panglobtm dengue rrt-pcr kit · 5150 el camino real, suite a-32 los altos, ca, 94022 us emergo...

PanGlobTM Dengue rRT-PCR Kit

Instructions For Use

Document # 3.1, Rev B

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Symbol Glossary

Definition

25 Sufficient for 25 reactions

Store at -20 0C

Consult instructions for use

Positive control

Use-by date

CE mark

Lot number

European Authorized Representative

IVD In vitro Diagnostic

Manufacturer

Globavir Biosciences, Inc. 5150 El Camino Real, Suite A-32 Los Altos, CA, 94022 US

Emergo Europe Molenstraat 15 2513 BH, The Hague The Netherlands

PanGlob is a TradeMark of Globavir Biosciences, Inc.

Doc. Rev. C, March 2018

-20°C

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Table of Contents 1 Overview.......................................................................................................................................... 3

1.1 Intended Use ............................................................................................................................ 3

1.2 Principle of Test ....................................................................................................................... 3

2 Materials and Reagents ................................................................................................................... 3

2.1 Reagents Provided in Kit .......................................................................................................... 3

2.2 Reagents Provided by User ...................................................................................................... 3

2.3 Supplies Provided by User ....................................................................................................... 4

2.4 Equipment Provided by User ................................................................................................... 4

2.5 Limitations................................................................................................................................ 4

2.6 Warnings and Precautions ....................................................................................................... 4

2.7 Specimen Collection, Shipping and Storage ............................................................................ 5

2.8 Preparation .............................................................................................................................. 6

2.8.1 Avoiding sample contamination ....................................................................................... 6

2.8.2 Equipment preparation .................................................................................................... 6

2.8.3 Reagent Preparation ......................................................................................................... 6

3 Procedure ........................................................................................................................................ 7

3.1 Protocol Use Limitations .......................................................................................................... 7

3.2 Quality control for each RT-PCR run ........................................................................................ 7

3.3 Reaction Setup ......................................................................................................................... 7

3.4 RT-PCR amplification conditions .............................................................................................. 9

3.5 Data Analysis/Interpretation ................................................................................................. 10

4 Performance Characterization ...................................................................................................... 11

4.1 Method Comparison .............................................................................................................. 11

4.2 Precision, Linearity and Limit of Detection ............................................................................ 11

4.3 Analytical Reactivity ............................................................................................................... 13

4.4 Cross Reactivity ...................................................................................................................... 13

5 References ..................................................................................................................................... 14

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1 Overview

1.1 Intended Use

Globavir Biosciences PanGlobTM real-time, reverse transcriptase polymerase chain reaction (rRT-PCR) kit is intended for the in vitro detection of Dengue virus (DENV) infection in human plasma. This test can detect all serotypes (DENV1 - 4) but does not distinguish between them.

1.2 Principle of Test

The PanGlobTM Dengue rRT-PCR Kit is a qualitative test for the detection of Dengue virus (DENV) RNA in human plasma. Specific portions of viral RNA are amplified with optimized primers and probes (provided in kit), reverse transcriptase, DNA polymerase, and optimized themocycling procedure. During amplification, the specially designed probes are hydrolyzed resulting in fluorescent signal generation.

The PanGlob assay is capable of detecting all four DENV serotypes by targeting conserved regions of the viral genome.

An RNase P internal control is included to validate the extraction, amplification and detection processes in every test.

2 Materials and Reagents

2.1 Reagents Provided in Kit

The following reagents are included in the PanGlob Dengue rRT-PCR Kit:

Reagent Amount Provided Dilution Stability and Storage

PanGlobTM Dengue Primer mix (Dpri, also includes RNase P Primers)

Amount sufficient for 25 reactions

Suspend in 100 μL of nuclease free water

2 year at -200C

PanGlobTM Dengue Probe mix (Dpro, also includes RNase P Probe mix)

Amount sufficient For 25 reactions


2 year at -200C

Four PanGlobTM positive controls (DENV1-4) (PC)



2 year at -200C

PanGlobTM Dengue One-Step Master Mix


1 year at -200C

2.2 Reagents Provided by User

The following reagents must be provided by the user of the PanGlob Dengue rRT-PCR Kit:

Reagent Concentration Dilution Stability and

Storage

Nuclease-Free Water (or equivalent) (NW) N/A

Blank Human Plasma (BP) N/A

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2.3 Supplies Provided by User

