laboratory diagnosis dengue infections - … diagnosis of dengue infections how should i select the...

LABORATORY DIAGNOSISOF DENGUE INFECTIONS

Cristina Domingo CarrascoRobert Koch Institut

ECDC training Workshop on laboratory diagnosis of dengue virus infectionsBerlin, 23‐27 January 2012

LABORATORY DIAGNOSIS OF DENGUE INFECTIONS

HOW SHOULD I SELECT THE DIAGNOSIS METHOD TO APPLY IN MY LAB?

WHO: Dengue guidelines for diagnosis, treatment, prevention and control (2010)


Tests with high sensitivity and specificity require more complextechnologies and technical expertise

Guzmán MG, Nature Reiews, 2010

• The choice of the diagnosis methods depends mostly on the days of illness at sample collection date

• Other points to consider are the time required to reach a definite diagnosis, equipment available…


WHO: Dengue guidelines for diagnosis, treatment, prevention and control (2010)

• The choice of the diagnosis methods depends mostly on the days of illness at sample collection date

• Other points to consider are the time required to reach a definite diagnosis, equipment available…


IT IS STRONGLY RECOMMENDED TO COMBINE THE USE OF SEROLOGICAL AND MOLECULAR /ANTIGEN DETECTION METHODS

0 1 2 3 4 5 6 7 8 9 10 15 30 90

Viremia IgM IgG

0 1 2 3 4 5 6 7 8 9 10 15 30 90

Viremia IgM IgG

MOLECULAR D.

SEROLOGY

PRIMARY INFECTIONS SECONDARY INFECTIONS

NS1 antigen D.

MOLECULAR D

SEROLOGY

NS1 antigen D.

VIRAL ISOLATION VIRAL ISOLATION


VIRAL ISOLATION

C6/36 cells non‐ infected infected

Preferred cell‐line: C6/36

Other cell lines: AP61, Vero E6, LLC‐MK2, BHK21

Only samples collected during the viremic phase (3‐5 days)

Keep control on the specimens transport temperatures

Specimens with dry ice should be placed in a sealed plastic‐bag

Detection by monoclonal antibodies IIF or PCR

Requires BSL‐3 facilities and trained personnel


FACILITIES Genome extraction area has to be separated of the amplification area,

nested PCR and detection areas have to be separated of RT‐PCR area Non‐shared reagents and instrumental Routine decontamination of the working areas

SAMPLES Positive results must be confirmed with a second method targeted to

a different region of the genome (dengue vs panflavirus)

REAGENTS Not every commercial master mix provides the same performance for

all assays: validation previous to use We recommend the use of silical‐based commercial kits for RNA

extraction

MOLECULAR DIAGNOSIS


CONTROLS

Suitable controls must be included in the assay:

positive control (the last positive dilution)

negative control (water or negative serum)

standard curve for quantification

• It is preferable to include the positive and negative controls from the

extraction step

QUALITY ASSESMENT

Each assay must be evaluated in terms of sensitivity and specificity in the

laboratory before use for diagnosis

The labs should participate in QA activities regularly


OVERVIEW ON AVAILABLE MOLECULAR DIAGNOSIS SYSTEMS• Reverse‐transcriptase PCR (RT‐PCR)

Lanciotti et al, J. Clin. Microbiol, 1992Harris et al, J. Clin. Microbiol, 1998

Nested protocols have an increased sensitivity but have higher risk of carry‐overcontamination

• Real‐time RT‐PCRs

rapidity

ability to provide quantitative measurements

lower contamination rate

high sensitivity

high specifictiy due to the use of internal probes (not in SYBR‐Green)

easyness of standardization with available commercial master mixes

C/preM: serotyping by size

GOLD STANDARD

Method Region Amplicon size (bp) Sensitivity Specificity# Clinical/Spiked samples

