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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: [email protected]
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: [email protected] .
© World Health Organization 2016
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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.
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The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or recommended
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Summary
An international collaborative study was conducted to assess the suitability of a candidate reference
panel for Dengue virus (DENV) types 1 to 4 RNA for use in nucleic acid amplification technology
(NAT)-based assays. Two reference panel candidates were prepared for each DENV type, one liquid
frozen (Panel 1) and one lyophilized (Panel 2). The reference panel candidates consisted of a DENV
prototype laboratory strain that was grown in cell culture in mosquito cells, subjected to inactivation
of infectivity by heat treatment and diluted in human plasma. Both reference panels 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 Panel 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 DENV4 (H241 GenBank# KR011349 )
strains be established as the 1st International Reference Panel for DENV RNA with a unitage of
13,500, 69,200, 23,400, and 33,900 International Units per ml for DENV-1 to 4, respectively. The
real-time and accelerated stability studies of the proposed International Reference Panel revealed that
the material is stable at the recommended storage temperature, i.e. at or below 4°C, for 12 months,
and is 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.
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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 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 panel candidates evaluated in this study. The aim
of this study is to establish the WHO 1st International Reference Panel for Dengue Virus types 1 to 4
RNA for NAT-based assays and demonstrate its suitability, assess its potency and finally, assign an
internationally agreed-upon unitage.
Preparation of Bulk Materials
The DENV WHO candidate reference panel was 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 at 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 panel, 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 panel, 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 candidate reference panel, 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 standards was assessed for all 4
panel members 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
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residual moisture was found to be 1.1% w/w. To determine if the lyophilization process had affected
the RNA titer of the DENV candidate reference panel, 10 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 WHO International Reference Panel is 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.
Reference panels 1 and 2 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 panel candidates
identifying the DENV type, e.g. vials were coded as “DENV-1 vial AA”, “DENV-1 vial BB”,
“DENV-1 vial CC”, etc. Thus, each testing laboratory received 72 vials, 24 vials per panel 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 panel 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 candidate reference panel member and negative controls
for each run. For laboratories that had a quantitative DENV NAT, we asked to test each DENV
reference panel candidate 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 panel 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.
Statistical Methods
Quantitative Assays
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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 panel candidate was estimated by pooling all measurements
collected in all labs.
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 panel members (DENV-1 AA, DENV-2 CC, DENV-3
BB, DENV-4 CC), were estimated relative to the corresponding lyophilized candidate reference
panel member (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 candidate DENV WHO reference panel was assessed through both
real-time and accelerated thermal degradation studies. Vials containing the lyophilized candidate
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WHO reference panel 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 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 panel candidate) and 18 (for the lyophilized reference panel candidate) qualitative and 4
quantitative datasets (for both frozen liquid and lyophilized reference panel 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 panel candidates. These laboratories only tested the undiluted candidates without
performing any further dilution. The mean estimates for each reference panel 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 lab 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 candidate reference panel member are shown in
Table 8. The mean estimates were calculated based on all measurements collected in the four labs
that performed quantitative assays. The overall means for the qualitative assays are shown in Table 9.
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The results obtained from the lyophilized candidate reference panel members only differ by 0.03 –
0.34 log10 from those observed for the liquid frozen reference panel candidate, which demonstrates
that the process of freeze-drying did not significantly affect the integrity of DENV RNA for
reference panel 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 labs that performed quantitative assays
resulting in wider dispersion of the results.
Relative Potencies
Based upon the mean estimates of titer values from quantitative and qualitative protocols, the WHO
lyophilized candidate reference panel members 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 candidate reference panel member compared to the values obtained directly from
quantitative and qualitative analysis.
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Results of Stability Studies
Vials of the lyophilized DENV reference panel candidate 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 candidate reference panel members 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 reference panel 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 panels, since the agreement of results markedly improved
when data was expressed relative to the lyophilized candidate reference panel members (in case of
DENV-1, there was a 4 log10 improvement) . This observation suggests that using the proposed
reference panel as a standard 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
candidate reference panel members 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 candidate reference panel consisting of DENV-1 BB, DENV-2
AA, DENV-3 CC and DENV-4 BB, should be established as the 1st International Reference Panel for
DENV-1, DENV-2, DENV-3 and DENV-4, with a unitage of 13,500, 69,200, 23,400, and 33,900
International Units/mL, respectively, based on the results from the international collaborative study.
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A total of 1,500 vials per standard 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.
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 a WHO International Reference Panel 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.
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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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.1537-
2995.2008.01772.x
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.1537-
2995.2008.01771.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.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.
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.
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Figure 1. Mean estimates for liquid reference panel member 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 panel member DENV-1 BB. White squares represent
number of copies (quantitative assays) and gray squares represent NDU (qualitative assays).
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log10 copies or NDU/mL
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Figure 3. Mean estimates for liquid reference panel member DENV-2 CC. 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 panel member DENV-2 AA. White squares represent
number of copies (quantitative assays) and gray squares represent NDU (qualitative assays).