The following supplies are required to utilize the PanGlob Dengue rRT-PCR Kit:

– Sterile, nucleotide-free and nuclease-free 1.5 mL microcentrifuge tubes – Sterile, nucleotide-free and nuclease-free 0.2 mL PCR reaction tubes – Cooling racks or block for 1.5 mL and 0.2 mL tubes – Optical PCR tubes strip caps – Disposable, powder-free gloves

2.4 Equipment Provided by User

The following equipment is required to perform the PanGlob Dengue rRT-PCR test:

– RotorGene-Q RT-PCR instrument (Qiagen, Venlo, NL) or equivalent RT-PCR instrument – Microcentrifuge – Vortex – Micropipettes, 20 μL and 200 μL

2.5 Limitations

Please note the following limitations on the use of the PanGlob Dengue Kit:

– For in vitro use only. – This assay has been validated with human plasma specimens, and has not been validated

using other specimen types. – A positive result does not rule out diseases caused by other pathogens, and should be used

in combination with clinical diagnoses. – False-negative results may occur if infecting viruses have genomic mutations, or if specimen

samples are misproperly collected and prepared. – Clinical Specificity has been tested using RNA viruses from the flaviviridae family such as

West Nile Virus (WNV), Japanese Encephalitis Virus (JEV), Tick-Borne Encephalitis Virus (TBEV) and Hepatitis-C virus (HCV). No detectable amplification was observed using this assay.

– As with other tests, false-positive results are possible. Repeat testing may distinguish some such cases.

– All specimen collection, reagent preparation, and amplification steps should be performed by qualified, trained personnel.

– Do not use reagents past the expiration date, and observe proper handling and storage guidelines. Note: Refer to Section 4 for further detail on assay performance characteristics

2.6 Warnings and Precautions

– All test specimens and waste consumables should be considered potentially infectious and handled accordingly as per institutional, regional and national regulations.

– Wear personal protective equipment, such as gloves and lab coats, when handling kit reagents or performing this assay. Wash hands thoroughly after performing test.

– Do not eat, drink, smoke, apply make-up, or handle contact lenses in areas where kit reagents and/or human specimens are being prepared or used.

– Do not pipette by mouth.

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– Dispose of kit reagents and test specimens according to institutional, regional and national regulations.

– Accurate test results are dependent on proper laboratory technique. To ensure quality results, the following precautions should be strictly adhered to:

o Work should proceed in a uni-directional manner, according to the following general outline: specimen extraction, RT-PCR instrument set-up, reagent preparation, RT-PCR amplification.

o Dedicated areas should be maintained for assay set-up and amplification/detection activities. Supplies and equipment (including Personal Protective Equipment (PPE) and micropipettes) should not be used in both areas.

o Supplies and equipment (including PPE and micropipettes) used for specimen extraction and preparation should not be used for reagent preparation or for processing amplified DNA.

o Surfaces and equipment should be decontaminated using 5% bleach (Sigma, #L099100), DNAzap™(Life Technologies, #AM9890), and/or Rnase AWAY™ (Life Technologies, #10328-011).

o Keep reagents and tubes capped when not in direct use.

– Due to the sensitivity of rRT-PCR assays, any contamination of specimens or reagents may produce inaccurate results. Use aseptic techniques, and pipette reagents carefully.

– All reagents and supplies should be maintained on a cold block or on ice during preparation and use to avoid thermal degradation of enzymes and oligonucleotides.

– Do not substitute reagents from different kit lots or other manufacturers other than those listed in this document.

– Protect fluorogenic probes from light.

– Reagents contain glycerol, which may cause irritation upon inhalation or contact with skin.

– See next section for additional storage and handling details.

2.7 Specimen Collection, Shipping and Storage

The PanGlob Dengue rRT-PCR assay has been optimized for the analysis of human plasma. Specimen collection, handling, and processing should be performed in accordance with all local, regional and national biological safety regulations.

Collect blood samples using approved venipuncture techniques by trained laboratory personnel. Transport specimens to the laboratory as quickly as possible and separate plasma (EDTA or ACD) within 6 hours. If transport exceeds 6 hours, whole blood should be refrigerated (2-8°C) and then delivered on a cold pack. Separated plasma should be kept frozen (-15 to -30°C).

