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.