Laboratory investigation of dengue in Jeddah

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Laboratory investigation of dengue in Jeddah

  1. 1. Laboratory Diagnosis of Dengue Hemorrhagic Fever Dr. Hosam H. A. Madani MBBS King Abdul-Aziz University 1987 Dip Bact Manchester University 1992 MsC Virology London university 1995 Jeddah Regional Laboratory.
  2. 2. Dengue Virus <ul><li>Arthropod – borne. </li></ul><ul><li>Genus: Flavivirus . </li></ul><ul><li>Family: Flavivirdae . </li></ul><ul><li>Virions: spherical, 40-50 nm Diameter, inner core ( C), lipid envelope with glycoprotein polymers (E), and membrane (M). </li></ul><ul><li>Genome: infectious,11kb ssRNA, +ve polarity </li></ul><ul><li>Virus is inactivated by heat and disinfectant, containing detergent or lipid solvent. </li></ul>
  3. 3. Laboratory Diagnosis of Dengue <ul><li>Virus Isolation and Culture </li></ul><ul><li>Serological : </li></ul><ul><ul><li>Elisa. </li></ul></ul><ul><ul><li>Haemagglutination-Inhibition Titers. </li></ul></ul><ul><ul><li>Complement-Fixation test. </li></ul></ul><ul><li>RT-PCR. </li></ul><ul><li>Antigen Detection. </li></ul><ul><li>Neutralization Tests. </li></ul><ul><li>Dot-Blot Immunoassay. </li></ul><ul><ul><li>DNA Array </li></ul></ul>
  4. 4. Jeddah Regional Laboratory <ul><li>Elisa : </li></ul><ul><ul><li>IgM. </li></ul></ul><ul><ul><li>IgG. </li></ul></ul><ul><li>RT-PCR: </li></ul><ul><ul><li>Screening, for samples taken within 5-7 days from appearance of symptoms. </li></ul></ul><ul><ul><li>Typing for dengue RNA detected samples. </li></ul></ul>
  5. 5. Virus isolation <ul><li>Gold Standard for most viral infection. </li></ul><ul><li>Sample: </li></ul><ul><ul><li>Serum. </li></ul></ul><ul><ul><li>Plasma. </li></ul></ul><ul><ul><li>Anticoaggulated whole blood (leukocyte culture). </li></ul></ul><ul><ul><li>CSF. </li></ul></ul><ul><ul><li>Tissues from dead patients (autopsy). </li></ul></ul><ul><ul><li>Mosquito. </li></ul></ul>
  6. 6. Virus Isolation <ul><li>Limitation: </li></ul><ul><ul><li>During Viraemia only (early 5 days). </li></ul></ul><ul><ul><li>Slow results (Days) vs. PCR (Hours). </li></ul></ul><ul><ul><li>Expensive to maintain the technique. </li></ul></ul><ul><ul><li>Strict storage and transportation needs. </li></ul></ul><ul><ul><ul><li>Heat-labile. </li></ul></ul></ul><ul><ul><ul><li>Refrigerated (4-8C) or wet ice for 24 hours. </li></ul></ul></ul><ul><ul><ul><li>Frozen at - 70 C, or more, for longer storage. </li></ul></ul></ul><ul><ul><ul><li>Never ever freeze in ordinary Freezer (-20C). </li></ul></ul></ul>
  7. 7. Virus isolation <ul><li>Inoculation of samples into: </li></ul><ul><ul><li>Mosquitos (head, squashed). </li></ul></ul><ul><ul><ul><li>Antigen detection by immunofluresence. </li></ul></ul></ul><ul><ul><li>Cell culture lines; </li></ul></ul><ul><ul><ul><li>Vertebrate cell-line: Vero, LLC-MK </li></ul></ul></ul><ul><ul><ul><li>Insect cell-line: C6/36,AP61. </li></ul></ul></ul><ul><ul><ul><li>Antigen detection by antibody staining. </li></ul></ul></ul><ul><ul><ul><li>Cytopathic effect. </li></ul></ul></ul><ul><ul><ul><li>Plaque formation. </li></ul></ul></ul><ul><ul><li>Suckling mice (intracranial) ; </li></ul></ul><ul><ul><ul><li>S & S of encephalitis. </li></ul></ul></ul><ul><ul><ul><li>Antigen detection by antibody staining. </li></ul></ul></ul><ul><ul><ul><li>Culture. </li></ul></ul></ul>