Plasma samples should remain unfrozen for no longer than 4 hours, during which time samples should be kept in a refrigerator or on ice. When not in use, samples should be stored below -20°C and multiple freeze/thaw cycles should be avoided. If transportation of samples is required, samples should be kept on dry ice and all applicable transport regulations should be adhered to. Before assaying samples, thaw plasma and mix well. Use 140 μL of plasma for extraction using QiaAmp viral RNA mini kit described below.

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2.8 Preparation

2.8.1 Avoiding sample contamination

Because of the high sensitivity of rRT-PCR assays, special precautions must be taken in order to avoid sample contamination and false-positive results. The following precautionary steps are recommended:

– Maintain separate areas for reagent preparation and nucleic acid handling. – Maintain separate, dedicated equipment and supplies (e.g., pipettes, microcentrifuges,

pipette tips) for reagent preparation and nucleic acid handling. – Change gloves whenever contamination is suspected, or between every sample. – Keep reagent and reaction tubes capped and covered as much as possible.

2.8.2 Equipment preparation

To reduce the chance of contamination, work surfaces, pipettes, microcentrifuges, and other equipment should be cleaned and decontaminated using a freshly prepared 5% bleach solution, DNAzap, and/or RNase AWAY.

2.8.3 Reagent Preparation

**Important: keep all reagents in a cold rack and/or on ice during assay setup.**

– Primers and Probes o DenV and Rnase P primers and probes are provided as pre-measured lyophilized

solids. Resolubilize by adding 100 μL of nuclease free water (NW). Store at -200C and away from light until needed.

o Thaw frozen aliquots of primers and probes prior to running the assay o Probes should always be kept in the dark during reagent preparation o Briefly vortex all primers o Briefly centrifuge primers and probes, and place in cold rack

– Master Mix, Enzymes, and Water o Master mix, enzymes, and water should be kept in the reagent preparation area o Thaw master mix, enzymes, and water o Place master mix, enzymes, and water in cold rack

– Controls and Analysis Specimens o For controls add 125 μL of nuclease free water and store at -20 0C until needed. o Controls and extracted nucleic acid specimens to be analyzed should kept in the

nucleic acid handling area o Thaw frozen samples and control aliquots o Vortex and briefly centrifuge all controls and samples o Place controls and specimens in cold rack

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3 Procedure

3.1 Protocol Use Limitations

This protocol was optimized using specific commercially available reagents and equipment, and product substitutions are not recommended. For specimen RNA extraction, the QIAamp viral RNA mini kit (Qiagen #52904 or #52906, Venlo, NL) should be used. Amplification should be carried out on the Rotor-Gene Q instrument (Qiagen, Venlo, NL), using the One-Step Master Mix provided.

The performance of the PanGlob Dengue rRT-PCR assay depends on the amount and quality of sample template RNA. The PanGlob Dengue assay has been validated on plasma (EDTA or ACD) using the QIAamp viral RNA mini kit (Qiagen, Venlo, NL). The use of other specimen types or extraction protocols may impact test performance.

3.2 Quality control for each RT-PCR run

For each RT-PCR run, the following controls are recommended:

– DENV positive control (PC) – DENV negative controls, Blank Plasma (BP) and Nuclease free Water (NW) – No Template Control using RNase Free H2O (NTC)

The positive and negative controls should be taken through the nucleic acid extraction process, using either the PanGlobTM PC (positive control reactions) or blank plasma (negative control reactions) as appropriate . It is recommended that the positive control be rotated among the DENV serotypes, though all 4 can be run on each run if desired.

The No Template Control (NTC) is not extracted but controls for contamination of reagents or contamination during PCR set-up. Use nuclease-free water for the NTC.

Four positive controls (PCs) are provided – one for each of the four DENV serotypes. For each RT-PCR run, it is recommended that a single positive control is used, and that the serotype control selected be rotated (so that the first run performed uses the DENV1 PC, the second run uses the DENV2 PC, the third run uses the DENV3 PC, the fourth run uses DENV4 PC, and this cycle is repeated for additional runs). However, all four PC can be used for each run, if desired.

3.3 Reaction Setup

It is recommended that reaction assay mixtures (RM) are created as a single master stock. This solution is then dispensed into a 96-well reaction plate or PCR tubes according to the procedure below. Add extracted nucleic acids or controls to the appropriate test reactions.

A. Label one 1.5 mL microcentrifuge tube for the Reaction Mix. The Reaction Mix, which contains all reagents, will be dispensed into all wells.

