WHO/BS/2016.2299 ENGLISH ONLY EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION Geneva, 17-21 October 2016

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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 Añez^1,#, Evgeniya Volkova¹, Rafaelle C.G. Fares¹, Zhen Jiang¹, Maria Rios^1,*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 2. 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

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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-derivedDENV-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 log10NAT-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. FDA¹. 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.

1 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

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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.

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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.1537- 2995.2008.01772.x

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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.1537-

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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.1537- 2995.2012.03566.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.

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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

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1 2 3 4 5 6 7 8

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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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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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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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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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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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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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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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Figure 9. Intra-laboratory variability in quantitative estimates for lyophilized candidate reference reagents.

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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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8

11 10 14b

15

1 2 3 4 5 6 7 8

9 10

2 2.5 3 3.5 4 4.5 5 5.5 6 6.5

1.5

13b 14a

7 16

19

(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.

(25)

Table 3. Stability and accelerated degradation studies. Titers are expressed as log10 NDU/mL.

DENV-1, lyophilized

Time Tested temperature

-20°C +4°C +20°C +37°C

1 week 5.3 n.d 5.1 5.1

1 month n.d 5.4 5.3 5.3

2 months n.d n.d 5.3 4.9

3 months 5.6 5.0 5.1 5.0

6 months 5.5 5.2 5.0 4.8

9 months 5.7 n.d 4.9 4.8

12 months 5.5 4.9 4.7 4.5

-20°C +4°C +20°C +37°C

1 week 6.1 n.d 5.8 5.9

1 month n.d 5.9 5.8 5.9

3 months 5.9 5.8 5.7 5.6

6 months 6.0 5.7 5.8 5.4

9 months 5.7 n.d 5.6 5.5

12 months 5.9 5.7 5.6 5.5

-20°C +4°C +20°C +37°C

1 week 5.4 n.d 5.1 5.4

1 month n.d 5.3 5.2 5.3

3 months 5.4 5.2 5.1 5.1

6 months 5.2 5.2 5.0 5.0

9 months 5.3 n.d 5.1 4.9

12 months 5.4 5.2 5.0 4.9

-20°C +4°C +20°C +37°C

1 week 5.1 n.d 5.1 5.4

1 month n.d 5.1 4.7 4.8

3 months 5.1 5.0 4.9 4.8

6 months 5.0 5.0 4.8 4.7

9 months 5.1 n.d 4.7 4.6

12 months 5.2 5.0 4.7 4.6

(26)

Table 4. Assays used by the participant laboratories Laboratory

code

Assay type (quantitative

or qualitative)

Extraction protocol

NAT type Assay

Target

Reference

1 qualitative QIAamp Viral RNA kit with QIACube (Qiagen)

Real-time RT-PCR (TaqMan)

3’ NCR Unpublished data

2 quantitative QIAamp DSP Virus kit (Qiagen)

LightMix kit DENV (Tib Molbiol)

3’ NCR

3 qualitative RNA extraction on automated instrument platform

Real-time RT-PCR *

4 qualitative Automated extraction on MagnaPure (Roche)

3’NCR (DENV-1 – 4)

Warrilow et al 2002

5 qualitative QIAamp Viral RNA Mini kit (Qiagen)

RT-PCR followed by multiplex nested PCR;

analysis by agarose gel electrophoresis

NS5 (DENV-1 – 4)

Bronzoni et al 2005

Conventional RT-PCR followed by

conventional PCR;

analysis by agarose gel electrophoresis

NS3 (DENV-1 – 4)

Seah et al 1995

7 qualitative NucliSENS – easyMAG automated system (BioMerieux)

3’NCR (DENV-1 – 4)

Kaiser et al (unpublishe

d data) Domingo et

al 2010 8 qualitative QIAamp Viral

RNA Mini kit (Qiagen)

Real-time RT-PCR DENV-1:

NS5;

DENV-2: E;

DENV-3 and 4: prM;

Johnson et al 2005

9 qualitative Magnetic-based target capture on automated system (Tigris)

Transcription-mediated amplification followed by hybridization protection assay

5’NCR and C

Unpublished data

DENV-1:

NS5;

DENV-2: E;

DENV-3:

M;

DENV-4: E

Johnson et al 2005

(27)

and M 11 quantitative QIAamp Viral

QuantiTect Probe RT- PCR (Qiagen)

8973-9084 1008-1605 740-813 904-992

Johnson et al 2005

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

C-prM

RT-nested PCR E/NS1

junction (DENV-1 – 4)

Domingo et al 2011;

Domingo et al 2006 13 quantitative QIAamp Viral

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

3’NCR Kaiser M, unpublished

DENV-1:

NS5;

DENV-2: E;

DENV-3 and 4: prM;

Johnson et al 2005

14 quantitative QIAamp Viral RNA Mini kit (Qiagen)

Real-time RT-PCR (TaqMan) using standards

DENV-1:

NS5;

DENV-2: E;

DENV-3 and 4: prM;

Johnson et al 2005

E Santiago et

al 2013 16 qualitative Qiagen columns

(Qiagen)

Various (DENV-1 – 4)

Santiago et al 2013 17 qualitative QIAamp Viral

Fourplex Real-time Reverse Transcriptase Real-time PCR Assay

DENV-1:

NS5;

DENV-2: E;

DENV-3:

M;

DENV-4: E and M

Johnson et al 2005

5’NCR or 3’NCR

Leparc- Goffart et al 2009 19 qualitative QIAamp Viral

3’NCR (DENV-1 – 4)

Gurukumar et al 2009;

Chien et al 2006 20 qualitative QIAamp Viral

Various (DENV-1 – 4)

Santiago et al 2013

(28)

21 qualitative High Pure Viral Nucleic Acid kit (Life

Science/Roche)

NS5 (DENV-1 – 4)

Chien et al 2006

*Information was not provided

Table 5. Mean estimates of DENV titers based on quantitative assays results (log10 copies/mL).

Lab Code

Sample

#1, DENV-1 AA (FRO)

Sample

#2, DENV-1 BB (LYO)

Sampl e #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)

2 2.84 3.38 3.67 3.25 3.34 3.32 4.21 3.85

11 7.05 6.69 6.51 6.64 6.79 6.66 6.21 6.65

13 5.12 4.98 6.16 5.21 5.41 5.17 6.13 6.33

14 5.74 5.45 5.6 5.82 5.69 5.47 5.14 4.94

FRO = liquid frozen candidates; LYO = lyophilized candidates

Table 6. Variation of estimates for lyophilized materials within laboratories where quantitative assays were performed, in copies/ml.

SD – standard deviation; n – number of replicates.

Lab Code

DENV-1 DENV-2 DENV-3 DENV-4

Mean SD n Mean SD n Mean SD n Mean SD n

2 2.77x10³ 1.97x10³ 4 5.07x10³ 2.48x10³ 4 2.14x10³ 6.25x10² 4 1.87x10⁴ 9.20x10³ 4 11 5.83x10⁶ 3.60x10⁶ 4 3.30x10⁶ 5.89x10⁵ 4 4.63x10⁶ 1.02x10⁶ 4 1.70x10⁶ 4.76x10⁵ 4 13 9.51x10⁴ 5.63x10³ 4 1.46x10⁶ 1.65x10⁵ 4 1.51x10⁵ 2.80x10⁴ 4 1.35x10⁶ 1.20x10⁵ 4 14 2.79x10⁵ 1.85x10⁴ 3 4.02x10⁵ 1.11x10⁴ 3 2.94x10⁵ 3.79x10³ 3 1.39x10⁵ 1.89x10⁴ 3