Laboratory Investigation

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
8
(No Transcript)
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_at_
• Non-flavivirus malaria, leptospirosis,
  toxoplasmosis, syphilis
• Connective Tissue Disorder RA

_at_


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
  (lt 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
6167-99
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 - 97.3) 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

21
(No Transcript)
22
Investigation of Post Mortem Case
Caution Massive blood transfusion may affect the
test result mentioned above
23
Collection of sample
24
(No Transcript)
25
(No Transcript)