)

      4. NCT02996461 situation on 12-06-2018: Active
      VRC 320: A Phase I, Randomized Clinical Trial to Evaluate the Safety and Immunogenicity of a Zika Virus DNA Vaccine, VRC-ZKADNA090-00-VP, Administered Via Needle and Syringe or Needle-free Injector, PharmaJet, inHealthy Adults
      Study Start Date: December 16, 2016
      Estimated Primary Completion Date: December 28, 2018
      Estimated Study Completion Date: December 28, 2018
      Preliminary Publication > Gaudinski M.R. et al (+35) Lancet 391: 552-562. (2018)

      NCT03110770 / situation on 12-06-2018: Recruiting
      VRC705: A Zika Virus DNA Vaccine in Healthy Adults and Adolescents  (VRC-ZKADNA090-00-VP  in  Phase 2)
      Study Start Date: 29 March, 2017
      Estimated Primary Completion Date: January, 2020
      Estimated Study Completion Date: January, 2020
      History > Dowd K.A. et al (+30): Science 354: 237-240 (2016)

      messenger RNA (mRNA) Vaccine coding for virus surface structural proteins
      1. NCT03014089situation on 12-06-2018: Active
      Safety, Tolerability, and Immunogenicity of mRNA-1325 in Healthy Adult Subjects
      Study Start Date: December, 2016
      Estimated Primary Completion Date: September, 2018
      Estimated Study Completion Date: September, 2018


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