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WHO/BS/2016.2299

ENGLISH ONLY

EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION

Geneva, 17-21 October 2016

Collaborative Study to Establish a World Health Organization

International Reference Panel for Dengue Virus types 1 to 4 RNA for Nucleic Acid

Amplification Technology (NAT)-Based Assays

Germán Añez1,#, Evgeniya Volkova1, Rafaelle C.G. Fares1, Zhen Jiang1, Maria Rios1,* and the

Collaborative Study Group2

1- U.S. Food and Drug Administration, 10903 New Hampshire Ave, Silver Spring, Maryland,

United States of America

# Current address: Sanofi Pasteur, Swiftwater, Pennsylvania, United States of America

* Principal contact: [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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WHO/BS/2016.2299

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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

http://www.r-project.org/

WHO/BS/2016.2299

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.

http://doi.org/10.1128/genomeA.01583-15

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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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11 8 10 14b

12

13a

18

19

1

2

3

4

5

6

7

8


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


WHO/BS/2016.2299

1

2

3 4

5

6

7

8

9

10

11

14a

14b

15

19

1

2

3

4

5

6

7

8


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


WHO/BS/2016.2299

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


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


WHO/BS/2016.2299

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

members.

WHO/BS/2016.2299

1 2 3

4

6

19

9

8 11

10

14b

15

1

2

3

4

5

6

7

8


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).

WHO/BS/2016.2299

1 2

3 4

6

19

9

8

11 10

14b

15

1

2

3

4

5

6

7

8


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



WHO/BS/2016.2299

1

2

3

4

6

5

9

8

11

10

14b

15

1

2

3

4

5

6

7

8


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



WHO/BS/2016.2299

1 2

3

4

6

5

9

8

11

10

14b

15

1

2

3

4

5

6

7

8


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



WHO/BS/2016.2299

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.

WHO/BS/2016.2299

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


-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

DENV-3, lyophilized


-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

DENV-4, lyophilized


-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

WHO/BS/2016.2299

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


RNA Mini kit

(Qiagen)

RT-PCR followed by

multiplex nested PCR;

analysis by agarose gel

electrophoresis

NS5

(DENV-1 –

4)

Bronzoni et

al 2005


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


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


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

WHO/BS/2016.2299

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


RNA Mini kit

(Qiagen)

Ready-to-Go RT-PCR

Beads (GE Healthcare)

C-prM


RNA Mini kit

(Qiagen)

RT-nested PCR E/NS1

junction

(DENV-1 –

4)

Domingo et

al 2011;

Domingo et

al 2006


RNA Mini kit

(Qiagen)

Real-time RT-PCR

(TaqMan) using in vitro

transcribed RNA as

standard curve

3’NCR Kaiser M,

unpublished


RNA Mini kit

(Qiagen)

Real-time RT-PCR

(TaqMan)

DENV-1:

NS5;

DENV-2: E;

DENV-3

and 4: prM;

Johnson et

al 2005


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


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


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


RNA Mini kit

(Qiagen)

Real-time RT-PCR

(TaqMan)

5’NCR or

3’NCR

Leparc-

Goffart et al

2009


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

WHO/BS/2016.2299

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

WHO/BS/2016.2299

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

WHO/BS/2016.2299

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

WHO/BS/2016.2299

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

WHO/BS/2016.2299

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

WHO/BS/2016.2299

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

WHO/BS/2016.2299

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

WHO/BS/2016.2299

Vanessa Suin/Aurélie Francart Re-emerging Viruses Program

Viral Diseases | Communicable and Infectious

Diseases

Scientific Institute of Public Health

Brussels, Belgium

WHO/BS/2016.2299

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

WHO/BS/2016.2299

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

WHO/BS/2016.2299

10. ACKNOWLEDGEMENTS

We would like to thank Dr. Robert Lanciotti from the CDC for

providing viral strains used in preparation of the reagents.

