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Collaborative study to establish a World Health Organization International Reference Panel for Dengue Virus types 1 to 4 RNA for Nucleic Acid Amplification Technology (NAT)-based assays

December 31, 2016 World Health Organization

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WHO/BS/2016.2299 ENGLISH ONLY EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION Geneva, 17-21 October 2016 Collaborative Study to Establish a World Health Organization International Reference Panel for Dengue Virus types 1 to 4 RNA for Nucleic Acid Amplification Technology (NAT)-Based Assays Germán Añez1,#, Evgeniya Volkova1, Rafaelle C.G. Fares1, Zhen Jiang1, Maria Rios1,* and the Collaborative Study Group2 1. U.S. Food and Drug Administration, 10903 New Hampshire Ave, Silver Spring, Maryland, United States of America, # Current address: Sanofi Pasteur, Swiftwater, Pennsylvania, United States of America * Principal contact: Maria.Rios@fda.hhs.gov See Appendix 1

NOTE: This document has been prepared for the purpose of inviting comments and suggestions on the proposals contained therein, which will then be considered by the Expert Committee on Biological Standardization (ECBS). Comments MUST be received by 16 September 2016 and should be addressed to the World Health Organization, 1211 Geneva 27, Switzerland, attention: Technologies, Standards and Norms (TSN). Comments may also be submitted electronically to the Responsible Officer: Dr C M Nübling at email: nueblingc@who.int. © World Health Organization 2016 All rights reserved. Publications of the World Health Organization are available on the WHO web site (www.who.int) or can be purchased from WHO Press, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel.: +41 22 791 3264; fax: +41 22 791 4857; e-mail: bookorders@who.int). Requests for permission to reproduce or translate WHO publications – whether for sale or for noncommercial distribution – should be addressed to WHO Press through the WHO web site: (http://www.who.int/about/licensing/copyright_form/en/index.html). The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full agreement. The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. All reasonable precautions have been taken by the World Health Organization to verify the information contained in this publication. However, the published material is being distributed without warranty of any kind, either expressed or implied. The responsibility for the interpretation and use of the material lies with the reader. In no event shall the World Health Organization be liable for damages arising from its use. The named authors alone are responsible for the views expressed in this publication.

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Summary An international collaborative study was conducted to assess the suitability of reference reagent candidates for Dengue virus (DENV) types 1 to 4 RNA for use in nucleic acid amplification technology (NAT)-based assays. Two sets of reference reagent candidates were prepared for each DENV type, one liquid frozen (Set 1) and one lyophilized (Set 2). The reference reagent candidates consisted of DENV prototype laboratory strains that were grown in cell culture in mosquito cells, subjected to inactivation of infectivity by heat treatment and diluted in human plasma. Both reference reagent sets were sent coded to the participants for testing in four independent runs utilizing the DENV NAT assay available in their laboratory. Results were communicated to the U.S. Food and Drug Administration (FDA) where the analyses were performed. A total of 28 laboratories from 20 countries accepted to participate in the study, of which 21 submitted the results for qualitative and quantitative assessments. It is proposed that materials comprising Set 1, which consists of heat-inactivated and lyophilized preparations of cell culture-derived DENV-1 (Hawaii, GenBank# KM204119), DENV-2 (New Guinea C, GenBank # KM204118), DENV-3 (H87, GenBank# KU050695 ), and DENV-4 (H241 GenBank# KR011349 ) strains be established as International Reference Reagents for DENV RNA with a unitage of 13,500, 69,200, 23,400, and 33,900 units per ml for DENV-1 to 4, respectively. The real-time and accelerated stability studies of the proposed International Reference Reagents revealed that the materials are stable at the recommended storage temperature, i.e. at or below 4°C, for a minimum of 12 months, and are therefore suitable for long term use.

Introduction Dengue is a mosquito-borne disease that afflicts more than 100 tropical and subtropical countries, causing an estimated 390 million infections per year (Bhatt et al 2013). Dengue is caused by any of four closely related flaviviruses (DENV-1 to 4) and is transmitted by mosquitoes from the genus Aedes, mainly Aedes aegypti. Infection with any of the four DENV can be asymptomatic in approximately 80% of infected individuals, or can result in dengue fever, an influenza-like illness that may progress to severe dengue, a potentially life-threatening condition (WHO, 2009). Although dengue primarily affects tropical and sub-tropical countries, the virus can be imported by infected travelers returning to non-endemic regions (Chuang et al., 2008; Anez et al., 2012; Anez et al., 2013). DENVs are transmissible by transfusion (TT-DENV) of blood and blood components, and by solid organ transplant, thus posing a risk for recipients of these products (Tambyah et al., 2008; Linnen et al., 2008; Waggoner et al., 2013). Prevalence studies conducted in endemic regions have found a high rate of asymptomatic DENV infection among blood donors, and TT-DENV has been reported in dengue endemic regions, including Puerto Rico (Mohammed et al., 2008; Stramer et al., 2012; Stramer et al., 2013). At this time there is no FDA-approved assay for the screening of blood for DENV. NAT assays are considered the most appropriate approach for blood donor screening for recent DENV infections (Johnson et al., 2005). During the epidemic seasons of 2011-2012, a transcription-mediated amplification (TMA) NAT test was used to test blood in Puerto Rico

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under an FDA-approved Investigational New Drug (IND) protocol, and was able to identify DENV RNA-positive donations that tested negative by an antigen screening assay (Matos et al., 2016; ClinicalTrials.gov). The proposal by the U.S. FDA to prepare standards for DENV RNA for use in NAT-based assays was endorsed by the WHO Expert Committee on Biological Standardization (ECBS) in 2009 (WHO/BS/09.2126). The prototype laboratory strains for each DENV type, i.e. DENV-1 strain Hawaii, DENV-2 strain New Guinea C, DENV-3 strain H87, and DENV-4 strain H241 were used to prepare the liquid frozen and lyophilized reference reagent candidates evaluated in this study. The aim of this study is to establish the WHO International Reference Reagents for Dengue Virus types 1 to 4 RNA for NAT-based assays and demonstrate their suitability, assess their potency and finally, assign an internationally agreed-upon unitage.

Preparation of Bulk Materials The four DENV WHO candidate reference reagents were prepared by growing prototype strains (kindly provided by Robert Lanciotti, CDC) of each of the four DENV types (DENV-1 to 4) in tissue culture using the susceptible mosquito cell line C6/36 (ATCC CRL-1660). The complete sequences of the four DENV serotypes were determined and made available in the GenBank (Table 1) (Añez et al., 2016). Cell culture supernatants were harvested, heat-inactivated and diluted in defibrinated, dialyzed, filtered human plasma that was acquired from a commercial source (Basematrix, SeraCare). According to the manufacturer’s certificate of analysis, the plasma diluent had tested negative for HBsAg, HCV/HIV-1 (NAT), anti-HIV 1/2, anti-HCV, and Syphilis. The viral stocks produced were pre-characterized at the Center for Biologics Evaluation and Research (CBER) of the U.S. FDA, as well as in four laboratories within the U.S. (data not published, available to ECBS upon request). Based on this initial study, a target concentration of 6 log10 NAT-detectable units per mL was chosen for the bulk preparation. To prepare the bulk material for the DENV WHO candidate reference reagents, 200 mL of each of the viral stocks were added to 1,800 mL of Basematrix, mixed, aliquoted, and stored at -80°C until further use. For the lyophilized DENV candidate reference reagents, a total of 2,000 vials for each (DENV-1, DENV-2, DENV-3 and DENV-4) candidate were filled and lyophilized at OCBQ/CBER/FDA, which is a testing facility accredited to ISO 17025. A volume of 1 mL was dispensed into 3 mL glass vials and sealed with rubber stoppers. The material was freeze-dried using a VirTis Benchmark Lyophilizer (SP Scientific). After lyophilization, the vials were stored at +4°C. For the liquid frozen DENV reference reagent candidates, 500 vials for each DENV type were prepared similarly to the lyophilized candidates, but in lieu of lyophilization, the liquid frozen vials were stored at -80°C until further use. The coefficient of variation of the fill volume for the lyophilized candidates was assessed for all 4 reagents and found to be 0.005% for DENV-1 and DENV-3 and 0.007% for DENV-2 and DENV-4. The residual moisture content was determined by methanol extraction using the Karl Fischer Coulometric Method with non-pyridine reagent and Mettler-Toledo Coulometric Titrator. Methanol extractions were performed on the lyophilized contents of two vials of DENV-1, and the residual moisture was found to be 1.1% w/w. To determine if the lyophilization process had affected the RNA titer of the DENV reference reagent candidates, 10

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vials were compared to aliquots of the bulk material that were stored at -80°C and no significant reduction of the respective DENV RNA titers was found (data not shown). The proposed DENV-1, DENV-2, DENV-3 and DENV-4 WHO International Reference Reagents are stored at CBER/FDA in Silver Spring, Maryland, United States at +4°C with continuous temperature monitoring. Manufacturing records are held at CBER/FDA and are available upon request by the ECBS.

Collaborative Study For the collaborative study, 28 laboratories from 20 countries initially accepted the invitation to participate. The results presented here comprise 21 laboratories from 15 countries that returned data. The participants in the collaborative study who provided data are listed in Appendix 1. Liquid Frozen and lyophilized reference reagents, sets 1 and 2 respectively, were sent coded to each participant, together with a vial containing only the liquid frozen plasma diluent (“negative” control), in replicates of six to ensure that enough material was available for testing. Since not all laboratories had a DENV NAT assay capable of discriminating between the four different DENV types, we sent the coded reference reagent candidates identifying the DENV type (e.g. vials were coded as “DENV-1 vial AA”, “DENV-1 vial BB”, “DENV-1 vial CC”, etc) to minimize cost. Thus, each testing laboratory received 72 vials: 24 vials per set and 24 negative controls (Table 2). Shipments which included liquid frozen and lyophilized vials were sent on dry ice; shipments to the four testing laboratories that received only the lyophilized candidates were sent at ambient temperature. The participating laboratories were asked to test all DENV reference reagent candidates using their DENV NAT assay(s) available, in four independent runs, at least one week apart and using a freshly thawed or reconstituted vial for each DENV reference reagent candidate and negative controls for each run. For laboratories that had a quantitative DENV NAT, we asked to test each DENV reference reagent candidates undiluted in at least triplicate and report the results in copies/mL calculated based on standard curves. For the qualitative testing of the samples, a two-step determination approach was used to determine the end-point for each DENV reference reagent candidate. First, each laboratory was asked to perform a series of 10-fold (1 log10) dilution steps using their assay diluent (i.e. plasma, nuclease-free water and phosphate buffered saline solution) and test each dilution to determine the initial end-point for the candidate in each respective assay. After that, the participants were asked to perform three independent tests of the material, diluting it 3.2-fold (½ log10) at least two dilutions above and below the previously determined end-point for the DENV RNA titer.

