SEROLOGICAL DIAGNOSIS OF DENGUE INFECTIONS

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

2 FACILITIES Neutralization assays must be performed in BSL 3 facilities BSL 2 facilities can be used when using inactivated samples SAMPLES Positive single IgM results must be confirmed with a second sample to demonstrate seroconversion or with a background assay It is better to assay the samples in duplicate

3 CONTROLS Suitable controls must be included in the assay: Commercial assays: positive control, negative control, and calibrators normally provided by the manufacturer Do not interchange controls from different batch kits In house assays: validated negative and positive sera must be included in each assay NT assays: negative serum, positive serum of known NT titer, cell control, and back titration control of the virus in each assay QUALITY ASSESMENT Each in house assay must be evaluated in terms of sensitivity and specificity in the laboratory before use for diagnosis Commercial assays have gone through extensive standardiyation process The labs should participate in QA activities regularly

4 ANTIGEN DETECTION IN THE DIAGNOSIS OF DENGUE Dengue antigens can be detected in tissues from fatal cases by specific mab NS1 detection is a potential means for the early diagnosis of dengue infection It does not allow serotype identification ANTIGEN CAPTURE ELISA Thepresenceof anti DENV antibodies, mostly IgG could diminish the sensitivity of the assay!!!!!

5 ANTIBODY DETECTION IN THE DIAGNOSIS OF DENGUE Mostcommonlyusedstrategyin the diagnosis of dengue Inexpensive Easy to perform Rather complicated to interpret: o Presence of multiple or sequential infections with the 4 dengue serotypes o Presence of antibodies against other flaviviruses due to previous infections or vaccinations o Cross reactivity against homologous and heterologous flavivirus antigens o Serodiagnosis identification of past, recent and acute dengue infection with long lasting IgG antibodies

6 FLAVIVIRUS SEROLOGICAL CROSS REACTIONS Tick borne encephalitis TBEV Japanese encephalitis JEV Yellow fever YFV West Nile fever WNV Dengue fever 1 4 DENV

7 FLAVIVIRUS SEROLOGICAL CROSS REACTIONS

8 DENGUE SEROLOGICAL DIAGNOSIS METHODOLOGIES LABORATORY DIAGNOSIS OF DENGUE INFECTIONS NEUTRALIZING ANTIBODIES DETECTION IN THE DIAGNOSIS OF DENGUE It is considered confirmatory of a dengue infection Theoretically can define the infecting serotype in primary infections The test is laborious and time consuming RequiresBSL 3 facilities PLAQUE REDUCTION NEUTRALIZATION ASSAY (PRNT) MICRONEUTRALIZATION ASSAY

9 IgM DETECTION IN THE DIAGNOSIS OF DENGUE Antibodies develop after 3 5 days in primoinfections in 50% patients Antibodies are present in the sera for 2 3months Less cross reactive with other flavivirus than IgG Specific anti dengue IgG and RF can give false negative/positive results False positives in autoimmune diseases Background assay, capture assays or use of RF absorbent IgM CAPTURE ELISA (MAC ELISA) Sensitivity 90% Specificity 98% UMELISA Anti chain antibody

10 DENGUE SEROLOGICAL DIAGNOSIS METHODOLOGIES LABORATORY DIAGNOSIS OF DENGUE INFECTIONS IgG DETECTION IN THE DIAGNOSIS OF DENGUE Indirect IgG ELISA Confirmation of seroconversion in paired sera Classification of primary vs secondary infections Lacks specificity with the flavivirus serocomplex Useful for sero epidemiological studies Identification of past dengue infection It is available a capture IgG ELISA for detection of secondary cases

11 LESSONS FROM EXTERNAL QUALITY ASSURANCE ACTIVITIES IgM results IgG results IgM 58% correct positive results/ IgG 71% correct positive results IgM 97% correct negative results/ IgG 89% correct negative results No variance in the performance of in house and commercial tests IIF showed lower sensitivity than ELISA in IgM detection Donoso Mantke O et al, JCV 2003

12 RAPID IMMUNOCRHOMATOGRAPHIC TESTS Easy to perform Expensive Point of care diagnostics Compromise between sensitivity/specificity NS1 antigen detection IgM/IgG detection BioRad PanBio Best results according to WHO SD WHO/TDR/PDVI laboratory network found that ELISA generally performs better than rapid tests

13 ASSAYS TO BE PERFORMED DURING THE WORKSHOP METHODS REAGENTS IgM capture ELISA IgG indirect ELISA PanBio NS1 antigen ELISA NS1 Antigen detection Rapid test IgM/IgG Duo detection Rapid test Dengue 1 4 Mosaic IIF Dx reagents (FOCUS Technologies) EUROIMMUN BSL 2 facilities Same samples for IgM and IgG assays Same samples for rapid test Independent samples fo serotyping Work individually

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