laboratory investigation of dengue in jeddah
TRANSCRIPT
Laboratory Diagnosis of Dengue Hemorrhagic Fever
Dr. Hosam H. A. MadaniMBBS King Abdul-Aziz University 1987Dip Bact Manchester University 1992MsC Virology London university 1995
Jeddah Regional Laboratory.
Dengue Virus
Arthropod – borne. Genus: Flavivirus. Family: Flavivirdae. Virions: spherical, 40-50 nm Diameter, inner core
( C), lipid envelope with glycoprotein polymers (E), and membrane (M).
Genome: infectious,11kb ssRNA, +ve polarity Virus is inactivated by heat and disinfectant,
containing detergent or lipid solvent.
Laboratory Diagnosis of Dengue
Virus Isolation and Culture Serological :
Elisa. Haemagglutination-Inhibition Titers. Complement-Fixation test.
RT-PCR. Antigen Detection. Neutralization Tests. Dot-Blot Immunoassay.
DNA Array
Jeddah Regional Laboratory
Elisa : IgM. IgG.
RT-PCR: Screening, for samples taken within 5-7 days from
appearance of symptoms. Typing for dengue RNA detected samples.
Virus isolation
Gold Standard for most viral infection. Sample:
Serum. Plasma. Anticoaggulated whole blood (leukocyte culture). CSF. Tissues from dead patients (autopsy). Mosquito.
Virus Isolation
Limitation: During Viraemia only (early 5 days). Slow results (Days) vs. PCR (Hours). Expensive to maintain the technique. Strict storage and transportation needs.
Heat-labile. Refrigerated (4-8C) or wet ice for 24 hours. Frozen at - 70 C, or more, for longer storage. Never ever freeze in ordinary Freezer (-20C).
Virus isolation
Inoculation of samples into: Mosquitos (head, squashed).
Antigen detection by immunofluresence. Cell culture lines;
Vertebrate cell-line: Vero, LLC-MK Insect cell-line: C6/36,AP61. Antigen detection by antibody staining. Cytopathic effect. Plaque formation.
Suckling mice (intracranial) ; S & S of encephalitis. Antigen detection by antibody staining. Culture.
Antigen Detection in tissues
Fluorescent antibody. Immunoperoxidase. Avin-biotin enzyme assay.
Haemagglutination-Inhibition test
Advantage: Simple. Sensitive. Cheap and easily prepared reagents.
Dissadvantage: Pretreatment of samples by acetone or kaolin to remove
inhibitors. Absorption in Gander or type O human blood to remove
non specific agglutinin. Paired sera must be used. Do not discriminate between other flaviviruses. known positive and negative samples should be used.
Complement fixation test
Less sensitive Complement- fixing antibodies appear latter
than IgM or HI antibodies. More specific. Not used anymore.
Neutralization test
Serum dilution, virus-constant, plaque- reduction test.
Neutralizing antibodies can be detected in early convalescence in primary infection.
Two or more of Dengue types, and other flaviviruses, can be found in case of secondary infection, with highest titer for the older infection.
Dot-blot immunoassay
New Chipron DNA array.
IgM Elisa
Primary Infection: 3-5 days after the onset of infection Few months (3-5month) up to 8month Some cases not detected for 7-10 days
Secondary Infection May be not detected or Low level in 70%.
IgG Elisa
Primary Infection: Few weeks after the onset of infection. Long lasting.
Secondary Infection Very high levels can be Detected 2 days of onset of infection Some cases not detected for 7-10 days.
IgM / IgG Elisa
Principles of the test: 96 wells (8x12 stripes) coated with dengue Antigen. Patient antibodies bind with the coated antigen. Conjugate, substrate, Optical Density (OD) > cutoff = POSITIVE Antibody Index (AI) = OD of sample / cutoff.
Sample: Serum stored 2-8 C for 48 hours -20 C for 1 year Icteric, Haemolysed, lipaemic sera are not suitable.
IgM/IgG Elisa
Cross Reaction: Across the flavivius group
Murray Valley encephalitis Japanese encephalitis St.Louis encephalitis Yellow fever West Nile
Malaria
Rheumatoid Factor
False positive with Hetrophilic antibodies (animal IgG)
IgM / IgG Elisa
Positive Elisa result does not mean the patient is mandatory suffering from Dengue Infection.
Elisa result should only be considered in association with the Clinical picture.
Cutoff should be adjusted in each country.
IgM Elisa in JRL
Pathozyme DENGUE M (Omega Diagnostics) Enzyme-immunoassay (EIA) for the detection of IgM antibodies to Dengue in Human serum.
