AN IMMUNOASSAY FOR THE DIAGNOSIS OF VIRAL INFECTIONS
20190383814 · 2019-12-19
Assignee
Inventors
- Katja Steinhagen (Gross Groenau, DE)
- Andrea Deerberg (Gross Groenau, DE)
- Erik Lattwein (Luebeck, DE)
- Christiane Radzimski (Reinfeld, DE)
- Jana Böthfür (Schlagsdorf, DE)
Cpc classification
C12N2770/24122
CHEMISTRY; METALLURGY
C12N2770/24134
CHEMISTRY; METALLURGY
G01N2333/185
PHYSICS
A61K39/00
HUMAN NECESSITIES
International classification
Abstract
A recombinant polypeptide can be used in the diagnosis of the presence of a Zika virus in a patient. The recombinant polypeptide includes SEQ ID NO1 or a variant thereof, where the recombinant polypeptide is a monomer, a dimer, or a hexamer.
Claims
1: A recombinant polypeptide, comprising: SEQ ID NO 1 or a variant thereof, wherein the recombinant polypeptide is a monomer, a dimer, or a hexamer.
2: The recombinant polypeptide according to claim 1, wherein the polypeptide is expressed in a eukaryotic cell.
3: A composition, comprising: the recombinant polypeptide according to claim 1, and a mammalian apolipoprotein or a variant thereof and/or a Zika virus envelope glycoprotein.
4: A diagnostically useful carrier, comprising: a binder compound for specifically capturing an antibody to SEQ ID NO1 in a sample from a subject, and a carrier, which is at least one member selected from the group consisting of a bead, a test strip, a microtiter plate, a blot, a lateral flow test, a glass surface, a slide, a biochip, and a membrane.
5: The diagnostically useful carrier according to claim 4, wherein the binder compound for specifically capturing an antibody to SEQ ID NO1 is a recombinant polypeptide, wherein the recombinant polypeptide comprises SEQ ID NO1 or a variant thereof, and wherein the polypeptide is a monomer, a dimer, or a hexamer.
6: The diagnostically useful carrier according to claim 4, further comprising: at least one binder compound for specifically capturing an antibody to an antigen selected from the group consisting of SEQ ID NO2, SEQ ID NO3, SEQ ID NO4, SEQ ID NO5, SEQ ID NO6, SEQ ID NO7, SEQ ID NO8, SEQ ID NO19, and SEQ ID NO9 and/or at least one binder compound for specifically capturing an antibody to an antigen selected from the group consisting of SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO13, SEQ NO 14, SEQ ID NO15, SEQ ID NO16, SEQ ID NO17, SEQ ID NO18, and SEQ ID NO27.
7: A kit, comprising: the diagnostically useful carrier according to claim 4.
8: A method, comprising obtaining a plasma sample from a subject detecting in the plasma sample the presence or absence of an antibody to SEQ ID NO1 by contacting the plasma sample with the diagnostically useful carrier according to claim 4 and detecting binding between the carrier and the antibody to SEQ ID NO1.
9. (canceled)
10: The method according to claim 8, further comprising: detecting in a sample from a subject the presence or absence of an antibody to one or more antigens selected from the group consisting of SEQ ID NO2, SEQ ID NO3, SEQ ID NO4, SEQ ID NO5, SEQ ID NO6, SEQ ID NO7, SEQ ID NO8, SEQ ID NO19, SEQ ID NO9, SEQ ID NO11, SEQ ID NO12, SEQ ID NO13, SEQ ID NO14, SEQ ID NO15, SEQ ID NO16, SEQ ID NO17, SEQ ID NO18, and SEQ ID NO27.
11: The method according to claim 10, wherein the presence or absence of an antibody to SEQ ID NO 1 and the presence or absence of an antibody to one or more further antigens is detected in spatially separate binding reactions.
12: The method according to claim 8, further comprising: monitoring an IgA, IgM and/or IgG class antibody to SEQ ID NO1 for a time period of at least three days.
13: The method according to claim 12, wherein a total concentration of IgM to SEQ ID NO1 is detected in addition.
14: The method according to claim 8, wherein the antibody is a mammalian antibody.
15-18. (canceled)
19: The composition according to claim 3, wherein the composition is a pharmaceutical composition, optionally comprising a pharmaceutically acceptable composition, or the composition is an immunogenic composition, optionally comprising an adjuvant.
20: The method according to claim 14, wherein the antibody is a human antibody.
21: The method according to claim 14, wherein the antibody is a human antibody and is at least one member selected from the group consisting of human IgA class antibody, human IgM class antibody, and human IgG class antibody.
22: The diagnostically useful carrier according to claim 4, further comprising: at least one binder compound for specifically capturing an antibody to SEQ ID NO2 and/or at least one binder compound for specifically capturing an antibody to SEQ ID NO 18.
23: The composition according to claim 3, which comprises: the recombinant polypeptide, and a polypeptide comprising SEQ ID NO 1 or a variant thereof.
Description
[0124] The present invention is further illustrated by the following examples, sequences and figures from which further features, embodiments, aspects and advantages of the present invention may be taken. All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, with suitable methods and materials being described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety, as are priority applications EP16000422.2, EP16000442.0, EP16000454.5 and EP16000454.5. Further, the materials, methods, and examples are illustrative only and are not intended to be limiting, unless otherwise specified.
[0125]
[0126]
[0127]
[0128]
[0129]
[0130]
[0131]
[0132]
[0133]
[0135]
[0137]
[0138]
[0139] SEQ ID NO1: Zika virus NS1 antigen
[0140] SEQ ID NO2: dengue virus 1 NS1 antigen
[0141] SEQ ID NO3: dengue virus 2 NS1 antigen
[0142] SEQ ID NO4: dengue virus 3 NS1 antigen
[0143] SEQ ID NO5: dengue virus 4 NS1 antigen
[0144] SEQ ID NO6: West Nile virus NS1 antigen
[0145] SEQ ID NO7: Tick-borne encephalitis virus NS1 antigen
[0146] SEQ ID NO8: Japanese encephalitis virus NS1 antigen
[0147] SEQ ID NO9: Yellow fever virus NS1 antigen
[0148] SEQ ID NO10: Zika virus NS1 antigen with C-terminal His tag
[0149] SEQ ID NO 11: Zika virus envelope glycoprotein
[0150] SEQ ID NO12: dengue virus 1 envelope glycoprotein
[0151] SEQ ID NO13: dengue virus 2 envelope glycoprotein
[0152] SEQ ID NO14: dengue virus 3 envelope glycoprotein
[0153] SEQ ID NO15: dengue virus 4 envelope glycoprotein
[0154] SEQ ID NO16: West Nile virus envelope glycoprotein
[0155] SEQ ID NO17: Tick-borne encephalitis virus envelope glycoprotein
[0156] SEQ ID NO18: Japanese encephalitis virus envelope glycoprotein
[0157] SEQ ID NO19: Powassan virus NS1 antigen
[0158] SEQ ID NO20: Zika virus NS1 antigen with C-terminal His tag and additional fused peptide
[0159] SEQ ID NO21: Zika virus NS1 epitope
[0160] SEQ ID NO22: Zika virus NS1 epitope
[0161] SEQ ID NO23: Zika virus NS1 epitope
[0162] SEQ ID NO24: Zika virus NS1 epitope
[0163] SEQ ID NO25: Zika virus NS1 epitope
[0164] SEQ ID NO26: Zika virus NS1 epitope
[0165] SEQ ID NO27: Yellow fever virus envelope glycoprotein
EXAMPLE 1: STUDYING THE DIAGNOSTIC PERFORMANCE OF ZIKA NS1-BASED ELISA
[0166] Methods
[0167] Human Serum Samples
[0168] Serum samples from patients with ZIKV infection (n=29) and patients with other flaviviral or non-flaviviral infections as well as yellow fever vaccinations (n=128) were examined in this study. Sera from healthy pregnant women (n=100) and blood donors living in flavivirus endemic and non-endemic areas (n=699) served as negative controls. Follow-up samples from a German patient with clinically and serologically confirmed ZIKV infection contracted during a stay in Colombia were tested by the WHO Collaborating Centre for Arbovirus and Haemorrhagic Fever Reference and Research (Hamburg, Germany) and used for time-course analysis of anti-ZIKV antibody levels. All sera were stored at 20 C. until assayed. The samples were used anonymously to maintain confidentiality and the study protocol conformed to the recommendations of the Central Ethical Committee of Germany.
[0169] Protein Expression and Purification
[0170] Recombinant NS1[ZIKV] was expressed in HEK293T cells using standard cloning an expression methods based on the pTriEx-1 plasmid with an artificial signal sequence and a C-terminal His tag (SEQ ID NO20). Transfected cells were cultured at 37 C. and 8.5% CO.sub.2 in Dulbecco's modified eagle's medium with 10% fetal calf serum, 100 U/ml penicillin and 0.1 mg/ml streptomycin for three to five days. Cells were harvested, resuspended in 20 mM Tris-HCl pH 7.4, 10% (w/v) sucrose, 5 mM EDTA, 1 mM PMSF and stored at 80 C. until further use.
