WHO Emergency Use Assessment and Listing for Zika IVDs PUBLIC REPORT. Product: RealStar Zika Virus RT-PCR Kit 1.0 EUAL Number: EAZ

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1 EAZ WHO EUAL ZIKV Public Report 08/2016, version 1.0 WHO Emergency Use Assessment and Listing for Zika IVDs PUBLIC REPORT Product: RealStar Zika Virus RT-PCR Kit 1.0 EUAL Number: EAZ Emergency Use Assessment and Listing of In Vitro Diagnostics Procedure WHO has developed an Emergency Use Assessment and Listing (EUAL) procedure to expedite the availability of in vitro diagnostics (IVDs) needed in public health emergency situations. This EUAL procedure will generate WHO recommendations in order to provide advice to procurement agencies and Member States on the acceptability of a specific IVD in the context of a public health emergency, based on a minimum set of available quality, safety, and performance data and an agreed plan for further evaluation. The EUAL procedure is comprised of three components that aim to assess the safety, quality and performance of the IVD: a review of the manufacturer s quality management system documentation; a review of the documentary evidence of safety and performance; and an independent performance evaluation RealStar Zika Virus RT-PCR Kit 1.0 with product code REF manufactured by altona Diagnostics GmbH, Mörkenstraße 12, Hamburg, Germany, CE marked regulatory version, was listed as eligible for WHO procurement on 05 August The RealStar Zika Virus RT-PCR Kit 1.0 is an in vitro diagnostic test, based on real-time PCR technology, for the qualitative detection of Zika virus specific RNA in human serum or urine. The assay includes a heterologous amplification system (Internal Control) to identify possible RT-PCR inhibition and to confirm the integrity of the reagents of the kit. The test is based on real-time RT-PCR technology, utilizing a reverse-transcriptase (RT) reaction to convert RNA into complementary DNA (cdna), polymerase chain reaction (PCR) for the amplification of specific target sequences and target specific probes for the detection of the amplified DNA. The probes are labelled with fluorescent reporter and quencher dyes. Probes specific for Zika virus RNA are labelled with the fluorophore FAM. The probe specific for the Internal Control (IC) is labelled with the fluorophore JOE. Using probes linked to distinguishable dyes enables the parallel detection of Zika virus specific RNA and the Internal Control in corresponding detector channels of the real-time PCR instrument. The test consists of three processes in a single tube assay: Reverse transcription of target RNA and Internal Control RNA to cdna PCR amplification of target cdna and Internal Control cdna Simultaneous detection of PCR amplicons by fluorescent dye labelled probes Page 1 of 9

2 EAZ WHO EUAL ZIKV Public Report 08/2016, version 1.0 Content of the kit Component Master A Master B Internal Positive PCR grade Control Control Water Number of Vials Volume [μl/vial] Lid Colour Blue Purple Green Red White Master A and Master B contain all components (buffer, enzymes, primers, and probes) to allow reverse transcription, PCR mediated amplification and target detection of Zika virus specific RNA and Internal Control in one reaction setup. Materials required but not provided Material Product name/description Instrumentation for Mx 3005P QPCR System (Stratagene) amplification VERSANT kpcr Molecular System AD (Siemens) The RealStar Zika Virus RT- ABI Prism 7500 SDS and 7500 Fast SDS (Applied PCR Kit 1.0 was developed Biosystems) and validated to be used LightCycler 480 Instrument II (Roche) with the following real-time Rotor-Gene 6000 (Corbett Research) PCR instruments: Rotor-Gene Q 5/6 plex Platform (QIAGEN) CFX96 Real-Time System and CFX96 Deep Well Real- Time System (Bio-Rad) Kits for nucleic acid extraction The following nucleic acid extraction kit is validated: QIAamp Viral RNA Mini Kit (QIAGEN). Additional materials Desktop centrifuge with a rotor for 2 ml reaction tubes Centrifuge with a rotor for microtiter plates, if using 96 well reaction plates Vortex mixer Appropriate 96 well reaction plates or reaction tubes with corresponding (optical) closing material Pipettes (adjustable) Pipette tips with filters (disposable) Powder-free gloves (disposable) Storage: The test kit should be stored at -25 o -15 C. Shelf-life upon manufacture: 9 months. Warnings/limitations: The performance observed using urine as only specimen type was lower than that observed when using paired specimens or serum only specimens. For this reason urine is not recommended as a sole specimen type at this present time. Page 2 of 9

3 EAZ WHO EUAL ZIKV Public Report 08/2016, version 1.0 WHO EUAL Assessment altona Diagnostics GmbH submitted an expression of interest for WHO emergency quality assessment of RealStar Zika Virus RT-PCR Kit 1.0 on 26 February Review of quality management documentation To establish the eligibility for WHO procurement, altona Diagnostics GmbH was asked to provide up-to-date information about the status of their quality management system. Based on the review of the submitted quality management system documentation, it was established that sufficient information was provided by altona Diagnostics GmbH to fulfil the requirements described in the Invitation to manufacturers of in vitro diagnostics for Zika virus to submit an application for emergency use assessment and listing by WHO. Quality management documentation for Emergency Use Assessment and Listing conclusion: Acceptable. Product dossier assessment altona Diagnostics GmbH submitted documentation in support of safety and performance for RealStar Zika Virus RT-PCR Kit 1.0 as per the Invitation to manufacturers of in vitro diagnostics for Zika virus to submit an application for emergency use assessment and listing by WHO 1. The information submitted in the product application was reviewed by WHO staff and external experts (reviewers) appointed by WHO. The findings of the reviews were reported in accordance with Instructions for Submission Requirements: In vitro diagnostics (IVDs) Detecting Zika Virus Nucleic Acid or Antigen (WHO document number WHO PQDx_240 v 2). Safety and performance documentation for Emergency Use Assessment and Listing conclusion: Acceptable with commitments to EUAL listing: Manufacturer to undertake additional testing in pregnant women from affected geographical regions. Laboratory evaluation Analytical testing The limit of detection of the assay was evaluated independently by the Bernhard Nocht Institute for Tropical Medicine (BNITM) in Hamburg, Germany which is a WHO Collaborating Centre for Arbovirus and Haemorrhagic Fever Reference and Research. Testing was conducted using a material prepared as a candidate international standard (IS) developed by the Paul-Ehrlich-Institut (PEI), Langen, Germany on behalf of WHO. The 1 Invitation to manufacturers of in vitro diagnostics for Zika virus to submit an application for emergency use assessment and listing by WHO. Page 3 of 9