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Statistical Methods Quantitative Assays For the quantitative testing, the undiluted material of each sample was tested in replicate by each laboratory that had an assay with quantitative capability. For comparison of laboratories, the replicate results of each laboratory were combined as the arithmetic mean of log10 copies/mL, as previously described by Baylis et al. (2013), and values were expressed as ‘copies’ per mL. Finally, the number of ‘copies’ per ml for each reference reagent candidate was estimated by pooling all measurements collected in all laboratories. Qualitative Assays We assumed that a single NAT-detectable unit (NDU) will be sufficient to provide a positive test result and that the probabilities of positive results are determined by the underlying number of NDUs, which follows a Poisson distribution (Collet D, 1991). For each laboratory and sample, results from four independent runs were pooled and a ratio of positive results to total number of replicates for each dilution was calculated. These ratios were then used with the maximum likelihood method to estimate NDU/mL for each laboratory and sample after correcting for differences in sample test volume between laboratories. These estimated NDU/ml values are not equivalent to a genuine viral copy number per mL (Saldanha et al., 1999; Saldanha et al., 2004). To confirm the results obtained using the maximum-likelihood method, the data were also analyzed using the Spearman–Karber method and probit regression with log dilution and its quadratic term as independent variables, as described by Baylis et al. (2013) (data not shown). Combination of Quantitative and Qualitative Data Quantitative results for 4 laboratories and qualitative results from 19 laboratories were combined to calculate an overall mean for each sample, which was estimated using a linear mixed model assuming random laboratories. Relative Potencies The potencies of liquid candidate reference reagents (DENV-1 AA, DENV-2 CC, DENV-3 BB, DENV-4 CC), were estimated relative to the corresponding lyophilized reference reagent candidates (DENV-1 BB, DENV-2 AA, DENV-3 CC, DENV-4 BB) for quantitative assays, qualitative assays, and combined quantitative and qualitative data. For the qualitative assays, the relative potencies were calculated using parallel line analysis assuming Poisson distribution. For the quantitative assays, the relative potencies were calculated using parallel line analysis with log transformed data. All statistical analyses were conducted using SAS/STAT 9.3 (SAS Institute, Cary, NC, USA) and R package 3.2.4 (http://www.r-project.org/).

Stability and Accelerated Degradation Studies The stability of the lyophilized DENV WHO reference reagent candidates was assessed through both real-time and accelerated thermal degradation studies. Vials containing the lyophilized WHO reference reagent candidates were stored at -20°C, -80°C (to provide a baseline if there is any suggestion of instability at higher temperatures) and +4°C (the recommended storage temperature) and have been tested for up to 12 months. For the accelerated thermal degradation, vials were incubated at +20°C and +37°C for up to 12 months (Table 3). After incubation at the

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respective temperatures, the contents of the vials were reconstituted in 1 mL of nuclease-free water and analyzed by real-time PCR (Johnson et al., 2005).

Results from Collaborative Studies Data were received from a total of 21 participating laboratories. A total of 13 (for the liquid frozen reference reagent candidates) and 18 (for the lyophilized reference reagent candidates) qualitative and 4 quantitative datasets (for both liquid frozen and lyophilized reference reagent candidates) were reported, and analysis was performed at the FDA. Details regarding the methods used for the RNA extraction and NAT assays are available in Table 4. Among the participants, 2 were commercial test developers and 19 were research laboratories who used their in-house NAT assays. These assays included real-time PCR assays, end-point PCR assays, and TMA assays. Each laboratory was randomly assigned a code (not corresponding to the order presented in Appendix 1) and all data was compiled and analyzed. Qualitative and quantitative results from the same laboratory were identified in the graphics with the same laboratory code and a letter “a” or “b” to differentiate between qualitative and quantitative results, respectively. Quantitative Assay Results Only four (19%) of the testing laboratories provided quantitative data for each of the DENV reference reagent candidates. These laboratories only tested the undiluted candidates without performing any further dilution. The mean estimates for each reference reagent candidate in each laboratory were expressed in log10 copies/mL. The results are shown in Table 5 and Figures 1-8. The variation of estimates for lyophilized materials within laboratories is shown in Figure 9 and Table 6 and demonstrates generally good reproducibility between runs. Qualitative Assay Results A total of 18 (86%) laboratories provided datasets from qualitative assay determinations. The results for each sample in each laboratory were expressed in log10 NAT-detectable units/mL and presented in Table 7 and Figures 1-8. The level of variation for qualitative assays is lower than that for quantitative assays, indicating a wider range of sensitivity for quantitative assays, which were performed by only 4 laboratories. Analyses of the datasets by the Spearman–Karber method and probit regression produced values similar to those obtained with the maximum likelihood method and are available from the authors upon request. Determination of Overall Laboratory Means For quantitative results, the overall means for each reference reagent candidate are shown in Table 8. The mean estimates were calculated based on all measurements collected in the four laboratories that performed quantitative assays. The overall means for the qualitative assays are shown in Table 9. The results obtained from the lyophilized candidate reference reagents only differ by 0.03 – 0.34 log10 from those observed for the liquid frozen reference reagents, which demonstrates that the process of freeze-drying did not significantly affect the integrity of DENV RNA for reference reagent usage. In general, the mean results from the qualitative and quantitative assays do not exhibit universal agreement, with mean estimates comparatively lower for qualitative assays. This can occur in part because of the relatively lower number of laboratories that performed quantitative assays resulting in wider dispersion of the results.

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Relative Potencies Based upon the mean estimates of titer values from quantitative and qualitative protocols, the WHO lyophilized reference reagent candidates were estimated to have potencies of 4.13 (95% CI 3.64 – 4.62), 4.84 (95% CI 4.39 – 5.30), 4.37 (95% CI 3.98 – 4.76), and 4.53 (95% CI 4.19 – 4.87) log10 units/mL for DENV-1 – DENV-4, respectively. These values were used to calculate relative potencies of liquid samples relative to lyophilized samples, shown in Tables 10-12 and in histograms in Figures 10-13. Agreement between protocols and participants was noticeably improved by representing the data in relative terms, with reduced 95% CIs and SDs for the relative potency for each liquid reference reagent candidate compared to the values obtained directly from quantitative and qualitative analysis.

Results of Stability Studies Vials of the lyophilized DENV reference reagent candidates were stored at -20°C, +4°C, +20°C, or +37°C for up to 12 months and tested in duplicate by real-time PCR for DENV RNA. Results suggested that the lyophilized preparation is stable when stored at +4°C (the recommended storage temperature) or lower temperatures. The most significant loss of titer was observed for DENV-1 (0.6 log10) when stored for 12 months at 37°C and DENV-4 (0.8 log10) when stored for 9 months at 37°C (Table 3).The effect on stability of freezing/thawing of the reconstituted samples was not investigated.

Conclusions In this collaborative study, various quantitative and qualitative assays were employed to determine the potency of reference reagent candidates for DENV types 1 to 4 RNA and evaluate their suitability for use in NAT-based assays. Collaborators used both proprietary and commercially available methods for RNA extraction and NAT testing. Both DENV generic NAT assays (capable of detecting all serotypes without discrimination) and DENV serotype-specific NAT assays were employed by collaborators. All assays were able to detect DENV RNA in the vials where it was present and correctly identify negative control vials (which contained only plasma diluent not spiked with DENV). When used, internal assay standards were comprised of serially diluted in vitro transcribed RNA, serially diluted DENV RNA of known concentration, or standard preparations provided with the commercial test kit. The vast differences in test methodologies including internal standards used by collaborating laboratories probably contributed to the levels of variation observed for quantitative results. Two sets of reference reagent candidates were prepared, one liquid frozen and one lyophilized, both containing all DENV serotypes; no significant difference in the RNA titer was observed between the two sample preparation methods. Stability studies have indicated that the lyophilized preparations are stable for at least 12 months when stored at the recommended storage temperature of +4°C. The relative potency data provide some evidence for commutability between the results of liquid frozen and lyophilized forms of reference reagents, since the agreement of results markedly improved when data was expressed relative to the lyophilized reference reagent candidates (in

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case of DENV-1, there was a 4 log10 improvement) . This observation suggests that using the proposed reference reagents as standards will harmonize results produced by different methods. The relative potency data also shows some improvement of agreement between quantitative and qualitative results, which suggests that the variability comes at least partially from the lack of standardization. Based upon the quantitative and qualitative results of the collaborative study, the lyophilized reference reagent candidates were estimated to have potencies of 4.13, 4.84, 4.37, and 4.53 log10 units/mL for DENV-1 to DENV-4, respectively.

Recommendations It is proposed that the lyophilized reference reagent candidates, namely DENV-1 BB, DENV-2 AA, DENV-3 CC and DENV-4 BB, should be established as International Reference Reagents for DENV-1, DENV-2, DENV-3 and DENV-4, with a unitage of 13,500, 69,200, 23,400, and 33,900 Units/mL, respectively, based on the results from the international collaborative study. A total of 1,500 vials per reference reagent are available to the WHO and the custodian laboratory is the Center for Biologics Evaluation and Research/U.S. FDA 1. The internal code for the candidate standards is DENV-1 (code DENV-1 BB), DENV-2 (code DENV-2 AA), DENV-3 (code DENV-3 CC) and DENV-4 (code DENV-4 BB). The recommended storage and shipment temperature is +4°C.

Acknowledgements To Dr. Robert Lanciotti from the CDC for kindly providing the DENV strains used to prepare the candidate standards, and to Stephen Kerby and Laure Juompan for their technical assistance. We thank especially all laboratories who participated in this study. This project has been funded in part by the Intramural CBER/FDA Modernizing Science Funding Program.

In previous discussion NIBSC has indicated that they could store part of the material so that it is kept at two sites.

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Comments from Participants The collaborative study report was distributed to the participants. All comments were addressed and corrections performed where appropriate. All laboratories who replied agreed that the materials are suitable to be established as WHO International Reference Reagents with the assigned unitage.