[0171] Cells were resuspended in 20 mmol/l tris chloride pH 8.0, 600 mmol/l sodium chloride, 20 mmol/l magnesium chloride, 20 mmol/l imidazole, 1 mmol/l PMSF, 0.5 mmol/l dithiotreitol, 0.1% Triton X-100 and lysed by homogenization. Cell debris was removed by centrifugation for 60 minutes at 100,000g, 4 C. The soluble protein fraction was applied to Nickel Rapid Run (Agarose Bead Technologies, Miami. Fla., USA) equilibrated with 5 mmol/l tris chloride pH 8.0, 150 mmol/l sodium chloride, 0.015% (w/v) Triton X-100, 0.5 mmol/l dithiotreitol, 20 mmol/l imidazole and eluted by increasing the imidazole concentration to 150 mmol/l. The eluates were pooled, diluted with two volumes 20 mmol/l tris chloride pH 8.5, 5 mmol/l EDTA, 1 mmol/l PMSF, 0.015% (w/v) Triton X-100, 1 mmol/l dithiotreitol and cleared by centrifugation at 100,000g and 4 C. for 60 minutes. The supernatant was loaded onto a HiTrap Q FF column (GE Lifesciences, Freiburg, Germany) equilibrated with 20 mmol/l tris chloride pH 8.5, 2.5 mmol/l EDTA, 1 mmol/l PMSF, 0.015% (w/v) Triton X-100, 1 mmol/l dithiotreitol, 50 mmol/l sodium chloride, washed and eluted with 20 mmol/l tris chloride pH 8.5, 2.5 mmol/l EDTA, 1 mmol/l PMSF, 0.015% (w/v) Triton X-100, 1 mmol/l dithiotreitol with stepwise increase of sodium chloride from 50 to 1000 mmol/l. All fractions containing NS1[ZIKV] were pooled and concentrated by ultrafiltration (VivaSpin, Sartorius, Gottingen, Germany). The final preparations were stored at 80 C. until further use.
[0172] Enzyme-Linked Immunosorbent Assay
[0173] NS1-coated microtiter-plates (Nunc, Roskilde, Denmark) and standardized reagents from the commercially available Anti-Zika Virus IgG and IgM ELISA (EUROIMMUN, Lbeck, Germany) were used as recommended by the manufacturer. In brief, sera diluted 1:101 in PBS plus 0.1% (w/v) casein were added to the wells and allowed to react for 60 minutes at 37 C. In case of IgM detection, sera were pre-incubated with rheumatoid factor absorbent for 10 minutes. Bound antibodies were detected applying rabbit anti-human IgG peroxidase conjugate or goat anti-human IgM peroxidase conjugate for 30 min, followed by staining with tetramethylbenzidine for 15 min. The enzymatic reaction was stopped by addition of one volume of 0.5 mol/l sulphuric acid. Optical density was determined photometrically at 450 nm (reference 620 nm). Unless indicated otherwise, all assay procedures were carried out at room temperature.
[0174] The cut-off for positivity was validated and optimized by receiver-operating characteristics (ROC). A highly positive index patient serum was diluted to generate a cut-off calibrator that was incubated in every experiment. A ratio of the extinction value of the patient sample over the extinction value of calibrator was calculated.
[0175] Statistics
[0176] Statistical analyses were performed using GraphPad Prism 6 (GraphPad Software Inc., La Jolla, Calif., USA) and SigmaPlot 13.0 analysis software (SSI, San Jose, Calif., USA). Confidence intervals (CI 95%) were calculated according to the modified Wald method.
[0177] Results
[0178] Eukaryotic Expression and Purification of ZIKV-Specific NS1
[0179] ZIKV-specific NS1 was expressed in the human cell line HEK293T and purified from the cell lysate (mNS1) or culture supernatant (sNS1). When separated by SDS-PAGE, mNS1 and sNS1 migrated essentially according to their predicted molecular mass (43.9 kDa;
[0180] Development of NS1-Based Anti-ZIKV ELISA
[0181] Purified recombinant mNS1 and sNS1 were used as solid phase antigens in ELISA for the detection of anti-ZIKV IgM and IgG, respectively. ROC analyses were performed based on 29 sera from patients with ZIKV infection and 908 controls, including 109 patients with flavivirus infections or vaccinations, 100 pregnant women and 699 blood donors. Areas under the curve (IgM, 0.979; IgG, 0.956) indicated excellent diagnostic performance (
[0182] Diagnostic Performance of the Anti-ZIKV ELISA
[0183] Sensitivity: Out of 29 serum samples from patients with clinically and serologically confirmed ZIKV infection, 24 (82.8%) were positive for anti-ZIKV IgM, 20 (69.0%) for IgG, and 28 (96.6%) for IgM and/or IgG (
[0184] Specificity: Among 799 healthy controls, only 1/99 (1.0%) Argentinian and 1/500 (0.2%) German blood donors were found anti-ZIKV IgM positive, while 100 healthy US-American blood donors and 100 healthy pregnant women were negative. Anti-ZIKV IgG was present in 1/100 (1.0%) US-American and 1/500 (0.2%) German blood donors, but absent in the cohorts of healthy Argentinian blood donors and pregnant women. Thus, overall specificity amounted to 99.7% for either Ig class (
[0185] Cross-reactivity: Serum panels from 128 clinically and serologically well characterized patients or vaccinees with high titers of antibodies of class IgM and/or IgG against flaviviruses (DENV, YFV, WNV or JEV) and CHIKV were analyzed. Anti-ZIKV IgM reactivity was detectable in 1/34 (2.9%) patients infected with WNV and anti-ZIKV IgG in 1/25 (4.0%) patients infected with JEV (
EXAMPLE 2: PREPARATION OF ZIKA NS1 ANTIGEN
[0186] Recombinant NS1[ZIKV] was expressed in HEK293T cells using standard cloning and expression methods based on the pTriEx-1 plasmid with an artificial signal sequence and a C-terminal His tag (SEQ ID NO20). Transfected cells were cultured at 37 C. and 8.5% CO2 in Dulbecco's modified eagle's medium with 10% fetal calf serum, 100 U/ml penicillin and 0.1 mg/ml streptomycin for three to five days. Cell culture supernatant was decanted and stored until further use. Cells were harvested, resuspended in 20 mM Tris-HCl pH 7.4, 10% (w/v) sucrose, 5 mM EDTA, 1 mM PMSF and stored at 80 C. until further use.
[0187] To prepare mNS1, cells were resuspended in 20 mmol/l tris chloride pH 8.0, 600 mmol/l sodium chloride, 20 mmol/l magnesium chloride, 20 mmol/l imidazole, 1 mmol/l PMSF, 0.5 mmol/l dithiotreitol, 0.1% Triton X-100 and lysed by homogenization. Cell debris was removed by centrifugation for 60 minutes at 100,000g, 4 C. The soluble protein fraction was applied to Nickel Rapid Run (Agarose Bead Technologies, Miami, Fla. USA) equilibrated with 5 mmol/l tris chloride pH 8.0, 150 mmol/l sodium chloride, 0.015% (w/v) Triton X-100, 0.5 mmol/l dithiotreitol, 20 mmol/l imidazole and eluted by increasing the imidazole concentration to 150 mmol/l. The eluates were pooled, diluted with two volumes 20 mmol/l tris chloride pH 8.5, 5 mmol/l EDTA, 1 mmol/l PMSF, 0.015% (w/v) Triton X-100, 1 mmol/l dithiotreitol and cleared by centrifugation at 100,000g and 4 C. for 60 minutes. The supernatant was loaded onto a HiTrap Q FF column (GE Lifesciences, Freiburg, Germany) equilibrated with 20 mmol/l tris chloride pH 8.5, 2.5 mmol/l EDTA, 1 mmol/l PMSF, 0.015% (w/v) Triton X-100, 1 mmol/l dithiotreitol, 50 mmol/l sodium chloride, washed and eluted with 20 mmol/l tris chloride pH 8.5, 2.5 mmol/l EDTA, 1 mmol/l PMSF, 0.015% (w/v) Triton X-100, 1 mmol/l dithiotreitol with stepwise increase of sodium chloride from 50 to 1000 mmol/l. All fractions containing NS1[ZIKV] were pooled and concentrated by ultrafiltration (VivaSpin, Sartorius, Gottingen, Germany). The final preparations were stored at 80 C. until further use.
[0188] To prepare sNS1, cell culture supernatant was adjusted to 5 mmol/l tris chloride pH 8.0, 164 mmol/l sodium chloride, 50 mmol/l magnesium chloride, 20 mmol/l imidazole, 0.1% Triton X-100, cleared by centrifugation for 30 minutes at 17,600g, 4 C., applied to Nickel Rapid Run (Agarose Bead Technologies, Miami. Fla., USA) equilibrated with 5 mmol/l tris chloride pH 8.0, 300 mmol/l sodium chloride, 20 mmol/l imidazole and eluted by increasing the imidazole concentration to 150 mmol/l. All fractions containing NS1 [ZIKV] were pooled and concentrated by ultrafiltration (VivaSpin, Sartorius, Gottingen, Germany). The final preparations were stored at 80 C. until further use.
[0189] To prepare sNS1 in complex with bovine apolipoprotein AI, cell culture supernatant was adjusted to 5 mmol/l tris chloride pH 8.0, 164 mmol/l sodium chloride, 50 mmol/l magnesium chloride, 20 mmol/l imidazole, cleared by centrifugation for 30 minutes at 17,600g, 4 C., applied to Nickel Rapid Run (Agarose Bead Technologies, Miami, Fla., USA) equilibrated with 5 mmol/l tris chloride pH 8.0, 300 mmol/l sodium chloride, 20 mmol/l imidazole and eluted by increasing the imidazole concentration to 150 mmol/l. All fractions containing NS1[ZIKV]/Apo AI[Bos taurus]-complexes were pooled and concentrated by ultrafiltration (VivaSpin, Sartorius, Gottingen, Germany). The final preparations were stored at 80 C. until further use.
[0190] When separated by SDS-PAGE, NS1 migrated essentially according to its predicted molecular mass (43.9 kDa). Protein identity was verified by mass spectrometry.