Veterinary samples Reference

Multiplex RT‐PCR 1‐step Cap‐prM 482/119/290/398 2500

copies/rxn JEV, WNV, YFV, CHKV Clinical No Saxena P. et al, 2008

Multiplex RT‐PCR 1‐step NS5 109/139/169/199 0.1‐0.001

PFU/rxn JEV, Plasmodium falciparum NA No Maneekan P. et al, 2009

Multiplex nested RT‐PCR E/NS1 400/486/416/418 NA JEV, YFV, TBEV, MVEV, SLEV Clinical No Domingo C. et al, 2011

Multiplex real‐time RT‐PCR

1‐step

3´‐UTR / 5´‐UTR‐Cap 79 NA NA Clinical No Dumoulin A. et al, 2008

Multiplex RT‐PCR ligase detection

reactionCap/E 76 to 92 0.004 ‐ 0.7

PFU/rxnSLE, MVFV, KUNV, PEV, YFV,

JEV, WNV Clinical No Das S. et al, 2008

Dry format real‐time RT‐PCR 1‐

step

3´‐UTR/ 5´‐UTR‐Cap NA 10 PFU/mL JEV, WNV, YFV, MVFV,

KUNV, SLE Clinical No Wu SJ. et al, 2008

Serotype specific real‐time RT‐PCR

1‐step5´‐UTR‐Cap 65/68/68/73 10 ‐ 100

GE/rxn JEV, WNV, YFV Clinical No Sadon N. et al, 2008

RT‐LAMP 3´‐UTR/ 5´‐UTR‐Cap 199/211/218/229 0.1 ‐ 1

PFU/rxn JEV, WNV, SLE Clinical No Parida M. et al,

TMA NA NA 15 copies/mL NA Clinical No Munoz‐Jordan JL. et al,

2009Multiplex real‐time RT‐PCR 1‐

step3´UTR 74 1 ‐ 0.01

PFU/rxn

JEV, WNV, YFV, CHKV, Leptospira, Plasmodium sp.,

RickettsiaClinical No Gurukumar KR. et al,

2009

Serotype specific real‐time RT‐PCR

1‐step

3´‐UTR/ 5´‐UTR‐Cap

108/150/203/85/113

0.1 ‐ 1 PFU/rxn LGTV, WNV, YFV, LIV, JEV Clinical No Leparc‐Goffart I. et al,

2009


OVERVIEW ON NEW MOLECULAR DIAGNOSIS DEVELOPMENTS

Domingo C. et al, Future Virology, 2011

LESSONS FROM EXTERNAL QUALITY ASSURANCE ACTIVITIESSamples no.

#2 #9 #12 #4 #14 #5 #13 #6 #10 #11 #3 #7

DENV‐1 DENV‐1 DENV‐1 DENV‐1 DENV‐1 DENV‐3 DENV‐3 DENV‐2 DENV‐4 JE/YFWN/TBE CHIK Negative

Copy no. [GE/mL]

