Laboratory Investigation

Dr Salmah IdrisMicrobiologist

Hospital Sungai Buloh

ObjectiveObjective

• Diagnostic Test for diagnosis of Diagnostic Test for diagnosis of Dengue Dengue

• Option and indication of the testOption and indication of the test

• Post mortem investigationPost mortem investigation

• Collection of samplesCollection of samples

Diagnostic TestDiagnostic Test

• Antibody detection- SerologyAntibody detection- Serology

• Viral IsolationViral Isolation

• Detection of virus genetic materialDetection of virus genetic material

• Dengue Virus Antigen or proteinDengue Virus Antigen or protein

Immune response

Dengue IgM testDengue IgM test

• IgM capture enzyme-linked immunosorbent assay IgM capture enzyme-linked immunosorbent assay (ELISA)(ELISA)

• Widely usedWidely used

• IgM titre is higher in primary compared to IgM titre is higher in primary compared to secondary infectionsecondary infection

• Peak at about 2 weeks and undetectable level by Peak at about 2 weeks and undetectable level by 60 – 90 days.60 – 90 days.

Dengue IgM captured ELISADengue IgM captured ELISA

• Primary DenguePrimary Dengue– 55% detectable between 4-7 days55% detectable between 4-7 days– 100% after day 7100% after day 7

• Secondary Secondary – 78% after day 778% after day 7

• 28% of secondary dengue infections were 28% of secondary dengue infections were undiagnosed when IgM was the only test undiagnosed when IgM was the only test

performedperformed

Level 7

Level 9

Level 8

Level 9

Level 8

Indirect IgG ELISAIndirect IgG ELISA

• Primary and secondary denguePrimary and secondary dengue– IgG was detected in 100% after day 7 of IgG was detected in 100% after day 7 of

illnessillness– Recommended if dengue IgM is still Recommended if dengue IgM is still

negative after day 7 in secondary negative after day 7 in secondary dengue dengue

Level 7

Level 8

Level 8

Haemagglutination Inhibition Test Haemagglutination Inhibition Test (HI)(HI)• Gold standardGold standard

• Labour intensive and gold standardLabour intensive and gold standard

• Paired samples for interpretation (Acute and Paired samples for interpretation (Acute and convelescant samples – 2 weeks apart) convelescant samples – 2 weeks apart)

• Research purposesResearch purposes

• Differentiate between primary and secondary Differentiate between primary and secondary dengue infectiondengue infection

Dengue rapid testDengue rapid test

• ImmunochromatographyImmunochromatography

• Qualitative detection of IgM and IgGQualitative detection of IgM and IgG

• Yield is higher when samples were Yield is higher when samples were collected later in the convalescent phase collected later in the convalescent phase of infectionof infection

• Good specificity and could be used when Good specificity and could be used when ELISA facilities were not availableELISA facilities were not available

Dengue Rapid testDengue Rapid test

• Recommended - Dengue IgM Capture ELISA after Recommended - Dengue IgM Capture ELISA after a rapid testa rapid test

• Result had to interpreted in the clinical context Result had to interpreted in the clinical context because of false positive or negative because of false positive or negative

Dengue Rapid testDengue Rapid test

• False positiveFalse positive– Cross react withCross react with

•Flavivirus- JEFlavivirus- JE@@

•Non-flavivirus – malaria, leptospirosis, Non-flavivirus – malaria, leptospirosis, toxoplasmosis, syphilistoxoplasmosis, syphilis**

•Connective Tissue Disorder – RAConnective Tissue Disorder – RA##

@ *

#

Virus Isolation

• Definitive test for dengue infectionDefinitive test for dengue infection• Lab equipped with tissue culture facilitiesLab equipped with tissue culture facilities• Useful only at early phase of illness , blood Useful only at early phase of illness , blood

collected before day 5 of illness ( before collected before day 5 of illness ( before the formation of Neutralizing antibodies)the formation of Neutralizing antibodies)

• During febrile illnessDuring febrile illness– Virus can be isolated from serum, plasma and Virus can be isolated from serum, plasma and

leucocytesleucocytes– Post mortem specimensPost mortem specimens

• Expensive and time consuming (2 weeks Expensive and time consuming (2 weeks to completeto complete

Virus Isolation

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

Polymerase Chain Reaction (PCR) (PCR)

• Eg. RT-PCR Eg. RT-PCR • Diagnosis of dengue infection in the early phase (< 5 Diagnosis of dengue infection in the early phase (< 5

days of illness)days of illness)• Sensitivity 100% in the first 5 days of illness, reduced Sensitivity 100% in the first 5 days of illness, reduced

to 70% by day 6to 70% by day 6

• Determine dengue serotypeDetermine dengue serotype

Overview of 3 Steps Used in Molecular Amplification Assays for Flaviviruses

1. RNA extraction RNA extraction from :

Serum, csf, tissue & mosquito pools

2. Amplification Standard PCR

TaqMan RT-PCR

SYBR Green RT-PCR

NASBA

3. Detection Agarose gel

Nucleic acid sequencing:

Southern Blot

TaqMan probe ABI iCycler Smart Cycler OPTICAN etc.

Melting curve analysis

NucliSensTM Readerr/ECL Molecular beacons

Adapted from Lanciotti, 2003; Adv Virus Res 61:67-99

LampMicroRT-PCRChip

TurbidometerReal Time

Gel

Polymerase Chain Reaction (PCR) (PCR)

• LimitationLimitation– Available in certain facilities and trained Available in certain facilities and trained

personalpersonal– Test is expensiveTest is expensive– Specimens requires special storage Specimens requires special storage

temperatures and short transportation , time temperatures and short transportation , time between collection and extractionbetween collection and extraction

• Use only be considered for in-patients who Use only be considered for in-patients who present with diagnostic challenges in the present with diagnostic challenges in the early phase of illnessearly phase of illness

Non-structural protein -1 (NS1 Ag)Non-structural protein -1 (NS1 Ag)

• Highly conserved glycoprotein that seems Highly conserved glycoprotein that seems to be essential for virus viabilityto be essential for virus viability

• Secretion of the NS1 protein is a hallmark Secretion of the NS1 protein is a hallmark of flavivirus infecting mammalian cellsof flavivirus infecting mammalian cells– Dengue infection, yellow fever and West NileDengue infection, yellow fever and West Nile

• High concentration in the sera of dengue High concentration in the sera of dengue infected patients during the early phase of infected patients during the early phase of the disease the disease

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

NS1 AntigenNS1 Antigen

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

Investigation of Post Mortem CaseInvestigation of Post Mortem Case

Caution: Massive blood transfusion may affect the test result mentioned above

Collection of sampleCollection of sample