EXAMPLE 3: PREPARATION OF NS1 OLIGOMERS
[0191] Protein preparations of mNS1 and sNS1 prepared as in Example 2 were analyzed by analytical gel filtration using a Superdex 200 g column (GE Healthcare, Munich, Germany), in 20 mmol/l tris chloride pH 8.5, 2.5 mmol/l EDTA, 1 mmol/l PMSF, 0.015% (w/v) Triton X-100, 1 mmol/l dithiotreitol, 300 mmol/l sodium chloride or 5 mmol/l tris chloride pH 8.0, 300 mmol/l sodium chloride, 150 mmol/l imidazole. Protein mixtures with known molecular weights were run separately and used as a calibrator. Retention times of individual peaks were used to calculate the molecular weight of the observed NS1 populations and eluate fractions were analyzed using denaturing/non-denaturing gel electrophoresis under reducing conditions followed by silver staining.
[0192]
[0193] Protein preparations of mNS1 and sNS1 were treated with or without 16 mmol/l dithiotreitol and incubated at 70 C. or at room temperature for 10 minutes, followed by SDS gel electrophoresis and Coomassie staining.
[0194] Dimeric mNS1 or sNS1 populations could also be generated by detergent treatment of the hexameric population, e.g. 0.1% Triton X-100 or 0.2% sodium dodecylsulfate. Analytical gel filtration clearly showed that mNS1 and sNS1 exist mainly as hexamers in vitro with a molecular mass of slightly above 200 kDa, however, only the SDS-resistant dimer, migrating with an apparent molecular mass of 70 kDa, could be visualized in an SDS-containing gel. The dimer was further converted into the monomer (MW[NS1.sub.monomeric]=43.9 kDa) by heat denaturation. This process is independent of disulfide bonds.
EXAMPLE 4: NS1 MONOMERS AND DIMERS MAY BE USED TO DETECT ANTIBODIES IN PATIENT SERUM BY WAY OF WESTERN BLOTTING
[0195] Protein preparations of mNS1 and sNS1 prepared as in Example 2 were treated with or without 16 mmol/l dithiotreitol and were incubated for 10 min at 70 C. or at room temperature to achieve monomeric or oligomeric NS1. Both populations were mixed and separated using SDS electrophoresis followed by transfer to a nitrocellulose membrane. Proteins were either stained unspecifically by Ponceau S staining to demonstrate NS1 in monomeric and dimeric form or were incubated with anti-His antibody as a positive control, buffer devoid of serum as negative control and four human sera (dilution 1:51), two of them from healthy blood donors and two from patients suffering from Zika virus infection.
[0196]
EXAMPLE 5: NS1 ANTIGEN STABILITY IS INCREASED IN THE PRESENCE OF BOVINE APOLIPOPROTEIN AI
[0197] The following experiment shows that a complex of a polypeptide comprising SEQ ID NO1 and mammalian apolipoprotein is more stable in a solution than the polypeptide by itself. Therefore, a mammalian apolipoprotein may be used to stabilize the polypeptide and devices and kits comprising it.
[0198] Protein preparations of mNS1, sNS1 and a complex consisting of sNS1 and bovine apolipoprotein AI, the latter made by preparing protein as in Example 1 followed by addition of chromatography fractions comprising the apolipoprotein, were transferred into 50 mmol/l sodium phosphate pH 7.4, 150 mmol/l sodium chloride using desalting spin columns (Zeba Spin, ThermoFisher Scientific, Waltham, USA). An aliquot of each preparation was kept on ice or at room temperature over night to allow for precipitation of non-PBS-soluble proteins. Aliquots were centrifuged for 30 min at 4 C. and 100,000g, and supernatants and pellets (resuspended in an equivalent volume of 50 mmol/l sodium phosphate pH 7.4, 150 mmol/l sodium chloride, 8 mol/l urea) were analyzed using denaturing gel electrophoresis under reducing conditions followed by Coomassie staining.
[0199] Harsh buffer exchange conditions were chosen to provoke aggregation of potentially unstable proteins.
[0200]
[0201] On the other hand, the entirety of sNS1 in complex with bovine apolipoprotein AI remains in the supernatant after centrifugation, indicating a stabilizing effect on sNS1.
EXAMPLE 6: COMPARING REACTIVITY OF VARIOUS NS1 PREPARATIONS
[0202] The following experiment was performed to evaluate the reactivity of different preparations of Zika Virus NS1 antigen in an indirect ELISA for the detection of anti-Zika virus antibodies in human sera. It shows that complexation with a mammalian apolipoprotein increases the reactivity of NS1 and thus the sensitivity of the assay.
[0203] Preparation of Coated Microtiter Plates
[0204] Three different Zika NS1 preparations were used: sNS1 (soluble NS1 purified from cell culture supernatant), mNS1 (membranous NS1 purified from cells), sNS1+ApoAI (soluble NS1 purified from cell culture supernatant, in complex with bovine ApoAI).
[0205] For use in microtiter ELISA these three NS1 preparations were diluted in PBS to final concentrations of 0.5, 1.0 and 2.0 g/ml, respectively. Microtiter plates were coated with 100 l antigen dilution per well.
[0206] Incubation of Samples:
[0207] A set of anti-Zika-NS1 IgM positive or negative human sera was used for evaluation of reactivity of the different antigen preparations. All reagents used during incubation are included in every EUROIMMUN IgM ELISA Test-Kit for infectious diagnostics (e.g. EI 2668-9601 M). Sera were diluted 1:101 in IgM sample buffer containing IgG/RF absorbent and incubated at room temperature for 10 min to absorb rheumatoid factors and IgG. Samples were applied to microtiter plates and incubated as described for commercial EUROIMMUN anti-Zika virus ELISA (IgM) (EI 2668-9601 M). In brief: 60 min at 37 C.; 3 washing steps using EUROIMMUN wash buffer; addition of 100 l of peroxidase-labelled anti-human IgM conjugate (goat) per well; incubation for 30 min at room temperature: 3 washing steps using EUROIMMUN wash buffer, addition of 100 l of chromogen/substrate solution (TMB/H2O2) per well; incubation for 15 min at room temperature; addition of 100 l stop-solution (0.5 M sulfuric acid); measurement of optical density at 450 nm.
[0208] Interpretation of Results:
[0209]
EXAMPLE 7: A MULTICOHORT STUDY OF NS1-BASED ELISA ASSAY PERFORMANCE SHOWS LACK OF CROSS-REACTIVITY WITH DENGUE VIRUS ANTIBODIES
[0210] The diagnostic performance of the assay according to the present invention was examined using sera from returning travellers and patients from ZIKV-endemic areas with laboratory-confirmed ZIKV infection, potentially cross-reactive samples from patients with flaviviral and other infections, as well as control panels from blood donors of different ages and geographical origin.
[0211] Methods
[0212] Human Sera
[0213] The study included serum samples from 27 patients who had tested positive for ZIKV RNA by reverse transcription PCR (RT-PCR); Group 1: travellers returning from endemic areas (n=8); Group 2: residents in ZIKV-endemic areas (n=19). On the basis of direct detection of the pathogen's genome, these cases were referred to as having RT-PCR-confirmed ZIKV infection. Samples from a further 85 patients had been pre-characterised by anti-ZIKV indirect immunofluorescence assay (IIFA; EUROIMMUN, Lbeck, Germany) based on whole virus antigen, showing reactivity for anti-ZIKV IgM and/or IgG; Group 3: travellers returning from endemic areas (n=26); Group 4: residents in ZIKV-endemic areas (n=59). Since false-positive results due to cross-reactivity of this IIFA cannot be excluded, these cases were referred to as having suspected ZIKV infection (Table 1).
[0214] Classification into three stages of ZIKV infection was according to the Pan American Health Organization (PAHO)/World Health Organization (WHO) recommendations on ZIKV surveillance in the Americas: 5 days post symptom onset, initial stage; 6-20 days post symptom onset, active stage; >20 days post symptom onset, late stage. Samples from travellers returning from endemic areas were provided by the diagnostic institutes (listed in Table 1) to which they had been sent for routine diagnostic testing. Samples from patients residing in Latin America (i.e. Dominican Republic and Colombia) were purchased from Boca Biolistics (Coconut Creek, Fla. United States (United States (US)), Allied Research Society (Miami Lakes, Fla., US) and Biomex GmbH (Heidelberg, Germany). As confirmed by these institutes and companies, written informed consent had been obtained from all patients, and there were no legal or ethical restrictions to using the samples.
[0215] To evaluate cross-reactivity, samples were used from 252 patients with either a post-YFV vaccination status (n=12), or with other flaviviral (DENV=93; WNV=34, JEV=25), non-flaviviral (CHIKV=19) and Plasmodium spp. (PLAS: n=69) infections. In samples from DENV-infected patients, the confirmation of DENV as the infectious agent was based on NS1 antigen detection. Sera from 1,015 healthy individuals (pregnant women, blood donors and children) living in flavivirus-endemic and non-endemic areas served as negative controls. Pre-characterisation data for all control cohorts are reported in Table 2. To the best of the authors' knowledge, none of these samples were analysed in previous studies.