4 EAZ WHO EUAL ZIKV Public Report 08/2016, version 1.0 candidate international standard has been assigned a potency of 7.70 log10 units/ml based on the results of an international collaborative study. The results of the study will be put forward to the Expert Committee on Biological Standardization (ECBS) for adoption in October The study was performed under the BSL-2 conditions. Reference material was reconstituted in 0.5 ml of nuclease-free water as recommended by PEI (WHO Zika Virus Collaborative Study Protocol Q2 2016). A 1-log10 step dilution series of the candidate IS spanning the claimed LoD of the assay under evaluation were prepared in both urine and serum. Two replicates of each dilution concentration were tested. In a second experiment, a 5 member 0.5-log step dilution series spanning the estimated LoD was tested using 8 replicates at each concentration level. Testing was performed using 10 μl of eluted RNA on the Roche LightCycler 480 instrument in accordance with manufacturer s recommendation and as per the instrument s user manual. The analytical sensitivity (95% limit of detection [LoD]) of RealStar Zika Virus RT-PCR Kit 1.0 was determined by Probit analysis in both urine and plasma specimens. Table 1 Dose for centile 95 (95% LoD) and confidence interval (CI) 95% LoD (CI) RNA IU/ml of specimen Serum (60.4 to 864.2) Urine (64.9 to 845.0) Clinical Testing: Clinical testing is ongoing. Results will be made available shortly. Laboratory evaluation for Emergency Use Assessment and Listing conclusion: Acceptable contingent on clinical evaluation results. Page 4 of 9

5 EAZ WHO EUAL ZIKV Public Report 08/2016, version 1.0 WHO Emergency Use Assessment and Listing Decision Based on the review of the manufacturer s submitted data, as well as data generated from the limited laboratory evaluation RealStar Zika Virus RT-PCR Kit 1.0 is eligible for WHO procurement. Post market surveillance to monitor the performance of RealStar Zika Virus RT-PCR Kit 1.0 is highly recommended. Scope and duration of procurement eligibility RealStar Zika Virus RT-PCR Kit 1.0 with product code REF , manufactured by altona Diagnostics GmbH is considered to be eligible for WHO procurement. This listing does not infer that the product meets WHO prequalification requirements and does not mean that the product is listed as WHO prequalified. As part of the on-going requirements for listing as eligible for WHO procurement altona Diagnostics GmbH must engage in post-market surveillance activities to ensure that the product continues to meet safety, quality and performance requirements. altona Diagnostics GmbH is required to notify WHO of any complaints, including adverse events related to the use of the product within 7 days of receipt. Furthermore, WHO will continue to monitor the performance of the assay in the field. WHO reserves the right to rescind eligibility for WHO procurement, if additional information on the safety, quality and performance comes to WHO s attention during post-market surveillance activities. Page 5 of 9

6 EAZ WHO EUAL ZIKV Public Report 08/2016, version 1.0 Labelling 1. Labels 2. Instructions for use Page 6 of 9

7 EAZ WHO EUAL ZIKV Public Report 08/2016, version Labels 1.2. Box labels Page 7 of 9

8 EAZ WHO EUAL ZIKV Public Report 08/2016, version Tube labels Page 8 of 9

9 EAZ WHO EUAL ZIKV Public Report 08/2016, version Instructions for use Version 08/2016 Page 9 of 9

10 Instructions for Use RealStar Zika Virus RT-PCR Kit /2016 EN

11 RealStar Zika Virus RT-PCR Kit 1.0 For use with Mx 3005P QPCR System (Stratagene) VERSANT kpcr Molecular System AD (Siemens) ABI Prism 7500 SDS (Applied Biosystems) ABI Prism 7500 Fast SDS (Applied Biosystems) Rotor-Gene 6000 (Corbett Research) Rotor-Gene Q5/6 plex Platform (QIAGEN) CFX96 Real-Time System (Bio-Rad) CFX96 Deep Well Real-Time System (Bio-Rad) LightCycler 480 Instrument II (Roche) MAN EN-S altona Diagnostics GmbH Mörkenstr. 12 D Hamburg

12 Content 1. Intended Use Kit Components Storage Material and Devices required but not provided Background Information Product Description Real-Time PCR Instruments Warnings and Precautions Procedure Sample Preparation Master Mix Setup Reaction Setup Programming the Real-Time PCR Instrument Settings Fluorescence Detectors (Dyes) Temperature Profile and Dye Acquisition Special Remarks on the Setup of Recommended Real-Time PCR Instruments Performance Evaluation Performance Evaluation without Nucleic Acid Extraction Analytical Sensitivity Analytical Specificity Precision Performance Evaluation including Nucleic Acid Extraction Serum Samples Urine Samples Clinical Performance Evaluation Limitations Quality Control Technical Assistance Literature Trademarks and Disclaimers Explanation of Symbols Data Analysis Validity of Diagnostic Test Runs Valid Diagnostic Test Run Invalid Diagnostic Test Run Interpretation of Results Qualitative Analysis