References Añez, G., Chancey C., Grinev A., and Rios M. 2012. Dengue and other arboviruses: a global view of risks. ISBT Sci Series. 7:274–282. Añez, G, Rios, M (2013) Dengue in the United States of America. A worsening scenario? Biomed Res Int 2013: 678645. Doi: 10.1155/2013/678645 Añez, G., Heisey, D. A., Volkova, E., & Rios, M. (2016). Complete Genome Sequences of Dengue Virus Type 1 to 4 Strains Used for the Development of CBER/FDA RNA Reference Reagents and WHO International Standard Candidates for Nucleic Acid Testing. Genome Announcements, 4(1), e01583–15. http://doi.org/10.1128/genomeA.01583-15 Baylis SA, Blümel J, Mizusawa S, et al.: World Health Organization International Standard to harmonize assays for detection of hepatitis E virus RNA. Emerg Infect Dis 2013; 19:729–735 Bhatt S, Gething PW, Brady OJ, Messina JP, Farlow AW, et al. (2013) The global distribution and burden of dengue. Nature 496:504-507. Doi: 10.1038/nature12060 Chuang V, Wong TY, Leung YH, Ma E, Law YL, et al. (2008) Review of dengue fever cases in Hong Kong during 1998 to 2005. Hong Kong Med J 14:170-177. ClinicalTrials.gov Registry: U.S. National Institutes of Health; 2015 [cited 2015 February 2]. Available from: https://www.clinicaltrials.gov/ct2/show/study/NCT01656174?term=Dengue+virus&rank=3. Collet D: Modeling Binary Data. London, Chapman & Hall, 1991 Johnson BW, Russell BJ, Lanciotti RS. (2005) Serotype-specific detection of dengue viruses in a fourplex real-time reverse transcriptase PCR assay. J Clin Microbiol 43: 4977-4983. Linnen JM, Vinelli E, Sabino EC, Tobler LH, Hyland C, et al. (2008) Dengue viremia in blood donors from Honduras, Brazil, and Australia. Transfusion 48:1355-1362. Doi: 10.1111/j.15372995.2008.01772.x

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Matos D, Tomashek KM, Perez-Padilla J, Munoz-Jordan J, Hunsperger E, Horiuchi K, et al. Probable and possible transfusion-transmitted dengue associated with NS1-antigen negative but RNA-confirmed-positive red blood cells. Transfusion. 2016. Jan;56(1):215-22. Doi: 10.1111/trf.13288. Epub 2015 Sep 7. Mohammed H, Linnen JM, Muñoz-Jordán JL, Tomashek K, Foster G, et al. (2008) Dengue virus in blood donations, Puerto Rico, 2005. Transfusion 48:1348-1354. Doi: 10.1111/j.15372995.2008.01771.x Saldanha J, Lelie N, Heath A, the WHO Collaborative Study Group: Establishment of the first International Standard for nucleic acid amplification technology (NAT) assays for HCV RNA. Vox Sang 1999; 76:149–158 Saldanha J, Shead S, Heath A, et al.: Collaborative study to evaluate a working reagent for West Nile virus RNA detection by nucleic acid testing. Transfusion 2004; 44:97–102 Stramer SL, Linnen JM, Carrick JM, Foster GA, Krysztof DE, et al. (2012) Dengue viremia in blood donors identified by RNA and detection of dengue transfusion transmission during the 2007 dengue outbreak in Puerto Rico. Transfusion 52:1657-1666. Doi: 10.1111/j.15372995.2012.03566.x Stramer SL, Foster GA, Brodsky J, Muñoz-Jordan JL, Hunsperger E, et al. (2013) Investigational dengue testing yields high rates of ribonucleic acid (RNA)-positive donors in Puerto Rico. Transfusion 53(S2): 216A. Tambyah PA, Koay ES, Poon ML, Lin RV, Ong BK et al. (2008) Dengue hemorrhagic fever transmitted by blood transfusion. N Engl J Med 359:1526-1527. Doi: 10.1056/NEJMc0708673 Waggoner JJ, Soda EA, Deresinski S. (2013) Rare and emerging viral infections in transplant recipients. Clin Infect Dis 57:1182-1188. Doi: 10.1093/cid/cit456 World Health Organization (2009) Dengue guidelines for diagnosis, treatment, prevention and control. Third edition. Geneva: World Health Organization. 147 p.

WHO/BS/2016.2299 Page 11 Figure 1. Mean estimates for liquid reference reagent candidate DENV-1 AA. White squares represent number of copies (quantitative assays) and gray squares represent NDU (qualitative assays).

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WHO/BS/2016.2299 Page 12 Figure 2. Mean estimates for lyophilized reference reagent candidate DENV-1 BB. White squares represent number of copies (quantitative assays) and gray squares represent NDU (qualitative assays).

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WHO/BS/2016.2299 Page 13 Figure 3. Mean estimates for liquid reference reagent candidate DENV-2 CC. White squares represent number of copies (quantitative assays) and gray squares represent NDU (qualitative assays).

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WHO/BS/2016.2299 Page 14 Figure 4. Mean estimates for lyophilized reference reagent candidate DENV-2 AA. White squares represent number of copies (quantitative assays) and gray squares represent NDU (qualitative assays).

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WHO/BS/2016.2299 Page 15 Figure 5. Mean estimates for liquid reference reagent candidate DENV-3 BB. White squares represent number of copies (quantitative assays) and gray squares represent NDU (qualitative assays).

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WHO/BS/2016.2299 Page 16 Figure 6. Mean estimates for lyophilized reference reagent candidate DENV-3 CC. White squares represent number of copies (quantitative assays) and gray squares represent NDU (qualitative assays).

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WHO/BS/2016.2299 Page 17 Figure 7. Mean estimates for liquid reference reagent candidate DENV-4 CC. White squares represent number of copies (quantitative assays) and gray squares represent NDU (qualitative assays).

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WHO/BS/2016.2299 Page 18 Figure 8. Mean estimates for lyophilized reference reagent candidate DENV-4 BB. White squares represent number of copies (quantitative assays) and gray squares represent NDU (qualitative assays).

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WHO/BS/2016.2299 Page 19 Figure 9. Intra-laboratory variability in quantitative estimates for lyophilized candidate reference reagents.

WHO/BS/2016.2299 Page 20 Figure 10. Relative potency of the DENV-1 liquid candidate to the lyophilized candidate. White squares represent number of copies (quantitative assays) and gray squares represent NDU (qualitative assays).

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WHO/BS/2016.2299 Page 21 Figure 11. Relative potency of the DENV-2 liquid candidate to the lyophilized candidate. White squares represent number of copies (quantitative assays) and gray squares represent NDU (qualitative assays).

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WHO/BS/2016.2299 Page 22 Figure 12. Relative potency of the DENV-3 liquid candidate to the lyophilized candidate. White squares represent number of copies (quantitative assays) and gray squares represent NDU (qualitative assays).

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WHO/BS/2016.2299 Page 23 Figure 13. Relative potency of the DENV-4 liquid candidate to the lyophilized candidate. White squares represent number of copies (quantitative assays) and gray squares represent NDU (qualitative assays).

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14b 8

14a 13b 10

Number of laboratories

7 6 5 4 3 2 1

19 16 9 7 6 2

8 5 4 3 1 11

1.5

2.5

3.5

4.5

5.5

6.5

log10 copies or NDU/mL

WHO/BS/2016.2299 Page 24 Table 1. DENV strains used to produce the reference reagents. Virus strain* Accession No DENV-1 Hawaii KM204119 DENV-2 New Guinea C KM204118 DENV-3 H87 KU050695 DENV-4 H241 KR011349 *Source: Dr. Robert Lanciotti, CDC, Fort Collins, CO, USA.

Table 2. DENV reference reagent candidates sent for testing in the International Collaborative Study. Set 1 Sample code Format DENV-1, AA Liquid frozen DENV-2, CC Liquid frozen DENV-3, BB Liquid frozen DENV-4, CC Liquid frozen Set 2 Sample Code DENV-1, BB* Lyophilized DENV-2, AA* Lyophilized DENV-3, CC* Lyophilized DENV-4, BB* Lyophilized Negative Controls DENV-1, CC Liquid frozen (negative sample) DENV-2, BB Liquid frozen (negative sample) DENV-3, AA Liquid frozen (negative sample) DENV-4, AA Liquid frozen (negative sample) * DENV candidates proposed as International Reference Reagents.

WHO/BS/2016.2299 Page 25 Table 3. Stability and accelerated degradation studies. Titers are expressed as log10 NDU/mL. DENV-1, lyophilized Tested temperature Time -20°C 1 week 5.3 1 month n.d 2 months n.d 3 months 5.6 6 months 5.5 9 months 5.7 12 months 5.5 DENV-2, lyophilized Tested temperature Time -20°C 1 week 6.1 1 month n.d 2 months n.d 3 months 5.9 6 months 6.0 9 months 5.7 12 months 5.9 DENV-3, lyophilized Tested temperature Time -20°C 1 week 5.4 1 month n.d 2 months n.d 3 months 5.4 6 months 5.2 9 months 5.3 12 months 5.4 DENV-4, lyophilized Tested temperature Time -20°C 1 week 5.1 1 month n.d 2 months n.d 3 months 5.1 6 months 5.0 9 months 5.1 12 months 5.2

+4°C n.d 5.4 n.d 5.0 5.2 n.d 4.9

+20°C 5.1 5.3 5.3 5.1 5.0 4.9 4.7

+37°C 5.1 5.3 4.9 5.0 4.8 4.8 4.5

+4°C n.d 5.9 n.d 5.8 5.7 n.d 5.7

+20°C 5.8 5.8 5.8 5.7 5.8 5.6 5.6

+37°C 5.9 5.9 5.8 5.6 5.4 5.5 5.5

+4°C n.d 5.3 n.d 5.2 5.2 n.d 5.2

+20°C 5.1 5.2 5.4 5.1 5.0 5.1 5.0

+37°C 5.4 5.3 5.4 5.1 5.0 4.9 4.9

+4°C n.d 5.1 n.d 5.0 5.0 n.d 5.0

+20°C 5.1 4.7 4.8 4.9 4.8 4.7 4.7

+37°C 5.4 4.8 4.8 4.8 4.7 4.6 4.6

WHO/BS/2016.2299 Page 26 Table 4. Assays used by the participant laboratories Laboratory code Assay type (quantitative or qualitative) qualitative Extraction protocol NAT type Assay Target Reference

quantitative

qualitative

QIAamp Viral RNA kit with QIACube (Qiagen) QIAamp DSP Virus kit (Qiagen) RNA extraction on automated instrument platform Automated extraction on MagnaPure (Roche) QIAamp Viral RNA Mini kit (Qiagen) QIAamp Viral RNA Mini kit (Qiagen)