TABLE-US-00001 TABLE 1 Characteristics of patients with RT-PCR-confirmed (n = 27) and suspected (n = 85) Zika virus infection, study evaluating a novel NS1-based ELISA, Germany 2016 Age Diagnostic ZIKV- ZIKV- Virus Case groups Sampling Phase of Clinical centre/provider RT-PCR assay/ RT-PCR neutralisation IIFA IgM IIFA IgG ID in years Sex Country of infection Current/former residence Dpso infection.sup.a symptoms.sup.b of samples performed at result.sup.c assay titre titre.sup.d titre.sup.d Group 1: RT-PCR-confirmed ZIKV infection, travellers returning from ZIKV-endemic areas (n = 8) 1 20-29 M NA Non-endemic 7 Active Yes WHOCC, RealStar Zika Pos NA .sup.1:3,200 1:3,200 2 30-39 F Haiti Non-endemic 4 Active Yes Hamburg, Virus RT-PCR Pos NA 1:320 1:32,000 3 50-59 M NA Non-endemic 3 Initial No Germany (Altona Pos NA NA NA 4 50-59 F NA Non-endemic <4 Initial NA Diagnostics, Pos NA 1:100 1:1,000 Hamburg, Germany)/ WHOCC 5 20-29 F NA Non-endemic 17 Active NA ITM, Antwerp, RealStar Zika Pos >1:640 NA NA 6 40-49 M NA Non-endemic 11 Active NA Belgium Virus RT-PCR Pos 1:243 NA NA 7 0-9 M NA Non-endemic 3 Initial NA (Altona Pos NA NA NA 8 20-29 F NA Non-endemic 11 Active NA Diagnostics, Pos 1:788 NA NA Hamburg, Germany)/ ITM Group 2: RT-PCR-confirmed ZIKV infection, residents in ZIKV-endemic areas (n = 19) 1 60-69 F Suriname The Netherlands/Suriname.sup.e 3 Initial NA AMC, In-house Zika Pos NA NA NA 2 50-59 M Suriname The Netherlands/Suriname.sup.e 5 Initial NA Amsterdam, RT-PCR/AMC Pos NA NA NA 3 40-49 F Suriname The Netherlands/Suriname.sup.e 11 Active NA the Netherlands Pos NA NA NA 4 40-49 M Suriname The Netherlands/Suriname.sup.e 9 Active NA Pos NA NA NA 5 50-59 F Suriname The Netherlands/Suriname.sup.e 6 Active NA Pos NA NA NA 6 50-59 M Suriname The Netherlands/Suriname.sup.e 6 Active NA Pos NA NA NA 7 50-59 F Suriname The Netherlands/Suriname.sup.e 53 Late NA Pos NA NA NA 8 50-59 F Suriname The Netherlands/Suriname.sup.e 17 Active NA Pos NA NA NA 9 60-69 F Suriname The Netherlands/Suriname.sup.e 24 Late NA Pos NA NA NA 10 70-79 M Suriname The Netherlands/Suriname.sup.e 6 Active NA Pos NA NA NA 11 0-9 M Dominican Republic The Netherlands 1 Initial NA Pos NA NA NA 12 50-59 F Dominican Republic Dominican Republic 20 Active Yes Boca Biolistics, Trioplex real- Pos NA 0 1:32,000 13 50-59 F Dominican Republic Dominican Republic 31 Late Yes Coconut Creek, time RT-PCR Pos NA 1:100 1:32,000 Florida, US (CDC, Atlanta, Georgia, US)/CDC 14 20-29 M Colombia Colombia 3 Initial Yes Allied Research Trioplex real- Pos NA 0 1:1,000 15 40-49 F Colombia Colombia 5 Initial Yes Society, Miami time RT-PCR Pos NA 0 1:1,000 16 50-59 F Colombia Colombia 4 Initial Yes Lakes, Florida, (CDC, Atlanta, Pos NA 1:10 1:3,200 17 10-19 M Colombia Colombia 3 Initial Yes US Georgia, Pos NA 0 1:3,200 US)/CDC 18 20-29 F Colombia Colombia 6 Active Yes Biomex GmbH, RealStar Zika Pos NA .sup.1:3,200 1:32,000 Heidelberg, Virus RT-PCR Germany (Altona Diagnostics, Hamburg, Germany)/ Altona Diagnostics 19 10-19 M Colombia Colombia/US 15 Active Yes Trioplex real- Pos NA 1:10 1:32,000 time RT-PCR (CDC, Atlanta, Georgia, US)/CDC Group 3: Suspected ZIKV infection, travellers returning from ZIKV-endemic areas (n = 26) 1 NA NA NA Non-endemic NA NA NA WHOCC, NA NA NA .sup.1:3,200 1:10,000 2 NA NA NA Non-endemic NA NA NA Hamburg, NA NA .sup.1:1,000 1:10,000 3 NA NA NA Non-endemic NA NA NA Germany NA NA .sup.1:3,200 1:10,000 4 NA NA Brazil Non-endemic NA NA NA NA NA .sup.1:1,000 1:32,000 5 NA NA Brazil Non-endemic NA NA NA NA NA .sup.1:1,000 1:3,200 6 NA NA Brazil Non-endemic NA NA NA NA NA .sup.1:3,200 1:10,000 7 NA NA Brazil Non-endemic NA NA NA NA NA 1:100 <1:100 8 NA NA NA Non-endemic NA NA NA NA NA .sup.1:1,000 1:100 9 NA NA NA Non-endemic NA NA NA NA NA 1:320 1:10,000 10 NA NA NA Non-endemic NA NA NA NA NA 1:320 1:32,000 11 NA NA Brazil Non-endemic 19 Active Yes NA NA 1:320 1:10,000 12 NA NA Brazil Non-endemic NA NA NA NA NA 1:100 1:100,000 13 NA NA Brazil Non-endemic NA NA NA NA NA .sup.1:1,000 1:320 14 NA NA Brazil Non-endemic NA NA NA NA NA 1:320 1:3,200 15 NA NA Brazil Non-endemic NA NA NA NA NA 1:320 1:1,000 16 NA NA Brazil Non-endemic NA NA NA NA NA .sup.1:1,000 1:10,000 17 NA NA Brazil Non-endemic NA NA NA NA NA 1:320 1:10,000 18 NA NA NA Non-endemic 32 Late NA NA NA 1:100 1:32,000 19 NA NA Colombia Non-endemic 45 Late NA NA NA 1:100 1:3,200 20 NA NA NA Non-endemic NA NA NA NA NA .sup.1:1,000 1:10,000 21 NA NA Denmark Non-endemic NA NA NA NA NA 1:100 1:32,000 22 NA NA NA Non-endemic NA NA NA NA NA .sup.1:3,200 1:32,000 23 NA NA Colombia Non-endemic NA NA NA NA NA 1:100 1:10,000 24 NA NA Brazil Non-endemic NA NA NA NA NA 1:320 1:32,000 25 NA NA Brazil Non-endemic NA NA NA NA NA 1:320 1:32,000 26 NA NA Colombia Non-endemic 15 Active NA NA NA .sup.1:3,200 1:10,000 Group 4: Suspected ZIKV infection, residents in ZIKV-endemic areas (n = 59) 1 30-39 F Colombia Colombia 6 Active Yes Allied Research NA NA NA .sup.1:1,000 1:320,000 2 20-29 M Colombia Colombia 8 Active Yes Society, Miami NA NA 1:100 1:1,000 3 30-39 F Colombia Colombia 11 Active Yes Lakes, Florida, NA NA 0 1:1,000 4 40-49 M Colombia Colombia 14 Active Yes US NA NA .sup.1:3,200 1:320,000 5 30-39 F Colombia Colombia 17 Active Yes NA NA .sup.1:3,200 1:320,000 6 80-89 M Colombia Colombia 20 Active Yes NA NA 1:320 1:320,000 7 50-59 F Colombia Colombia 23 Late Yes NA NA 1:320 1:10,000 8 30-39 M Colombia Colombia 30 Late Yes NA NA .sup.1:3,200 1:320,000 9 40-49 F Colombia Colombia 49 Late Yes NA NA 1:100 1:10,000 10 10-19 F Colombia Colombia 54 Late Yes NA NA 1:10 1:1,000 11 50-59 F Colombia Colombia 6 Active Yes NA NA 0 1:3,200 12 40-49 F Colombia Colombia 4 Initial Yes NA NA 0 1:1,000 13 10-19 M Colombia Colombia 66 Late Yes NA NA 0 1:32,000 14 40-49 F Colombia Colombia 68 Late Yes NA NA 1:10 1:32,000 15 50-59 F NA Colombia 70 Late Yes NA NA 0 1:32,000 16 40-49 F NA Colombia 2 Initial Yes NA NA 0 1:10,000 17 20-29 F Colombia Colombia 7 Active Yes NA NA 1:100 1:320,000 18 50-59 F NA Colombia 4 Initial Yes NA NA 1:100 1:100,000 19 40-49 M Colombia Colombia 3 Initial Yes NA NA 1:10,000 1:32,000 20 40-49 F Colombia Colombia 4 Initial Yes NA NA 1:32 1:32,000 21 30-39 M Colombia Colombia 4 Initial Yes NA NA 1:32 1:32,000 22 40-49 F Colombia Colombia 4 Initial Yes NA NA 0 1:100,000 23 30-39 M Colombia Colombia 4 Initial Yes NA NA 0 1:32,000 24 20-29 F Colombia Colombia 5 Initial Yes NA NA 1:10 1:10,000 25 40-49 F Colombia Colombia 5 Initial Yes NA NA .sup.1:1,000 1:100,000 26 30-39 F Colombia Colombia 3 Initial Yes NA NA 0 1:3,200 27 40-49 F Colombia Colombia 4 Initial Yes NA NA 0 1:32,000 28 20-29 F Colombia Colombia 3 Initial Yes NA NA 0 1:320 29 50-59 F Colombia Colombia 4 Initial Yes NA NA 0 1:10,000 30 20-29 F Colombia Colombia 3 Initial Yes NA NA 1:32 1:10,000 31 30-39 F Colombia Colombia 3 Initial Yes Biomex GmbH, NA NA NA 0 1:32,000 32 20-29 F Colombia Colombia 4 Initial Yes Heidelberg, NA NA 1:100 1:32,000 33 10-19 F Colombia Colombia 9 Active Yes Germany NA NA 1:100 1:32,000 34 20-29 F Colombia Colombia 12 Active Yes NA NA 0 1:32,000 35 10-19 F Colombia Colombia 20 Active Yes NA NA 1:100 1:10,000 36 20-29 F Colombia Colombia 27 Late Yes NA NA 1:320 1:10,000 37 30-39 F Colombia Colombia 36 Late Yes NA NA 1:10 1:32,000 38 10-19 F Colombia Colombia 56 Late Yes NA