13 1. Intended Use The is an in vitro diagnostic test, based on real-time PCR technology, for the qualitative detection of Zika virus specific RNA in human serum or urine. 2. Kit Components Lid Color Component Number of Vials Volume [µl/vial] Blue Master A 8 60 Purple Master B Green Internal Control Red Positive Control White Water (PCR grade) Material and Devices required but not provided Appropriate real-time PCR instrument (see chapter 6.1 Real-Time PCR Instruments) Appropriate nucleic acid extraction system or kit Desktop centrifuge with a rotor for 2 ml reaction tubes Centrifuge with a rotor for microtiter plates, if using 96 well reaction plates Vortex mixer Appropriate 96 well reaction plates or reaction tubes with corresponding (optical) closing material Pipettes (adjustable) Pipette tips with filters (disposable) Powder-free gloves (disposable) 3. Storage The is shipped on dry ice. The components of the kit should arrive frozen. If one or more components are not frozen upon receipt, or if tubes have been compromised during shipment, contact altona Diagnostics GmbH for assistance. All components should be stored between -25 C and -15 C upon arrival. Repeated thawing and freezing of Master reagents (more than twice) should be avoided, as this might affect the performance of the assay. The reagents should be frozen in aliquots, if they are to be used intermittently. Storage between +2 C and +8 C should not exceed a period of two hours. Protect Master A and Master B from light. NOTE NOTE Please ensure that all instruments used have been installed, calibrated, checked and maintained according to the manufacturer s instructions and recommendations. It is highly recommended to use the 72-well rotor with the appropriate 0.1 ml reaction tubes, if using the Rotor-Gene 6000 (Corbett Research) or the Rotor-Gene Q 5/6 plex (QIAGEN). 6 7

14 5. Background Information Zika virus is an enveloped, single stranded (+) RNA virus of the family Flaviviridae. Like many other members of the family it is mainly transmitted by mosquitoes of the genus Aedes. In 1947, the virus was isolated from a rhesus monkey in Uganda for the first time. The first human case of Zika virus infection was discovered in 1968 in Nigeria. Initially, evidence for Zika virus infections has only been found in patients from Africa and South-East Asia. In 2007 the virus caused a large outbreak in Micronesia and other islands in the Pacific Ocean. French Polynesia, Easter Islands and Cook Islands were affected in Since 2015 the virus is also endemic to South America, namely Brazil, where since then large numbers of suspected cases were recorded. Fever, rash and arthralgia are common symptoms and signs of Zika virus infections which nevertheless usually are mild and self-limiting. Because Zika, dengue and chikungunya virus are endemic to the same geographical regions and cause similar symptoms, definite identification of the etiological agent is only possible with laboratory testing. Zika virus detection by means of realtime RT-PCR should be done early (up to 10 days) after illness onset. Antibody assays commonly cross-react with closely related flaviviruses and detection of neutralising antibodies by plaque reduction assays is troublesome and can only be done in specialised laboratories. 6. Product Description The is an in vitro diagnostic test, based on real-time PCR technology, for the qualitative detection of Zika virus specific RNA. The assay includes a heterologous amplification system (Internal Control) to identify possible RT-PCR inhibition and to confirm the integrity of the reagents of the kit. Real-time RT-PCR technology utilizes reverse-transcriptase (RT) reaction to convert RNA into complementary DNA (cdna), polymerase chain reaction (PCR) for the amplification of specific target sequences and target specific probes for the detection of the amplified DNA. The probes are labelled with fluorescent reporter and quencher dyes. Probes specific for Zika virus RNA are labelled with the fluorophore FAM. The probe specific for the Internal Control (IC) is labelled with the fluorophore JOE. Using probes linked to distinguishable dyes enables the parallel detection of Zika virus specific RNA and the Internal Control in corresponding detector channels of the real-time PCR instrument. The test consists of three processes in a single tube assay: NOTE Due to the relatively fast molecular evolution of RNA viruses, there is an inherent risk for any RT-PCR based test system that accumulation of mutations over time may lead to false negative results. Reverse transcription of target and Internal Control RNA to cdna PCR amplification of target and Internal Control cdna Simultaneous detection of PCR amplicons by fluorescent dye labelled probes 8 9

