ImmunoWELL Zika Virus Serology

Introduction ImmunoWELL Zika Test consists of two Zika virus serology assays, one detecting specific IgG and the other specific IgM. The Zika virus IgG test detects anti-non-structural protein 1 (NS1). The Zika virus IgM test uses gradient purified, inactivated virus lysate as its antigen. It is expected that nucleic acid test provides the most accurate result during early infection while serology becomes useful as the nucleic acid detection wanes. This means that IgG is typically present. Our studies show that the IgG amount is typically very high in Zika infected patients. Based on this information, GenBio designed the serology interpretation to require IgG to be positive before interpreting the IgM result.

Cross-Reactivity Significant serology cross-reactivity to related viruses (for example, Dengue virus, West Nile Virus, Yellow Fever virus, Chikgunya virus etc.) is expected. Therefore, to identify the specific causative agent requires use of one or more additional test methods (for example, specific nucleic acid detection or neutralization serology). Even so, by using a combination of IgG and IgM results, serology test results are useful to detect current and recent infection. Besides our initial IgG and IgM kits, GenBio continues to develop an additional test that better differentiates Zika infection from other Flavivirus infections.

Procedure The procedure is optimized for use in the larger laboratory. Incubation steps are extended to assure reactions are complete. The assay does not require significant temperature control and internal calibration (run standardization) adjusts for equipment and technical differences. After validation, the test system may be used by any laboratory with expertise to perform microwell immunoassays. The test may be used manually or incorporated into open source automated equipment.

Process Control GenBio has developed and manufactured serology assays since 1988 and its development staff has assay development expertise back to the late 1970’s. GenBio has provided assays into the US market including its largest reference laboratories since the 1990’s. This includes several IgM serology assays. The Zika virus assays use the same common component reagents and corresponding, validated manufacturing processes. Only the antigens and control reagents are unique to the new assay.

Interpretation of Results Diagnostic accuracy is best when both IgG and IgM results are used. This result is defined as “Total Activity, TA”. Total Activity ratio = IgG Activity (GA) * IgM Activity (MA) A ratio of 0.75 to 1.00 is equivocal A ratio above 1.00 represent a significant reaction If GA is higher than 2.50, testing a 10-fold dilution is suggested IgM may be present, but is not always present. IgG is detected about the same time as IgM is detected, but IgG remains for months while IgM remains for weeks.

IgG Reactivity IgG Titer is the best indicator of recent Flavivirus infection IgG Activity above 2.5 ratio indicates an acute flavivirus infection Additional titration is useful to confirm recent flavivirus infection Measurement of anti-NS1 cannot distinguish Zika virus from other Flavivirus infections. Under development at GenBio is an additional IgG test to better distinguish Zika virus from other Flavivirus serology reactivity.

IgM Reactivity IgM reactivity is significantly lower than IgG reactivity Never interpret IgM as positive if the IgG activity is less than 1.0 ratio. It is best to use the Total Activity (TA) ratio to interpret significant IgM activity. The TA ratio combines IgG and IgM reactivity results.

High IgG Testing Zika Virus infections cause exceptionally high IgG amount We recommend testing IgG total higher than 2.5 ratio at a 1:10 dilution. Use specimen diluent to make the 10-fold dilution. Calculate a “Total 10” ratio by multiplying the 1:10 IgG and original IgM result. This will result in a higher Total ratio that is used to better identify a serum from an active infection from a past infection.

Serial Samples from Zika Nucleic Acid Patients Zika Patient Results Serial Samples from Zika Nucleic Acid Patients

Serial Bleed Patient Studies Serial samples collected from Dominican Republic subjects At least one specimen reported positive nucleic acid result By 20 days after first symptoms, IgG is very high Results and subject information are reported in the following slides Below is an Excel file containing data for the following charts

10730 Male

10740 Female

10745 Female, 2nd Trimester

10752 Female, 2nd Trimester

10758 Female

10793 Female

10795 Female, end of 1st Trimester

10797 Female, 1st Trimester, Miscarriage at 31 days

10803 Male

13790 Female, 1st Trimester

Zika Serology Results of Endemic and nonendemic Groups Population Studies Zika Serology Results of Endemic and nonendemic Groups

Pregnant US Subjects Specimens collected from two hundred sixty four (264) pregnant New York City subjects were tested. All specimens were collected prior to 2010 and therefore no Zika virus exposure is expected. Five specimens reported greater than 1.00 IgG Activity (GA) No specimens reported greater than 1.00 IgM Activity (MA) No specimens reported greater than 1.00 Total Activity (TA) Below is an Excel file containing the results.

South Florida Blood Donor Specimens collected from fifty (50) subjects in Florida were tested. All specimens were collected in April 2018, after the 2017 epidemic season. Because the subjects are donating blood, it is presumed that the subject is healthy without disease symptoms. One specimen reported greater than 1.00 IgG Activity (GA) No specimens reported greater than 1.00 IgM Activity (MA) No specimens reported greater than 1.00 Total Activity (TA) Below is an Excel file containing the results.

Puerto Rico Blood Donor Specimens collected from fifty (50) subjects in Puerto Rico were tested. All specimens were collected in April 2018, after the 2017 epidemic season. Because the subjects are donating blood, it is presumed that the subject is healthy without disease symptoms. Fifteen specimens reported greater than 1.00 IgG Activity (GA) One specimen reported greater than 1.00 IgM Activity (MA) Seven specimens reported greater than 1.00 Total Activity (1:10 titration) One specimen (#24) report > 10.00 Total Activity. Below is an Excel file containing the results.

Columbia Blood Donor Specimens collected from ninety-five (95) subjects in Columbia were tested. All specimens were collected in April 2018, after the 2017 epidemic season. Because the subjects are donating blood, it is presumed that the subject is healthy without disease symptoms. Fifty-one specimens reported greater than 1.00 IgG Activity (GA) No specimens reported greater than 1.00 IgM Activity (MA) Fifteen specimens reported greater than 1.00 Total Activity (1:10 titration) One specimen (#81) reports > 10.00 Total Activity. Below is an Excel file containing the results.

Summary Zika Virus Serology

Summary Information During active infection, IgG activity is very high and usually a 10-fold dilution of the specimen is required to differentiate active infection from past flavivirus infections. Recent, active flavivirus infection usually reports greater than 10.00 antibody activity. The presence of IgM improves the accuracy, but IgM is not always present.

Flavivirus Cross-reactivity GenBio has limited information about Dengue or Chikgunya cross- reactivity with Zika reactivity. Our limited studies do show that greater than 10.00 Total activity can occur during active Dengue or Chikgunya infections. The serial bleed specimens of Zika infected subjects (nucleic acid confirmed) show that IgM is not always present or sometimes show low activity. It is unknown why, but previous Flavivirus history may be a factor. Methods such as cell culture neutralization or nucleic acid tests are useful to distinguish Flavivirus types and should be used if clinically indicated.