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7
8
log10 copies or NDU/mL
Nu
mb
er o
f la
bora
tori
es
9
10
2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 2
14b
16 8
Figure 5. Mean estimates for liquid reference panel member DENV-3 BB. White squares represent
number of copies (quantitative assays) and gray squares represent NDU (qualitative assays).
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1 2 3
5 6
7
16 9
11 8 10 14b
12
13a
18
19
1
2
3
4
5
6
7
8
log10 copies or NDU/mL
Nu
mb
er o
f la
bora
tori
es
9
10
2.5 3 3.5 4 4.5 5 5.5 6 6.5 2
13b
14a
20 4
15
17
21
Figure 6. Mean estimates for lyophilized reference panel member DENV-3 CC. White squares represent
number of copies (quantitative assays) and gray squares represent NDU (qualitative assays).
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1
2
3 4
5
6
7
8
9
10
11
14a
14b
15
19
1
2
3
4
5
6
7
8
log10 copies or NDU/mL
Nu
mb
er o
f la
bora
tori
es
9
10
2.5 3 3.5 4 4.5 5 5.5 6 6.5 2
13b
16
8
Figure 7. Mean estimates for liquid reference panel member DENV-4 CC. White squares represent
number of copies (quantitative assays) and gray squares represent NDU (qualitative assays).
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1
2
3 4
5
6
7 18
9
8
11
10
14a 14b
15
17 12
13a 1
2
3
4
5
6
7
8
log10 copies or NDU/mL
Nu
mb
er o
f la
bora
tori
es
9
10
2.5 3 3.5 4 4.5 5 5.5 6 6.5 2
13b 19
20
16
21
Figure 8. Mean estimates for lyophilized reference panel member 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 panel
members.
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1 2 3
4
6
19
9
8 11
10
14b
15
1
2
3
4
5
6
7
8
log10 copies or NDU/mL
Nu
mb
er o
f la
bora
tori
es
9
10
2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 1.5
13b
14a
7
16
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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1 2
3 4
6
19
9
8
11 10
14b
15
1
2
3
4
5
6
7
8
log10 copies or NDU/mL
Nu
mb
er o
f la
bora
tori
es
9
10
2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 1.5
13b
14a
7 16
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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Page 22
1
2
3
4
6
5
9
8
11
10
14b
15
1
2
3
4
5
6
7
8
log10 copies or NDU/mL
Nu
mb
er o
f la
bora
tori
es
9
10
2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 1.5
13b
14a
7
16
19
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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1 2
3
4
6
5
9
8
11
10
14b
15
1
2
3
4
5
6
7
8
log10 copies or NDU/mL
Nu
mb
er o
f la
bora
tori
es
9
10
2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 1.5
13b
14a
7
16
19
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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Table 1. DENV strains used to produce the reference panel candidates.
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 panel candidates sent for testing in the International Collaborative Study.
Panel 1 Sample code Format
DENV-1, AA Liquid frozen
DENV-2, CC Liquid frozen
DENV-3, BB Liquid frozen
DENV-4, CC Liquid frozen
Panel 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 Panel.
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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
DENV-2, lyophilized
Time Tested temperature
-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
2 months n.d n.d 5.8 5.8
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
DENV-3, lyophilized
Time Tested temperature
-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
2 months n.d n.d 5.4 5.4
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
DENV-4, lyophilized
Time Tested temperature
-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
2 months n.d n.d 4.8 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
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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)
Real-time RT-PCR
(TaqMan)
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
6 qualitative QIAamp Viral
RNA Mini kit
(Qiagen)
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)
Real-time RT-PCR
(TaqMan)
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
10 qualitative QIAamp Viral
RNA Mini kit
(Qiagen)
Real-time RT-PCR
(TaqMan)
DENV-1:
NS5;
DENV-2: E;
DENV-3:
M;
DENV-4: E
Johnson et
al 2005