Real-time RT-PCR (TaqMan)

3’ NCR

Unpublished data

LightMix kit DENV (Tib Molbiol) Real-time RT-PCR

3’ NCR

Real-time RT-PCR (TaqMan)

3’NCR (DENV-1 – 4) NS5 (DENV-1 – 4) NS3 (DENV-1 – 4)

Warrilow et al 2002

qualitative

NucliSENS – easyMAG automated system (BioMerieux) QIAamp Viral RNA Mini kit (Qiagen)

RT-PCR followed by multiplex nested PCR; analysis by agarose gel electrophoresis Conventional RT-PCR followed by conventional PCR; analysis by agarose gel electrophoresis Real-time RT-PCR (TaqMan)

Bronzoni et al 2005

Seah et al 1995

3’NCR (DENV-1 – 4)

Real-time RT-PCR

qualitative

Magnetic-based target capture on automated system (Tigris) QIAamp Viral RNA Mini kit (Qiagen)

Transcription-mediated amplification followed by hybridization protection assay Real-time RT-PCR (TaqMan)

DENV-1: NS5; DENV-2: E; DENV-3 and 4: prM; 5’NCR and C

Kaiser et al (unpublishe d data) Domingo et al 2010 Johnson et al 2005

Unpublished data

DENV-1: NS5; DENV-2: E; DENV-3: M; DENV-4: E

Johnson et al 2005

WHO/BS/2016.2299 Page 27 and M 8973-9084 1008-1605 740-813 904-992 C-prM

quantitative

QIAamp Viral RNA Mini kit (Qiagen) QIAamp Viral RNA Mini kit (Qiagen) QIAamp Viral RNA Mini kit (Qiagen) QIAamp Viral RNA Mini kit (Qiagen) QIAamp Viral RNA Mini kit (Qiagen)

QuantiTect Probe RTPCR (Qiagen)

Johnson et al 2005

qualitative

Ready-to-Go RT-PCR Beads (GE Healthcare) RT-nested PCR

qualitative

quantitative

qualitative

Real-time RT-PCR (TaqMan) using in vitro transcribed RNA as standard curve Real-time RT-PCR (TaqMan)

E/NS1 junction (DENV-1 – 4) 3’NCR

Domingo et al 2011; Domingo et al 2006 Kaiser M, unpublished

quantitative

QIAamp Viral RNA Mini kit (Qiagen)

Real-time RT-PCR (TaqMan) using standards

qualitative

QIAamp Viral RNA Mini kit (Qiagen) Qiagen columns (Qiagen) QIAamp Viral RNA Mini kit (Qiagen)

Real-time RT-PCR (TaqMan) Real-time RT-PCR (TaqMan) Fourplex Real-time Reverse Transcriptase Real-time PCR Assay

DENV-1: NS5; DENV-2: E; DENV-3 and 4: prM; DENV-1: NS5; DENV-2: E; DENV-3 and 4: prM; E

Johnson et al 2005

Santiago et al 2013 Santiago et al 2013 Johnson et al 2005

qualitative

QIAamp Viral RNA Mini kit (Qiagen) QIAamp Viral RNA Mini kit (Qiagen) QIAamp Viral RNA Mini kit (Qiagen)

Real-time RT-PCR (TaqMan) Real-time RT-PCR (TaqMan)

Various (DENV-1 – 4) DENV-1: NS5; DENV-2: E; DENV-3: M; DENV-4: E and M 5’NCR or 3’NCR 3’NCR (DENV-1 – 4) Various (DENV-1 – 4)

qualitative

Real-time RT-PCR (TaqMan)

LeparcGoffart et al 2009 Gurukumar et al 2009; Chien et al 2006 Santiago et al 2013

WHO/BS/2016.2299 Page 28 High Pure Viral Nucleic Acid kit (Life Science/Roche) *Information was not provided 21 qualitative Real-time RT-PCR (TaqMan) NS5 (DENV-1 – 4) Chien et al 2006

Table 5. Mean estimates of DENV titers based on quantitative assays results (log10 copies/mL). Lab Code Sample Sample Sampl Sample Sample #1, #2, e #4, #6, #8, DENV-1 DENV-1 DENV DENV-2 DENV-3 AA BB -2 AA CC BB (FRO) (LYO) (LYO) (FRO) (FRO) 2 2.84 3.38 3.67 3.25 3.34 11 7.05 6.69 6.51 6.64 6.79 13 5.12 4.98 6.16 5.21 5.41 14 5.74 5.45 5.6 5.82 5.69 FRO = liquid frozen candidates; LYO = lyophilized candidates Sample #9, DENV-3 CC (LYO) 3.32 6.66 5.17 5.47 Sample #11, DENV-4 BB (LYO) 4.21 6.21 6.13 5.14 Sample #12, DENV-4 CC (FRO) 3.85 6.65 6.33 4.94

Table 6. Variation of estimates for lyophilized materials within laboratories where quantitative assays were performed, in copies/ml. Lab Code 2 11 13 14 DENV-1 DENV-2 Mean SD n Mean SD 3 3 3 2.77x10 1.97x10 4 5.07x10 2.48x103 5.83x106 3.60x106 4 3.30x106 5.89x105 9.51x104 5.63x103 4 1.46x106 1.65x105 2.79x105 1.85x104 3 4.02x105 1.11x104 SD – standard deviation; n – number of replicates. DENV-3 n Mean SD 3 4 2.14x10 6.25x102 4 4.63x106 1.02x106 4 1.51x105 2.80x104 3 2.94x105 3.79x103 DENV-4 n Mean SD 4 4 1.87x10 9.20x103 4 1.70x106 4.76x105 4 1.35x106 1.20x105 3 1.39x105 1.89x104

n 4 4 4 3

WHO/BS/2016.2299 Page 29 Table 7. Mean values of DENV titers based on qualitative assays results (log10 NDU/mL) estimated by Poisson/Maximum likelihood method. Lab Code Sample Sample Sample Sample Sample Sample Sample #1, #2, #4, #6, #8, #9, #11, DENV-1 DENV-1 DENV-2 DENV-2 DENV-3 DENV-3 DENV-4 AA BB AA CC BB CC BB (FRO) (LYO) (LYO) (FRO) (FRO) (LYO) (LYO) 4.16 3.03 4.1 4.11 3.03 2.79 4.03 4.43 4.82 5.5 5.22 5.12 4.78 4.91 3.62 3.67 4.54 4.55 4.06 3.46 4.31 3.9 NA* 3.9 NA* 3.96 2.88 3.9 4.57 4.57 5.76 5.27 5.07 4.86 3.99 0.96 1.7 3.82 2.86 4.67 4.85 4.18 3.74 4.27 4.73 4.77 4.39 4.05 4.74 4.25 3.92 5.64 4.88 5.13 4.88 5.31 4.94 4.71 5.27 5.1 5.16 4.71 4.45 NA** 3.85 6.27 NA** NA** 3.75 4.45 NA** 4.26 5.77 NA** NA** 4.83 3.63 4.71 4.57 5.35 5.57 5.25 4.91 5.26 4.03 4.03 5.05 4.86 4.61 3.98 4.94 3.31 3.83 4.78 3.67 3.67 4.07 4.04 NA** 5.03 5.87 NA** NA** 4.87 5.22 NA** 4.36 5.13 NA** NA** 3.47 4.41 4.12 3.65 3.48 3.91 4.13 3.53 3.44 NA** 5.28 5.8 NA** NA** 4.7 5.41 NA** 2.21 2.37 NA** NA** 3.36 3.42 *: Model does not converge **: Sample not tested FRO = liquid frozen candidates; LYO = lyophilized candidates; NA = Not applicable Sample #12, DENV-4 CC (FRO) 4.11 4.98 4.39 3.9 2.78 3.85 4.93 4.77 4.59 NA** NA** 5.19 5.05 3.67 NA** NA** 3.13 NA** NA**

1 3 4 5 6 7 8 9 10 12 13 14 15 16 17 18 19 20 21

WHO/BS/2016.2299 Page 30 Table 8. Overall mean estimates for quantitative assays (log10 copies/mL). Sample #1, DENV-1 AA (FRO) Sample #2, DENV1 BB (LYO) Sample #4, DENV-2 AA (LYO) Sample #6, DENV-2 CC (FRO) Sample #8, DENV-3 BB (FRO) Sample #9, DENV-3 CC (LYO) Sample #11, DENV-4 BB (LYO) Sample #12, DENV-4 CC (FRO)

14 15 15 15 15 15 15 15 n 5.02 5.10 5.48 5.19 5.27 5.13 5.44 5.41 Mean 1.61 1.28 1.18 1.41 1.39 1.28 0.88 1.23 SD 4.39 4.83 4.38 4.47 4.42 4.95 4.71 Lowerci 4.08 5.81 6.13 6.00 6.08 5.84 5.93 6.12 Upperci 5.95 2009 648 485 970 929 651 225 557 cv geo n = number of replicate measurements collected in all labs; sd = standard deviation; lowerci/upperci = 95% confidence limits of the mean; cv geo = geometric coefficient of variation (%); FRO = liquid frozen candidates; LYO = lyophilized candidates

Table 9. Overall mean estimates for qualitative assays (log10 NDU/mL).