Lab. no. RT‐PCR technique 7,0E+05 7,0E+04 7,5E+03 7,0E+02 7,0E+01 3,0E+04 3,0E+03 1,0E+05 1,0E+05 neg. neg. neg. Score* Classification #8 Heminested[17] ++ ++ ++ + + ++ ++ ++ ++ ++ ‐ ‐ ‐ 22 OPTIMAL7 TaqMan[12] ++ ++ ++ ++ (‐) ++ ++ ++ ++ ‐ ‐ ‐ 22 OPTIMAL13 SYBR‐Green[18] ++ ++ ++ ++ (‐) ++ ++ ++ ++ ‐ ‐ ‐ 22 OPTIMAL17a TaqMan[14] ++ ++ ++ ++ (‐) ++ ++ ++ ++ ‐ ‐ ‐ 22 OPTIMAL12 TaqMan[19] ++ ++ ++ + ++ + (‐) ++ ++ ‐ ‐ ‐ 20 IMPROVE21 SYBR‐Greena ++ ++ ++ ++ (‐) ++ ++ ++ ++ (+) ‐ ‐ 20 IMPROVE2a Nested[11] ++ ++ ++ (‐) (‐) ++ ++ ++ ++ ‐ ‐ ‐ 20 OPTIMAL2b TaqMana ++ ++ ++ (‐) (‐) ++ (‐) ++ ++ ‐ ‐ ‐ 18 IMPROVE4b Nested[28] ++ ++ ++ (‐) (‐) ++ (‐) ++ ++ ‐ ‐ ‐ 18 IMPROVE28a Nestedb ++ ++ ++ (‐) (‐) ++ (‐) ++ ++ ‐ ‐ ‐ 18 IMPROVE15 TaqMan[20] ++ ++ (‐) (‐) ++ ++ (‐) ++ ++ ‐ ‐ ‐ 18 IMPROVE5 TaqMan[21] ++ ++ ++ ++ (‐) (‐) (‐) ++ ++ ‐ ‐ ‐ 18 IMPROVE20 TaqMan[21] ++ ++ ++ ++ (‐) (‐) (‐) ++ ++ ‐ ‐ ‐ 18 IMPROVE14 Nested[12] + + + + (‐) ++ ++ ++ + ‐ ‐ ‐ 17 ACCEPTABLE27 Nestedb + ++ ++ ++ (‐) ++ (‐) ++ ++ (+) ‐ ‐ 17 IMPROVE28b TaqMana ++ ++ (‐) (‐) (‐) ++ (‐) ++ ++ ‐ ‐ ‐ 16 IMPROVE29 TaqMan[18] ++ ++ ++ (‐) (‐) ++ (‐) ++ (‐) ‐ ‐ ‐ 16 IMPROVE31 TaqMana + + + + + + + + + ‐ ‐ ‐ 15 ACCEPTABLE23b TaqMana + + (‐) (‐) (‐) ++ (‐) ++ ++ ‐ ‐ ‐ 14 IMPROVE19a Nested[14] ++ ++ (‐) (‐) (‐) ++ (‐) (‐) ++ ‐ ‐ ‐ 14 IMPROVE1 TaqManc ++ ++ (‐) (‐) (‐) + + ++ (‐) ‐ ‐ ‐ 14 IMPROVE36 Nested[28] + + + (‐) (‐) + + + + ‐ ‐ ‐ 13 ACCEPTABLE10 TaqMan[17] + + + (‐) (‐) + + + + ‐ ‐ ‐ 13 ACCEPTABLE19b TaqMan[19] + + + (‐) + + + + (‐) ‐ ‐ ‐ 13 IMPROVE25b Nested[15] ++ + (‐) (‐) (‐) ++ (‐) ++ (‐) ‐ ‐ ‐ 12 IMPROVE9a Nestedb ++ ++ ++ (‐) (‐) ++ ++ ++ (‐) (+) ‐ ‐ 12 IMPROVE9b TaqMana + + + + (‐) + + + + (+) ‐ ‐ 12 IMPROVE22 TaqMan[13] ++ ++ (‐) (‐) (‐) (‐) (‐) (‐) ++ ‐ ‐ ‐ 12 IMPROVE4a TaqMana + + + (‐) (‐) + (‐) + + ‐ ‐ ‐ 12 IMPROVE30 Nested[28] (‐) ++ (‐) ++ ++ (‐) (‐) + (‐) ‐ (+) ‐ 11 IMPROVE17b TaqMan[19] + + (‐) (‐) + (‐) + (‐) (‐) ‐ ‐ ‐ 11 IMPROVE37 TaqMan[22] + + (‐) (‐) (‐) + + + (‐) ‐ ‐ ‐ 11 IMPROVE3 SYBR‐Greena + (‐) (‐) (‐) (‐) + (‐) + + ‐ ‐ ‐ 10 IMPROVE16 TaqManc ++ + (‐) ++ + + + + + (+) (+) (+) 10 IMPROVE18 TaqMan[19] + + (‐) (‐) (‐) (‐) (‐) + + ‐ ‐ ‐ 10 IMPROVE24 RT‐PCRb + + (‐) (‐) (‐) (‐) (‐) + + ‐ ‐ ‐ 10 IMPROVE6 TaqManc + + (‐) (‐) (‐) + (‐) + (‐) ‐ ‐ ‐ 10 IMPROVE25a TaqMan[19] + + (‐) (‐) (‐) + (‐) + + ‐ ‐ ‐ 10 IMPROVE11 Nested[15] ++ ++ (‐) (‐) (‐) + (‐) + (‐) (+) ‐ ‐ 10 IMPROVE35a Nested[12] ++ ++ (‐) (‐) (‐) (‐) (‐) (‐) (‐) ‐ ‐ ‐ 10 IMPROVE34 TaqMan[23] + + (‐) (‐) (‐) (‐) (‐) (‐) + ‐ ‐ ‐ 9 IMPROVE23a SYBR‐Green[29] + (‐) (‐) (‐) (‐) (‐) (‐) + (‐) ‐ ‐ ‐ 8 IMPROVE32 Heminested[17] ++ (‐) (‐) (‐) (‐) (‐) (‐) (‐) (‐) ‐ ‐ ‐ 8 IMPROVE33 TaqMan[19] + + + (‐) (‐) (‐) (‐) + + ‐ ‐ ‐ 8 IMPROVE26 Nestedb (‐) (‐) (‐) (‐) (‐) (‐) (‐) (‐) (‐) ‐ ‐ ‐ 6 IMPROVE35b Nested[16] (‐) (‐) (‐) (‐) (‐) (‐) (‐) (‐) + ‐ ‐ (+) 5 IMPROVE