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and M
11 quantitative QIAamp Viral
RNA Mini kit
(Qiagen)
QuantiTect Probe RT-
PCR (Qiagen)
8973-9084
1008-1605
740-813
904-992
Johnson et
al 2005
12 qualitative QIAamp Viral
RNA Mini kit
(Qiagen)
Ready-to-Go RT-PCR
Beads (GE Healthcare)
C-prM
13 qualitative QIAamp Viral
RNA Mini kit
(Qiagen)
RT-nested PCR E/NS1
junction
(DENV-1 –
4)
Domingo et
al 2011;
Domingo et
al 2006
13 quantitative QIAamp Viral
RNA Mini kit
(Qiagen)
Real-time RT-PCR
(TaqMan) using in vitro
transcribed RNA as
standard curve
3’NCR Kaiser M,
unpublished
14 qualitative QIAamp Viral
RNA Mini kit
(Qiagen)
Real-time RT-PCR
(TaqMan)
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
15 qualitative QIAamp Viral
RNA Mini kit
(Qiagen)
Real-time RT-PCR
(TaqMan)
E Santiago et
al 2013
16 qualitative Qiagen columns
(Qiagen)
Real-time RT-PCR
(TaqMan)
Various
(DENV-1 –
4)
Santiago et
al 2013
17 qualitative QIAamp Viral
RNA Mini kit
(Qiagen)
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
18 qualitative QIAamp Viral
RNA Mini kit
(Qiagen)
Real-time RT-PCR
(TaqMan)
5’NCR or
3’NCR
Leparc-
Goffart et al
2009
19 qualitative QIAamp Viral
RNA Mini kit
(Qiagen)
Real-time RT-PCR
(TaqMan)
3’NCR
(DENV-1 –
4)
Gurukumar
et al 2009;
Chien et al
2006
20 qualitative QIAamp Viral Real-time RT-PCR Various Santiago et
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RNA Mini kit
(Qiagen)
(TaqMan) (DENV-1 –
4)
al 2013
21 qualitative High Pure Viral
Nucleic Acid kit
(Life
Science/Roche)
Real-time RT-PCR
(TaqMan)
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 1.97x10
3 4 5.07x10
3 2.48x10
3 4 2.14x10
3 6.25x10
2 4 1.87x10
4 9.20x10
3 4
11 5.83x106 3.60x10
6 4 3.30x10
6 5.89x10
5 4 4.63x10
6 1.02x10
6 4 1.70x10
6 4.76x10
5 4
13 9.51x104 5.63x10
3 4 1.46x10
6 1.65x10
5 4 1.51x10
5 2.80x10
4 4 1.35x10
6 1.20x10
5 4
14 2.79x105 1.85x10
4 3 4.02x10
5 1.11x10
4 3 2.94x10
5 3.79x10
3 3 1.39x10
5 1.89x10
4 3
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Table 7. Mean values of DENV titers based on qualitative assays results (log10 NDU/mL) estimated by
Poisson/Maximum likelihood method.
Lab
Code
Sample
#1,
DENV-1
AA
(FRO)
Sample
#2,
DENV-1
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)
1 4.16 3.03 4.1 4.11 3.03 2.79 4.03 4.11
3 4.43 4.82 5.5 5.22 5.12 4.78 4.91 4.98
4 3.62 3.67 4.54 4.55 4.06 3.46 4.31 4.39
5 3.9 NA* 3.9 NA* 3.96 2.88 3.9 3.9
6 4.57 4.57 5.76 5.27 5.07 4.86 3.99 2.78
7 0.96 1.7 3.82 2.86 4.67 4.85 4.18 3.85
8 3.74 4.27 4.73 4.77 4.39 4.05 4.74 4.93
9 4.25 3.92 5.64 4.88 5.13 4.88 5.31 4.77
10 4.94 4.71 5.27 5.1 5.16 4.71 4.45 4.59
12 NA** 3.85 6.27 NA** NA** 3.75 4.45 NA**
13 NA** 4.26 5.77 NA** NA** 4.83 3.63 NA**
14 4.71 4.57 5.35 5.57 5.25 4.91 5.26 5.19
15 4.03 4.03 5.05 4.86 4.61 3.98 4.94 5.05
16 3.31 3.83 4.78 3.67 3.67 4.07 4.04 3.67
17 NA** 5.03 5.87 NA** NA** 4.87 5.22 NA**
18 NA** 4.36 5.13 NA** NA** 3.47 4.41 NA**
19 4.12 3.65 3.48 3.91 4.13 3.53 3.44 3.13
20 NA** 5.28 5.8 NA** NA** 4.7 5.41 NA**
21 NA** 2.21 2.37 NA** NA** 3.36 3.42 NA**
*: Model does not converge
**: Sample not tested
FRO = liquid frozen candidates; LYO = lyophilized candidates; NA = Not applicable
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Table 8. Overall mean estimates for quantitative assays (log10 copies/mL).
Sample
#1,
DENV-1
AA
(FRO)
Sample
#2,
DENV-
1 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)
n 14 15 15 15 15 15 15 15
Mean 5.02 5.10 5.48 5.19 5.27 5.13 5.44 5.41
SD 1.61 1.28 1.18 1.41 1.39 1.28 0.88 1.23
Lowerci 4.08 4.39 4.83 4.38 4.47 4.42 4.95 4.71
Upperci 5.95 5.81 6.13 6.00 6.08 5.84 5.93 6.12
cv geo 2009 648 485 970 929 651 225 557
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
#1,
DENV-1
AA
(FRO)
Sample
#2,
DENV-1
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)
n 13 18 19 12 13 19 19 13
Mean 3.90 3.99 4.90 4.56 4.48 4.14 4.42 4.26
SD 0.99 0.92 1.00 0.79 0.68 0.73 0.63 0.76
Lowerci 3.35 3.55 4.45 4.11 4.10 3.81 4.14 3.83
Upperci 4.45 4.42 5.35 5.02 4.86 4.48 4.71 4.68
cv geo 294 248 297 177 139 157 122 168
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
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Table 10. Potencies of liquid reference panel candidate relative to lyophilized reference panel candidate
based on data from quantitative assays.
