What are Viral Hemorrhagic         Fevers (VHFs)? A group of illnesses that are caused by several distinct  families of v...
Viral hemorrhagic fever (…contd) The prototypical viral hemorrhagic fever is Yellow Fever Not all viral hemorrhagic feve...
Viral hemorrhagic fever (…contd) Acute infection:  fever, myalgia, malaise; progression to prostration Small vessel inv...
Viral hemorrhagic fever (…contd) Viruses of four distinct families    Arenaviruses    Filoviruses    Bunyaviruses    ...
Viral hemorrhagic fever (…contd)  Arenaviridae      Bunyaviridae      Filoviridae     Flaviviridae ClassificationJunin    ...
Shapes of the above viruses
DengueDengue is the biggest Arbovirus problem in the world today with over 2 million cases per yearDengue is found in SE...
Dengue (….contd)Classically, dengue presents with a high fever, lymphadenopathy, myalgia, bone and joint pains, headache,...
Global Occurrence of Dengue
Approximate actual and potential distribution of               Aedes aegypti.    The band between the 10° C isotherms repr...
Magnitude of ProblemThe reasons for this dramatic global emergence of Dengue as a major public health problem are: Increa...
Indian Scenario
Indian ScenarioThe    first recorded epidemic of clinically Dengue like illness occurred at Madras in 1780. First    out...
Indian ScenarioRecent Dengue epidemic occurred in 1996, 2003 & 2006.In 2008, 12,419 Dengue cases and     80 deaths were ...
Indian Scenario….sharing experiences from                             our centre                                       Pri...
Indian Scenario….sharing experiences from                          our centre     Month          Dengue-specific Antibody ...
Ref: J Infect Dev Countries 2011; 5(4):239-247The National figure also corroborates with the studyfrom our institute, carr...
Ref: J Infect Dev Countries 2011; 5(4):239-247
Ref: J Infect Dev Countries 2011; 5(4):239-247
The Vector: Aedes mosquito Aedes        (Stegomyia)  aegypti Breeds       in     small  accumulating standing  water Eg...
Dengue Transmission1. Mosquitoes transmitDengue virus to human dendriticcells.                                   12. Virus...
Steps required for any Flaviviruses infection and          transmission by a mosquito
Dengue - Virology                                                                 Ref: Goodsell DS.                       ...
Dengue – Virology (… Contd) The Deadly Switch                                                                Ref: Goodsell...
Dengue – Virology (… Contd)Ref: Goodsell DS.RCSB Protein DataBank. July, 2008.Each of these enzymes performs a different p...
Dengue – Virology (… Contd)                                                     Ref: Goodsell DS.                         ...
Clinical Presentation Of Dengue                                   Dengue Virus InfectionAsymptomatic                      ...
Clinical spectrum, pathophysiology, and        classification of dengue hemorrhagic fever. At the top are key clinical fin...
Dengue             (cont…) Dengue haemorrhagic fever and shock syndrome  appear most often in patients previously infecte...
Clinical Case Definition for Dengue Hemorrhagic Fever         4 Necessary Criteria:1. Fever, or recent history of acute fe...
Dengue Clinical SyndromesUndifferentiated feverClassic dengue feverDengue hemorrhagic feverDengue shock syndrome
Hemorrhagic Manifestations          of DengueSkin : petechiae, purpura, ecchymosesGingival bleedingNasal bleedingGastr...
Four Grades of DHF Grade 1   Fever and nonspecific constitutional symptoms   Positive tourniquet test is only hemorrhag...
Laboratory Tests in Dengue Fever Clinical laboratory tests    CBC--WBC, platelets, hematocrit   Albumin   Liver functi...
Laboratory Diagnosis of Dengue       Fever: Virus detection Detection of virus by culture is obviously the  definitive di...
Laboratory Diagnosis of Dengue     Fever: Virus detection Drawbacks and limitations of Viral isolation The period of illn...
Laboratory Diagnosis of Dengue    Fever: Virus detection
Laboratory Diagnosis of Dengue Fever:                Virus detection               Inoculation into mosquitos Most sensit...
Laboratory Diagnosis of Dengue Fever:             Virus detection            Inoculation into mosquitos      Toxorhynchite...
Laboratory Diagnosis of Dengue Fever:             Virus detection       Inoculation into mosquito cell lines C6/36 and AP...
Laboratory Diagnosis of Dengue Fever:                Virus detection        Inoculation into vertebrate cell lines VERO a...