15 The consists of: Two Master reagents (Master A and Master B) Internal Control (IC) Positive Control PCR grade water Master A and Master B contain all components (buffer, enzymes, primers, and probes) to allow reverse transcription, PCR mediated amplification and target detection of Zika virus specific RNA and Internal Control in one reaction setup. 6.1 Real-Time PCR Instruments The was developed and validated to be used with the following real-time PCR instruments: Mx 3005P QPCR System (Stratagene) VERSANT kpcr Molecular System AD (Siemens) ABI Prism 7500 SDS (Applied Biosystems) ABI Prism 7500 Fast SDS (Applied Biosystems) Rotor-Gene 6000 (Corbett Research) Rotor-Gene Q5/6 plex Platform (QIAGEN) CFX96 Real-Time System (Bio-Rad) CFX96 Deep Well Real-Time System (Bio-Rad) LightCycler 480 Instrument II (Roche) 7. Warnings and Precautions Read the Instructions for Use carefully before using the product. Before first use check the product and its components for: Integrity Completeness with respect to number, type and filling (see chapter 2. Kit Components) Correct labelling Frozenness upon arrival Use of this product is limited to personnel specially instructed and trained in the techniques of real-time PCR and in vitro diagnostic procedures. Specimens should always be treated as infectious and/or biohazardous in accordance with safe laboratory procedures. Wear protective disposable powder-free gloves, a laboratory coat and eye protection when handling specimens. Avoid microbial and nuclease (DNase/RNase) contamination of the specimens and the components of the kit. Always use DNase/RNase-free disposable pipette tips with aerosol barriers. Always wear protective disposable powder-free gloves when handling kit components. Use separated and segregated working areas for (i) sample preparation, (ii) reaction setup and (iii) amplification/detection activities. The workflow in the laboratory should proceed in unidirectional manner. Always wear disposable gloves in each area and change them before entering a different area. Dedicate supplies and equipment to the separate working areas and do not move them from one area to another. Store positive and/or potentially positive material separated from all other components of the kit. Do not open the reaction tubes/plates post amplification, to avoid contamination with amplicons

16 Additional controls may be tested according to guidelines or requirements of local, state and/or federal regulations or accrediting organizations. Do not autoclave reaction tubes after the PCR, since this will not degrade the amplified nucleic acid and will bear the risk to contaminate the laboratory area. Do not use components of the kit that have passed their expiration date. Discard sample and assay waste according to your local safety regulations. 8. Procedure 8.1 Sample Preparation The quality of the extracted RNA has a profound impact on the performance of the entire test system. It has to be ensured that the system used for nucleic acid extraction is compatible with real-time PCR technology. The following kit was validated in combination with the for nucleic acid extraction: QIAamp Viral RNA Mini Kit (QIAGEN) Cat. No for 50 extractions Cat. No for 250 extractions The extraction of the RNA using the QIAamp Viral RNA Mini Kit has to be performed following the manufacturer s instructions using 140 µl of specimen as starting material. For elution of the extracted RNA 60 µl AVE buffer should be used. Alternative nucleic acid extraction systems and kits might also be appropriate. The suitability of the nucleic acid extraction procedure for use with RealStar Zika Virus RT-PCR Kit 1.0 has to be validated by the user. If using a spin column based sample preparation procedure including washing buffers containing ethanol, it is highly recommended to perform an additional centrifugation step for 10 min at approximately x g (~ rpm), using a new collection tube, prior to the elution of the nucleic acid. CAUTION If your sample preparation system is using washing buffers containing ethanol, make sure to eliminate any traces of ethanol prior to elution of the nucleic acid. Ethanol is a strong inhibitor of real-time PCR. CAUTION The use of carrier RNA is crucial for extraction efficiency and stability of the extracted nucleic acid. For additional information and technical support regarding pre-treatment and sample preparation please contact our Technical Support (see chapter 14. Technical Assistance). 8.2 Master Mix Setup All reagents and samples should be thawed completely, mixed (by pipetting or gentle vortexing) and centrifuged briefly before use. The contains a heterologous Internal Control (IC), which can either be used as a RT-PCR inhibition control or as a control of the sample preparation procedure (nucleic acid extraction) and as a RT-PCR inhibition control

17 If the IC is used as a RT-PCR inhibition control, but not as a control for the sample preparation procedure, set up the Master Mix according to the following pipetting scheme: Number of Reactions (rxns) 1 12 Master A 5 µl 60 µl Master B 15 µl 180 µl Internal Control 1 µl 12 µl CAUTION CAUTION If the IC (Internal Control) was added during the sample preparation procedure, the Master Mix for the controls must be prepared including the IC. Never add the IC directly to the specimen. Volume Master Mix 21 µl 252 µl If the IC is used as a control for the sample preparation procedure and as a RT-PCR inhibition control, add the IC during the nucleic acid extraction procedure. The IC must not be added directly to the specimen. The IC should always be added to the specimen/lysis buffer mixture. The volume of the IC which has to be added, always and only depends on the elution volume. It represents 10% of the elution volume. E.g. when using the QIAamp Viral RNA Mini Kit the RNA with an volume of 60 μl AVE buffer, 6 μl of IC per sample must be added to the specimen/lysis buffer mixture. If the IC was added during the sample preparation procedure, set up the Master Mix according to the following pipetting scheme: 8.3 Reaction Setup Pipette 20 µl of the Master Mix into each required well of an appropriate optical 96-well reaction plate or an appropriate optical reaction tube. Add 10 µl of the sample (eluate from the nucleic acid extraction) or 10 µl of the controls (Positive or Negative Control). Reaction Setup Master Mix 20 µl Sample or Control 10 µl Total Volume 30 µl Number of Reactions (rxns) 1 12 Master A 5 µl 60 µl Master B 15 µl 180 µl Volume Master Mix 20 µl 240 µl Make sure that at least one Positive and one Negative Control is used per run. Thoroughly mix the samples and controls with the Master Mix by pipetting up and down. Close the 96-well reaction plate with appropriate lids or optical adhesive film and the reaction tubes with appropriate lids. Centrifuge the 96-well reaction plate in a centrifuge with a microtiter plate rotor for 30 seconds at approximately 1000 x g (~ 3000 rpm)