B. Determine the number of reactions (n) to be performed using the reaction mix, including the number of samples to be tested and the 3 controls (PC, BP or NW, and NTC). – If the number of samples including controls (n) is < 15, then N = n + 1 – If the number of samples including controls (n) is ≥ 15, then N = n + 2

C. Reaction Mix (RM): Calculate the amount of each reagent required. Add the appropriate volumes of each reagent to the 1.5 mL RM tube. This step should be performed on ice.

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Table 1 - Reagent Dilutions for Reaction Mix (for one reaction)

Reagent Volume

Dengue Primer Mix (Dpri) N * 3.5 μL

Dengue Probe Mix (Dpro) N * 3.5 μL

PanGlobTM One-Step Master Mix N * 12.5 μL

Extracted Sample N * 5 μL

Nuclease free water N * 0.5 μL

Total Volume N * 25 μL

D. Add all reagents carefully, and then gently mix the RM by pipetting up and down. Do not vortex.

E. Centrifuge briefly to collect contents at bottom of tube, and return RM to cold rack.

F. Place reaction tubes or 96-well plate in cold rack.

G. Dispense 20 μL of each RM into the all tubes/wells.

The following two tables are examples for reaction mix and sample setup in 8-tube PCR reaction strips, and are intended as a guide to aid researcher planning.

Table 2 - Example Setup for Reaction Mix in an 8 tube reaction.

Tube 1 2 3 4 5 6 7 8

Mix (sample)

RM

(PC)

RM

(BP or NW)

RM

(S1)

RM

(S2)

RM

(S3)

RM

(S4)

RM

(S5)

RM (NTC)

Table 3 -Example Setup for nucleotide additions in a 8 tube reaction.

Tube 1 2 3 4 5 6 7 8

Nucleotide (sample)

PC BP

(S5)

S1

(S1)

S2

(S2)

S3

(S3)

S4

(S4)

S5

(S5)

NW

(NTC)

NOTE: The nuclease-free water (NW) for the no template controls (NTC) should be added first before any of the samples are added to check for contamination in the Reaction Mix (RM). Extracted nucleotide solutions for positive and negative controls should be added last after all samples and NTCs are sealed.

H. Before moving the plate to the nucleic acid handling area, set up the NTC reaction in the assay setup area.

I. Pipette 5 μL of nuclease-free water into NTC wells. Cap the wells.

J. Cover the reaction tubes/wells and move the strip/plate to the nucleic acid handling area.

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K. Vortex the tubes containing the samples and control nucleic acids for 5 sec. Centrifuge for 5 sec.

L. Set up the extracted nucleic acid samples in the cold rack.

M. Pipette 5 μL of each sample into the tube/well labeled for that sample. Change pipette tips after each addition.

N. Cap the tubes/wells after addition of each sample. This will help to prevent sample cross contamination.

O. Change gloves frequently, and whenever contamination is suspected to have occurred.

P. Repeat steps M - O for each sample.

Q. For the positive control, transfer 5 μL of the positive control sample to the PC tube/well. Cap the PC tube/well.

R. Finally, pipette 5 μL of the blank plasma into the BP negative control tube/well. Cap the BP tube/well.

S. If using PCR tube strips, label the TAB of each strip to indicate sample position (Do not label the tops of tubes). Briefly centrifuge tube strips for 10-15 sec., and return tubes to cold rack.

T. If using 96-well plates, centrifuge at 500 x g for 30 sec. at 4OC. Return to cold rack.

3.4 RT-PCR amplification conditions

The final reaction volume is 25 μL/tube. Program the RotoGene-Q RT-PCR thermocycler using the parameters provided in Table 4, repeating the final 3 amplification steps for a total of 45 cycles.

Table 4 - Temperature Program for RT-PCR Reactions

Reaction Temperature Duration

Reverse Transcription 52 OC 15 min

Taq Inhibitor Activation 94 OC 2 min

PCR Amplification 1 94 OC 15 sec



Fluorescence data should be collected during the 55OC incubation step. Both Dengue and RNase P probes should be measured in each assay using dual wavelength measurements. The excitation and emission wavelengths for the Dengue and RNase P probes are given follows in Table 5:

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Table 5 - Excitation and Emission Wavelengths for PanGlob Probes

Probe Excitation Wavelength Emission Wavelength

Dengue Probes 495 nm 520 nm

RNase P Probes 590 nm 610 nm

When RotorGene-Q RT-PCR instrument has initialized, transfer PCR tube strip or 96-well plate into the machine and begin run.