Sample Sample Sample Sample Sample Sample Sample Sample #1, #2, #4, #6, #8, #9, #11, #12, DENV-1 DENV-1 DENV-2 DENV-2 DENV-3 DENV-3 DENV-4 DENV-4 AA BB AA CC BB CC BB CC (FRO) (LYO) (LYO) (FRO) (FRO) (LYO) (LYO) (FRO) 13 18 19 12 13 19 19 13 n 3.90 3.99 4.90 4.56 4.48 4.14 4.42 4.26 Mean 0.99 0.92 1.00 0.79 0.68 0.73 0.63 0.76 SD 3.55 4.45 4.11 4.10 3.81 4.14 3.83 Lowerci 3.35 4.42 5.35 5.02 4.86 4.48 4.71 4.68 Upperci 4.45 294 248 297 177 139 157 122 168 cv geo n = number of laboratories; sd = standard deviation; lowerci/upperci = 95% confidence limits of the mean; cv geo = geometric coefficient of variation (%); FRO = liquid frozen candidates; LYO = lyophilized candidates

WHO/BS/2016.2299 Page 31 Table 10. Potencies of liquid reference reagent candidates relative to lyophilized reference reagent candidates based on data from quantitative assays. Lab Code DENV-1, FRO Relative to LYO Relative Potency (log10) 3.60 4.49 4.27 4.42 DENV-2, FRO Relative to LYO DENV-3, FRO Relative to LYO DENV-4, FRO Relative to LYO

95% Relative 95% Relative 95% Relative 95% Confidence Potency Confidence Potency Confidence Potency Confidence Interval (log10) Interval (log10) Interval (log10) Interval 2 (3.23, 3.96) 4.42 (4.06,4.78) 4.39 (4.06, 4.72) 4.17 (3.58, 4.77) 11 (3.53, 5.45) 4.97 (4.80, 5.14) 4.50 (4.19, 4.81) 4.96 (4.72, 5.20) 13 (4.21, 4.33) 3.89 (3.08, 3.69) 4.61 (4.28, 4.94) 4.73 (4.52,4.93) 14 (4.36, 4.48) 5.05 (5.02, 5.07) 4.60 (4.57, 4.62) 4.32 (4.17, 4.48) Mean 4.20 4.58 4.53 4.55 95% CI (3.80, 4.59) (4.05,5.11) (4.42, 4.63) (4.19, 4.90) SD 0.41 0.54 0.10 0.36 FRO = liquid frozen candidates; LYO = lyophilized candidates; 95% CI: 95% confidence intervals Table 11. Potencies of liquid reference reagent candidates relative to lyophilized reference reagent candidates based on data from qualitative assays. DENV-1, FRO Relative to LYO Lab Code Relative Potency (log10) 5.26 3.75 4.08 NA** 4.13 3.39 3.61 4.46 4.36 4.28 4.13 4.60 3.61 DENV-2, FRO Relative to LYO DENV-3, FRO Relative to LYO DENV-4, FRO Relative to LYO

95% Relative 95% Relative 95% Relative 95% Confidence Potency Confidence Potency Confidence Potency Confidence Interval (log10) Interval (log10) Interval (log10) Interval 1 (4.89,5.63) 4.84 (4.46,5.23) 4.61 (4.25,4.97) 4.61 (4.23,5.00) 3 (3.36,4.12) 4.56 (4.20,4.94) 4.71 (4.36, 5.06) 4.60 (4.14,5.15) 4 (3.72,4.44) 4.85 (4.48,5.21) 4.96 (4.63,5.29) 4.61 (4.27,4.96) 5 NA** NA** NA** 5.45 (5.04,5.88) 4.53 (4.14,4.92) 6 (3.76,4.50) 4.35 (3.99,4.71) 4.57 (4.21,4.92) 3.33 (2.98,3.67) 7 (2.93,3.80) 3.88 (3.53,4.23) 4.19 (3.87,4.51) 4.19 (3.80,4.58) 8 (3.24,3.97) 4.88 (4.49,5.26) 4.71 (4.33,5.08) 4.72 (4.36,5.07) 9 (4.11,4.81) 4.08 (3.73,4.43) 4.62 (4.27,4.97) 3.99 (3.61,4.38) 10 (3.98,4.75) 4.68 (4.30,5.05) 4.82 (4.46,5.18) 4.67 (4.30,5.03) 14 (3.92,4.64) 5.07 (4.70,5.44) 4.71 (4.35,5.07) 4.45 (4.11,4.79) 15 (3.75,4.51) 4.64 (4.27,5.01) 5.00 (4.64,5.36) 4.64 (4.25,5.03) 19 (4.29,4.92) 5.27 (4.95,5.59) 4.97 (4.65,5.29) 4.22 (3.78,4.64) 16 (3.24,3.98) 3.73 (3.36,4.11) 3.97 (3.59,4.34) 4.16 (3.80,4.53) Mean 4.14 4.57 4.71 4.36 95% CI (3.85,4.43) (4.30,4.84) (4.51,4.91) (4.15,4.57) SD 0.51 0.47 0.37 0.39 FRO = liquid frozen candidates; LYO = lyophilized candidates; 95% CI: 95% confidence intervals; NA = Not applicable, sample not tested

WHO/BS/2016.2299 Page 32 Table 12. Potencies of liquid reference reagent candidates relative to lyophilized reference reagent candidates based on combined data from quantitative and qualitative assays DENV-1, FRO DENV-2, FRO DENV-3, FRO DENV-4, FRO Relative to LYO Relative to LYO Relative to LYO Relative to LYO Combined Mean 4.15 4.57 4.67 4.41 95% CI (3.92, 4.39) (4.34, 4.80) (4.51, 4.83) (4.23, 4.59) SD 0.48 0.47 0.33 0.38 FRO = liquid frozen candidates; LYO = lyophilized candidates; 95% CI: 95% confidence intervals

WHO/BS/2016.2299 Page 33 Appendix 1. List of participants in the DENV WHO IRR study, in alphabetical order (by Principal Investigator). Participants Wenjun Liu/John A. Aaskov Affiliation Australian Army Malaria Institute/Queensland University of Technology Brisbane, Australia Maria João Alves/Líbia Zé-Zé Center for Vectors and Infectious Diseases Research, National Institute of Health Águas de Moura, Portugal Maria Rosario Z. Capeding/Edelwisa SegubreMercado/Mary Ann U. Igoy Alzira Batista Cecílio/Ana Caroline Zampiroli Ataíde Research Institute for Tropical Medicine Muntinlupa City, Philippines Ezequiel Dias Foundation Belo Horizonte, Brazil Cristina Domingo Robert Koch Institute Centre for Biological Threats and Special Pathogens Berlin, Germany Zoonotic Diseases and Special Pathogens, National Microbiology Laboratory, Public Health Agency of Canada Winnipeg, Manitoba, Canada Slav Dugenny/Dave Petrich Roche Molecular Systems, Inc. Pleasanton, United States Myrielle Dupont-Rouzeyrol/D. Girault/O.O’Connor Institute Pasteur of New Caledonia New Caledonia Delia Enria/Cintia Fabbri/María Alejandra Morales National Institute of Human Viral Diseases Pergamino, Argentina Duane J Gubler/ October M Sessions/Eng Eong Ooi Program on Emerging Infectious Diseases

Michael Drebot /Kimberly Holloway

WHO/BS/2016.2299 Page 34 Duke-NUS Graduate Medical School Singapore, Singapore Vu Thi Que Huong/ Nguyen Thi Cong Dung Pasteur Institute in Ho Chi Minh City Ho Chi Minh City, Vietnam Isabelle Leparc-Goffart/Christine Prat Institute of Biomedical Research of the Army Marseille, France Jeffrey M. Linnen/Edgar Ong/Robin Cory Hologic, Inc. San Diego, United States Jorge Muñoz-Jordán/Gilberto Santiago Centers for Disease Control and Prevention San Juan, Puerto Rico Mauricio L Nogueira/Tatiana E Colombo Faculty of Medicine of São José do Rio Preto São José do Rio Preto, Brazil Micha Nuebling/Julia Kress/Michael Chudy Paul Ehrlich Institute Langen (Hesse), Germany David Perera Institute of Health & Community Medicine University of Malaysia, Sarawak Sarawak, Malaysia Maria Rios/Germán Añez/Zhen Jiang/Daniel Heisey U.S. Food and Drug Administration Silver Spring, United States María Paz Sánchez-Seco/Leticia Franco Institute of Health “Carlos III” Madrid, Spain Jonas Schmidt-Chanasit Bernhard Nocht Institute for Tropical Medicine WHO Collaborating Centre for Arbovirus and Haemorrhagic Fever Reference and Research Hamburg, Germany Vanessa Suin/Aurélie Francart Re-emerging Viruses Program

WHO/BS/2016.2299 Page 35 Viral Diseases | Communicable and Infectious Diseases Scientific Institute of Public Health Brussels, Belgium

WHO/BS/2016.2299 Page 36 Appendix 2. List of participants in the characterization of DENV stocks used to prepare the reference reagent candidates. Participants Ana P. Goncalvez/Ronald E. Engle/Robert H. Purcell Laura D. Kramer/Susan Jones/Alan P. Dupuis Robert Lanciotti Maria Rios/Germán Añez Affiliation National Institutes of Health Bethesda, United States New York State Department of Health Albany, United States Centers for Disease Control and Prevention Fort Collins, United States U.S. Food and Drug Administration Silver Spring, United States

WHO/BS/2016.2299 Page 37 Appendix 3. Draft Instructions for Use for the Dengue Virus types 1 to 4 RNA Reference Reagents for NAT-based assays U.S. Food and Drug Administration Center for Biologics Evaluation and Research for Biological Standardization

A WHO Collaborating Center

Store at +4°C.

WHO International Reference Reagents International Reference Reagents for Dengue virus types 1 to 4 for Nucleic Acid Amplification Techniques (NAT)-based assays CBER/FDA codes: DENV-1 BB, DENV-2 AA, DENV-3 CC and DENV-4 BB

Please note: because of the inherent stability of lyophilized material, these materials may ship at ambient temperature. 6. DIRECTIONS FOR OPENING Vials have a “flip-up” circular cap. Either on the cap or the collar of the vial, there is an indication of the point at which to lever off the cap. This exposes an area of the rubber stopper through which reconstitution and withdrawal of the preparation can be made using a hypodermic needle and syringe. If use of a pipette is preferred, then fully remove the metal collar using, for example, forceps, taking care to avoid cuts by wearing appropriate gloves. Remove the stopper for access. Care should be taken to prevent loss of the contents. 7. USE OF MATERIAL No attempt should be made to weigh out any portion of the freeze-dried material prior to reconstitution. Each vial should be reconstituted with 1 mL of nuclease-free water. The product should be reconstituted just prior to use. Once reconstituted, freeze-thawing of the product is not recommended, since it has not been evaluated. 8. STABILITY It is the policy of WHO not to assign an expiry date to their international reference materials. They remain valid as the assigned potency and status until withdrawn or amended. The reference materials are held at CBER/FDA within assured, temperature-controlled storage facilities. Reference Materials should be stored on receipt as indicated on the label. Once diluted or aliquoted, users should determine the stability of the material according to their own method of preparation, storage and use. Users who have data supporting deterioration in the characteristics of any reference preparation are encouraged to contact CBER/FDA. 9. REFERENCES Añez G., Volkova E., Fares R.C.G., Jiang Z., Rios M. Collaborative Study to Establish a World Health Organization International Reference Panel for Dengue Virus types 1 to 4 RNA for Nucleic Acid Amplification Technology (NAT)-Based Assays. WHO Report 2016, WHO/BS/2016.2299. 10. ACKNOWLEDGEMENTS We would like to thank Dr. Robert Lanciotti from the CDC for providing viral strains used in preparation of the reagents.