Laboratory Diagnosis of Dengue Fever:     Antigen detection in fixed tissueSample: Peripheral Blood Leukocyte Autopsy Lu...
Laboratory Diagnosis of Dengue Fever: Reverse transcriptase-PCR amplification of               Dengue RNA High  potential...
Laboratory Diagnosis of Dengue Fever:         Serology: IgM capture ELISA The IgM Capture or the MAC-ELISA is the most wi...
Laboratory Diagnosis of Dengue Fever:         Serology: IgM capture ELISA                Experience at our centre MAC-ELI...
Laboratory Diagnosis of Dengue Fever:           Serology: IgM capture ELISA The Interpretation of MAC-ELISA resultsRef: De...
Laboratory Diagnosis of Dengue Fever:           Rapid NS1 Antigen detection Extensive study taking place to establish rap...
Laboratory Diagnosis of Dengue Fever:   Serology: Haemagglutination-Inhibition                 test (HAI) Simple, sensiti...
Laboratory Diagnosis of Dengue Fever:     Serology: Haemagglutination-Inhibition                   test (HAI)        The I...
Progress toward a Dengue Vaccine Control of dengue by widespread vaccination has been a priority of WHO for three decades...
Progress toward a Dengue Vaccine  Vaccine development and the issue of ImmunopathogenesisThe fear:The pathogenesis of seve...
Progress toward a Dengue Vaccine                        LEADING
Progress toward a Dengue Vaccine
Progress toward a Dengue Vaccine The leading candidate vaccine in clinical trials at  present is the ChimeriVax dengue va...
Kyasanur forest disease Kyasanur forest disease is a tick-borne viral  hemorrhagic fever endemic to South Asia The disea...
Kyasanur forest disease (…contd) The reservoir hosts for the disease are porcupines, rats and mice. The vector for diseas...
Crimean Congo Hemorrhagic Fever:           India affected Index case: Ameena Momin (Case A), 32yr old woman from Korat vi...
Crimean Congo Hemorrhagic Fever:         India affected(Ref: Mishra AC, Mehta M, Mourya DT, Gandhi S. Crimean-Congo haemor...
Crimean Congo Hemorrhagic Fever:            India affected                        DIAGNOSIS Only Nucleic acid Amplificati...
Crimean Congo Hemorrhagic Fever:            India affected                          IMPORTANCE: Many severely ill patient...
Novel Bunyavirus identified in         China (SFTS)      Why do we discuss it here in context of                     India...
Novel bunyavirus identified in         China (…contd) Surveillance for infectious disease in China has advanced in  recen...
Novel bunyavirus identified in        China(…contd) In June 2009, a patient in Xinyang City in Henan  Province (central C...
Novel bunyavirus identified in        China(…contd) In these cases, serum or white blood cells were inoculated  onto Vero...
Novel bunyavirus identified in        China(…contd) The investigators isolated a novel pathogen, which they  named SFTS b...
All that are round and spiculated are             not Dengue      Thanks for your attention
Viral Haemorrhagic Fevers with special reference to Dengue
Viral Haemorrhagic Fevers with special reference to Dengue
Viral Haemorrhagic Fevers with special reference to Dengue
Viral Haemorrhagic Fevers with special reference to Dengue
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Viral Haemorrhagic Fevers with special reference to Dengue

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  • Good presentation.Enrich it by incorporating slides describing pathogenesis/immunopathogenesis of dengue.Alittle more information about NS1 Ag is desirable.Why do you suggest CBC---WBC count and LFT ? What help you will get as regard diagnosis or treatment modalities? Include the constrains for development of vaccine.
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Viral Haemorrhagic Fevers with special reference to Dengue

  1. 1. What are Viral Hemorrhagic Fevers (VHFs)? A group of illnesses that are caused by several distinct families of viruses A severe multisystem syndrome (multiple organ systems in the body are affected Vascular system damaged : SHOCK syndromes Body’s ability to regulate itself (Homeostasis) is impaired Many cause severe and life-threatening disease.