NA 1:100 1:10,000 39 30-39 F Colombia Colombia 67 Late Yes NA NA 1:10 1:32,000 40 10-19 F Colombia Colombia 2 Initial Yes Allied Research NA NA NA 0 1:10,000 41 30-39 F Colombia Colombia 5 Initial Yes Society, Miami NA NA 1:320 1:10,000 42 20-29 F Colombia Colombia 6 Active Yes Lakes, Florida, NA NA 1:100 1:10,000 43 20-29 F Colombia Colombia 8 Active Yes US NA NA 1:100 1:32,000 44 30-39 F Colombia Colombia 15 Active Yes NA NA 0 1:10,000 45 20-29 F Colombia Colombia 21 Late Yes NA NA 1:10 1:100,000 46 20-29 F Colombia Colombia 29 Late Yes NA NA 1:320 1:32,000 47 20-29 F Colombia Colombia 38 Late Yes NA NA .sup.1:1,000 1:320,000 48 10-19 F Colombia Colombia 50 Late Yes NA NA 1:10 1:10,000 49 20-29 F Colombia Colombia 88 Late Yes NA NA 0 1:1,000 50 40-49 F Colombia Colombia 2 Initial Yes NA NA 0 1:3,200 51 20-29 M Colombia Colombia 5 Initial Yes NA NA .sup.1:1,000 1:32,000 52 30-39 F Colombia Colombia 6 Active Yes NA NA 0 1:1,000 53 20-29 M Colombia Colombia 8 Active Yes NA NA 0 1:10,000 54 30-39 F Colombia Colombia 15 Active Yes NA NA 1:320 1:320,000 55 30-39 M Colombia Colombia 21 Late Yes NA NA 1:100 1:32,000 56 40-49 M Colombia Colombia 29 Late Yes NA NA 1:32,000 1:32,000 57 40-49 F Colombia Colombia 38 Late Yes NA NA 0 1:320 58 50-59 F Colombia Colombia 50 Late Yes NA NA 0 1:100,000 59 50-59 M Colombia Colombia 85 Late Yes NA NA 0 1:32,000 AMC: Academic Medical Center; CDC: Centers for Disease Control and Prevention; dpso: days post symptom onset; F: female; IIFA: indirect immunofluorescence assay; ITM: Institute of Tropical Medicine, M: male; NA: not available; NS: non-structural protein; Pos: positive; US: United States; WHOCC: World Health Organization Collaborating Centre (for Arbovirus and Haemorrhagic Fever Reference and Research); ZIKV: Zika virus. .sup.aPhase of infection at the time of sample collection: initial phase: 5 dpso; active phase: 6 to 20 dpso; late phase: >20 dpso. .sup.bFever, skin rash, joint pain, myalgia, headache, conjunctivitis, eye pain, diarrhoea and malaise. .sup.cZIKV-RT-PCR results can also refer to serum or urine samples taken at an earlier date than the samples used for anti-ZIKV serological testing. .sup.dIIFA was performed at EUROIMMUN, Lbeck, Germany, using the Anti-Zika Virus IIFA test kit (EUROIMMUN). Cut-off IgM: 1:10; IgG: 1:100. .sup.eSera from Dutch residents who were born and raised in Suriname and/or had visited their country of origin occasionally.
TABLE-US-00002 TABLE 2 Characteristics of control cohorts, study evaluating a novel NS1-based ELISA, Germany 2016 Origin of sample Diagnostic centre Sample Cohort n donors Type (provider of samples) receipt Pre-characterisation Flavivirus infection or vaccination DENVa 47 Germany, Returning travellers MVZ Diamedes GmbH 2011-2014 Panbio or BIO-RAD DENV-NS1 ELISA.sup.a,b: 47/47 (100%) DENV-NS1 positive (high IgM) Italy from endemic areas Bielefeld, Germany; DENV-RT-PCR (only 8/47 tested).sup.b: n = 4 subtype DENV-1, n = 2 subtype with DENV infection University of Bologna, DENV-2, n = 2 subtype DENV-3 (contracted e.g. in Bologna, Italy; EUROIMMUN Anti-DENV ELISA (IgM, IgG).sup.c: 40/47 (85%) anti-DENV IgM Brazil, Bali, Thailand, WHOCC, Hamburg, Germany positive, 30/47 (64%) anti-DENV IgG positive, 37/47 (79%) anti-DENV IgM Laos, Philippines, India, ratio 3.0, 10/47 (21%) anti-DENV IgM ratio <3.0, anti-DENV IgM median Cambodia, Taiwan) ratio = 3.9 DENVb 46 Germany, Returning travellers MVZ Diamedes GmbH 2011-2014 DENV-NS1 ELISA.sup.a,b: 46/46 (100%) DENV-NS1 positive (high IgG) Italy from endemic areas Bielefeld, Germany; DENV-RT-PCR (only 1/46 tested).sup.b: n = 1 subtype DENV-4 with DENV infection University of Bologna, EUROIMMUN Anti-DENV ELISA (IgM, IgG).sup.c: 35/46 (76%) anti-DENV IgM (contracted e.g. in Bologna, Italy positive, 40/46 (87%) anti-DENV IgG positive, 37/46 (80%) anti-DENV IgG Brazil, Bali, Thailand, ratio 3.0, 9/46 (20%) anti-DENV IgG ratio <3.0, Laos, Philippines, India, anti-DENV IgG median ratio = 3.9 Cambodia, Taiwan) YFV 12 France Individuals vaccinated Cerba Specimen Services, 2015 YFV seroneutralisation test.sup.d: 12/12 (100%) anti-YFV positive against YFV Saint-Ouen I'Aumne, EUROIMMUN Anti-WNV ELISA (IgM, IgG).sup.c: 0/12 (0%) anti-WNV IgM positive, France 0/12 (0%) anti-WNV IgG positive EUROIMMUN Anti-CHIKV ELISA (IgM, IgG).sup.c: 0/12 (0%) anti-CHIKV IgM positive, 1/12 (8%) anti-CHIKV IgG positive WNV 34 US Patients from endemic MAYO Clinic, Scottsdale, 2014 WNV PRNT.sup.e: 34/34 (100%) anti-WNV positive areas with WNV infection Arizona, US EUROIMMUN Anti-WNV ELISA (IgM, IgG).sup.c: 23/34 (68%) anti-WNV IgM positive, 26/34 (76%) anti-WNV IgG positive JEV 25 Vietnam Patients from endemic National Hospital of Tropical 2016 DRG JE IgM capture ELISA.sup.f: 25/25 (100%) anti-JEV IgM positive areas with JEV infection Disease, Hanoi, Vietnam EUROIMMUN Anti-JEV ELISA (IgM, IgG).sup.c: 25/25 (100%) anti-JEV IgM positive, 19/25 (76%) anti-JEV IgG positive Non-flavivirus infection CHIKV 19 Reunion Patients from endemic Cerba Specimen Services, 2015 CHIKV VRP neutralisation test.sup.g: 19/19 (100%) anti-CHIKV positive areas with CHIKV Saint-Ouen I'Aumne, EUROIMMUN Anti-CHIKV ELISA (IgM, IgG).sup.c: 0/19 (0%) anti-CHIKV IgM infection France positive, 19/19 (100%) anti-CHIKV IgG positive Parasite infection PLAS 69 France Blood donors living in TheBindingSite, 2016 BioMrieux Plasmodium IFA (IgM, IgG).sup.d,h: 1/15 (7%) anti-Plasmodium IgM (including and travellers returning Schwetzingen, Germany positive, 15/15 (100%) anti-Plasmodium IgG positive overseas from Plasmodium- Cerba Specimen Services, BIO-RAD Malaria ELISA (IgG).sup.i: 54/54 (100%) anti-Plasmodium positive department endemic areas, acute or Saint-Ouen I'Aumne, and region past Plasmodium France Mayotte), infection Swiss Red Cross, Bern, French Switzerland Guiana, Tunisia, Madagascar, Switzerland Healthy controls: pregnant women, blood donors and children PREG 100 Germany Pregnant women from Laboratory Schottdorf, 2007 EUROIMMUN Anti-DENV ELISA (IgM, IgG).sup.c: 2/100 (2%) anti-DENV IgM non-flavivirus endemic Augsburg, Germany positive, 7/100 (7%) anti-DENV IgG positive areas without clinical EUROIMMUN Anti-WNV ELISA (IgM, IgG).sup.c 3/100 (3%) anti-WNV IgM positive, symptoms 4/100 (4%) anti-WNV IgG positive EUROIMMUN Anti-JEV ELISA (IgM, IgG).sup.c: 2/100 (2%) anti-JEV IgM positive, 14/100 (14%) anti-JEV IgG positive EUROIMMUN Anti-CHIKV ELISA (IgM, IgG).sup.c: 0/100 (0%) anti-CHIKV IgM positive, 0/100 (0%) anti-CHIKV IgG positive ZIM 128 Zimbabwe Blood donors from National Blood Transfusion 2003 EUROIMMUN Anti-DENV ELISA (IgG).sup.c: 4/128 (3%) anti-DENV IgG positive flavivirus and parasite Service, Zimbabwe, Africa EUROIMMUN Anti-CHIKV ELISA (IgG).sup.c: 3/128 (2%) anti-CHIKV IgG positive endemic areas without EUROIMMUN Anti-Plasmodium ELISA (IgG).sup.c: 36/128 (28%) anti-Plasmodium clinical symptoms IgG positive ARG 99 Argentina Blood donors from IACA Laboratory, Buenos 2014 EUROIMMUN Anti-DENV ELISA (IgM, IgG).sup.c: 2/99 (2%) anti-DENV IgM flavivirus endemic areas Aires, Argentina positive, 4/99 (4%) anti-DENV IgG positive without signs of viral EUROIMMUN Anti-WNV ELISA (IgM, IgG).sup.c: 2/99 (2%) anti-WNV IgM positive, infection (routine 3/99 (3%) anti-WNV IgG positive samples for EUROIMMUN Anti-CHIKV