  8. 8. Antigen Detection in tissues <ul><li>Fluorescent antibody. </li></ul><ul><li>Immunoperoxidase. </li></ul><ul><li>Avin-biotin enzyme assay. </li></ul>
  9. 9. Haemagglutination-Inhibition test <ul><li>Advantage: </li></ul><ul><ul><li>Simple. </li></ul></ul><ul><ul><li>Sensitive. </li></ul></ul><ul><ul><li>Cheap and easily prepared reagents. </li></ul></ul><ul><li>Dissadvantage: </li></ul><ul><ul><li>Pretreatment of samples by acetone or kaolin to remove inhibitors. </li></ul></ul><ul><ul><li>Absorption in Gander or type O human blood to remove non specific agglutinin. </li></ul></ul><ul><ul><li>Paired sera must be used. </li></ul></ul><ul><ul><li>Do not discriminate between other flaviviruses. </li></ul></ul><ul><ul><li>known positive and negative samples should be used. </li></ul></ul>
  10. 10. Complement fixation test <ul><li>Less sensitive </li></ul><ul><li>Complement- fixing antibodies appear latter than IgM or HI antibodies. </li></ul><ul><li>More specific. </li></ul><ul><li>Not used anymore. </li></ul>
  11. 11. Neutralization test <ul><li>Serum dilution, virus-constant, plaque- reduction test. </li></ul><ul><li>Neutralizing antibodies can be detected in early convalescence in primary infection. </li></ul><ul><li>Two or more of Dengue types, and other flaviviruses, can be found in case of secondary infection, with highest titer for the older infection. </li></ul>
  12. 12. Dot-blot immunoassay <ul><li>New </li></ul><ul><li>Chipron DNA array. </li></ul>
  13. 13. IgM Elisa <ul><li>Primary Infection: </li></ul><ul><ul><li>3-5 days after the onset of infection </li></ul></ul><ul><ul><li>Few months (3-5month) up to 8month </li></ul></ul><ul><ul><li>Some cases not detected for 7-10 days </li></ul></ul><ul><li>Secondary Infection </li></ul><ul><ul><li>May be not detected </li></ul></ul><ul><ul><li>or Low level in 70%. </li></ul></ul>
  14. 14. IgG Elisa <ul><li>Primary Infection: </li></ul><ul><ul><li>Few weeks after the onset of infection. </li></ul></ul><ul><ul><li>Long lasting. </li></ul></ul><ul><li>Secondary Infection </li></ul><ul><ul><li>Very high levels can be Detected </li></ul></ul><ul><ul><li>2 days of onset of infection </li></ul></ul><ul><ul><li>Some cases not detected for 7-10 days. </li></ul></ul>
  15. 15. IgM / IgG Elisa <ul><li>Principles of the test : </li></ul><ul><ul><li>96 wells (8x12 stripes) coated with dengue Antigen. </li></ul></ul><ul><ul><li>Patient antibodies bind with the coated antigen. </li></ul></ul><ul><ul><li>Conjugate, substrate, </li></ul></ul><ul><ul><li>Optical Density (OD) > cutoff = POSITIVE </li></ul></ul><ul><ul><li>Antibody Index (AI) = OD of sample / cutoff. </li></ul></ul><ul><li>Sample: </li></ul><ul><ul><li>Serum stored 2-8 C for 48 hours </li></ul></ul><ul><ul><li>-20 C for 1 year </li></ul></ul><ul><ul><li>Icteric, Haemolysed, lipaemic sera are not suitable. </li></ul></ul>