Lab
Code
DENV-1, FRO
Relative to LYO
DENV-2, FRO
Relative to LYO
DENV-3, FRO
Relative to LYO
DENV-4, FRO
Relative to LYO
Relative
Potency
(log10)
95%
Confidence
Interval
Relative
Potency
(log10)
95%
Confidence
Interval
Relative
Potency
(log10)
95%
Confidence
Interval
Relative
Potency
(log10)
95%
Confidence
Interval
2 3.60 (3.23, 3.96) 4.42 (4.06,4.78) 4.39 (4.06, 4.72) 4.17 (3.58, 4.77)
11 4.49 (3.53, 5.45) 4.97 (4.80, 5.14) 4.50 (4.19, 4.81) 4.96 (4.72, 5.20)
13 4.27 (4.21, 4.33) 3.89 (3.08, 3.69) 4.61 (4.28, 4.94) 4.73 (4.52,4.93)
14 4.42 (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 panel candidates relative to lyophilized reference panel candidates
based on data from qualitative assays.
Lab
Code
DENV-1, FRO
Relative to LYO
DENV-2, FRO
Relative to LYO
DENV-3, FRO
Relative to LYO
DENV-4, FRO
Relative to LYO
Relative
Potency
(log10)
95%
Confidence
Interval
Relative
Potency
(log10)
95%
Confidence
Interval
Relative
Potency
(log10)
95%
Confidence
Interval
Relative
Potency
(log10)
95%
Confidence
Interval
1 5.26 (4.89,5.63) 4.84 (4.46,5.23) 4.61 (4.25,4.97) 4.61 (4.23,5.00)
3 3.75 (3.36,4.12) 4.56 (4.20,4.94) 4.71 (4.36, 5.06) 4.60 (4.14,5.15)
4 4.08 (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** NA** 5.45 (5.04,5.88) 4.53 (4.14,4.92)
6 4.13 (3.76,4.50) 4.35 (3.99,4.71) 4.57 (4.21,4.92) 3.33 (2.98,3.67)
7 3.39 (2.93,3.80) 3.88 (3.53,4.23) 4.19 (3.87,4.51) 4.19 (3.80,4.58)
8 3.61 (3.24,3.97) 4.88 (4.49,5.26) 4.71 (4.33,5.08) 4.72 (4.36,5.07)
9 4.46 (4.11,4.81) 4.08 (3.73,4.43) 4.62 (4.27,4.97) 3.99 (3.61,4.38)
10 4.36 (3.98,4.75) 4.68 (4.30,5.05) 4.82 (4.46,5.18) 4.67 (4.30,5.03)
14 4.28 (3.92,4.64) 5.07 (4.70,5.44) 4.71 (4.35,5.07) 4.45 (4.11,4.79)
15 4.13 (3.75,4.51) 4.64 (4.27,5.01) 5.00 (4.64,5.36) 4.64 (4.25,5.03)
19 4.60 (4.29,4.92) 5.27 (4.95,5.59) 4.97 (4.65,5.29) 4.22 (3.78,4.64)
16 3.61 (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
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Table 12. Potencies of liquid reference panel candidates relative to lyophilized reference panel candidates
based on combined data from quantitative and qualitative assays
DENV-1, FRO
Relative to LYO
DENV-2, FRO
Relative to LYO
DENV-3, FRO
Relative to LYO
DENV-4, FRO
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
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Appendix 1. List of participants in the DENV WHO IRP study, in alphabetical order (by Principal
Investigator).
Participants Affiliation
Wenjun Liu/John A. Aaskov 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 Segubre-
Mercado/Mary Ann U. Igoy
Research Institute for Tropical Medicine
Muntinlupa City, Philippines
Alzira Batista Cecílio/Ana Caroline Zampiroli Ataíde
Ezequiel Dias Foundation
Belo Horizonte, Brazil
Cristina Domingo Robert Koch Institute
Centre for Biological Threats and Special
Pathogens
Berlin, Germany
Michael Drebot /Kimberly Holloway 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
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Duane J Gubler/ October M Sessions/Eng Eong Ooi Program on Emerging Infectious Diseases
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 (Hessen), 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
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Vanessa Suin/Aurélie Francart Re-emerging Viruses Program
Viral Diseases | Communicable and Infectious
Diseases
Scientific Institute of Public Health
Brussels, Belgium
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Appendix 2. List of participants in the characterization of DENV stocks used to prepare the candidate
reference panels.