ELISA (IgM, IgG).sup.c: 3/99 (3%) anti-CHIKV IgM parasitology) positive, 1/99 (1%) anti-CHIKV IgG positive EUROIMMUN Anti-Trypanosoma ELISA (IgM, IgG).sup.c: 2/99 (2%) anti- Trypanosoma IgM positive, 1/99 (1%) anti-Trypanosoma IgG positive US 100 US Blood donors without Serologix, New Hope, 2014 EUROIMMUN Anti-DENV ELISA (IgM, IgG).sup.c: 1/100 (1%) anti-DENV IgM clinical symptoms (n): Pasadena, US positive, 6/100 (6%) anti-DENV IgG positive Hispanic (25), African EUROIMMUN Anti-WNV ELISA (IgM, IgG).sup.c: 0/100 (0%) anti-WNV IgM American (30), positive, 4/100 (4%) anti-WNV IgG positive Caucasian (43), Asian EUROIMMUN Anti-CHIKV ELISA (IgM, IgG).sup.c: 0/100 (0%) anti-CHIKV IgM (1), Colombian (1) positive, 4/100 (4%) anti-CHIKV IgG positive GER 500 Germany Blood donors from non- University Medical Center 2012 NA flavivirus endemic areas Schleswig-Holstein, Campus without clinical Lbeck, Lbeck, Germany symptoms CHIL 88 Germany Children (10 years) Praxis Dr Fischer-Wassels, 2007-2008 EUROIMMUN Anti-DENV ELISA (IgM, IgG).sup.c: 0/100 (0%) anti-DENV IgM form non-flavivirus Dortmund, Germany positive, 0/100 (0%) anti-DENV IgG positive endemic areas without EUROIMMUN Anti-WNV ELISA (IgM, IgG).sup.c: 1/100 (1%) anti-WNV IgM clinical symptoms positive, 0/100 (0%) anti-WNV IgG positive EUROIMMUN Anti-JEV ELISA (IgM, IgG).sup.c: 0/100 (0%) anti-JEV IgM positive, 0/100 (0%) anti-JEV IgG positive EUROIMMUN Anti-CHIKV ELISA (IgM, IgG).sup.c: 0/100 (0%) anti-CHIKV IgM positive, 0/100 (0%) anti-CHIKV IgG positive ARG: Argentina; CHIKV: chikungunya virus; CHIL: children; DENV: dengue virus; IFA: immunofluorescence assay; GER: Germany; JEV: Japanese encephalitis virus; IIFA: indirect immunofluorescence assay; NA: not available; PLAS: Plasmodium; PREG: pregnant women; PRNT: plaque reduction neutralisation test; RT-PCR: reverse transcription-PCR; US: United States; WHOCC: World Health Organization Collaborating Centre (for Arbovirus and Haemorrhagic Fever Reference and Research); WNV: West Nile virus; YFV: yellow fever virus; ZIKV: Zika virus; ZIM: Zimbabwe. .sup.aPerformed at MVZ Diamedis GmbH, Bielefeld, Germany. .sup.bPerformed at the University of Bologna, Italy. .sup.cPerformed at EUROIMMUN, Lbeck, Germany. .sup.dPerformed at Cerba Specimen Services, Saint-Ouen I'Aumne, France. .sup.ePerformed at the University of Leipzig, Germany. .sup.fPerformed at the National Hospital of Tropical Disease, Hanoi, Vietnam. .sup.gPerformed at the University of Bonn, Germany. .sup.hPerformed at TheBindingSite, Schwetzingen, Germany. .sup.iPerformed at the Swiss Red Cross, Bern, Switzerland.
anonymised to the Institute for Experimental Immunology (affiliated to EUROIMMUN). All sera were stored at 20 C. until assayed. The study was performed according to the recommendations of the Central Ethical Committee of Germany [29].
[0216] Enzyme-Linked Immunosorbent Assays
[0217] Anti-Zika Virus IgM and IgG ELISA (EUROIMMUN) were used as recommended by the manufacturer. These kit assays are based on standardised reagents and microtitre plates coated with recombinant ZIKV-NS1. Briefly, sera diluted 1:101 in sample buffer were added to the wells and allowed to react for 60 min at 37 C. Before IgM detection, sera were pre-incubated with sample buffer containing IgG/rheumatoid factor (RF) absorbent (EUROIMMUN) to remove class IgG antibodies and class IgM RF from the sample. This step prevents specific IgG from displacing IgM from the antigen (leading to false IgM-negative results) and RF-IgM from reacting with specifically bound IgG (leading to false IgM-positive results). Bound antibodies were detected by applying goat anti-human IgM peroxidase conjugate or rabbit anti-human IgG peroxidase conjugate for 30 min at room temperature, followed by staining with tetramethylbenzidine for 15 min. The enzymatic reaction was stopped by addition of one volume 0.5 mol/L sulphuric acid. A calibrator (chicken-human chimeric ZIKV antibody with a concentration adjusted to give an extinction value defining the upper limit of the reference range of non-infected persons) as well as positive and negative controls were provided with the test kit and assayed with each test run. Colour intensity of the enzymatic reactions was determined photometrically at 450 nm (reference 620 nm), resulting in extinction values. A signal-to-cut-off ratio (extinction.sub.sample/extinction.sub.calibrator) was calculated for each sample.
[0218] Receiver-operating characteristics (ROC) analysis based on the initial validation dataset of positive and negative samples was done by the manufacturer to evaluate assay performance at each possible cut-off, demonstrating optimal sensitivity and specificity at ratio values of 0.8 (IgM) and 0.6 (IgG). To ensure high specificity, the borderline range (0.8 to <1.1) was established between the highest negative and the lowest positive validation sample, resulting in a positivity cut-off of 1.1.
[0219] Anti-dengue Virus IgM and IgG ELISA (EUROIMMUN) were used.
[0220] Statistics
[0221] Statistical analyses were performed using GraphPad Prism 6 (GraphPad Software Inc., La Jolla, Calif., US) and SigmaPlot 13.0 (SSI, San Jose, Calif., US). Sensitivity was calculated as the proportion of ZIKV patients (referring to groups 1 to 4 as indicated) identified as positive by the assay. Specificity was calculated as the proportion of negative test results obtained among healthy controls. We calculated 95% confidence intervals (Cis) according to the modified Wald method. The study was performed in compliance with the Standards for Reporting of Diagnostic accuracy (STARD) statement.
[0222] Results
[0223] Sensitivity of the Enzyme-Linked Immunosorbent Assay
[0224] The sensitivity of the novel NS1-based anti-ZIKV ELISA was evaluated in sera from 27 patients with RT-PCR-confirmed ZIKV infection that had been sub-grouped into travellers returning from ZIKV-endemic areas and endemic-area residents. Among eight infected travellers returning from ZIKV-endemic areas (group 1), positive anti-ZIKV IgM and IgG reactivity was found in seven (87.5%) and three (37.5%) cases, respectively. Of 19 infected residents in endemic-areas (group 2), six (31.6%) were positive for anti-ZIKV IgM and 15 (79.0%) for IgG. In addition, sera from 85 patients with suspected ZIKV infection were examined. Here, of 26 infected travellers returning from ZIKV-endemic areas (group 3) 21 (80.8%) were positive for anti-ZIKV IgM and 18 (69.2%) for IgG, while among 59 infected residents in endemic-areas (group 4), six (10.2%) showed positive reactivity for anti-ZIKV IgM and 53 (89.9%) for IgG. For the total of RT-PCR-confirmed and suspected cases, the combined ELISA sensitivity (IgM and/or IgG) amounted to 23/27 (85.2%) and 78/85 (91.8%), respectively.
[0225] Confining the time point of serological evaluation to the active and late phase of ZIKV infection, i.e. 6 days after symptom onset, anti-ZIKV IgM reactivity was observed in 10/17 (58.8%) patients with positive ZIKV-RT-PCR and 3/38 (7.9%) patients with suspected ZIKV infection, while anti-ZIKV IgG was detectable in 15/17 (88.2%) and 34/38 (89.5%) cases, respectively. Thus, the combined sensitivity (IgM and/or IgG) reached 17/17 (100%) among RT-PCR-confirmed cases and 34/38 (89.5%) among suspected cases (Table 3).