Instructions for use (Version 1.0, July 2016) 1. INTENDED USE The International Reference Reagents for Dengue virus (DENV) types 1 to 4 RNA are intended to be used either individually or together as a panel in the standardization of nucleic acid amplification technique (NAT)-based assays for DENV. Each reference reagent was prepared by diluting a heat-inactivated, cell culture derived laboratory prototype strain (DENV-1 Hawaii, DENV-2 New Guinea C, DENV-3 H87 and DENV-4 H241) in defibrinated, dialyzed, filtered human plasma. The reference reagents were lyophilized in 1 mL aliquots and stored at +4°C, and evaluated in an international collaborative study involving 21 laboratories which used a variety of NAT assays for testing. 2. CAUTION This preparation is not for administration to humans. The material is of human origin, as well as cell culture supernatant from mosquito C6/36 cells infected with DENV. DENV infectivity has been inactivated by heat treatment. As with all materials of biological origin, this preparation should be regarded as potentially hazardous to health. It should be used and discarded according to your own laboratory's safety procedures. Such safety procedures should include the wearing of protective gloves and avoiding the generation of aerosols. Care should be exercised in opening ampoules or vials, to avoid cuts. 3. UNITAGE These standards have been assigned a unitage of 13,500 (DENV-1), 69,200 (DENV-2), 23,400 (DENV-3) and 33,900 (DENV-4) Units/mL. 4. CONTENTS Each vial contains 1 mL of lyophilized plasma containing heat-inactivated DENV. Country of origin of biological material: United States. 5. STORAGE

WHO/BS/2016.2299 Page 38 11. FURTHER INFORMATION Further information can be obtained as follows; This material: Maria.Rios@fda.hhs.gov WHO Biological Standards: http://www.who.int/biologicals/en/ 12. CUSTOMER FEEDBACK Customers are encouraged to provide feedback on the suitability or use of the material provided or other aspects of our service. Please send any comments to Maria.Rios@fda.hhs.gov 13. CITATION In all publications, including data sheets, in which this material is referenced, it is important that the preparation's title, its status, the CBER/FDA code number, and the name and address of CBER/FDA are cited correctly. 14. MATERIAL SAFETY SHEET Physical and Chemical properties Physical appearance: Corrosive: Stable: Oxidizing: Hygroscopic: Irritant: Flammable: Handling: Other (specify): Lyophilized powder No Yes No No No No See caution, Section 2 Contains material of human origin (plasma) and heatinactivated, non-infectious DENV grown in mosquito C6/36 cells Toxicological properties 15. LIABILITY AND LOSS Information provided by the Institute is given after the exercise of all reasonable care and skill in its compilation, preparation and issue, but it is provided without liability to the Recipient in its application and use. It is the responsibility of the Recipient to determine the appropriateness of the standards or reference materials supplied by the Institute to the Recipient (“the Goods”) for the proposed application and ensure that it has the necessary technical skills to determine that they are appropriate. Results obtained from the Goods are likely to be dependent on conditions of use by the Recipient and the variability of materials beyond the control of the Institute. All warranties are excluded to the fullest extent permitted by law, including without limitation that the Goods are free from infectious agents or that the supply of Goods will not infringe any rights of any third party. The Institute shall not be liable to the Recipient for any economic loss whether direct or indirect, which arise in connection with this agreement. The total liability of the Institute in connection with this agreement, whether for negligence or breach of contract or otherwise, shall in no event exceed 120% of any price paid or payable by the Recipient for the supply of the Goods. If any of the Goods supplied by the Institute should prove not to meet their specification when stored and used correctly (and provided that the Recipient has returned the Goods to the Institute together with written notification of such alleged defect within seven days of the time when the Recipient discovers or ought to have discovered the defect), the Institute shall either replace the Goods or, at its sole option, refund the handling charge provided that performance of either one of the above options shall constitute an entire discharge of the Institute’s liability under this Condition.

Effects of inhalation:

Not established, avoid inhalation Effects of ingestion: Not established, avoid ingestion Effects of skin Not established, avoid absorption: contact with skin Suggested First Aid Inhalation: Ingestion: Contact with eyes: Seek medical advice Seek medical advice Wash with copious amounts of water. Seek medical advice Wash thoroughly with water.

Contact with skin:

Action on Spillage and Method of Disposal Spillage of ampoule contents should be taken up with absorbent material wetted with an appropriate disinfectant. Rinse area with an appropriate disinfectant followed by water. Absorbent materials used to treat spillage should be treated as biological waste.

WHO/BS/2016.2299 Page 39 Appendix 4. Study protocol, results and methods forms

World Health Organization collaborative study to assess the suitability of a candidate standard for dengue virus types 1 to 4 (DENV-1 to -4) RNA for detection by nucleic acid tests

Germán Añez, M.D. and Maria Rios, Ph.D. Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Office of Blood Evaluation and Research, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration

STUDY PROTOCOL

ASSAY PERFORMANCE AND REPORTING Participants should test the dengue virus types 1 to 4 (DENV-1 to 4) working reagents as they usually test samples suspected to contain DENV, using the nucleic acid test assay(s) system(s) available to them, and perform both qualitative and quantitative assays, if available. Results should be recorded clearly on the results form.

If more than one type of assay is performed by a particular laboratory (for example, two different extraction methods or different RT-PCR protocols), separate qualitative Result

WHO/BS/2016.2299 Page 40

tables and Method forms should be completed for each method. Results reported on a single set of Result tables will be treated as replicate results of the same assay run. RESULTS A datasheet is provided so that all relevant information can be recorded. A separate datasheet should be completed for each assay. Volumes of sample extracted, final volume of extracted RNA and volume of RNA used per amplification reaction should be included. Note that results for different assay methods performed by a laboratory must be reported on separate Result forms and Method forms. Please indicate on each reporting sheet the Laboratory and Name of the Investigator(s). All completed forms should be returned preferably by email, within two (2) months from the receipt of the material. The results and completed Method forms should be returned to:

Maria Rios, Ph.D. and Germán Añez, M.D. LEP/DETTD/OBRR/CBER/FDA 29 Lincoln Dr, HFM-310 Bethesda MD, 20892 Tel: +1-301-435 4585 (MR) and +1-301-827-2467 (GA) E-mail: Maria.Rios@fda.hhs.gov (MR), German.Anez@fda.hhs.gov (GA)

WHO/BS/2016.2299 Page 41

TESTING PROTOCOL 1. Each participant will receive 6 vials of lyophilized and 12 vials of liquid frozen working reagents for each of the four DENV types (total of 72 vials), see Table 1. Freshly reconstituted vials (for lyophilized reagents) and freshly thawed vials (for liquid frozen reagents) should be used for each independent run of the assay. Please reconstitute each lyophilized vial with 1 ml of deionized, nuclease-free water immediately before use. The reconstituted vials should be shaken gently over a period of approximately 20 minutes to dissolve the contents. The liquid frozen working reagents should be thawed immediately before use.

Table 1. DENV working reagents (DENV International Standard candidates). Testing material Sample # / Candidate name 1) DENV-1, AA 2) DENV-1, BB 3) DENV-1, CC 4) DENV-2, AA 5) DENV-2, BB 6) DENV-2, CC 7) DENV-3, AA 8) DENV-3, BB 9) DENV-3, CC 10) DENV-4, AA 11) DENV-4, BB 12) DENV-4, CC Vials sent 6 6 6 6 6 6 6 6 6 6 6 6

Format Liquid frozen Lyophilized Liquid frozen Lyophilized Liquid frozen Liquid frozen Liquid frozen Liquid frozen Lyophilized Liquid frozen Lyophilized Liquid frozen

2. Please perform at least four (4) independent runs of the DENV NAT assay available in your laboratory for each working reagent (i.e. liquid frozen and lyophilized materials for each DENV type). Each independent run should be performed on a different day (preferably one week apart). For each round of testing, a single-use vial should be reconstituted (lyophilized material) or thawed (liquid frozen material) and serial dilutions prepared using nuclease-free water and nuclease-free tubes; the undiluted reagent and each of the dilutions prepared from the liquid

WHO/BS/2016.2299 Page 42 frozen and lyophilized materials should be also subjected to RNA extraction followed by nucleic acid amplification assay(s) (i.e. RT-PCR, TaqMan and/or any other NAT assay available) to determine the DENV RNA end-point.

3. We ask that on the first day of testing (first run of the assay), the participants extract RNA from the undiluted material and from each member of an initial series of eight (8) 10-fold dilutions that should be tested in triplicates, to determine the end-point for each material (i.e. liquid frozen and lyophilized for each DENV type): Testing material Sample # Description 1 Undiluted reagent 2 10-1 3 10-2 4 10-3 5 10-4 6 10-5 7 10-6 8 10-7 9 10-8

For the remaining three rounds of testing (in different days, preferably one week apart), please dilute the reference reagents two half-log (100.5) dilutions (i.e. 1:3.2 dilutions) on either side of the end-point determined on the first round of testing, extract RNA and test with your NAT assay in triplicates. For example, if during the first round of testing the dilution 10-6 is found to be the end-point: Testing material 4 5 6 10-3 10-4 10-5

Sample # X Dilution

1 Undiluted reagent

2 10-1

3 10-2

7 10-6

8 10-7

9 10-8

2/3 # positive 3/3 3/3 3/3 3/3 3/3 3/3 0/3 0/3 replicates End-point Then, for the subsequent three testing rounds the participant should test each of the samples (i.e. liquid frozen and lyophilized material candidates for each DENV type), as follows: Testing material 3 4 10-6 -5.5 10 End-point

Sample # Description

1 Undiluted reagent

2 10 -5

5 10 -6.5

6 10-7

WHO/BS/2016.2299 Page 43 4. If in addition to the qualitative assay described above, your laboratory have a quantitative NAT assay capability (i.e. your assay is performed including a DENV RNA standard curve), we ask that the collaborators report to us the results of the four independent determinations of the undiluted DENV working reagent only (both liquid frozen and lyophilized), tested in triplicates.