Participants Affiliation
Ana P. Goncalvez/Ronald E. Engle/Robert H. Purcell National Institutes of Health
Bethesda, United States
Laura D. Kramer/Susan Jones/Alan P. Dupuis New York State Department of Health
Albany, United States
Robert Lanciotti Centers for Disease Control and Prevention
Fort Collins, United States
Maria Rios/Germán Añez U.S. Food and Drug Administration
Silver Spring, United States
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Appendix 3. Draft Instructions for Use for the Dengue Virus types 1 to 4 RNA Reference Material for
NAT-based assays
U.S. Food and Drug Administration A WHO Collaborating Center Center for Biologics Evaluation and Research for Biological Standardization
WHO International Reference Panel 1st International Reference Panel 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
Instructions for use
(Version 1.0, July 2016)
1. INTENDED USE
The 1st International Reference Panel for Dengue virus
(DENV) types 1 to 4 RNA is intended to be used to in the
standardization of nucleic acid amplification technique
(NAT)-based assays for DENV. The reference panel 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 delipidated,
defibrinated human plasma. The panel members 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 assay 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
This standard has been assigned a unitage of 13,500
(DENV-1), 69,200 (DENV-2), 23,400 (DENV-3) and 33,900
(DENV-4) International 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
Store at +4°C.
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.
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/XX.XXXX
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10. ACKNOWLEDGEMENTS
We would like to thank Dr. Robert Lanciotti from the CDC for
providing viral strains used in preparation of the reagents.
11. FURTHER INFORMATION
Further information can be obtained as follows;
This material: [email protected]
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 [email protected]
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: Lyophilized powder
Corrosive: No
Stable: Yes
Oxidizing: No
Hygroscopic: No
Irritant: No
Flammable: No
Handling: See caution, Section 2
Other (specify): Contains material of human
origin (plasma) and heat-
inactivated, non-infectious
DENV grown in mosquito
C6/36 cells
Toxicological properties
Effects of inhalation: Not established, avoid
inhalation
Effects of ingestion: Not established, avoid
ingestion
Effects of skin
absorption:
Not established, avoid
contact with skin
Suggested First Aid
Inhalation: Seek medical advice
Ingestion: Seek medical advice
Contact with eyes: Wash with copious amounts
of water. Seek medical
advice
Contact with skin:
Wash thoroughly with water.
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.
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.
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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
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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: [email protected] (MR), [email protected] (GA)
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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 Vials sent
Sample # / Candidate name Format
1) DENV-1, AA Liquid frozen 6
2) DENV-1, BB Lyophilized 6
3) DENV-1, CC Liquid frozen 6
4) DENV-2, AA Lyophilized 6
5) DENV-2, BB Liquid frozen 6
6) DENV-2, CC Liquid frozen 6
7) DENV-3, AA Liquid frozen 6
8) DENV-3, BB Liquid frozen 6
9) DENV-3, CC Lyophilized 6
10) DENV-4, AA Liquid frozen 6
11) DENV-4, BB Lyophilized 6
12) DENV-4, CC Liquid frozen 6
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
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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 # 1 2 3 4 5 6 7 8 9
Description Undiluted
reagent 10
-1 10
-2 10
-3 10
-4 10
-5 10
-6 10
-7 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
Sample # X 1 2 3 4 5 6 7 8 9
Dilution Undiluted
reagent 10
-1 10
-2 10
-3 10
-4 10
-5 10
-6 10
-7 10
-8
# positive
replicates 3/3 3/3 3/3 3/3 3/3 3/3
2/3 0/3 0/3
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
Sample # 1 2 3 4 5 6
Description Undiluted
reagent 10
-5 10
-5.5
10-6
10
-6.5 10
-7
End-point
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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.
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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)
* As determined in the first round of testing (test day 1).
Comments:
Sample #1, liquid frozen
(DENV-1, AA)
Test day 1
Dilutions Replicate 1 Replicate 2 Replicate 3 Result
(# positive replicates)
1 Undiluted +, Ct 15 +, Ct 15 +, Ct 15 3/3, Ct Av. 15
2 10-1
+, Ct 17 +, Ct 17 +, Ct 17 3/3, Ct Av. 17
3 10-2
+, Ct 19 +, Ct 19 +, Ct 19 3/3, Ct Av. 19
4 10-3
+, Ct 21 +, Ct 21 +, Ct 21 3/3, Ct Av. 21
5 10-4
+, Ct 23 +, Ct 24 +, Ct 23 3/3, Ct Av. 23.3
6 10-5
+, Ct 25 +, Ct 25 +, Ct 26 3/3, Ct Av. 25.3
7 10-6
+, Ct 28 +, Ct 29 – 2/3, Ct Av. 28.5
8 10-7
+, Ct 32 – – 1/3, Ct Av. 32
9 10-8
– – – 0/3
Sample #1, liquid
frozen (DENV-1, AA)
Test day 2 Test day 3 Test day 4
Dilutions Rep 1 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)
1 Undiluted +, Ct 15 +, Ct 15 +, Ct 15 3/3, Ct
Av. 15
+, Ct 15 +, Ct 15 +, Ct 15 3/3, Ct
Av. 15
+, Ct 15 +, Ct 15 +, Ct 15 3/3, Ct
Av. 15
2 101 +, Ct 23 +, Ct 24 +, Ct 23 3/3, Ct
Av. 23.3
+, Ct 23 +, Ct 24 +, Ct 23 3/3, Ct
Av. 23.3
+, Ct 23 +, Ct 24 +, Ct 23 3/3, Ct
Av. 23.3
3 100.5
+, Ct 25 +, Ct 25 +, Ct 26 3/3, Ct
Av. 25.3
+, Ct 25 +, Ct 25 +, Ct 26 3/3, Ct
Av. 25.3
+, Ct 25 +, Ct 25 +, Ct 26 3/3, Ct
Av. 25.3
4 End-point* +, Ct 28 +, Ct 29 – 2/3, Ct
Av. 28.5
+, Ct 28 +, Ct 29 – 2/3, Ct
Av. 28.5
+, Ct 28 +, Ct 29 – 2/3, Ct
Av. 28.5
5 10-0.5
+, Ct 32 – – 1/3, Ct
Av. 32
+, Ct 32 – – 1/3, Ct
Av. 32
+, Ct 32 – – 1/3, Ct
Av. 32
6 10-1
– – – 0/3 – – – 0/3 – – – 0/3
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* As determined in the first round of testing (test day 1).