TABLE-US-00003 TABLE 3 Anti-ZIKV reactivity in patients with RT-PCR-confirmed (n = 27) and suspected (n = 85) ZIKV infection as determined by ELISA for IgM and IgG, study evaluating a novel NS1-based ELISA, Germany 2016 Anti-ZIKV ELISA reactivity Anti-ZIKV ELISA reactivity (1 day post symptom onset).sup.c (6 days post symptom onset).sup.d,e Group Characteristics n IgM IgG IgM/IgG n IgM IgG IgM/IgG 1 RT-PCR-confirmed Positive 8 7 3 7 5 5 3 5 ZIKV infection, Sensitivity 87.5 37.5 87.5 100 60.0 100 travellers returning %.sup.b (50.8-99.9) (13.5-69.6) (50.8-99.9) (51.1-100) (22.9-88.4) (51.1-100) from ZIKV-endemic (95% CI) areas 2 RT-PCR-confirmed Positive 19 6 15 16 12 5 12 12 ZIKV infection, Sensitivity 31.6 78.9 84.2 41.7 100 100 residents in ZIKV- %.sup.b (15.2-54.2) (56.1-92.1) (61.6-95.3) (19.3-68.1) (71.8-100) (71.8-100) endemic areas.sup.a (95% CI) Total RT-PCR-confirmed Positive 27 13 18 23 17 10 15 17 1 + 2 ZIKV infection Sensitivity 48.1 66.7 85.2 58.8 88.2 100 %.sup.b (30.7-66.0) (47.7-81.5) (66.9-94.7) (36.0-78.4) (64.4-98.0) (78.4-100) (95% CI) 3 Suspected ZIKV Positive 26 21 18 25 NA.sup.e infection, travellers Sensitivity 80.8 69.2 96.2 returning from %.sup.b (61.7-92.0) (49.9-83.7) (79.6-100) ZIKV-endemic areas (95% CI) 4 Suspected ZIKV Positive 59 6 53 53 38 3 34 34 infection, residents Sensitivity 10.2 89.9 89.9 7.9 89.5 89.5 ZIKV-endemic areas %.sup.b (4.4-20.8) (79.2-95.6) (79.2-95.6) (2.0-21.5) (75.3-96.4) (75.3-96.4) (95% CI) Total Suspected ZIKV Positive 85 27 71 78 38.sup.e 3 34 34 3 + 4 infection Sensitivity 31.8 83.5 91.8 7.9 89.5 89.5 %.sup.b (22.8-42.3) (74.1-90.1) (83.7-96.2) (2.0-21.5) (75.3-96.4) (75.3-96.4) (95% CI) CI: confidence interval; NA: not available or not applicable; NS: non-structural protein; RT-PCR: reverse transcription-PCR; ZIKV: Zika virus. .sup.aThis group contains 10 sera from residents of the Netherlands who were born and raised in Suriname and/or had visited their country of origin occasionally. .sup.bReferring to the total number of samples in the respective patient group during the indicated sampling period. .sup.cReferring to the whole study population of ZIKV-infected patients, i.e. samples (one per patient) taken between day 1 and day 88 post symptom onset, representing the initial (day 1-5 post symptom onset), active (day 6-20) and late phase (>20 days) of infection. .sup.dSamples (one per patient) taken between day 6 and day 88 post symptom onset, representing the active (day 6 to 20 post symptom onset) and late phase (>20 days post symptom onset) of infection. .sup.eGroup 3 is not represented in the sampling period 6 days post symptom onset, because the sampling date was available for only four out of a total of 26 samples in this group.
TABLE-US-00004 TABLE 4 Anti-ZIKV reactivity in potentially cross-reactive specimens (n = 252) and healthy controls (n = 1,015) as determined by ELISA for IgM and IgG, study evaluating a novel NS1-based ELISA, Germany 2016 Prevalence % (CI 95%).sup.c Specificity (CI 95%).sup.c Cohort Characteristics IgM IgG IgM IgG DENVa Dengue viris infection (high median anti-DENV IgM).sup.a 47 0 (0-9.0) 0 (0-9.0) 100 (91.0-100) 100 (91.0-100) DENVb Dengue viris infection (high median anti-DENV IgG.sup.b 46 0 (0-9.2) 0 (0-9.2) 100 (90.8-100) 100 (90.8-100) YFV Yellow fever virus vaccination 12 0 (0-28.2) 0 (0-28.2) 100 (71.8-100) 100 (71.8-100) WNV West Nile virus infection 34 2.9 (0-16.2) 0 (0-12.1) 97.1 (83.8-100) 100 (87.9-100) JEV Japanese encephalitis virus infection 25 0 (0-15.8) 4.0 (0-21.1) 100 (84.2-100) 96.0 (78.9-100) CHIKV Chikungunya virus infection 19 0 (0-19.8) 0 (0-19.8) 100 (80.2-100) 100 (80.2-100) PLAS Plasmodium spp. Infection 69 1.4 (0-8.5) 0 (0-6.3) 98.6 (91.5-100) 100 (93.7-100) Total Potentially cross-reactive samples 252 0.8 (0-3.0) 0.4 (0-24) 99.2 (97.0-100) 99.6 (97.6-100) PREG German pregnant women 100 0 (0-4.4) 0 (0-14) 100 (95.6-100) 100 (95.6-100) ZIM Zimbabwean blood donors 128 0 (0-3.5) 0 (0-3.5) 100 (96.5-100) 100 (96.5-400) ARG Argentinian blood donors 99 1.0 (0-6.1) 0 (0-4.5) 99.0 (94.0-100) 100 (95.5-100) US US-American blood donors 100 0 (0-4.4) 1.0 (0-6.0) 100 (95.6-100) 99.0 (94.0-100) GER German blood donors 500 0.2 (0-1.2) 0.2 (0-1.2) 99.8 (98.8-100) 99.8 (98.8-400) CHIL German children 88 0 (0-5.0) 0 (0-5.0) 100 (95.0-100) 100 (95.0-100) Total Healthy control samples 1,015 0.2 (0-0.8) 0.2 (0-0.8) 99.8 (99.2-100) 99.8 (99.2-100) ARG: Argentina; CHIKV: chikungunya virus; CHIL: children; DENV: dengue virus; GER: Germany; JEV: Japanese encephalitis virus; PLAS: Plasmodium; PREG: pregnant women; US: United States; WNV: West Nile virus; YFV: yellow fever virus; ZIKV: Zika virus; ZIM: Zimbabwe. .sup.aMedian anti-DENV IgM ratio 3.9 (79% of samples with anti-DENV IgM ratio 3.0), as indicated in the inset of FIG. 9A. .sup.bMedian anti-DENV IgG ratio 3.9 (80% of samples with anti-DENV IgG ratio 3.0), as indicated in the inset of FIG. 9B. .sup.cReferring to the total number of samples in the individual cohorts.
[0226] Comparing ZIKV-infected travellers returning from endemic areas (groups 1 and 3) with infected residents in these areas (groups 2 and 4), a tendency of distinct ZIKV antibody kinetics could be observed: in most returning travellers, high IgM ratio values (median 5.6; interquartile range (IQR): 4.6-6.9) and moderate IgG ratios (median 2.2; IQR 0.9-2.8) were detectable in the active phase of infection (cut-off ratio: 1.1). By contrast, the majority of endemic-area residents had infections with very high IgG ratios (median 4.8; IQR 3.3-5.9) during the active phase, while IgM ratios were variable, but predominantly negative or low (median 0.5; IQR 0.2-1.3) (
[0227] Time course analysis of a German patient who showed clinical symptoms after returning from a stay in Colombia revealed very high anti-ZIKV IgM ratios on first testing (day 10 after symptom onset), while IgG ratios increased to moderate levels during the acute phase of infection and thereafter (
[0228] Cross-Reactivity of the Enzyme-Linked Immunosorbent Assay
[0229] Cross-reactivity was analysed first in sera from 93 DENV-infected patients whose diagnosis had been secured by positive DENV-NS1 detection. This cohort was divided into one group (DENVa) with high anti-DENV IgM (median ratio 3.9) and another group (DENVb) with high anti-DENV IgG (median ratio 3.9), ensuring the presence of high levels of potentially cross-reactive antibodies. In both groups, anti-ZIKV reactivity was below the threshold, indicating absence of cross-reactivity in these specimens. Further testing, on a supplementary basis, included 159 sera from patients positive for IgM and/or IgG against YFV, WNV, JEV, CHIKV or PLAS. Anti-ZIKV IgM was positive in 1/34 (2.9%) patients infected with WNV and 1/69 (1.4%) patients infected with PLAS. Anti-ZIKV IgG was found in 1/25 (4.0%) patients infected with JEV (Figure G). For the total of 252 potentially cross-reactive samples, the overall positivity rate amounted to 2/252 (0.8%) for IgM and 1/252 (0.4%) for IgG (Table 4).
[0230] Specificity of the Enzyme-Linked Immunosorbent Assay
[0231] Assay specificity was assessed by testing 1,015 sera from healthy controls. Only 1/99 (1.0%) Argentinian and 1/500 (0.2%) German blood donors were found anti-ZIKV IgM positive, while all 128 Zimbabwean and 100 US American blood donors as well as 100 German pregnant women and 88 children in Germany were negative. Anti-ZIKV IgG was present in 1/100 (1.0%) US American and 1/500 (0.2%) German blood donors, but absent in the cohorts of Zimbabwean and Argentinian blood donors, pregnant women and children. Thus, overall specificity amounted to 99.8% for either Ig class (Table 4,
[0232] Discussion
[0233] The serological diagnosis of ZIKV infections has been challenging due to cross-reactions with other flaviviruses, secondary infections and previous vaccinations, which complicate interpretation, sometimes leading to unreliable or false-positive results. Here, we evaluated a newly-developed ELISA with recombinant ZIKV-NS1 protein as solid-phase antigen. Huzly et al. (2016 Apr. 21; 21(16). doi: 10.2807/1560-7917) recently provided evidence that this assay is highly specific, as demonstrated on a limited number of European patients with DENV, YFV, tick-borne encephalitis virus (TBEV) or hepatitis C virus infection. In the present study, testing on specimens collected 6 days after onset of symptoms (i.e. after the viraemic phase) revealed a combined sensitivity (IgM/IgG) of 100% for RT-PCR-confirmed cases of ZIKV infection at 99.8% specificity. Among suspected ZIKV cases, the combined sensitivity amounted to 89.5%. Notably, we included only one serum sample for each of the studied patients in our analysis, except for the time course analysis. For the serological diagnosis of patients, however, the evaluation of follow-up samples is important and recommended to demonstrate seroconversion or a 4-fold increase in antibody titre. In four of 27 RT-PCR-confirmed ZIKV cases, samples were negative for both IgM and IgG against ZIKV-NS1, presumably because all of them were taken only s 4 days after symptom onset, i.e. when antibodies had not yet reached detectable levels. Among 85 suspected ZIKV patients, too early sampling may account for two cases with negative IgM and IgG, while the remaining five double-negative cases could be due to the absence of ZIKV infection (deficits in pre-characterisation) or to false-negative results.