Welles are coated with purified dengue type 2 antigen. IgG is allowed to be absorbed from samples before testing. Sensitivity = 98.11 % Specificity = 89.47 %
Dengue IgM Capture elisa: (Panbio diagnostics):
Wells are coated by anti-human IgM antibodies. Dengue 1-4 Antigen + conjugated monoclonal antibodies = antigen-MAb complex Sandwich of anti-human IgM antibody/Serum Antibody/antigen-MAb complex
Sensitivity (pry): 85.4 – 98.9% Sensitivity (Sec): 46.6 – 64.7% Specificity : 95.7 – 100 %
IgG Elisa in JRL
Pathozyme DENGUE G (Omega Diagnostics) Enzyme-immunoassay (EIA) for the detection of IgG antibodies to Dengue in Human serum.
Welles are coated with purified dengue type 2 antigen. AI =1-2 suggest Primary infection AI > 2 suggest Secondary infection Sensitivity = 98.11 % Specificity = 92.1 %
Dengue IgM Capture Elisa: (Panbio Diagnostics):
For the Detection of Secondary dengue Infection
Wells are coated by anti-human IgG antibodies. Dengue 1-4 Antigen + conjugated monoclonal antibodies = antigen-MAb complex Sandwich of anti-human IgG antibody/Serum Antibody/antigen-MAb complex AI > 2.2 (or 22 panbio units) suggest Secondary infection
Sensitivity (Secondary): 73.8 – 93.6% Specificity (Primary): 83.8 – 98.6 % Specificity (Negative): 88.4 - 100 %
IgG in Secondary infection
Paired sera (minimum 7days)
Rising titers (four folds)
Haemagglutination-Inhibition Titers: >1:2560 for acute sample. > 2.2 AI (or 22 panbio units). >2 AI by: Omega.
Why PCR?
To cover the Window period before IgM can be detected in Primary infection.
Usually, RNA can not be detected after the formation of the antibodies.
To confirm Secondary infection when IgM level is low or not detected.
To detect the four types of dengue.
Molecular Diagnostics of dengue RNA extraction. Reverse transcription.
RNA to cDNA
Polymerase Chain reaction (PCR) Amplification, (Hybridization).
Target sequences amplified into millions copies Repeated Heating and cooling cycles.
Detection. Probes are used to detect presence of dengue RNA.
RNA extraction
50-100 ul of RNA, is extracted from Patients serum before PCR examination. Manual extraction:
RNA is extracted by the use of any manual commercial kits.
High Pure Viral RNA Kit from Roche. QIAamp Viral RNA mini kits from Qiagen.
Automated extraction: MagNA pure LC RNA Isolation Kit
RT-PCR
Instrument used in JRL
LightCycler instrument (Roche)
Screening for samples within 5-7 days of appearance of symptoms.
Typing of dengue RNA detected samples.
One step- RT-PCR
Artus dengue LC PCR Kit. (very Expensive)
Tib-molbiol TaqMan probes used with:
LightCycler RNA Amplification Kit HybProbe.
(Roche)
Probes for dengue Screening
TIB- Molbiol (germany). Den S2:
Forward primer.
Den AS1-3 and Den As4: Reverse primer.
Den P1: Taqman probes.
1
2
3
4
Probes for dengue typing
D 4 TMD 3 TM
Den 4 RDen 3 R
Den 4 sDen 3 s
Type 4:Type 3:
D 2 TMD 1 TM
Den 2 RDen 1 R
Den 2 sDen 1 s
Type 2:Type 1:
Positive Dengue in JRL
0
50
100
150
200
250
300
350
400
Zul H
eja 14
26
Muh
aram
14
27
Saf
ar 14
27
Rab
ie I 1
427
Rab
ieII 1
427
Jum
ad I 1
427
Jum
ad II
1427
Rajab
1427
Shab
an 142
7
Ram
adan 142
7
Sha
wal 142
7
PCR
IgM
Total
Percentage of Positive results
59.40151446.80119312.593212549Total
20.971320.97130.00062Shawal 1427
37.042033.33183.70254Ramadan 1427
38.892838.89280.00072Shaban 1427
22.641222.64120.00053Rajab 1427
55.424634.942920.481783Jumad II 1427
69.8925345.0316324.8690362Jumad I 1427
57.9533948.032819.9158585RabieII 1427
56.6727246.2522210.4250480Rabie I 1427
66.1429551.5723014.5765446Safar 1427
68.7514360.101258.6518208Muharam 1427
64.589350.007214.5821144Zul Heja 1426
%Total%IgM%PCR SusbectedDate
Reference
Thomas Laue et al. Detection of Dengue Virus RNA in Patients after Primary or Secondary Dengue Infection by using the TaqMan Automated Amplification System.. J Clin microbiol,Aug.1999;2543-7.
Drosten C et al. Rapid detection and quantification of RNA of Ebola and Marburge viruses, Lassa virus and, yellow fever virus by real-time reverse transcription-PCR. J Clin Microbiol 2002; 40: 2323-30.
World health organization Guide: Dengue haemorrhagic fever, diagnosis, treatment,
prevention and control.1997.