  16. 16. IgM/IgG Elisa <ul><li>Cross Reaction: </li></ul><ul><ul><li>Across the flavivius group </li></ul></ul><ul><ul><ul><li>Murray Valley encephalitis </li></ul></ul></ul><ul><ul><ul><li>Japanese encephalitis </li></ul></ul></ul><ul><ul><ul><li>St.Louis encephalitis </li></ul></ul></ul><ul><ul><ul><li>Yellow fever </li></ul></ul></ul><ul><ul><ul><li>West Nile </li></ul></ul></ul><ul><ul><li>Malaria </li></ul></ul><ul><ul><li>Rheumatoid Factor </li></ul></ul><ul><ul><li>False positive with Hetrophilic antibodies (animal IgG) </li></ul></ul>
  17. 17. IgM / IgG Elisa <ul><li>Positive Elisa result does not mean the patient is mandatory suffering from Dengue Infection. </li></ul><ul><li>Elisa result should only be considered in association with the Clinical picture. </li></ul><ul><li>Cutoff should be adjusted in each country. </li></ul>
  18. 18. IgM Elisa in JRL <ul><li>Pathozyme DENGUE M (Omega Diagnostics ) </li></ul><ul><li>Enzyme-immunoassay (EIA) for the detection of IgM antibodies to Dengue in Human serum . </li></ul><ul><ul><li>Welles are coated with purified dengue type 2 antigen. </li></ul></ul><ul><ul><li>IgG is allowed to be absorbed from samples before testing. </li></ul></ul><ul><ul><li>Sensitivity = 98.11 % </li></ul></ul><ul><ul><li>Specificity = 89.47 % </li></ul></ul><ul><li>Dengue IgM Capture elisa : (Panbio diagnostics ): </li></ul><ul><ul><li>Wells are coated by anti-human IgM antibodies. </li></ul></ul><ul><ul><li>Dengue 1-4 Antigen + conjugated monoclonal antibodies = antigen-MAb complex </li></ul></ul><ul><ul><li>Sandwich of anti-human IgM antibody/ Serum Antibody /antigen-MAb complex </li></ul></ul><ul><ul><li>Sensitivity (pry): 85.4 – 98.9% </li></ul></ul><ul><ul><li>Sensitivity (Sec): 46.6 – 64.7% </li></ul></ul><ul><ul><li>Specificity : 95.7 – 100 % </li></ul></ul>
  19. 19. IgG Elisa in JRL <ul><li>Pathozyme DENGUE G (Omega Diagnostics ) </li></ul><ul><li>Enzyme-immunoassay (EIA) for the detection of IgG antibodies to Dengue in Human serum . </li></ul><ul><ul><li>Welles are coated with purified dengue type 2 antigen. </li></ul></ul><ul><ul><li>AI =1-2 suggest Primary infection </li></ul></ul><ul><ul><li>AI > 2 suggest Secondary infection </li></ul></ul><ul><ul><li>Sensitivity = 98.11 % </li></ul></ul><ul><ul><li>Specificity = 92.1 % </li></ul></ul><ul><li>Dengue IgM Capture Elisa : (Panbio Diagnostics ): </li></ul><ul><li>For the Detection of Secondary dengue Infection </li></ul><ul><ul><li>Wells are coated by anti-human IgG antibodies. </li></ul></ul><ul><ul><li>Dengue 1-4 Antigen + conjugated monoclonal antibodies = antigen-MAb complex </li></ul></ul><ul><ul><li>Sandwich of anti-human IgG antibody/ Serum Antibody /antigen-MAb complex </li></ul></ul><ul><ul><li>AI > 2.2 (or 22 panbio units) suggest Secondary infection </li></ul></ul><ul><ul><li>Sensitivity ( Secondary ): 73.8 – 93.6% </li></ul></ul><ul><ul><li>Specificity (Primary): 83.8 – 98.6 % </li></ul></ul><ul><ul><li>Specificity (Negative): 88.4 - 100 % </li></ul></ul>
  20. 20. IgG in Secondary infection <ul><li>Paired sera (minimum 7days) </li></ul><ul><ul><li>Rising titers (four folds) </li></ul></ul><ul><li>Haemagglutination-Inhibition Titers: </li></ul><ul><ul><li>>1:2560 for acute sample. </li></ul></ul><ul><ul><li>> 2.2 AI (or 22 panbio units). </li></ul></ul><ul><ul><li>>2 AI by: Omega. </li></ul></ul>
  21. 21. Why PCR? <ul><li>To cover the Window period before IgM can be detected in Primary infection. </li></ul><ul><li>Usually, RNA can not be detected after the formation of the antibodies. </li></ul><ul><li>To confirm Secondary infection when IgM level is low or not detected. </li></ul><ul><li>To detect the four types of dengue. </li></ul>