Correct positive/total results (%) 43/46(93.5)

41/46(89) 23/46 (50)

14/46(30.4) 8/46 (17.4)

32/46 (69.5) 17/46 (37)

38/46 (82.6)

32/46 (69.5) 40/46 (87)

44/46(95.6)

44/46(95.6)

Domingo C et al, PLoSNTD 2010

LESSONS FROM EXTERNAL QUALITY ASSURANCE ACTIVITIES

Domingo C et al, PLoSNTD 2010


LESSONS FROM EXTERNAL QUALITY ASSURANCE ACTIVITIES

ASSAYS TO BE PERFORMED DURING THE COURSE

METHOD REGION AMPLICON SIZE SENSITIVITY SPECIFICITY

RT‐nested PCRDomingo et al, 2011

E/NS1 400/486/416/418 NA JEV, YFV, TBEV, MVEV, SLEV

RT‐heminested PCRScaramozzino et al, 2001

NS5 2505‐ 21.000 GE/rxn Mammalian cell lines

C6/36 cell line

*Dengue RT‐qPCRTiBMolBiol 3´‐UTR ‐ 5 GE/rxn FLAVIVIRUSES

Panflavi RT‐qPCR TiBMolBiol NS5 ‐ 16 flaviviruses

10‐100 GE/rxnCHIK, Sindbis, RVF,

Influenza A


ASSAYS TO BE PERFORMED DURING THE WORKSHOP

METHODS REAGENTS

Dengue RT‐nested PCR

RT‐PCR One Step (QIAGEN)

Panflavi RT‐heminested PCR

Dengue RT‐qPCRTiBMolBiol

Ag‐One Path (AMBION)Panflavi RT‐qPCR

TiBMolBiol


PCR facilities at RKI (Ground floor and 4th floor)Same samples for all the assaysWorking teams of 2 people

ASSAYS TO BE PERFORMED DURING THE WORKSHOP

METHODS REAGENTSDengue RT‐nested PCR

RT‐PCR One Step (QIAGEN)Panflavi RT‐heminested PCR

**Dengue RT‐qPCRTiBMolBiol Ag‐One Path (AMBION)Panflavi RT‐qPCR TiBMolBiol


** RT‐qPCR with excellent sensitivity and specificity, validated with clinicalsamples but not published

Also we recommend the RT‐qPCR of Callahan, 2001 (you will find in your folder the original article). You can order to TiB MolBiol or to your normal provider

TiBMolBiol offers also the possibility of Light Cycler kit for Roche instrumentswith hybridation probes (see package insert in yout folder)


WHO: Dengue guidelines for diagnosis, treatment, prevention, and control, 2010

laboratory diagnosis dengue infections - … diagnosis of dengue infections how should i select the...

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