1. Laboratory Investigation
Dr Salmah Idris
Microbiologist
Hospital Sungai Buloh

2. Objective Diagnostic Test for diagnosis of Dengue
Option and indication of the test
Post mortem investigation
Collection of samples

3. Diagnostic Test Antibody detection- Serology Viral Isolation
Detection of virus genetic material
Dengue Virus Antigen or protein

4. Immune response

5. Dengue IgM test IgM capture enzyme-linked immunosorbent assay (ELISA)
Widely used
IgM titre is higher in primary compared to secondary infection
Peak at about 2 weeks and undetectable level by 60 – 90 days.

6. Dengue IgM captured ELISA
Primary Dengue
55% detectable between 4-7 days
100% after day 7
Secondary
78% after day 7
28% of secondary dengue infections were undiagnosed when IgM was the only test performed
Level 7
Level 9
Level 9
Level 8
Level 8

7. Indirect IgG ELISA Primary and secondary dengue
IgG was detected in 100% after day 7 of illness
Recommended if dengue IgM is still negative after day 7 in secondary dengue
Level 7
Level 8
Level 8

9. Haemagglutination Inhibition Test (HI)
Gold standard
Labour intensive and gold standard
Paired samples for interpretation (Acute and convelescant samples – 2 weeks apart)
Research purposes
Differentiate between primary and secondary dengue infection

10. Dengue rapid test Immunochromatography
Qualitative detection of IgM and IgG
Yield is higher when samples were collected later in the convalescent phase of infection
Good specificity and could be used when ELISA facilities were not available

11. Dengue Rapid test
Recommended - Dengue IgM Capture ELISA after a rapid test
Result had to interpreted in the clinical context because of false positive or negative

12. Dengue Rapid test False positive Cross react with Flavivirus- JE@
Non-flavivirus – malaria, leptospirosis, toxoplasmosis, syphilis*
Connective Tissue Disorder – RA# @ * #

13. Virus Isolation Definitive test for dengue infection
Lab equipped with tissue culture facilities
Useful only at early phase of illness , blood collected before day 5 of illness ( before the formation of Neutralizing antibodies)
During febrile illness
Virus can be isolated from serum, plasma and leucocytes
Post mortem specimens
Expensive and time consuming (2 weeks to complete

14. Virus Isolation
Virus isolation has a poor yield if compared with molecular test. IT most probably due to the viability of the virus and the quality of samples.

15. Polymerase Chain Reaction (PCR)
Eg. RT-PCR
Diagnosis of dengue infection in the early phase (< 5 days of illness)
Sensitivity 100% in the first 5 days of illness, reduced to 70% by day 6
Determine dengue serotype

16. Overview of 3 Steps Used in Molecular Amplification Assays for Flaviviruses
RNA extraction from :
Serum, csf, tissue & mosquito pools
1. RNA extraction
Micro
RT-PCR
Chip
2. Amplification
Standard PCR
TaqMan RT-PCR
SYBR Green RT-PCR
NASBA
Lamp
3. Detection
Agarose gel
Melting curve analysis
NucliSensTM Readerr/ECL Molecular beacons
TaqMan probe
ABI
iCycler
Smart Cycler
OPTICAN etc.
Gel
Nucleic acid sequencing:
Southern Blot
Turbidometer
Real Time
Adapted from Lanciotti, 2003; Adv Virus Res 61:67-99

17. Polymerase Chain Reaction (PCR)
Limitation
Available in certain facilities and trained personal
Test is expensive
Specimens requires special storage temperatures and short transportation , time between collection and extraction
Use only be considered for in-patients who present with diagnostic challenges in the early phase of illness

18. Non-structural protein -1 (NS1 Ag)
Highly conserved glycoprotein that seems to be essential for virus viability
Secretion of the NS1 protein is a hallmark of flavivirus infecting mammalian cells
Dengue infection, yellow fever and West Nile
High concentration in the sera of dengue infected patients during the early phase of the disease

19. Detection rate is much better in acute sera of primary infection (75% %) when compared to acute sera of secondary infection (60%-70%)

20. NS1 Antigen
The sensitivity drops from day 4-5 of illness and usually becomes undetectable in the convalescence phase

22. Investigation of Post Mortem Case
Caution: Massive blood transfusion may affect the test result mentioned above

23. Collection of sample