Comments:
Sample #2, lyophilized
(DENV-1, BB)
Test day 1
Dilutions Replicate 1 Replicate 2 Replicate 3 Result
(# positive replicates)
1 Undiluted
2 10-1
3 10-2
4 10-3
5 10-4
6 10-5
7 10-6
8 10-7
9 10-8
Sample #2,
lyophilized
(DENV-1, BB)
Test day 2 Test day 3 Test day 4
Dilutions Rep 1 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)
1 Undiluted
2 101
3 100.5
4 End-point*
5 10-0.5
6 10-1
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* As determined in the first round of testing (test day 1).
Comments:
Sample #3, liquid frozen
(DENV-1, CC)
Test day 1
Dilutions Replicate 1 Replicate 2 Replicate 3 Result
(# positive replicates)
1 Undiluted
2 10-1
3 10-2
4 10-3
5 10-4
6 10-5
7 10-6
8 10-7
9 10-8
Sample #3, liquid
frozen (DENV-1, CC)
Test day 2 Test day 3 Test day 4
Dilutions Rep 1 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)
1 Undiluted
2 101
3 100.5
4 End-point*
5 10-0.5
6 10-1
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* As determined in the first round of testing (test day 1).
Comments:
Sample #4, lyophilized
(DENV-2, AA)
Test day 1
Dilutions Replicate 1 Replicate 2 Replicate 3 Result
(# positive replicates)
1 Undiluted
2 10-1
3 10-2
4 10-3
5 10-4
6 10-5
7 10-6
8 10-7
9 10-8
Sample #4,
lyophilized
(DENV-2, AA)
Test day 2 Test day 3 Test day 4
Dilutions Rep 1 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)
1 Undiluted
2 101
3 100.5
4 End-point*
5 10-0.5
6 10-1
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* As determined in the first round of testing (test day 1).
Comments:
Sample #5, liquid frozen
(DENV-2, BB)
Test day 1
Dilutions Replicate 1 Replicate 2 Replicate 3 Result
(# positive replicates)
1 Undiluted
2 10-1
3 10-2
4 10-3
5 10-4
6 10-5
7 10-6
8 10-7
9 10-8
Sample #5, liquid
frozen (DENV-2, BB)
Test day 2 Test day 3 Test day 4
Dilutions Rep 1 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)
1 Undiluted
2 101
3 100.5
4 End-point*
5 10-0.5
6 10-1
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* As determined in the first round of testing (test day 1).
Comments:
Sample #6, liquid frozen
(DENV-2, CC)
Test day 1
Dilutions Replicate 1 Replicate 2 Replicate 3 Result
(# positive replicates)
1 Undiluted
2 10-1
3 10-2
4 10-3
5 10-4
6 10-5
7 10-6
8 10-7
9 10-8
Sample #6, liquid
frozen (DENV-2, CC)
Test day 2 Test day 3 Test day 4
Dilutions Rep 1 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)
1 Undiluted
2 101
3 100.5
4 End-point*
5 10-0.5
6 10-1
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* As determined in the first round of testing (test day 1).
Comments:
Sample #7, liquid frozen
(DENV-3, AA)
Test day 1
Dilutions Replicate 1 Replicate 2 Replicate 3 Result
(# positive replicates)
1 Undiluted
2 10-1
3 10-2
4 10-3
5 10-4
6 10-5
7 10-6
8 10-7
9 10-8
Sample #7, liquid
frozen (DENV-3, AA)
Test day 2 Test day 3 Test day 4
Dilutions Rep 1 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)
1 Undiluted
2 101
3 100.5
4 End-point*
5 10-0.5
6 10-1
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* As determined in the first round of testing (test day 1).