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.


http://www.who.int/biologicals/en/


WHO/BS/2016.2299

Appendix 4. Study protocol, results and methods forms

World Health Organization collaborative study to assess the suitability of a

candidate standard for dengue virus types 1 to 4 (DENV-1 to -4) RNA for

detection by nucleic acid tests

Germán Añez, M.D. and Maria Rios, Ph.D.

Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Office of Blood

Evaluation and Research, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration

STUDY PROTOCOL

ASSAY PERFORMANCE AND REPORTING

Participants should test the dengue virus types 1 to 4 (DENV-1 to 4) working reagents as they

usually test samples suspected to contain DENV, using the nucleic acid test assay(s) system(s)

available to them, and perform both qualitative and quantitative assays, if available. Results

should be recorded clearly on the results form.

If more than one type of assay is performed by a particular laboratory (for example, two

different extraction methods or different RT-PCR protocols), separate qualitative Result

WHO/BS/2016.2299

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)



WHO/BS/2016.2299

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



8) DENV-3, BB Liquid frozen 6

9) DENV-3, CC Lyophilized 6


11) DENV-4, BB Lyophilized 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

WHO/BS/2016.2299

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

WHO/BS/2016.2299

4. If in addition to the qualitative assay described above, your laboratory have a quantitative NAT

assay capability (i.e. your assay is performed including a DENV RNA standard curve), we ask

that the collaborators report to us the results of the four independent determinations of the

undiluted DENV working reagent only (both liquid frozen and lyophilized), tested in

triplicates.

5. Datasheets, Method forms and Result forms are provided for recording of all relevant information

about the assay used. Please return the completed forms to us for analysis of the results.

WHO/BS/2016.2299

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)


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

WHO/BS/2016.2299


Comments:

Sample #2, lyophilized

(DENV-1, BB)

Test day 1



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)



(# positive

replicates)


replicates)


replicates)

1 Undiluted

2 101

3 100.5

4 End-point*

5 10-0.5

6 10-1

WHO/BS/2016.2299


Comments:


(DENV-1, CC)

Test day 1



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)



(# positive

replicates)


replicates)


replicates)

1 Undiluted

2 101

3 100.5

4 End-point*

5 10-0.5

6 10-1

WHO/BS/2016.2299


Comments:


(DENV-2, AA)

Test day 1



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)



(# positive

replicates)


replicates)


replicates)

1 Undiluted

2 101

3 100.5

4 End-point*

5 10-0.5

6 10-1

WHO/BS/2016.2299


Comments:


(DENV-2, BB)

Test day 1



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)



(# positive

replicates)


replicates)


replicates)

1 Undiluted

2 101

3 100.5

4 End-point*

5 10-0.5

6 10-1

WHO/BS/2016.2299


Comments:


(DENV-2, CC)

Test day 1



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)



(# positive

replicates)


replicates)


replicates)

1 Undiluted

2 101

3 100.5

4 End-point*

5 10-0.5

6 10-1

WHO/BS/2016.2299


Comments:


(DENV-3, AA)

Test day 1



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)



(# positive

replicates)


replicates)


replicates)

1 Undiluted

2 101

3 100.5

4 End-point*

5 10-0.5

6 10-1

WHO/BS/2016.2299


Comments:


(DENV-3, BB)

Test day 1



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)



(# positive

replicates)


replicates)


replicates)

1 Undiluted

2 101

3 100.5

4 End-point*

5 10-0.5

6 10-1

WHO/BS/2016.2299


Comments:


(DENV-3, CC)

Test day 1



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)



(# positive

replicates)


replicates)


replicates)

1 Undiluted

2 101

3 100.5

4 End-point*

5 10-0.5

6 10-1

WHO/BS/2016.2299


Comments:


(DENV-4, AA)

Test day 1



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)



(# positive

replicates)


replicates)


replicates)

1 Undiluted

2 101

3 100.5

4 End-point*

5 10-0.5

6 10-1

WHO/BS/2016.2299


Comments:


(DENV-4, BB)

Test day 1



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)



(# positive

replicates)


replicates)


replicates)