3.5 Data Analysis/Interpretation

The no template control (NTC) should not exhibit fluorescence growth curves that cross the threshold line. If a false positive occurs with one or both of the negative control reactions, sample contamination may have occurred. Invalidate the run and repeat with stricter adherence to the procedure guidelines.

All clinical samples, and Dengue negative control (BP or NW) should exhibit RNase P reaction curves that cross the threshold line at or before 40 cycles, indicating the presence of sufficient RNA from the human RNase P gene and verifying the specimen is of acceptable quality. Failure to detect RNase P in any of the clinical samples may indicate:

– Improper extraction of nucleic acid from clinical materials. – Absence of sufficient human cellular material in sample to enable detection. – Improper assay setup and/or execution. – Reagent or equipment malfunction.

Dengue positive control (PC) should produce a positive result with the DENV primers/probes before 40 cycles. If the positive reaction is not achieved, invalidate the run and repeat the assay with stricter adherence to procedure guidelines. Do not use PC reagents that repeatedly fail to generate the expected result.

When all controls meet stated requirements, a specimen is considered positive for DENV if the DENV reaction curves cross the threshold line within 40 cycles.

When all controls meet stated requirements, a specimen is considered negative for DENV if the DENV reaction curves do not cross the threshold within 40 cycles.

Table 6 – Summary of Control Results

Control Result

Dengue Positive Control (PC) +

Dengue Negative Control (BP or NW) -

No-Template Control (NTC) -

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4 Performance Characterization

4.1 Method Comparison

A panel of 200 clinical samples were obtained from Sri Lanka and Nicaragua. The samples were tested with the PanGlob assay using SuperScript III Reverse Transcriptase (Invitrogen # 18080044, Carlsbad, CA US), Platinum Taq DNA Polymerase (Invitrogen # 10966018, Carlsbad, CA US), and compared to 2 additional Dengue diagnostic assays (Table 7 and Table 8). The PanGlob assay’s clinical sensitivity was determined to be 98% (148/151), Table 7.

Table 7 - Comparison of the PanGlob, Altona and heminested assays with a composite reference.

Assay Assay result Composite reference results (n)

Positive Negative Total

PanGlob Positive 148 7 155

Negative 3 42 45

Total 151 49 200

RealStar Dengue RT-PCR (altona Diagnostics GmbH)

Positive 94 0 94

Negative 36 21 57

Total 130 21 151

Heminested Positive 119 3 122 Negative 32 46 78

Total 151 49 200

Table 8 – Clinical samples with detectable DENV RNA stratified by the day of illness in sample collection with comparison to available Dengue diagnostic assays

Day of illness PanGlob altona Heminested

2 27/27 (100) 27/27 (100) 27/27 (100) 3 14/16 (87.5) 12/14 (85.7) 13/16 (81.3) 4 20/24 (83.3) 15/20 (75.0) 19/24 (79.2) 5 35/42 (83.3) 17/31 (54.8) 24/42 (57.1)

>5 46/49 (93.9) 18/36 (50.0) 28/49 (57.1)

Total 142/158 (89.9) 89/128 (69.5) 111/158 (70.2) a Results are reported as number detected/total number (%).

4.2 Precision, Linearity and Limit of Detection

4.2.1 Precision:

The precision of the PanGlob assay was determined using three (3) dilutions of RNA Controls (high positive, low positive, and limit of quantitation). These were performed five (5) times on three (3) separate days. Fresh dilutions were made on the day of each run from aliquots of high-concentration stocks (1 ng/μL, high positive). Intra- and inter-run variability were calculated from the log10 concentration of the samples (Table 9).