  22. 22. Molecular Diagnostics of dengue <ul><li>RNA extraction. </li></ul><ul><li>Reverse transcription. </li></ul><ul><ul><li>RNA to cDNA </li></ul></ul><ul><li>Polymerase Chain reaction (PCR) </li></ul><ul><ul><li>Amplification, (Hybridization). </li></ul></ul><ul><ul><ul><ul><li>Target sequences amplified into millions copies </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Repeated Heating and cooling cycles. </li></ul></ul></ul></ul><ul><ul><li>Detection. </li></ul></ul><ul><ul><ul><ul><li>Probes are used to detect presence of dengue RNA. </li></ul></ul></ul></ul>
  23. 23. RNA extraction <ul><li>50-100 ul of RNA, is extracted from Patients serum before PCR examination. </li></ul><ul><ul><li>Manual extraction: </li></ul></ul><ul><ul><ul><li>RNA is extracted by the use of any manual commercial kits. </li></ul></ul></ul><ul><ul><ul><li>High Pure Viral RNA Kit from Roche. </li></ul></ul></ul><ul><ul><ul><li>QIAamp Viral RNA mini kits from Qiagen. </li></ul></ul></ul><ul><ul><li>Automated extraction: </li></ul></ul><ul><ul><ul><li>MagNA pure LC RNA Isolation Kit </li></ul></ul></ul>
  24. 24. RT-PCR <ul><li>Instrument used in JRL </li></ul><ul><li>LightCycler instrument (Roche) </li></ul><ul><ul><ul><li>Screening for samples within 5-7 days of appearance of symptoms. </li></ul></ul></ul><ul><ul><ul><li>Typing of dengue RNA detected samples. </li></ul></ul></ul>
  25. 25. One step- RT-PCR <ul><li>Artus dengue LC PCR Kit. </li></ul><ul><ul><li>(very Expensive) </li></ul></ul><ul><li>Tib-molbiol TaqMan probes used with: </li></ul><ul><li>LightCycler RNA Amplification Kit HybProbe.(Roche) </li></ul>
  26. 26. Probes for dengue Screening <ul><li>TIB- Molbiol (germany). </li></ul><ul><li>Den S2: </li></ul><ul><ul><li>Forward primer. </li></ul></ul><ul><li>Den AS1-3 and Den As4: </li></ul><ul><ul><li>Reverse primer. </li></ul></ul><ul><li>Den P1: </li></ul><ul><ul><li>Taqman probes. </li></ul></ul>
  27. 27. Probes for dengue typing D 4 TM D 3 TM Den 4 R Den 3 R Den 4 s Den 3 s Type 4 : Type 3 : D 2 TM D 1 TM Den 2 R Den 1 R Den 2 s Den 1 s Type 2 : Type 1:
  28. 28. Positive Dengue in JRL
  29. 29. Percentage of Positive results 59.40 1514 46.80 1193 12.59 321 2549 Total 20.97 13 20.97 13 0.00 0 62 Shawal 1427 37.04 20 33.33 18 3.70 2 54 Ramadan 1427 38.89 28 38.89 28 0.00 0 72 Shaban 1427 22.64 12 22.64 12 0.00 0 53 Rajab 1427 55.42 46 34.94 29 20.48 17 83 Jumad II 1427 69.89 253 45.03 163 24.86 90 362 Jumad I 1427 57.95 339 48.03 281 9.91 58 585 RabieII 1427 56.67 272 46.25 222 10.42 50 480 Rabie I 1427 66.14 295 51.57 230 14.57 65 446 Safar 1427 68.75 143 60.10 125 8.65 18 208 Muharam 1427 64.58 93 50.00 72 14.58 21 144 Zul Heja 1426 % Total % IgM % PCR   Susbected Date
  30. 30. Reference <ul><li>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. </li></ul><ul><li>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. </li></ul><ul><li>World health organization Guide: </li></ul><ul><li>Dengue haemorrhagic fever, diagnosis, treatment, prevention and control.1997. </li></ul>

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