5. Datasheets, Method forms and Result forms are provided for recording of all relevant information about the assay used. Please return the completed forms to us for analysis of the results.

WHO/BS/2016.2299 Page 44 RESULTS FORMS

Investigator: Date: Volume of sample extracted: Volume of final RNA preparation: Volume of RNA used for amplification/RT-PCR:

Qualitative assay: (Please note that we have completed the first table as an example. If Ct values are available please include them in this table, as shown below)

Sample #1, liquid frozen (DENV-1, AA) Dilutions 1 2 3 4 5 6 7 8 9 Undiluted 10-1 10-2 10-3 10-4 10-5 10-6 10-7 10-8 Test day 2 Replicate 1 Replicate 2

Test day 1 Replicate 3 Result (# positive replicates)

+, Ct 15 +, Ct 17 +, Ct 19 +, Ct 21 +, Ct 23 +, Ct 25 +, Ct 28 +, Ct 32 –

+, Ct 15 +, Ct 17 +, Ct 19 +, Ct 21 +, Ct 24 +, Ct 25 +, Ct 29 – – Test day 3

+, Ct 15 +, Ct 17 +, Ct 19 +, Ct 21 +, Ct 23 +, Ct 26 – – –

3/3, Ct Av. 15 3/3, Ct Av. 17 3/3, Ct Av. 19 3/3, Ct Av. 21 3/3, Ct Av. 23.3 3/3, Ct Av. 25.3 2/3, Ct Av. 28.5 1/3, Ct Av. 32 0/3 Test day 4

Sample #1, liquid frozen (DENV-1, AA) Dilutions 1 2 3 4 5 6 Undiluted 101 100.5 End-point* 10-0.5 10-1 Rep 1 +, Ct 15 +, Ct 23 +, Ct 25 +, Ct 28 +, Ct 32 –

Rep 2 +, Ct 15 +, Ct 24 +, Ct 25 +, Ct 29 – –

Rep 3 +, Ct 15 +, Ct 23 +, Ct 26 – – –

Result (# positive replicates) 3/3, Ct Av. 15 3/3, Ct Av. 23.3 3/3, Ct Av. 25.3 2/3, Ct Av. 28.5 1/3, Ct Av. 32 0/3

Rep 1 +, Ct 15 +, Ct 23 +, Ct 25 +, Ct 28 +, Ct 32 –

Rep 2 +, Ct 15 +, Ct 24 +, Ct 25 +, Ct 29 – –

Rep 3 +, Ct 15 +, Ct 23 +, Ct 26 – – –

Result (# positive replicates) 3/3, Ct Av. 15 3/3, Ct Av. 23.3 3/3, Ct Av. 25.3 2/3, Ct Av. 28.5 1/3, Ct Av. 32 0/3

Rep 1 +, Ct 15 +, Ct 23 +, Ct 25 +, Ct 28 +, Ct 32 –

Rep 2 +, Ct 15 +, Ct 24 +, Ct 25 +, Ct 29 – –

Rep 3 +, Ct 15 +, Ct 23 +, Ct 26 – – –

Result (# positive replicates) 3/3, Ct Av. 15 3/3, Ct Av. 23.3 3/3, Ct Av. 25.3 2/3, Ct Av. 28.5 1/3, Ct Av. 32 0/3

* As determined in the first round of testing (test day 1). Comments:

WHO/BS/2016.2299 Page 45

Sample #2, lyophilized (DENV-1, BB) Dilutions 1 2 3 4 5 6 7 8 9 Undiluted 10-1 10-2 10-3 10-4 10-5 10-6 10-7 10-8 Test day 2 Replicate 1 Replicate 2

Test day 1 Replicate 3 Result (# positive replicates)

Sample #2, lyophilized (DENV-1, BB) Dilutions 1 2 3 4 5 6 Rep 1

Test day 3

Test day 4

Rep 2

Rep 3

Result (# positive replicates)

Rep 1

Rep 2

Rep 3

Result (# positive replicates)

Rep 1

Rep 2

Rep 3

Result (# positive replicates)

Undiluted 101 100.5 End-point* 10-0.5 10-1 * As determined in the first round of testing (test day 1). Comments:

WHO/BS/2016.2299 Page 46

Sample #3, liquid frozen (DENV-1, CC) Dilutions 1 2 3 4 5 6 7 8 9 Undiluted 10-1 10-2 10-3 10-4 10-5 10-6 10-7 10-8 Test day 2 Replicate 1 Replicate 2

Test day 1 Replicate 3 Result (# positive replicates)

Sample #3, liquid frozen (DENV-1, CC) Dilutions 1 2 3 4 5 6 Rep 1

Test day 3

Test day 4

Rep 2

Rep 3

Result (# positive replicates)

Rep 1

Rep 2

Rep 3

Result (# positive replicates)

Rep 1

Rep 2

Rep 3

Result (# positive replicates)

Undiluted 101 100.5 End-point* 10-0.5 10-1 * As determined in the first round of testing (test day 1). Comments:

WHO/BS/2016.2299 Page 47

Sample #4, lyophilized (DENV-2, AA) Dilutions 1 2 3 4 5 6 7 8 9 Undiluted 10-1 10-2 10-3 10-4 10-5 10-6 10-7 10-8 Test day 2 Replicate 1 Replicate 2

Test day 1 Replicate 3 Result (# positive replicates)

Sample #4, lyophilized (DENV-2, AA) Dilutions 1 2 3 4 5 6 Rep 1

Test day 3

Test day 4

Rep 2

Rep 3

Result (# positive replicates)

Rep 1

Rep 2

Rep 3

Result (# positive replicates)

Rep 1

Rep 2

Rep 3

Result (# positive replicates)

Undiluted 101 100.5 End-point* 10-0.5 10-1 * As determined in the first round of testing (test day 1). Comments:

WHO/BS/2016.2299 Page 48

Sample #5, liquid frozen (DENV-2, BB) Dilutions 1 2 3 4 5 6 7 8 9 Undiluted 10-1 10-2 10-3 10-4 10-5 10-6 10-7 10-8 Test day 2 Replicate 1 Replicate 2

Test day 1 Replicate 3 Result (# positive replicates)

Sample #5, liquid frozen (DENV-2, BB) Dilutions 1 2 3 4 5 6 Rep 1

Test day 3

Test day 4

Rep 2

Rep 3

Result (# positive replicates)

Rep 1

Rep 2

Rep 3

Result (# positive replicates)

Rep 1

Rep 2

Rep 3

Result (# positive replicates)

Undiluted 101 100.5 End-point* 10-0.5 10-1 * As determined in the first round of testing (test day 1). Comments:

WHO/BS/2016.2299 Page 49

Sample #6, liquid frozen (DENV-2, CC) Dilutions 1 2 3 4 5 6 7 8 9 Undiluted 10-1 10-2 10-3 10-4 10-5 10-6 10-7 10-8 Test day 2 Replicate 1 Replicate 2

Test day 1 Replicate 3 Result (# positive replicates)

Sample #6, liquid frozen (DENV-2, CC) Dilutions 1 2 3 4 5 6 Rep 1

Test day 3

Test day 4

Rep 2

Rep 3

Result (# positive replicates)

Rep 1

Rep 2

Rep 3

Result (# positive replicates)

Rep 1

Rep 2

Rep 3

Result (# positive replicates)

Undiluted 101 100.5 End-point* 10-0.5 10-1 * As determined in the first round of testing (test day 1). Comments:

WHO/BS/2016.2299 Page 50

Sample #7, liquid frozen (DENV-3, AA) Dilutions 1 2 3 4 5 6 7 8 9 Undiluted 10-1 10-2 10-3 10-4 10-5 10-6 10-7 10-8 Test day 2 Replicate 1 Replicate 2

Test day 1 Replicate 3 Result (# positive replicates)

Sample #7, liquid frozen (DENV-3, AA) Dilutions 1 2 3 4 5 6 Rep 1

Test day 3

Test day 4

Rep 2

Rep 3

Result (# positive replicates)

Rep 1

Rep 2

Rep 3

Result (# positive replicates)

Rep 1

Rep 2

Rep 3

Result (# positive replicates)

Undiluted 101 100.5 End-point* 10-0.5 10-1 * As determined in the first round of testing (test day 1). Comments:

WHO/BS/2016.2299 Page 51

Sample #8, liquid frozen (DENV-3, BB) Dilutions 1 2 3 4 5 6 7 8 9 Undiluted 10-1 10-2 10-3 10-4 10-5 10-6 10-7 10-8 Test day 2 Replicate 1 Replicate 2

Test day 1 Replicate 3 Result (# positive replicates)

Sample #8, liquid frozen (DENV-3, BB) Dilutions 1 2 3 4 5 6 Rep 1

Test day 3

Test day 4

Rep 2

Rep 3

Result (# positive replicates)

Rep 1

Rep 2

Rep 3

Result (# positive replicates)

Rep 1

Rep 2

Rep 3

Result (# positive replicates)

Undiluted 101 100.5 End-point* 10-0.5 10-1 * As determined in the first round of testing (test day 1). Comments:

WHO/BS/2016.2299 Page 52

Sample #9, lyophilized (DENV-3, CC) Dilutions 1 2 3 4 5 6 7 8 9 Undiluted 10-1 10-2 10-3 10-4 10-5 10-6 10-7 10-8 Test day 2 Replicate 1 Replicate 2

Test day 1 Replicate 3 Result (# positive replicates)

Sample #9, lyophilized (DENV-3, CC) Dilutions 1 2 3 4 5 6 Rep 1

Test day 3

Test day 4

Rep 2

Rep 3

Result (# positive replicates)

Rep 1

Rep 2

Rep 3

Result (# positive replicates)

Rep 1

Rep 2

Rep 3

Result (# positive replicates)

Undiluted 101 100.5 End-point* 10-0.5 10-1 * As determined in the first round of testing (test day 1). Comments:

