LABORATORY DIAGNOSIS DENGUE INFECTIONS - … DIAGNOSIS OF DENGUE INFECTIONS HOW SHOULD I SELECT THE...
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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)
LABORATORY DIAGNOSIS OF DENGUE INFECTIONS
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…
LABORATORY DIAGNOSIS OF DENGUE INFECTIONS
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…
LABORATORY DIAGNOSIS OF DENGUE INFECTIONS
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
LABORATORY DIAGNOSIS OF DENGUE INFECTIONS
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
LABORATORY DIAGNOSIS OF DENGUE INFECTIONS
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
LABORATORY DIAGNOSIS OF DENGUE INFECTIONS
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
LABORATORY DIAGNOSIS OF DENGUE INFECTIONS
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
LABORATORY DIAGNOSIS OF DENGUE INFECTIONS
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
LABORATORY DIAGNOSIS OF DENGUE INFECTIONS
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
LABORATORY DIAGNOSIS OF DENGUE INFECTIONS
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
LABORATORY DIAGNOSIS OF DENGUE INFECTIONS
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
LABORATORY DIAGNOSIS OF DENGUE INFECTIONS
** 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)
LABORATORY DIAGNOSIS OF DENGUE INFECTIONS
WHO: Dengue guidelines for diagnosis, treatment, prevention, and control, 2010