  2. 2. Viral hemorrhagic fever (…contd) The prototypical viral hemorrhagic fever is Yellow Fever Not all viral hemorrhagic fevers are however arboviruses Hemorrhagic fever with Renal Syndrome (HFRS) are also considered in relation to VHF HFRS Caused by:  Hantaan  Seoul  Dobrava  Puumala viruses (Ref: Mandell, Douglas and Bennett’s “Principles and Practice of Infectious Disease, 7th Ed)
  3. 3. Viral hemorrhagic fever (…contd) Acute infection: fever, myalgia, malaise; progression to prostration Small vessel involvement: increased permeability, cellular damage Multisystem compromise  (varies with pathogen) Hemorrhage may be small in volume (indicates small vessel involvement, thrombocytopenia) Poor prognosis associated with: shock, encephalopathy, extensive hemorrhage
  4. 4. Viral hemorrhagic fever (…contd) Viruses of four distinct families  Arenaviruses  Filoviruses  Bunyaviruses  Flaviviruses RNA viruses  Enveloped in lipid coating Survival dependent on an animal or insect host, for the natural reservoir
  5. 5. Viral hemorrhagic fever (…contd) Arenaviridae Bunyaviridae Filoviridae Flaviviridae ClassificationJunin Crimean- Congo Ebola Kyasanur HF Forest DiseaseMachupo Hantavirus Marburg Omsk HFSabia Rift Valley fever Yellow FeverGuanarito SFTS (China, Dengue 2011)LassaArgentine HF (In block Red: Diseases prevalent in INDIA)Venezuelan HF
  6. 6. Shapes of the above viruses
  7. 7. DengueDengue is the biggest Arbovirus problem in the world today with over 2 million cases per yearDengue is found in SE Asia, Africa and the Caribbean and South America.4 serotypes: DEN 1,2,3,4Human infections arise from a human- mosquito-human cycle
  8. 8. Dengue (….contd)Classically, dengue presents with a high fever, lymphadenopathy, myalgia, bone and joint pains, headache, and a maculopapular rash.Severe cases may present with haemorrhagic fever and shock with a mortality of 5-10%. {Dengue haemorrhagic fever (DHF)or Dengue shock syndrome (DSS)}
  9. 9. Global Occurrence of Dengue
  10. 10. Approximate actual and potential distribution of Aedes aegypti. The band between the 10° C isotherms represents potential distribution between 35 ° North – 35° South(Ref: World Health Organization. Technical Guide for Diagnosis, Treatment,Surveillance, Prevention, and Control of Dengue Haemorrhagic Fever, 2nd ed.Geneva: World Health Organization; 1997.)
  11. 11. Magnitude of ProblemThe reasons for this dramatic global emergence of Dengue as a major public health problem are: Increased Air Travel Extensive vector infestations with declining vector control: effective mosquito control is virtually non existent in most Dengue endemic countries Unreliable Water supply and drainage systems Increasing non bio-degradable contaivers and poor solid Waste disposal Major global demographic changes: Urbanization with increasing population density in urban areas
  12. 12. Indian Scenario
  13. 13. Indian ScenarioThe first recorded epidemic of clinically Dengue like illness occurred at Madras in 1780. First outbreak in Indian subcontinent: 1812.First Dengue virus isolation- Kolkata in 1943– 1944.First outbreak in India: 1963 in Kolkata.Ref: Jatanasen S and Thongcharoen P (1993) Dengue hemorrhagic fever in South East-Asian countries. Monograph on dengue/dengue haemorrhagic fever. NewDelhi: WHO 23-30.
  14. 14. Indian ScenarioRecent Dengue epidemic occurred in 1996, 2003 & 2006.In 2008, 12,419 Dengue cases and 80 deaths were reported.Delhi shares ~25% of dengue disease burden of country.