WHO/BS/2016.2299 Page 53

Sample #10, liquid frozen (DENV-4, AA) Dilutions 1 2 3 4 5 6 7 8 9 Undiluted 10-1 10-2 10-3 10-4 10-5 10-6 10-7 10-8 Test day 2 Replicate 1 Replicate 2

Test day 1 Replicate 3 Result (# positive replicates)

Sample #10, liquid frozen (DENV-4, AA) Dilutions 1 2 3 4 5 6 Rep 1

Test day 3

Test day 4

Rep 2

Rep 3

Result (# positive replicates)

Rep 1

Rep 2

Rep 3

Result (# positive replicates)

Rep 1

Rep 2

Rep 3

Result (# positive replicates)

Undiluted 101 100.5 End-point* 10-0.5 10-1 * As determined in the first round of testing (test day 1). Comments:

WHO/BS/2016.2299 Page 54

Sample #11, lyophilized (DENV-4, BB) Dilutions 1 2 3 4 5 6 7 8 9 Undiluted 10-1 10-2 10-3 10-4 10-5 10-6 10-7 10-8 Test day 2 Replicate 1 Replicate 2

Test day 1 Replicate 3 Result (# positive replicates)

Sample #11, lyophilized (DENV-4, BB) Dilutions 1 2 3 4 5 6 Rep 1

Test day 3

Test day 4

Rep 2

Rep 3

Result (# positive replicates)

Rep 1

Rep 2

Rep 3

Result (# positive replicates)

Rep 1

Rep 2

Rep 3

Result (# positive replicates)

Undiluted 101 100.5 End-point* 10-0.5 10-1 * As determined in the first round of testing (test day 1). Comments:

WHO/BS/2016.2299 Page 55

Sample #12, liquid frozen (DENV-4, CC) Dilutions 1 2 3 4 5 6 7 8 9 Undiluted 10-1 10-2 10-3 10-4 10-5 10-6 10-7 10-8 Test day 2 Replicate 1 Replicate 2

Test day 1 Replicate 3 Result (# positive replicates)

Sample #12, liquid frozen (DENV-4, CC) Dilutions 1 2 3 4 5 6 Rep 1

Test day 3

Test day 4

Rep 2

Rep 3

Result (# positive replicates)

Rep 1

Rep 2

Rep 3

Result (# positive replicates)

Rep 1

Rep 2

Rep 3

Result (# positive replicates)

Undiluted 101 100.5 End-point* 10-0.5 10-1 * As determined in the first round of testing (test day 1). Comments:

WHO/BS/2016.2299 Page 56 Quantitative assay (if available): (Please note that we have completed the first part of the table as an example. If Ct values are available please include them in this table, as shown below)

Assay Sample #1, liquid frozen (DENV-1, AA) Undiluted

Test day 1 Rep 1 3.5e6, Ct 16 Rep 2 4.5e6, Ct 15 Rep 3 4.0e6, Ct. 15.5 Average Result* 4e6 c/ml, Ct Av. 15.5 Rep 1 3.5e6, Ct 16

Test day 2 Rep 2 4.5e6, Ct 15 Rep 3 4.0e6, Ct. 15.5 Average Result* 4e6 c/ml, Ct Av. 15.5

Assay Sample #1, liquid frozen (DENV-1, AA) Undiluted

Test day 3 Rep 1 3.5e6, Ct 16 Rep 2 4.5e6, Ct 15 Rep 3 4.0e6, Ct. 15.5 Average Result* 4e6 c/ml, Ct Av. 15.5 Rep 1 3.5e6, Ct 16

Test day 4 Rep 2 4.5e6, Ct 15 Rep 3 4.0e6, Ct. 15.5 Average Result* 4e6 c/ml, Ct Av. 15.5

Assay Sample #2, lyophilized (DENV-1, BB) Undiluted

Test day 1 Rep 1 Rep 2 Rep 3 Average Result* Rep 1

Test day 2 Rep 2 Rep 3 Average Result*

Assay Sample #2, lyophilized (DENV-1, BB) Undiluted

Test day 3 Rep 1 Rep 2 Rep 3 Average Result* Rep 1

Test day 4 Rep 2 Rep 3 Average Result*

Assay Sample #3, liquid frozen (DENV-1, CC) Undiluted

Test day 1 Rep 1 Rep 2 Rep 3 Average Result* Rep 1

Test day 2 Rep 2 Rep 3 Average Result*

Assay Sample #3, liquid frozen (DENV-1, CC) Undiluted

Test day 3 Rep 1 Rep 2 Rep 3 Average Result* Rep 1

Test day 4 Rep 2 Rep 3 Average Result*

* If TaqMan qRT-PCR is used for the quantitative determination, please explain how the DENV RNA standard curve was generated and report both the raw data (Ct values), as well as the calculated values based on the standard curve (i.e. copies/ml, GEq/ml, PDU/ml, etc.).

Comments:

WHO/BS/2016.2299 Page 57

Assay Sample #4, lyophilized (DENV-2, AA) Undiluted

Test day 1 Rep 1 Rep 2 Rep 3 Average Result* Rep 1

Test day 2 Rep 2 Rep 3 Average Result*

Assay Sample #4, lyophilized (DENV-2, AA) Undiluted

Test day 3 Rep 1 Rep 2 Rep 3 Average Result* Rep 1

Test day 4 Rep 2 Rep 3 Average Result*

Assay Sample #5, liquid frozen (DENV-2, BB) Undiluted

Test day 1 Rep 1 Rep 2 Rep 3 Average Result* Rep 1

Test day 2 Rep 2 Rep 3 Average Result*

Assay Sample #5, liquid frozen (DENV-2, BB) Undiluted

Test day 3 Rep 1 Rep 2 Rep 3 Average Result* Rep 1

Test day 4 Rep 2 Rep 3 Average Result*

Assay Sample #6, liquid frozen (DENV-2, CC) Undiluted

Test day 1 Rep 1 Rep 2 Rep 3 Average Result* Rep 1

Test day 2 Rep 2 Rep 3 Average Result*

Assay Sample #6, liquid frozen (DENV-2, CC) Undiluted

Test day 3 Rep 1 Rep 2 Rep 3 Average Result* Rep 1

Test day 4 Rep 2 Rep 3 Average Result*

Comments:

WHO/BS/2016.2299 Page 58

Assay Sample #7, liquid frozen (DENV-3, AA) Undiluted

Test day 1 Rep 1 Rep 2 Rep 3 Average Result* Rep 1

Test day 2 Rep 2 Rep 3 Average Result*

Assay Sample #7, liquid frozen (DENV-3, AA) Undiluted

Test day 3 Rep 1 Rep 2 Rep 3 Average Result* Rep 1

Test day 4 Rep 2 Rep 3 Average Result*

Assay Sample #8, lyophilized (DENV-3, BB) Undiluted

Test day 1 Rep 1 Rep 2 Rep 3 Average Result* Rep 1

Test day 2 Rep 2 Rep 3 Average Result*

Assay Sample #8, lyophilized (DENV-3, BB) Undiluted

Test day 3 Rep 1 Rep 2 Rep 3 Average Result* Rep 1

Test day 4 Rep 2 Rep 3 Average Result*

Assay Sample #9, lyophilized (DENV-3, CC) Undiluted

Test day 1 Rep 1 Rep 2 Rep 3 Average Result* Rep 1

Test day 2 Rep 2 Rep 3 Average Result*

Assay Sample #9, lyophilized (DENV-3, CC) Undiluted

Test day 3 Rep 1 Rep 2 Rep 3 Average Result* Rep 1

Test day 4 Rep 2 Rep 3 Average Result*

Comments:

WHO/BS/2016.2299 Page 59

Assay Sample #10, liquid frozen (DENV-4, AA) Undiluted

Test day 1 Rep 1 Rep 2 Rep 3 Average Result* Rep 1

Test day 2 Rep 2 Rep 3 Average Result*

Assay Sample #10, liquid frozen (DENV-4, AA) Undiluted

Test day 3 Rep 1 Rep 2 Rep 3 Average Result* Rep 1

Test day 4 Rep 2 Rep 3 Average Result*

Assay Sample #11, lyophilized (DENV-4, BB) Undiluted Sample #11, lyophilized (DENV-4, BB) Undiluted

Test day 1 Rep 1 Rep 2 Rep 3 Average Result* Rep 1

Test day 2 Rep 2 Rep 3 Average Result*

Assay Test day 3 Rep 1 Rep 2 Rep 3 Average Result* Rep 1

Test day 4 Rep 2 Rep 3 Average Result*

Assay Sample #12, liquid frozen (DENV-4, CC) Undiluted

Test day 1 Rep 1 Rep 2 Rep 3 Average Result* Rep 1

Test day 2 Rep 2 Rep 3 Average Result*

Assay Sample #12, liquid frozen (DENV-4, CC) Undiluted

Test day 3 Rep 1 Rep 2 Rep 3 Average Result* Rep 1

Test day 4 Rep 2 Rep 3 Average Result*

Comments:

WHO/BS/2016.2299 Page 60

METHODS FORM 1) RNA extraction method employed. Method for concentration of viral particles from sample before RNA extraction (if applicable):____________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ Brief description of RNA extraction method: ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ Volume of sample extracted:______________________________________________________ Final volume of extracted RNA:____________________________________________________ Volume of extracted RNA tested:____________________________________________________

2) RNA amplification method – Qualitative assays Amplification protocol: _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ Please provide a brief description of other procedure including methodology and amplification cycling conditions: _______________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________

WHO/BS/2016.2299 Page 61

Brief description of protocol for detection of amplified DNA: ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________

3) RNA amplification method – Quantitative assays Brief description of quantitative assay (if applicable): ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ Brief description of method for calculating concentration of DENV: ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________

4) Quality control Internal control included in assay: ______________________________________________________________________________ Inclusion of weak positive samples in each run for monitoring sensitivity:___________________ ______________________________________________________________________________ Inclusion of negative samples in each run for monitoring specificity:_______________________ ______________________________________________________________________________ Inclusion of run control:__________________________________________________________

WHO/BS/2016.2299 Page 62

Estimated DENV RNA concentration of run control:___________________________________ Inclusion of method for prevention of PCR carry-over:__________________________________ _____________________________________________________________________________________

Key facts

Organisation World Health Organization (WHO)

Document type Technical Documents

Adoption date December 31, 2016

Source World Health Organization