Laboratory Investigation Dr Salmah Idris Microbiologist Hospital Sungai Buloh

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

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

Immune response

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.

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

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

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

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

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

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

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

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.

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

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

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

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

Detection rate is much better in acute sera of primary infection (75% - 97.3%) when compared to acute sera of secondary infection (60%-70%)

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

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

Collection of sample