  15. 15. Indian Scenario….sharing experiences from our centre Primary Secondary Suspected Total Serologically infection infection secondary Month Suspected Positive cases (IgM (IgM+ IgG infection (IgG cases (%) Positivity) Positivity) Positivity) August 12 3 (0.34%) 1 (0.5%) 1 (0.26%) 1 (0.32%)September 157 68 (7.6%) 17 (8.6%) 24 (6.3%) 27 (8.6%) October 982 583 (65.3%) 126 (63.3%) 246 (64.57%) 211 (67.4%)November 362 230 (25.76%) 49 (24.6%) 110 (28.87%) 71 (22.68%)December 37 9 (1%) 6 (3%) 0 (0%) 3 (1%) Total 1550 893 (57.36%) 199 (22.28%) 381 (42.67%) 313 (35.05%)Ref: AnitaChakravarti* and RajniKumaria: Virology Journal 2005, 2:3
  16. 16. Indian Scenario….sharing experiences from our centre Month Dengue-specific Antibody Positive cases Children Adults (Positivity Total (Positivity %) %) August 3 0 3 (25%) A Clear SeptemberCut PEAK of incidence of (41.7%) 68 18 (48.6%) 50 new cases were seen in the (69.6%) October 583 133 Post-monsoon 450 (57%) November during the months of October season 230 54 (44.3%) 176 (83.8%) and November in cases ocurring8in or December 9 1 (11.1%) (28.6%) near Delhi 893 Total 206 (56.4%) 687 (58%)Ref: AnitaChakravarti* and RajniKumaria: Virology Journal2005, 2:3
  17. 17. Ref: J Infect Dev Countries 2011; 5(4):239-247The National figure also corroborates with the studyfrom our institute, carried out in Delhi
  18. 18. Ref: J Infect Dev Countries 2011; 5(4):239-247
  19. 19. Ref: J Infect Dev Countries 2011; 5(4):239-247
  20. 20. The Vector: Aedes mosquito Aedes (Stegomyia) aegypti Breeds in small accumulating standing water Eggs resist drying Domesticated mosquito Found within or close-by human environments, often biting indoors biting is predominantly by day
  21. 21. Dengue Transmission1. Mosquitoes transmitDengue virus to human dendriticcells. 12. Virus targets areaswith high WBC counts 2(liver, spleen, lymphnodes, bone marrow, 4And glands) 333. Virus entersWBCs & lymphaticTissue4. Dengue virus enters bloodCirculation. http://phil.cdc.gov/PHIL_Images/08051999/00004/dengue_phf/sld006.htm
  22. 22. Steps required for any Flaviviruses infection and transmission by a mosquito
  23. 23. Dengue - Virology Ref: Goodsell DS. RCSB Protein Data Bank. July, 2008.Dengue virus is a small virus that carries a single strand of RNA as its genome. Thegenome encodes only ten proteins. Three of these are structural proteins that formthe coat of the virus and deliver the RNA to target cells, and seven of them arenonstructural proteins that orchestrate the production of new viruses once the virusgets inside the cell. The outermost structural protein, termed the envelope protein, isshown here from PDB entry 1k4r
  24. 24. Dengue – Virology (… Contd) The Deadly Switch Ref: Goodsell DS. RCSB Protein Data Bank. July, 2008.When the virus is carried into the cell and into lysozomes, the acidic environmentcauses the protein to snap into a different shape, assembling into trimeric spike, asshown above from PDB entry 1ok8. Several hydrophobic amino acids at the tip of thisspike, colored bright red here, insert into the lysozomal membrane and cause the virusmembrane to fuse with lysozome. This releases the RNA into the cell and infectionstarts.
  25. 25. Dengue – Virology (… Contd)Ref: Goodsell DS.RCSB Protein DataBank. July, 2008.Each of these enzymes performs a different part of the life cycle. The polymerasebuilds new RNA strands based on the viral RNA, the helicase helps to separate thesestrands, and the methyltransferase adds methyl groups to the end of them, protectingthe RNA strands and coaxing the cells ribosomes to create viral proteins based onthem. The viral proteins are created in one long polyprotein chain, which is finallyclipped into the functional units by the protease. The little chain coloured blue is aportion of another viral protein, NS2B, that assists with the protease activity.
  26. 26. Dengue – Virology (… Contd) Ref: Goodsell DS. RCSB Protein Data Bank. July, 2008.The one shown here, from PDB entry 2r6p6, shows the envelopeprotein on the surface of the virus (in white) with many antibody Fabfragments (in blue) bound to the viral proteins. By looking carefully atthis structure, researchers have discovered that the antibodies distortthe arrangement of the envelope proteins, blocking their normalaction in infection.
  27. 27. Clinical Presentation Of Dengue Dengue Virus InfectionAsymptomatic Symptomatic Dengue hemorrhagic Undifferentiated Dengue fever fever fever syndrome (plasma leakage) (viral syndrome) Without With unusual No shock Dengue shock hemorrhage hemorrhage syndrome Dengue fever Dengue hemorrhagic FeverWHO 95629
  28. 28. Clinical spectrum, pathophysiology, and classification of dengue hemorrhagic fever. At the top are key clinical findings; in the center, pathophysiologic mechanisms; and on the side, the World Health Organization classification of cases: Grade 1: Fever accompanied by nonspecific constitutional symptoms; the only hemorrhagic manifestations are a positive tourniquet test result, easy bruising, or both. Grade 2: Spontaneous bleeding in addition to the manifestations of grade 1, usually in the form of skin hemorrhages or other hemorrhages. Grade 3: Circulatory failure manifested by a rapid, weak pulse and narrowing of pulse pressure or hypotension, with the presence of cold, clammy skin and restlessness. Grade 4: Profound shock with undetectable blood pressure or pulse.(Ref: WHO. Technical Guide for Diagnosis, Treatment, Surveillance, Prevention, andControl of Dengue Haemorrhagic Fever, 2nd ed. Geneva: 1997.)