[0234] Cross-reactivity with high-level DENV antibodies was not detectable and, according to preliminary analysis with a limited amount of samples, there was no indication for DENV serotype-dependent differences in cross-reactivity (data not shown). To better judge assay performance in endemic areas, samples from endemic residents who experienced multiple DENV (and other flavivirus) infections should be included in further assessments, as these samples have a potential for increased cross-reactivity. Analysis of all potentially cross-reactive specimens resulted in positive rates of 0.8% (IgM) and 0.4% (IgG) caused by one case each with WNV and PLAS with low-level anti-ZIKV IgM and one JEV case with low-level anti-ZIKV IgG. In these cases, however, double infections cannot be excluded, so it remains unclear if ELISA positivity resulted from the presence of ZIKV antibodies due to co-infection with ZIKV (true-positive) or from cross-reactivity (false-positive). In case of PLAS infection, PLAS-induced polyclonal B-cell activation may cause the production of potentially cross-reactive antibodies. Among patients with current PLAS infection, up to 30% false-positive or borderline reactions were reported using the presented NS1-based ELISA, which is in contrast to only 1.4% in the present study and probably explained by the fact that our cohort was comprised mainly of individuals with past PLAS infection status. Possible interferences should thus be considered when applying the assay.
[0235] In sera from travellers returning from ZIKV-endemic areas, we observed a tendency of ZIKV-specific IgM to appear at high ratios during the active phase of infection, paralleled by a moderate rise in IgG. In contrast, most residents in endemic areas had high anti-ZIKV IgG and low/negative IgM ratio values, irrespective of whether their samples were taken during the initial, active or late phase of infection. IgM responses in travellers returning from ZIKV-endemic areas tended to be higher compared with residents in such areas, whereas the IgG-positivity rate was higher in the latter subgroup. Such differences in ZIKV antibody kinetics were also illustrated by time course analysis of antibody levels in two representative patients, possibly reflecting that travellers returning from ZIKV-endemic countries predominantly had a primary flavivirus/ZIKV infection, while most residents probably contracted ZIKV as a secondary flavivirus infection. Similar kinetics have been described for primary and secondary infections in the Micronesian ZIKV epidemic and for DENV-infected patients, suggesting that the detection of both specific IgM and IgG is diagnostically important and relevant for differentiating primary from secondary infections. Regarding our comparison of patients residing in endemic countries vs travellers, however, systematic differences in the background of these populations (e.g. genetic, ethnic) cannot be excluded.
[0236] Another limitation of our study is that it does not comprise side-by-side testing with additional assays, such as the Zika MAC-ELISA (Centers for Disease Control and Prevention (CDC), Atlanta, Ga., US) or PRNT, to provide comparative data on these current tests. In addition, the non-deliberate absence of a uniform serological reference standard for the pre-characterisation of all ZIKV samples resulted in a high number of suspected cases of ZIKV infection.
[0237] Although ZIKV usually causes rather mild infections, there has been convincing evidence of a causal link to neuronal impairment, such as newborn microcephaly and GBS [37].
[0238] Furthermore, there have been studies showing that DENV NS1 antibodies have the potential of inducing autoantibodies in secondary infections, probably mediated by cross-reactive binding of antigens on platelets and endothelial cells, followed by cellular damage and inflammatory activation. Basic research is needed to fully elucidate the causal relations between neuronal disorders and ZIKV infection. Epidemiologic assessment of pregnant women and their babies, and of travellers returning from endemic areas, the surveillance of donated blood and the investigation of ZIKV prevalence in endemic and non-endemic areas may provide crucial information. These studies need reliable, fast, and easy-to-handle diagnostic tests that have low cross-reactivity and allow a definite diagnosis.
[0239] In conclusion, our study revealed that the NS1-based anti-ZIKV ELISA is a sensitive and highly specific tool for the serodiagnosis of ZIKV infections, eliminating cross-reactions with antibodies to DENV and other flaviviurses. The assay format is suitable for use in routine laboratories worldwide enabling high-throughput testing in epidemic settings. Serological identification of ZIKV infections is maximised by parallel testing for IgM and IgG. Further studies will be necessary to determine the accuracy of this and other current assays in a larger set of well-defined samples, and to clarify how ZIKV infection triggers GBS, newborn microcephaly and other neurological manifestations.
EXAMPLE 8: ANTI-ZIKA VIRUS IGA MAY INDICATE AN ACUTE INFECTION IN ANTI-ZIKA VIRUS IGM-NEGATIVE PATIENTS
[0240] This example shows that IgA to SEQ ID NO1 and related reagents and methods may be used for distinguishing an acute infection from a past and thus a primary from a secondary infection.
[0241] Methods
[0242] Serum samples were taken at several time points from two Columbians with a background of past flavivirus infections and from two German travellers, all presenting with confirmed ZIKV infections. Titers of anti-ZIKV IgM and IgG were measured using a commercial NS1-based Anti-Zika virus ELISA (Euroimmun AG, Germany). An indirect immunofluorescence test (Arbovirus Fever Mosaic 2, IgM, cut-off a 1:10, Euroimmun AG, Germany) based on cells infected with ZIKV was used additionally for IgM measurement. For determination of anti-ZIKV IgA, a corresponding ELISA was adapted, applying an anti-human IgA conjugated with peroxidase. In all assays, the cut-off was set to a ratio of 1.1.
[0243] Results
[0244] In the German travellers, anti-ZIKV IgM was detected at day 9 and day 16, respectively, irrespective of the method. Active infections were subsequently confirmed by anti-ZIKV IgG seroconversion. IgA measurements were above 1.1 in all samples except for one, showing an initial increase and a subsequent decrease (Table 5)
TABLE-US-00005 Anti-ZIKV Anti-ZIKV Anti-ZIKV Country of Country of Days after IgA ratio; IgM ratio; IgG ratio; Patient origin infection symptoms pos: >1.1 pos: >1.1 pos: >1.1 1 Colombia Colombia 16 0.1 0.0 0.6 6 0.6 0.0 2.0 24 3.4 0.0 4.9 66 0.6 0.0 3.3 2 Colombia Colombia 3 0.2 0.1 2.0 15 2.9 0.5 5.9 38 0.7 0.2 5.2 52 0.6 0.1 5.0 66 0.5 0.1 4.9 76 0.6 0.1 5.1 95 0.5 0.1 4.7 3 Germany Martinique 11 4.1 1.0 0.1 16 9.0 2.7 1.4 36 1.5 1.2 2.5 4 Germany Nicaragua 4 0.3 0.2 0.2 9 7.6 2.4 1.0 30 2.4 0.9 3.0
[0245] In the sequential samples of the two Colombian patients (results shown in
[0246] Conclusion
[0247] When specific IgM is not detectable neither with NS1- nor full virus-based assays as observed in the Colombian patients, measurement of anti-ZIKV IgA may allow discrimination of acute from past infections.
EXAMPLE 9: ABSENCE OF SPECIFIC IGM IN WEEK SIX POST SYMPTOM ONSET IN A PATIENT WITH CONFIRMED ZIKA VIRUS INFECTION
[0248] This example shows that detecting the presence or absence of both IgG and IgM to SEQ ID NO1 and related reagents and methods may be used for increasing the diagnostic reliability of an assay for diagnosing a Zika virus infection compared to assays based on the detection of Ig from one class only.
[0249] Introduction
[0250] Subsequently to the severe outbreak of Zika virus (ZIKV) infections in the Americas, CDC recommends nucleic acid testing in samples collected within two weeks after symptom onset. Serum samples collected after day 14 and up to day 84 should be tested for anti-ZIKV IgM antibodies assuming that these are present any time from near day four post symptom onset to up to twelve weeks.
[0251] However, absence of specific IgM has been frequently reported in patients with secondary Dengue virus (DENV) infections, since DENV and ZIKV are related flaviviruses immunological response may be comparable in ZIKV infections.
[0252] Methods
[0253] A Colombian woman of 42 years with ZIKV infection as confirmed by RT_PCR five days after appearance of the first symptoms was additionally tested for specific IgM and IgG antibodies. Serum samples taken at day five and day 41 post symptom onset were analysed using ZIKV IgM Capture ELISA, based on the full virus antigen (cut-off ratio 1.8; InBios, USA) and NS1-based Anti-Zika virus ELISA IgM as well as IgG (cut-off ratio 1.1; Euroimmun AG, Germany) according to instructions of the manufacturer.
[0254] Results
[0255] Results are shown in
[0256] Conclusion
[0257] Both available serum samples of this patient dated from the suggested anti-ZIKV IgM-positive time frame (12 weeks) but were tested IgM-negative independently of the antigenic substrate usedfull virus or NS1.
[0258] Instead, the two samples revealed an increasing IgG titer suggesting that parallel testing for anti-ZIKV IgM and IgG in two consecutive serum samples should be performed to detect either seroconversion or a significant IgG titer increase in order to avoid missing patients tested anti-ZIKV IgM-negative.
TABLE-US-00006 TABLE 6 Days Ratio after IgM Capture NS1-based NS1-based onset of ELISA ELISA IgM ELISA IgG symptoms Cut-off: 1.8 Cut-off: 1.1 Cut-off: 1.1 5 0.81 0.1 1.9 41 0.12 0.4 5.6