1 Undiluted

2 101

3 100.5

4 End-point*

5 10-0.5

6 10-1

WHO/BS/2016.2299


Comments:


(DENV-4, CC)

Test day 1



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)



(# positive

replicates)


replicates)


replicates)

1 Undiluted

2 101

3 100.5

4 End-point*

5 10-0.5

6 10-1

WHO/BS/2016.2299

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)


Result*


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


(DENV-1, BB)


Result*


Result*

Undiluted

Assay


(DENV-1, BB)


Result*


Result*

Undiluted

Assay

Sample #3, liquid

frozen (DENV-1, CC)


Result*


Result*

Undiluted

Assay

Sample #3, liquid

frozen (DENV-1, CC)


Result*


Result*

Undiluted

WHO/BS/2016.2299




Comments:

Assay


(DENV-2, AA)


Result*


Result*

Undiluted

Assay


(DENV-2, AA)


Result*


Result*

Undiluted

Assay

Sample #5, liquid

frozen (DENV-2, BB)


Result*


Result*

Undiluted

Assay

Sample #5, liquid

frozen (DENV-2, BB)


Result*


Result*

Undiluted

Assay

Sample #6, liquid

frozen (DENV-2, CC)


Result*


Result*

Undiluted

Assay

Sample #6, liquid

frozen (DENV-2, CC)


Result*


Result*

Undiluted

WHO/BS/2016.2299




Comments:

Assay

Sample #7, liquid

frozen (DENV-3, AA)


Result*


Result*

Undiluted

Assay

Sample #7, liquid

frozen (DENV-3, AA)


Result*


Result*

Undiluted

Assay


(DENV-3, BB)


Result*


Result*

Undiluted

Assay


(DENV-3, BB)


Result*


Result*

Undiluted

Assay


(DENV-3, CC)


Result*


Result*

Undiluted

Assay


(DENV-3, CC)


Result*


Result*

Undiluted

WHO/BS/2016.2299




Comments:

Assay

Sample #10, liquid

frozen (DENV-4, AA)


Result*


Result*

Undiluted

Assay

Sample #10, liquid

frozen (DENV-4, AA)


Result*


Result*

Undiluted

Assay

Sample #11,

lyophilized (DENV-4,

BB)


Result*


Result*

Undiluted

Assay

Sample #11,

lyophilized (DENV-4,

BB)


Result*


Result*

Undiluted

Assay

Sample #12, liquid

frozen (DENV-4, CC)


Result*


Result*

Undiluted

Assay

Sample #12, liquid

frozen (DENV-4, CC)


Result*


Result*

Undiluted

WHO/BS/2016.2299

METHODS FORM

1) RNA extraction method employed.

Method for concentration of viral particles from sample before RNA extraction (if

applicable):____________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

Brief description of RNA extraction method:

______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

Volume of sample extracted:______________________________________________________

Final volume of extracted RNA:____________________________________________________

Volume of extracted RNA tested:____________________________________________________

2) RNA amplification method – Qualitative assays

Amplification protocol:

_____________________________________________________________________________

_____________________________________________________________________________

_____________________________________________________________________________

Please provide a brief description of other procedure including methodology and amplification

cycling conditions: _______________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

WHO/BS/2016.2299

Brief description of protocol for detection of amplified DNA:

______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

3) RNA amplification method – Quantitative assays

Brief description of quantitative assay (if applicable):

______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

Brief description of method for calculating concentration of DENV:

______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

4) Quality control

Internal control included in assay:

______________________________________________________________________________

Inclusion of weak positive samples in each run for monitoring sensitivity:___________________

______________________________________________________________________________

Inclusion of negative samples in each run for monitoring specificity:_______________________

______________________________________________________________________________

Inclusion of run control:__________________________________________________________

WHO/BS/2016.2299

Estimated DENV RNA concentration of run control:___________________________________

Inclusion of method for prevention of PCR carry-over:__________________________________

_____________________________________________________________________________________

= = =

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