18 9. Programming the Real-Time PCR Instrument 9.3 Temperature Profile and Dye Acquisition For basic information regarding the setup and programming of the different realtime PCR instruments, please refer to the user manual of the respective instrument. For detailed programming instructions regarding the use of the RealStar Zika Virus RT-PCR Kit 1.0 on specific real-time PCR instruments please contact our Technical Support (see chapter 14. Technical Assistance). 9.1 Settings Define the temperature profile and dye acquisition: Reverse Transcription Stage Cycle Repeats Acquisition Temperature [ C] Time [min:sec] Hold :00 Denaturation Hold :00 Define the following settings: Amplification Cycling :15 yes 55 00:45 Settings :15 Reaction Volume 30 µl Ramp Rate Passive Reference Default ROX 9.2 Fluorescence Detectors (Dyes) Define the fluorescence detectors (dyes): Target Detector Name Reporter Quencher Zika virus specific RNA Zika virus FAM (None) Internal Control IC JOE (None) 16 17

19 9.4 Special Remarks on the Setup of Recommended Real-Time PCR Instruments Please find below special remarks on the setup of LightCycler 480 Instrument II (Roche), CFX96 Real-Time PCR Detection System (Bio-Rad), CFX96 Deep Well Real-Time PCR Detection System (Bio-Rad), ABI Prism 7500 SDS and ABI Prism 7500 Fast SDS (Applied Biosystems), Rotor-Gene 6000 (Corbett Research) and Rotor-Gene Q 5/6 plex Platform (QIAGEN), VERSANT kpcr Molecular System AD (Siemens Healthcare) and Mx 3005P QPCR System.(Stratagene) LightCycler 480 Instrument II 1. In the Experiment settings, select Detection Format: Dual Color Hydrolysis Probe / UPL Probe. 2. Make sure by checking the Customize field that the setting shown for the Filter Combinations are FAM ( ) and VIC /HEX/Yellow555 ( ). ABI Prism 7500 SDS Go to Plate Setup, Define Targets and Samples, Assign Targets and Samples : 1. Select the whole plate. 2. Click the assign-boxes for both targets. The targets should appear in the wells in the plate layout. 3. Make sure to choose none in the Select the dye to use as the passive reference (default setting is ROX ). ABI Prism 7500 SDS Fast The same settings for Plate Setup as for the ABI Prism 7500 SDS apply (see above). For the Fast version, go to Experiment properties. The ramp speed has to be set to Standard (~2 hours to complete a run). The is not compatible with the fast cycling conditions and the increased ramp rates. CFX96 Real-Time PCR Detection System and CFX96 Deep Well Real-Time PCR Detection System Open the Plate Editor window and select all wells of the 96 well-plate. Click Select Fluorophores. For Channel 1 check the box behind FAM and for Channel 2 check the box behind VIC. Assign samples to the wells by selecting the appropriate Sample Type and afterwards Load FAM and VIC to the wells. The target name of FAM should be set to Zika virus and the target name of VIC to Internal Control. Rotor-Gene 6000 and Rotor-Gene Q 5/6 plex/mdx Platform Chose the 72-Well-Rotor and the appropriate reaction volume. The Gain optimization should be performed before 1 st acquisition. VERSANT kpcr Molecular System AD Under the Instrument drop down menu select the Filter Set Gain Settings. A pop up window will appear. Set the following configurations: Cy 5 x8, ROX x4, JOE x8 and FAM x8. Close the pop-up window by clicking the OK button. Mx 3005P QPCR System Under the Instrument drop down menu select the Filter Set Gain Settings. A pop up window will appear. Set the following configurations: Cy 5 x8, ROX x4, JOE x8 and FAM x8. Close the pop-up window by clicking the OK button

20 10. Data Analysis For basic information regarding data analysis on specific real-time PCR instruments, please refer to the user manual of the respective instrument. For detailed instructions regarding the analysis of the data generated with the on different real-time PCR instruments please contact our Technical Support (see chapter 14. Technical Assistance) Interpretation of Results Qualitative Analysis Detection Channel Result Interpretation FAM JOE + +* Zika virus specific RNA detected Validity of Diagnostic Test Runs Valid Diagnostic Test Run For a valid diagnostic test run, the following control conditions must be met: No Zika virus specific RNA detected. Sample does not contain detectable amounts of Zika virus specific RNA. RT-PCR inhibition or reagent failure. Repeat testing from original sample or collect and test a new sample. Detection Channel Control ID FAM JOE Positive Control + + * Detection of the Internal Control in the JOE detection channel is not required for positive results in the FAM detection channel. A high Zika virus RNA load in the sample can lead to a reduced or absent Internal Control signal. Negative Control Invalid Diagnostic Test Run A diagnostic test run is invalid, (i) if the run has not been completed or (ii) if any of the control conditions for a valid diagnostic test run are not met. In case of an invalid diagnostic test run, repeat testing by using the remaining purified nucleic acids or start from the original samples again

21 11. Performance Evaluation 11.1 Performance Evaluation without Nucleic Acid Extraction Analytical Sensitivitiy The analytical sensitivity of the is defined as the concentration (copies/µl of the eluate) of Zika virus specific RNA molecules that can be detected with a positivity rate of 95%. The analytical sensitivity was determined by analysis of dilution series of quantified Zika virus RNA Analytical Specificity The analytical specificity of the is ensured by the thorough selection of the oligonucleotides (primers and probes). The oligonucleotides were checked by sequence comparison analysis against publicly available sequences to ensure that all relevant Zika virus genotypes will be detected. The analytical specificity of the was evaluated by testing a panel of genomic RNA extracted from non-zika virus, alphaviruses and other pathogens. Table 1: RT-PCR results used for the calculation of the analytical sensitivity with respect to the detection of Zika virus specific RNA Input Conc. [copies/µl] Number of Replicates Number of Positives Hit Rate [%] The analytical sensitivity of the was determined by Probit analysis: For the detection of Zika virus specific RNA, the analytical sensitivity is 0.61 copies/μl eluate [95% confidence interval (CI): copies/μl] The did not cross-react with any of the following pathogens: Dengue virus serotype 1 Dengue virus serotype 2 Dengue virus serotype 3 Dengue virus serotype 4 Japanese encephalitis virus Marburg virus (MARV) Sudan ebolavirus (SEBOV) St. Louis encephalitis virus West Nile virus Yellow fever virus Zaire ebolavirus (ZEBOV) Human parvovirus B19 Chikungunya virus Murray Valley encephalitis virus Plasmodium falciparum 22 23