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Table 9 – Precision of PanGlob Assay

DENV Serotype

Dilution

Interrun precision Intrarun precision

Concentration Concentration

log10 CDNA equivalents/μL

SD %

CoV

Range, log10 CDNA equivalents

/μL

SD % CoV

DENV-1

High positive 5.05 0.17 3.4 4.95 - 5.16 0.06 - 0.18 1.2 - 37 Low positive 3.25 0.16 4.8 3.05 - 3.38 0.04 - 0.07 1.3 - 2.3

Limit of quantitation

1.20 0.15 12.2 1.08 - 1.32 0.05 - 0.10 4.0 - 9.5

DENV-2

High positive 4.61 0.19 4.1 4.43 - 4.83 0.02 - 0.10 0.1 - 2.0 Low positive 2.62 0.08 3.2 2.57 - 2.74 0.051 - 0.054 1.9 - 2.1


0.57 0.28 48.1 0.39 - 0.76 0.18 - 0.29 23.9 - 56.0

DENV-3

High positive 5.02 0.18 3.7 4.89 - 5.03 0.07 - 0.21 1.3 - 4.2

Low positive 2.35 0.13 5.5 2.18 - 2.44 2.8 - 7.8 1.2 - 3.2


0.32 0.24 7.6 0.20 - 0.45 0.27 - 0.17 38.0 -127

DENV-4

High positive 4.04 0.14 3.6 3.96 - 4.16 0.06 - 0.18 1.4 - 4.6

Low positive 2.32 0.12 5.1 2.20 - 2.39 0.07 - 0.09 3.1 - 3.8


1.43 0.18 12.6 1.27 - 1.60 0.08 - 0.16 4.9 - 12.8

4.2.2 Linearity of Assay:

For each serotype, linearity studies were performed on serial 10-fold dilutions of both quantified plasmid DNA and reference virus RNA. The linear range was established by fitting a best-fit line to the data by regression analysis and included the range where the R2 value for this line was ≥ 0.99 (Figure 1).

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Figure 1 - Linearity of the PanGlob Assay for each serotype.

4.2.3 Limits of Detection:

To establish the lower limit of 95% detection (95% LLOD), the lowest concentrations of RNA at which all replicates were detectable during the linear range study were used as the starting point. Ten (10) replicates of four (4) 2-fold dilutions from this concentration were tested on a single run. The 95% LLOD was then calculated using probit analysis. The 95% LLOD was calculated to be 1.7 for DENV-1, 1.7 for DENV-2, 2.2 for DENV-3, and 7.6 cDNA equivalents/μL for DENV-4.

4.3 Analytical Reactivity

Genomic RNA from reference strains of the four DENV serotypes, DENV-1 Hawaii 1944, DENV-2 New Guinea C strain, DENV-3 strain H87, and DENV-4 strain H241 were obtained from Vircell (Granada, SP) and all were amplified in this test.

4.4 Cross Reactivity

No amplification was observed using RNA from other closely related flaviviruses, including genomic RNA of three strains of West Nile Virus (WNV, NY 1999; clinical isolate, previously reported as NAL strain; and B956), and a single strain each of Japanese Encephalitis Virus (JEV) and Tick-Borne Encephalitis Virus (TBEV). Sixty (60) HCV-positive clinical samples were also tested and no amplification was observed; these samples had a median HCV viral load of 6.05 log10 IU/mL of patient plasma (range <1.63 to 7.66).

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5 References

– Bhatt S, Gething P, Brady O, Messina J, Farlow A (2013) The global distribution and burden of Dengue. Nature 496: 504-507.

– Guzman MG, Halstead SB, Artsob H, Buchy P, Farrar J, Gubler DJ, Hunsperger E, Kroeger A, Margolis HS, Martínez E, Nathan MB, Pelegrino JL, Simmons C, Yoksan S, Peeling RW. Dengue: a continuing global threat. Nat Rev Microbiol, 2010 ;8(12 Suppl):S7-16.

– Kuan G, Gordon A, Aviles W, Ortega O, Hammond S, et al. (2009) The Nicaraguan Pediatric Dengue Cohort Study: Study Design, Methods, Use of Information Technology, and Extension to Other Infectious Diseases. American Journal of Epidemiology. pp. 120-129.

– Narvaez F, Gutierrez G, Pérez M, Elizondo D, Nuñez A, et al. (2011) Evaluation of the Traditional and Revised WHO Classifications of Dengue Disease Severity. PLoS Negl Trop Dis. pp. e1397.

– Nenguke T, Selway D, Redkar A. Quantitative two-step RT-PCR analysis using LUX Primers and reverse transcriptases stored at various temperatures. FOCUS (2003) 25:33-35.

– Wayland-Smith A, Dhariwal G, Smith MD, Rashtchian RA, Gerard GF, Potter J, Lee JE.

Optimized RT-PCR with Invitrogen™ Cloned AMV Reverse Transcriptase. Focus (2002)

24:14-15.

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