  29. 29. Dengue (cont…) Dengue haemorrhagic fever and shock syndrome appear most often in patients previously infected by a different serotype of dengue, thus suggesting an immunopathological mechanism. Diagnosis is made by serology. No specific antiviral therapy is available. Prevention of dengue in endemic areas depends on mosquito eradication. The population should remove all containers from their premises which may serve as vessels for egg deposition.
  30. 30. Clinical Case Definition for Dengue Hemorrhagic Fever 4 Necessary Criteria:1. Fever, or recent history of acute fever2. Hemorrhagic manifestations3. Low platelet count (100,000/mm3 or less)4. Objective evidence of “leaky capillaries” :  elevated hematocrit (20% or more over baseline)  low albumin  pleural or other serosal cavity effusions
  31. 31. Dengue Clinical SyndromesUndifferentiated feverClassic dengue feverDengue hemorrhagic feverDengue shock syndrome
  32. 32. Hemorrhagic Manifestations of DengueSkin : petechiae, purpura, ecchymosesGingival bleedingNasal bleedingGastro-intestinal bleeding: hematemesis, melenaHematuriaIncreased menstrual flow
  33. 33. Four Grades of DHF Grade 1  Fever and nonspecific constitutional symptoms  Positive tourniquet test is only hemorrhagic manifestation Grade 2  Grade 1 manifestations + spontaneous bleeding Grade 3  Signs of circulatory failure (rapid/weak pulse, narrow pulse pressure, hypotension, cold/clammy skin) Grade 4  Profound shock (undetectable pulse and BP)
  34. 34. Laboratory Tests in Dengue Fever Clinical laboratory tests  CBC--WBC, platelets, hematocrit  Albumin  Liver function tests  Urine--check for microscopic hematuria Dengue-specific tests  Virus isolation  Serology
  35. 35. Laboratory Diagnosis of Dengue Fever: Virus detection Detection of virus by culture is obviously the definitive diagnostic test. By the time a person infected with Dengue develops fever, the infection is widely disseminated. The virus is found in serum or plasma, in circulating blood cells and in selected tissues, especially those of the immune system, for approx. 2-7 days, roughly corresponding to the period of fever. Detection of dengue RNA using specific oligonucleotide primers, reverse transcriptase and thermostable polymerase are Faster and are applied in many Laboratories.
  36. 36. Laboratory Diagnosis of Dengue Fever: Virus detection Drawbacks and limitations of Viral isolation The period of illness when the dengue virus can be successfully detected is brief Within a day or 2 after subsidence of fever, the rising level of antibody interfere with virus culture Dengue virus is heat-labile and special precautions must be taken against the thermal inactivation of specimens. Laboratories equipped and staffed to culture viruses are expensive to develop and maintain.
  37. 37. Laboratory Diagnosis of Dengue Fever: Virus detection
  38. 38. Laboratory Diagnosis of Dengue Fever: Virus detection Inoculation into mosquitos Most sensitive dengue viral culture technique Serum, Plasma, CSF, Pleural fluid, Peripheral blood leucocytes & tissue homogenates can be used Toxorhynchites mosquitos generally used They are not hematophagus and their large size facilitates inoculation Infection is detected by Immunofluorescence of a tissue smear prepared from the crushed head of the mosquito (Head Squash) High sensitive culture requires 5-20 mosquitos per specimen adult male Aedes aegypti & Ae. Albopictus can also be used.