22 CAUTION Due to sequence homology between Usutu virus RNA and the target region used for the detection of Zika virus specific RNA, cross-reactivity with Usutu virus RNA cannot be ruled out. Usutu virus is a bird virus rarely infecting humans. It does not cause severe or fatal disease in human patients and an infection usually remains asymptomatic. Table 2: Precision data for the detection of Zika virus specific RNA Zika virus Average Threshold Cycle ( ) Standard Deviation Coefficient of Variation [%] Intra-Assay Variability Inter-Assay Variability Inter-Lot Variability Total Variability Precision Precision of the was determined as intraassay variability (variability within one experiment), inter-assay variability (variability between different experiments) and inter-lot variability (variability between different production lots). Total variability was calculated by combining the three analyses. The variability data are expressed in terms of standard deviation and coefficient of variation based on threshold cycle ( ) - values. At least eight replicates per sample were analysed for intra-assay variability, inter-assay and inter-lot variability. Table 3: Precision data for the detection of the Internal Control Internal Control Average Threshold Cycle ( ) Standard Deviation Coefficient of Variation [%] Intra-Assay Variability Inter-Assay Variability Inter-Lot Variability Total Variability

23 11.2 Performance Evaluation including Nucleic Acid Extraction Table 4: Determination of the tentative LoD for the Serum Samples Target Concentration geq/ml rate Replicate 1 (FAM ) Replicate 2 (FAM ) Replicate 3 (FAM ) Estimation of the Limit of Detection (LoD): Serial dilutions of Zika virus strain H/PF/2013 (stock concentration TCID50/ml: ) obtained from the European Virus Archive (Marseille, France) were prepared. Using qrt-pcr and quantified in vitro transcribed RNA the number of Zika virus genome equivalents (geq) per milliliter stock was determined to be 1.26E+09. For nucleic acid extraction, 126 µl of pooled serum were combined with 560 µl AVL buffer, containing 6 µl of the Internal Control (provided with the RealStar Zika Virus RT-PCR Kit 1.0) and spiked with 14 µl diluted Zika virus stock. The final mix was subjected to the extraction procedure following the manufacturer s instructions for the QIAamp Viral RNA Mini Kit (QIAGEN). Elution was performed in 60 µl AVE buffer. Each sample was extracted in triplicate and tested with the on the CFX96 Deep Well Real-Time PCR Detection System (Bio-Rad). The lowest concentration at which all three replicates were tested positive was treated as the tentative LoD. The results can be found in Table 4: Zika virus (strain H/PF/2013) / / / / / / / The in conjunction with the QIAamp Viral RNA Mini Kit manual extraction system and the CFX96 Deep Well Real- Time PCR Detection System detected 3/3 replicates with a concentration of geq/ml serum. Confirmation of the Limit of Detection (LoD): Based on the tentative LoD, diluted Zika virus stock was spiked into 20 individual serum samples to a final concentration of geq/ml. Nucleic acids were extracted with the QIAamp Viral RNA Mini Kit (QIAGEN) as described above. The obtained eluates were tested with the on the following instruments: CFX96 Deep Well Real-Time PCR Detection System (Bio-Rad) Rotor-Gene Q (QIAGEN) ABI Prism 7500 SDS (Applied Biosystems) LightCycler 480 Instrument II (Roche) The results can be found in Tables 5, 6, 7 and

24 Table 5: LoD confirmation on CFX96 Deep Well Real-Time PCR Detection System (Bio-Rad) Zika virus concentration = geq/ml Specimen Pos/Neg (FAM ) (VIC ) Zika virus concentration = geq/ml Specimen Pos/Neg (FAM ) (VIC ) Mean (n=19) SD Statistics CV % Result 19/20 The in conjunction with the QIAamp Viral RNA Mini Kit manual extraction system and the CFX96 Deep Well Real-Time PCR Detection System detected 19/20 replicates at a concentration of geq/m.l Therefore, the confirmed LoD is geq/ml serum. Table 6: LoD confirmation on Rotor-Gene Q (QIAGEN) Zika virus concentration = geq/ml Specimen Pos/Neg (FAM ) (VIC )

25 Zika virus concentration = geq/ml Specimen Pos/Neg (FAM ) (VIC ) Mean (n=20) Zika virus concentration = geq/ml Specimen Pos/Neg (FAM ) (VIC ) Statistics SD CV % Result 20/ At the concentration of geq/ml 20/20 replicates were detected positive and thereby confirm the LoD to be geq/ml serum for the RealStar Zika Virus RT-PCR Kit 1.0 in conjunction with the QIAamp Viral RNA Mini Kit manual extraction system and the Rotor-Gene Q (QIAGEN). Table 7: LoD confirmation on ABI Prism 7500 SDS (Applied Biosystems) Mean (n=20) Zika virus concentration = geq/ml Specimen Pos/Neg (FAM ) (VIC ) Statistics SD CV % Result 20/ At the concentration of geq/ml 20/20 replicates were detected positive and thereby confirm the LoD to be geq/ml serum for the RealStar Zika Virus RT-PCR Kit 1.0 in conjunction with the QIAamp Viral RNA Mini Kit manual extraction system and the ABI Prism 7500 SDS (Applied Biosystems)