Comments:
Sample #8, liquid frozen
(DENV-3, BB)
Test day 1
Dilutions Replicate 1 Replicate 2 Replicate 3 Result
(# positive replicates)
1 Undiluted
2 10-1
3 10-2
4 10-3
5 10-4
6 10-5
7 10-6
8 10-7
9 10-8
Sample #8, liquid
frozen (DENV-3, BB)
Test day 2 Test day 3 Test day 4
Dilutions Rep 1 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)
1 Undiluted
2 101
3 100.5
4 End-point*
5 10-0.5
6 10-1
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* As determined in the first round of testing (test day 1).
Comments:
Sample #9, lyophilized
(DENV-3, CC)
Test day 1
Dilutions Replicate 1 Replicate 2 Replicate 3 Result
(# positive replicates)
1 Undiluted
2 10-1
3 10-2
4 10-3
5 10-4
6 10-5
7 10-6
8 10-7
9 10-8
Sample #9,
lyophilized
(DENV-3, CC)
Test day 2 Test day 3 Test day 4
Dilutions Rep 1 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)
1 Undiluted
2 101
3 100.5
4 End-point*
5 10-0.5
6 10-1
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* As determined in the first round of testing (test day 1).
Comments:
Sample #10, liquid frozen
(DENV-4, AA)
Test day 1
Dilutions Replicate 1 Replicate 2 Replicate 3 Result
(# positive replicates)
1 Undiluted
2 10-1
3 10-2
4 10-3
5 10-4
6 10-5
7 10-6
8 10-7
9 10-8
Sample #10, liquid
frozen (DENV-4, AA)
Test day 2 Test day 3 Test day 4
Dilutions Rep 1 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)
1 Undiluted
2 101
3 100.5
4 End-point*
5 10-0.5
6 10-1
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* As determined in the first round of testing (test day 1).
Comments:
Sample #11, lyophilized
(DENV-4, BB)
Test day 1
Dilutions Replicate 1 Replicate 2 Replicate 3 Result
(# positive replicates)
1 Undiluted
2 10-1
3 10-2
4 10-3
5 10-4
6 10-5
7 10-6
8 10-7
9 10-8
Sample #11,
lyophilized
(DENV-4, BB)
Test day 2 Test day 3 Test day 4
Dilutions Rep 1 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)
1 Undiluted
2 101
3 100.5
4 End-point*
5 10-0.5
6 10-1
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* As determined in the first round of testing (test day 1).
Comments:
Sample #12, liquid frozen
(DENV-4, CC)
Test day 1
Dilutions Replicate 1 Replicate 2 Replicate 3 Result
(# positive replicates)
1 Undiluted
2 10-1
3 10-2
4 10-3
5 10-4
6 10-5
7 10-6
8 10-7
9 10-8
Sample #12, liquid
frozen (DENV-4, CC)
Test day 2 Test day 3 Test day 4
Dilutions Rep 1 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)
1 Undiluted
2 101
3 100.5
4 End-point*
5 10-0.5
6 10-1
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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)
* 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:
Assay
Sample #1, liquid
frozen (DENV-1, AA)
Test day 1 Test day 2 Rep 1 Rep 2 Rep 3 Average
Result*
Rep 1 Rep 2 Rep 3 Average
Result*
Undiluted 3.5e6,
Ct 16
4.5e6,
Ct 15
4.0e6,
Ct. 15.5 4e6 c/ml,
Ct Av. 15.5
3.5e6,
Ct 16
4.5e6,
Ct 15
4.0e6,
Ct. 15.5 4e6 c/ml,
Ct Av. 15.5
Assay
Sample #1, liquid
frozen (DENV-1, AA)
Test day 3 Test day 4 Rep 1 Rep 2 Rep 3 Average
Result*
Rep 1 Rep 2 Rep 3 Average
Result*
Undiluted 3.5e6,
Ct 16
4.5e6,
Ct 15
4.0e6,
Ct. 15.5 4e6 c/ml,
Ct Av. 15.5
3.5e6,
Ct 16
4.5e6,
Ct 15
4.0e6,
Ct. 15.5 4e6 c/ml,
Ct Av. 15.5
Assay
Sample #2, lyophilized
(DENV-1, BB)
Test day 1 Test day 2 Rep 1 Rep 2 Rep 3 Average
Result*
Rep 1 Rep 2 Rep 3 Average
Result*
Undiluted
Assay
Sample #2, lyophilized
(DENV-1, BB)
Test day 3 Test day 4 Rep 1 Rep 2 Rep 3 Average
Result*
Rep 1 Rep 2 Rep 3 Average
Result*
Undiluted
Assay
Sample #3, liquid
frozen (DENV-1, CC)
Test day 1 Test day 2 Rep 1 Rep 2 Rep 3 Average
Result*
Rep 1 Rep 2 Rep 3 Average
Result*
Undiluted
Assay
Sample #3, liquid
frozen (DENV-1, CC)
Test day 3 Test day 4 Rep 1 Rep 2 Rep 3 Average
