LABORATORY DIAGNOSIS OF DENGUE INFECTIONS

Transcription

1 ECDC training Workshop on laboratory diagnosis of dengue virus infections Berlin, January 2012 LABORATORY DIAGNOSIS OF DENGUE INFECTIONS Cristina Domingo Carrasco Robert Koch Institut

2 HOW SHOULD I SELECT THE DIAGNOSIS METHOD TO APPLY IN MY LAB? WHO: Dengue guidelines for diagnosis, treatment, prevention and control (2010)

3 Guzmán MG, Nature Reiews, 2010 Tests with high sensitivity and specificity require more complex technologies and technical expertise

4 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)

5 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 NS1 antigen D. MOLECULAR D. SEROLOGY NS1 antigen D. SEROLOGY MOLECULAR D VIRAL ISOLATION VIRAL ISOLATION Viremia IgM IgG PRIMARY INFECTIONS Viremia IgM IgG SECONDARY INFECTIONS

6 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

7 MOLECULAR DIAGNOSIS 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

8 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

9 OVERVIEW ON AVAILABLE MOLECULAR DIAGNOSIS SYSTEMS Reverse transcriptase PCR (RT PCR) Lanciotti et al, J. Clin. Microbiol, 1992 Harris et al, J. Clin. Microbiol, 1998 C/preM: serotyping by size Nested protocols have an increased sensitivity but have higher risk of carry over contamination Real time RT PCRs rapidity ability to provide quantitative measurements lower contamination rate high sensitivity GOLD STANDARD high specifictiy due to the use of internal probes (not in SYBR Green) easyness of standardization with available commercial master mixes

10 OVERVIEW ON NEW MOLECULAR DIAGNOSIS DEVELOPMENTS Method Region Amplicon size (bp) Sensitivity Specificity# Multiplex RT PCR 1 step Multiplex RT PCR 1 step Multiplex nested RT PCR Multiplex real time RT PCR 1 step Multiplex RT PCR ligase detection reaction Dry format realtime RT PCR 1 step Serotype specific real time RT PCR 1 step RT LAMP Cap prm 482/119/290/398 NS5 109/139/169/ copies/rxn PFU/rxn Clinical/Spiked samples Veterinary samples Reference JEV, WNV, YFV, CHKV Clinical No Saxena P. et al, 2008 JEV, Plasmodium falciparum NA No Maneekan P. et al, 2009 E/NS1 400/486/416/418 NA JEV, YFV, TBEV, MVEV, SLEV Clinical No Domingo C. et al, UTR / 5 UTR Cap Cap/E 76 to 92 3 UTR/ 5 UTR Cap 79 NA NA Clinical No Dumoulin A. et al, 2008 NA 5 UTR Cap 65/68/68/73 3 UTR/ 5 UTR Cap 199/211/218/229 TMA NA NA Multiplex realtime RT PCR 1 step Serotype specific real time RT PCR 1 step 3 UTR 74 3 UTR/ 5 UTR Cap 108/150/203/85/ PFU/rxn 10 PFU/mL GE/rxn PFU/rxn 15 copies/ml PFU/rxn PFU/rxn SLE, MVFV, KUNV, PEV, YFV, JEV, WNV JEV, WNV, YFV, MVFV, KUNV, SLE Clinical No Das S. et al, 2008 Clinical No Wu SJ. et al, 2008 JEV, WNV, YFV Clinical No Sadon N. et al, 2008 JEV, WNV, SLE Clinical No Parida M. et al, NA Clinical No JEV, WNV, YFV, CHKV, Leptospira, Plasmodium sp., Rickettsia Clinical No LGTV, WNV, YFV, LIV, JEV Clinical No Munoz Jordan JL. et al, 2009 Gurukumar KR. et al, 2009 Leparc Goffart I. et al, 2009 Domingo C. et al, Future Virology, 2011