26 Table 8: LoD confirmation on LightCycler 480 Instrument II (Roche) Zika virus concentration = geq/ml Specimen Pos/Neg (FAM ) (VIC ) Zika virus concentration = geq/ml Specimen Pos/Neg (FAM ) (VIC ) Mean (n=20) SD Statistics CV % Result 20/20 At the concentration of geq/ml 20/20 replicates were detected positive and thereby confirm the LoD to be geq/ml serum for the RealStar Zika Virus RT-PCR Kit 1.0 in conjunction with the QIAamp Viral RNA Mini Kit manual extraction system and the LightCycler 480 Instrument II (Roche) Urine Samples Estimation of the Limit of Detection (LoD): Serial dilutions of Zika virus strain H/PF/2013 (stock concentration TCID50/ml: ) obtained from the European Virus Archive (Marseille, France) were prepared. Using qrt-pcr and quantified in vitro transcribed RNA the number of Zika virus genome equivalents (geq) per milliliter stock was determined to be 1.26E+09. For nucleic acid extraction, 126 µl of pooled urine were combined with 560 µl AVL buffer, containing 6 µl of the Internal Control (provided with the RealStar Zika Virus RT-PCR Kit 1.0) and spiked with 14 µl diluted Zika virus stock. The final mix was subjected to the extraction procedure following the manufacturer s instructions for the QIAamp Viral RNA Mini Kit (QIAGEN). Elution was performed in 60 µl AVE buffer. Each sample was extracted in triplicate and tested with the RealStar Zika Virus RT-PCR Kit 1.0 on the CFX96 Deep Well Real-Time PCR Detection System (Bio-Rad). The lowest concentration at which all three replicates were tested positive was treated as the tentative LoD. The results can be found in Table 9: 32 33

27 Table 9: Determination of the tentative LoD for the Target Zika virus (strain H/PF/2013) Concentration geq/ml rate Replicate 1 (FAM ) Replicate 2 (FAM ) Replicate 3 (FAM ) / / / / / / / The in conjunction with the QIAamp Viral RNA Mini Kit manual extraction system and the CFX96 Deep Well Real-Time PCR Detection System detected 3/3 replicates with a concentration of geq/ ml urine. Confirmation of the Limit of Detection (LoD): Based on the tentative LoD, diluted Zika virus stock was spiked into 20 individual urine samples to a final concentration of geq/ml. Nucleic acids were extracted with the QIAamp Viral RNA Mini Kit (QIAGEN) as described above. The obtained eluates were tested with the on the following instruments: CFX96 Deep Well Real-Time PCR Detection System (Bio-Rad) Rotor-Gene Q (QIAGEN) ABI Prism 7500 SDS (Applied Biosystems) LightCycler 480 Instrument II (Roche) The results can be found in Tables 10, 11, 12 and 13. Table 10: LoD confirmation on CFX96 Deep Well Real-Time PCR Detection System (Bio-Rad) Zika virus concentration = geq/ml Specimen Pos/Neg (FAM ) (VIC )

28 Zika virus concentration = geq/ml Specimen Pos/Neg (FAM ) (VIC ) Mean (n=19) SD Statistics CV % Result 19/20 The in conjunction with the QIAamp Viral RNA Mini Kit manual extraction system and the CFX96 Deep Well Real-Time PCR Detection System detected 19/20 replicates at a concentration of geq/ ml. Therefore, the confirmed LoD is geq/ml urine. Table 11: LoD confirmation on Rotor-Gene Q (QIAGEN) Zika virus concentration = geq/ml Specimen Pos/Neg (FAM ) (JOE ) Zika virus concentration = geq/ml Specimen Pos/Neg (FAM ) (JOE ) Mean (n=19) SD Statistics CV % Result 19/20 At the concentration of geq/ml 19/20 replicates were detected positive and thereby confirm the LoD to be geq/ml urine for the RealStar Zika Virus RT-PCR Kit 1.0 in conjunction with the QIAamp Viral RNA Mini Kit manual extraction system and the Rotor-Gene Q (QIAGEN)

29 Table 12: LoD confirmation on ABI Prism 7500 SDS (Applied Biosystems) Zika virus concentration = geq/ml Specimen Pos/Neg (FAM ) (JOE ) Zika virus concentration = geq/ml Specimen Pos/Neg (FAM ) (JOE ) Mean (n=20) SD Statistics CV % Result 20/20 At the concentration of geq/ml 20/20 replicates were detected positive and thereby confirm the LoD to be geq/ml urine for the RealStar Zika Virus RT-PCR Kit 1.0 in conjunction with the QIAamp Viral RNA Mini Kit manual extraction system and the ABI Prism 7500 SDS (Applied Biosystems). Table 13: LoD confirmation on LightCycler 480 Instrument II (Roche) Zika virus concentration = geq/ml Specimen Pos/Neg (FAM ) (JOE )