Result*
Rep 1 Rep 2 Rep 3 Average
Result*
Undiluted
Page 57
WHO/BS/2016.2299
Page 57
* 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:
Assay
Sample #4, lyophilized
(DENV-2, AA)
Test day 1 Test day 2 Rep 1 Rep 2 Rep 3 Average
Result*
Rep 1 Rep 2 Rep 3 Average
Result*
Undiluted
Assay
Sample #4, lyophilized
(DENV-2, AA)
Test day 3 Test day 4 Rep 1 Rep 2 Rep 3 Average
Result*
Rep 1 Rep 2 Rep 3 Average
Result*
Undiluted
Assay
Sample #5, liquid
frozen (DENV-2, BB)
Test day 1 Test day 2 Rep 1 Rep 2 Rep 3 Average
Result*
Rep 1 Rep 2 Rep 3 Average
Result*
Undiluted
Assay
Sample #5, liquid
frozen (DENV-2, BB)
Test day 3 Test day 4 Rep 1 Rep 2 Rep 3 Average
Result*
Rep 1 Rep 2 Rep 3 Average
Result*
Undiluted
Assay
Sample #6, liquid
frozen (DENV-2, CC)
Test day 1 Test day 2 Rep 1 Rep 2 Rep 3 Average
Result*
Rep 1 Rep 2 Rep 3 Average
Result*
Undiluted
Assay
Sample #6, liquid
frozen (DENV-2, CC)
Test day 3 Test day 4 Rep 1 Rep 2 Rep 3 Average
Result*
Rep 1 Rep 2 Rep 3 Average
Result*
Undiluted
Page 58
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Page 58
* 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:
Assay
Sample #7, liquid
frozen (DENV-3, AA)
Test day 1 Test day 2 Rep 1 Rep 2 Rep 3 Average
Result*
Rep 1 Rep 2 Rep 3 Average
Result*
Undiluted
Assay
Sample #7, liquid
frozen (DENV-3, AA)
Test day 3 Test day 4 Rep 1 Rep 2 Rep 3 Average
Result*
Rep 1 Rep 2 Rep 3 Average
Result*
Undiluted
Assay
Sample #8, lyophilized
(DENV-3, BB)
Test day 1 Test day 2 Rep 1 Rep 2 Rep 3 Average
Result*
Rep 1 Rep 2 Rep 3 Average
Result*
Undiluted
Assay
Sample #8, lyophilized
(DENV-3, BB)
Test day 3 Test day 4 Rep 1 Rep 2 Rep 3 Average
Result*
Rep 1 Rep 2 Rep 3 Average
Result*
Undiluted
Assay
Sample #9, lyophilized
(DENV-3, CC)
Test day 1 Test day 2 Rep 1 Rep 2 Rep 3 Average
Result*
Rep 1 Rep 2 Rep 3 Average
Result*
Undiluted
Assay
Sample #9, lyophilized
(DENV-3, CC)
Test day 3 Test day 4 Rep 1 Rep 2 Rep 3 Average
Result*
Rep 1 Rep 2 Rep 3 Average
Result*
Undiluted
Page 59
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Page 59
* 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:
Assay
Sample #10, liquid
frozen (DENV-4, AA)
Test day 1 Test day 2 Rep 1 Rep 2 Rep 3 Average
Result*
Rep 1 Rep 2 Rep 3 Average
Result*
Undiluted
Assay
Sample #10, liquid
frozen (DENV-4, AA)
Test day 3 Test day 4 Rep 1 Rep 2 Rep 3 Average
Result*
Rep 1 Rep 2 Rep 3 Average
Result*
Undiluted
Assay
Sample #11,
lyophilized (DENV-4,
BB)
Test day 1 Test day 2 Rep 1 Rep 2 Rep 3 Average
Result*
Rep 1 Rep 2 Rep 3 Average
Result*
Undiluted
Assay
Sample #11,
lyophilized (DENV-4,
BB)
Test day 3 Test day 4 Rep 1 Rep 2 Rep 3 Average
Result*
Rep 1 Rep 2 Rep 3 Average
Result*
Undiluted
Assay
Sample #12, liquid
frozen (DENV-4, CC)
Test day 1 Test day 2 Rep 1 Rep 2 Rep 3 Average
Result*
Rep 1 Rep 2 Rep 3 Average
Result*
Undiluted
Assay
Sample #12, liquid
frozen (DENV-4, CC)
Test day 3 Test day 4 Rep 1 Rep 2 Rep 3 Average
Result*
Rep 1 Rep 2 Rep 3 Average
Result*
Undiluted
Page 60
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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: _______________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
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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:__________________________________________________________
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Page 62
Estimated DENV RNA concentration of run control:___________________________________
Inclusion of method for prevention of PCR carry-over:__________________________________
_____________________________________________________________________________________
= = =