11 LESSONS FROM EXTERNAL QUALITY ASSURANCE ACTIVITIES Samples 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/YF WN/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] OPTIMAL 7 TaqMan[12] ( ) OPTIMAL 13 SYBR Green[18] ( ) OPTIMAL 17a TaqMan[14] ( ) OPTIMAL 12 TaqMan[19] ( ) IMPROVE 21 SYBR Green a ( ) (+) 20 IMPROVE 2a Nested[11] ( ) ( ) OPTIMAL 2b TaqMan a ( ) ( ) ++ ( ) IMPROVE 4b Nested[28] ( ) ( ) ++ ( ) IMPROVE 28a Nested b ( ) ( ) ++ ( ) IMPROVE 15 TaqMan[20] ( ) ( ) ( ) IMPROVE 5 TaqMan[21] ( ) ( ) ( ) IMPROVE 20 TaqMan[21] ( ) ( ) ( ) IMPROVE 14 Nested[12] ( ) ACCEPTABLE 27 Nested b ( ) ++ ( ) (+) 17 IMPROVE 28b TaqMan a ( ) ( ) ( ) ++ ( ) IMPROVE 29 TaqMan[18] ( ) ( ) ++ ( ) ++ ( ) 16 IMPROVE 31 TaqMan a ACCEPTABLE 23b TaqMan a + + ( ) ( ) ( ) ++ ( ) IMPROVE 19a Nested[14] ( ) ( ) ( ) ++ ( ) ( ) IMPROVE 1 TaqMan c ( ) ( ) ( ) ( ) 14 IMPROVE 36 Nested[28] ( ) ( ) ACCEPTABLE 10 TaqMan[17] ( ) ( ) ACCEPTABLE 19b TaqMan[19] ( ) ( ) 13 IMPROVE 25b Nested[15] ++ + ( ) ( ) ( ) ++ ( ) ++ ( ) 12 IMPROVE 9a Nested b ( ) ( ) ( ) (+) 12 IMPROVE 9b TaqMan a ( ) (+) 12 IMPROVE 22 TaqMan[13] ( ) ( ) ( ) ( ) ( ) ( ) IMPROVE 4a TaqMan a ( ) ( ) + ( ) IMPROVE 30 Nested[28] ( ) ++ ( ) ( ) ( ) + ( ) (+) 11 IMPROVE 17b TaqMan[19] + + ( ) ( ) + ( ) + ( ) ( ) 11 IMPROVE 37 TaqMan[22] + + ( ) ( ) ( ) ( ) 11 IMPROVE 3 SYBR Green a + ( ) ( ) ( ) ( ) + ( ) IMPROVE 16 TaqMan c ++ + ( ) (+) (+) (+) 10 IMPROVE 18 TaqMan[19] + + ( ) ( ) ( ) ( ) ( ) IMPROVE 24 RT PCR b + + ( ) ( ) ( ) ( ) ( ) IMPROVE 6 TaqMan c + + ( ) ( ) ( ) + ( ) + ( ) 10 IMPROVE 25a TaqMan[19] + + ( ) ( ) ( ) + ( ) IMPROVE 11 Nested[15] ( ) ( ) ( ) + ( ) + ( ) (+) 10 IMPROVE 35a Nested[12] ( ) ( ) ( ) ( ) ( ) ( ) ( ) 10 IMPROVE 34 TaqMan[23] + + ( ) ( ) ( ) ( ) ( ) ( ) + 9 IMPROVE 23a SYBR Green[29] + ( ) ( ) ( ) ( ) ( ) ( ) + ( ) 8 IMPROVE 32 Heminested[17] ++ ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) 8 IMPROVE 33 TaqMan[19] ( ) ( ) ( ) ( ) IMPROVE 26 Nested b ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) 6 IMPROVE 35b Nested[16] ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) + (+) 5 IMPROVE Correct positive/total results (%) 43/46 41/46 14/46 32/46 38/46 32/46 44/46 44/46 (93.5) (89) 23/46 (50) (30.4) 8/46 (17.4) (69.5) 17/46 (37) (82.6) (69.5) 40/46 (87) (95.6) (95.6) Domingo C et al, PLoSNTD 2010

12 LESSONS FROM EXTERNAL QUALITY ASSURANCE ACTIVITIES LABORATORY DIAGNOSIS OF DENGUE INFECTIONS LESSONS FROM EXTERNAL QUALITY ASSURANCE ACTIVITIES Domingo C et al, PLoSNTD 2010

13 ASSAYS TO BE PERFORMED DURING THE COURSE METHOD REGION AMPLICON SIZE SENSITIVITY SPECIFICITY RT nested PCR Domingo et al, 2011 RT heminested PCR Scaramozzino et al, 2001 E/NS1 400/486/416/418 NA NS GE/rxn JEV, YFV, TBEV, MVEV, SLEV Mammalian cell lines C6/36 cell line *Dengue RT qpcr TiBMolBiol 3 UTR 5 GE/rxn FLAVIVIRUSES Panflavi RT qpcr TiBMolBiol NS5 16 flaviviruses GE/rxn CHIK, Sindbis, RVF, Influenza A

14 ASSAYS TO BE PERFORMED DURING THE WORKSHOP METHODS REAGENTS Dengue RT nested PCR Panflavi RT heminested PCR Dengue RT qpcr TiBMolBiol Panflavi RT qpcr TiBMolBiol RT PCR One Step (QIAGEN) Ag One Path (AMBION) PCR facilities at RKI (Ground floor and 4th floor) Same samples for all the assays Working teams of 2 people

15 ASSAYS TO BE PERFORMED DURING THE WORKSHOP METHODS Dengue RT nested PCR Panflavi RT heminested PCR **Dengue RT qpcr TiBMolBiol Panflavi RT qpcr TiBMolBiol REAGENTS RT PCR One Step (QIAGEN) Ag One Path (AMBION) ** RT qpcr with excellent sensitivity and specificity, validated with clinical samples 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 instruments with hybridation probes (see package insert in yout folder)

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