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
Level 7
– 78% after day 7
• 28% of secondary dengue infections were undiagnosed
when IgM was the only test performed
Level
Level
9
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 :
1. RNA extraction
Serum, csf, tissue & mosquito pools
2. Amplification
3. Detection
Standard
PCR
Agarose gel
Nucleic acid
sequencing:
Southern Blot
TaqMan
RT-PCR
TaqMan probe
ABI
iCycler
Smart Cycler
OPTICAN etc.
SYBR Green
RT-PCR
Melting curve
analysis
NASBA
Lamp
Micro
RT-PCR
Chip
NucliSensTM
Readerr/ECL
Molecular
beacons
Gel
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