30 Zika virus concentration = geq/ml Specimen Pos/Neg (FAM ) (JOE ) Clinical Performance Evaluation To evaluate the clinical performance of the. a total of 208 clinical specimens from 153 patients with signs and symptoms of Zika virus infection (106 female (F) and 47 male (M)) were analyzed retrospectively in a blinded fashion. From the 208 samples tested 103 were serum and 105 were urine specimens. For RNA extraction the QIAamp Viral RNA Mini Kit (QIAGEN) was used. 140 µl of each urine or serum sample were combined with 560 µl AVL buffer, containing 6 µl of the Internal Control provided with the. The sample/avl buffer mix was subjected to the extraction procedure following the manufacturer s instructions. Elution was performed in 60 µl AVE buffer. Statistics Mean (n=20) SD CV % Result 20/20 Eluates were tested with the. as well as with the real-time RT-PCR as described by Lanciotti et. al (Lanciotti RS, Kosoy OL, Laven JJ, Velez JO, Lambert AJ, et al. (2008) Genetic and Serologic Properties of Zika Virus Associated with an Epidemic, Yap State, Micronesia, 2007; Emerg Infect Dis Aug; 14(8): ). At the concentration of geq/ml 20/20 replicates were detected positive and thereby confirm the LoD to be geq/ml urine for the RealStar Zika Virus RT-PCR Kit 1.0 in conjunction with the QIAamp Viral RNA Mini Kit manual extraction system and the LightCycler 480 Instrument II (Roche). For the execution of the real-time RT-PCR described by Lanciotti et al. the following oligonucleotides; Table 14 have been used: Table 14: Oligonucleotides used for real-time RT-PCR described by Lanciotti et al. Primer/Probe Sequence 5'-> 3' Zika virus 1086 Zika virus 1162c Zika virus 1107-FAM CCGCTGCCCAACACAAG CCACTAACGTTCTTTTGCAGACAT AGCCTACCTTGACAAGCAGTCAGACACTCAA 40 41

31 Both tests, the. as well as the real-time RT- PCR described by Lanciotti et al., have been performed on a CFX96 Deep Well Real-Time PCR Detection System (Bio-Rad). Results generated for the different samples tested are shown in Table 15: Table 15: Results from testing clinical samples Table 15: (continuation) Days after Patient Gender Onset of ID Symptoms Specimen Type RealStar Zika Virus RT- (FAM TM ) PCR Kit 1.0 (VIC TM ) Real-time RT-PCR described by Lanciotti et al. (FAM TM ) Patient ID Gender Days after Onset of Symptoms Specimen Type RealStar Zika Virus RT- (FAM TM ) PCR Kit 1.0 (VIC TM ) Real-time RT-PCR described by Lanciotti et al. (FAM TM ) 13 F 4 Urine F 2 Serum Serum M 5 Urine M 2 Serum F 2 Serum Urine Urine F 3 Serum F 5 Urine F 3 Urine F 1 Serum Urine F 2 Serum Urine F 5 F 6 F 1 Serum Urine Serum Urine Serum Urine F 6 Urine Serum M 2 Urine Serum M 1 Urine F 2 Serum Serum M 4 Urine Serum M 0 Urine F 6 Urine F 7 Urine Serum F 3 Urine Serum F 3 Urine F 11 F 12 M 4 Serum Urine Serum Urine Serum Urine F 0 Urine Serum M 5 Urine F 7 Urine

32 Table 15: (continuation) Table 15: (continuation) Patient ID Gender Days after Onset of Symptoms Specimen Type RealStar Zika Virus RT- (FAM TM ) PCR Kit 1.0 (VIC TM ) Real-time RT-PCR described by Lanciotti et al. (FAM TM ) Patient ID Gender Days after Onset of Symptoms Specimen Type RealStar Zika Virus RT- (FAM TM ) PCR Kit 1.0 (VIC TM ) Real-time RT-PCR described by Lanciotti et al. (FAM TM ) 30 M 31 F 32 M 1 Serum Urine Serum Urine Serum Urine M 2 Urine Serum M 4 Urine F 6 Urine F 2 Serum F 3 Serum F 2 Serum F 0 Urine M 5 Serum Urine M 7 Urine F 2 Serum F 1 Serum F 2 Urine F 2 Urine M 8 Urine F 6 Serum Urine F 6 Serum F 7 Urine M 3 Serum Urine F 4 Serum Urine M 3 Serum Urine M 1 Serum Urine F 0 Serum M 6 Urine M 0 Serum Urine F 1 Serum Urine F 2 Serum F 7 Urine M 6 Urine Serum M 4 Urine Serum F 0 Urine F 7 Urine M 3 Urine F 2 Serum Urine

33 Table 15: (continuation) Table 15: (continuation) Patient ID Gender Days after Onset of Symptoms Specimen Type RealStar Zika Virus RT- (FAM TM ) PCR Kit 1.0 (VIC TM ) Real-time RT-PCR described by Lanciotti et al. (FAM TM ) Patient ID Gender Days after Onset of Symptoms Specimen Type RealStar Zika Virus RT- (FAM TM ) PCR Kit 1.0 (VIC TM ) Real-time RT-PCR described by Lanciotti et al. (FAM TM ) 66 F 2 Serum F 4 Serum M 1 Serum M 2 Serum F 3 Urine F 1 Serum M 1 Serum F 3 Urine Serum F 4 Urine F 3 Serum M 3 Serum F 3 Urine Serum F 6 Urine Serum F 2 Urine Serum M 3 Urine Serum F 4 Urine F 1 Serum Serum F 8 Urine * F 3 Urine M 5 Urine M 6 Urine Serum M 3 Urine Serum F 6 Urine F 3 Serum F 2 Serum F 1 Serum F 3 Urine M 3 Serum M 6 Urine F 3 Serum F 5 Urine M 3 Serum Serum F 2 Urine F 1 Urine F 4 Urine M 7 Urine F 0 Serum F 3 Urine Serum F 2 Urine F 0 Serum F 1 Urine F 2 Serum