  39. 39. Laboratory Diagnosis of Dengue Fever: Virus detection Inoculation into mosquitos Toxorhynchites Ae. aegypti & Ae. Albopictus Large, easy to inoculate Small, difficult to inoculate Raising is labour Easier to maintainintensive, as the larvae arecarnivorous & needs asecond mosquito specieslarvae as food source Non Hematophagus, Female spp can’t be usedhence safe to handle due to ability to act as vector
  40. 40. Laboratory Diagnosis of Dengue Fever: Virus detection Inoculation into mosquito cell lines C6/36 and AP-61 cell lines can be used Less sensitive than direct inoculation into live mosquitoes Cell cultures to be screened for specific evidence of infection by an immunoassay as the cytopathic effects might be absent in many dengue virus isolates As mosquito cell lines are propagable in ambient tropical temperatures (25-34° C), it is easier to maintain and practice
  41. 41. Laboratory Diagnosis of Dengue Fever: Virus detection Inoculation into vertebrate cell lines VERO and LLC-MK2 cell lines can be used Least sensitive than other direct inoculation methodsAll cultures are examined using serotype-specific anti- Dengue monoclonal Abs tagged to a second labelled Ab.Positive control: Dengue-complex-reactibe MAb Intracerebral inoculation into newborn mice is also tried in certain laboratories : but have proven to be very less sensitive
  42. 42. Laboratory Diagnosis of Dengue Fever: Antigen detection in fixed tissueSample: Peripheral Blood Leukocyte Autopsy Lung, Liver specimen Less commonly: Autopsy Thymus, Spleen, Lymph node, Bone marrowMainly for epidemiological purpose and confirmation of epidemic / outbreak.Immunohistochemistry examined using serotype- specific anti-Dengue monoclonal Abs tagged to a second labelled Ab.
  43. 43. Laboratory Diagnosis of Dengue Fever: Reverse transcriptase-PCR amplification of Dengue RNA High potential to detect dengue virus during convalescence, when circulating antibodies otherwise preclude its detection 2 step nested RT-PCR and 1 tube multiplex RT-PCR are among the most widely used methodsExperience at our centre have shown that the 1 tube multiplex RT-PCR is more sensitive and specific than the other available methods.(Ref: Kumaria R, Chakravarti A. Diagn Microbiol Infect Dis 2005)
  44. 44. Laboratory Diagnosis of Dengue Fever: Serology: IgM capture ELISA The IgM Capture or the MAC-ELISA is the most widely used serological test Serum, Saliva, dried blood sample collected in Filter paper and CSF can be used as sample Can even detect a rise in dengue-specific IgM in acute phase at 1-day to 2-day interval Specimens collected at an interval of 2-3 days spanning the day of defervescence are usually diagnostic
  45. 45. Laboratory Diagnosis of Dengue Fever: Serology: IgM capture ELISA Experience at our centre MAC-ELISA is regularly practiced at our centre IgM and IgG detection from non-invasive Saliva samples were carried out at our centre which yielded wonderful reproducible results:  Salivary IgM antibodies were detected in 100% of the serum IgM-positive samples and in 30% of the serum samples that were negative for IgM antibodies. Salivary IgG antibodies were detected in 93.3% of the serum samples that were positive for anti-dengue IgG antibodies and in none of the serum IgG-negative cases (Ref: Chakravarti A, Matlani M, Jain M. 2007, Curr Microbiol) IgM/IgG detection from reconstitution of dried blood samples from clinically suspected cases collected in filter paper are being carried out at present.
  46. 46. Laboratory Diagnosis of Dengue Fever: Serology: IgM capture ELISA The Interpretation of MAC-ELISA resultsRef: Dengue haemorrhagic fever: diagnosis, treatment, prevention and control.2nd edition. Geneva : World Health Organization
  47. 47. Laboratory Diagnosis of Dengue Fever: Rapid NS1 Antigen detection Extensive study taking place to establish rapid diagnosis and shorten the window period of misdiagnosis by detecting the NS1 antigen of dengue virus Most of the studies have shown that a combination rapid test comprising immunochromatographic assay for detection of both the NS1 Antigen and the anti-dengue Igm together yields satisfactory clinical results, instead of sole NS1 antigen detection.(Ref: 1. Tontulawat P et al, Southeast Asian J Trop Med Public Health. May, 2011.2. Fry SR et al, PLoS Negl Trop Dis. June, 2011.)
  48. 48. Laboratory Diagnosis of Dengue Fever: Serology: Haemagglutination-Inhibition test (HAI) Simple, sensitive and reproducible Reagents may be prepared locally Disadvantages:  Pretreatment of serum samples reqd with acetone/ kaolin and then adsorbed with type O human RBCs to remove non-specific inhibitors of agglutinin and non- specific agglutinins.  Paired sera are required with a gap of at least 7 days.  Can’t reliably distinguish between closely related Flaviviruses: Between Dengue and Jap Encephalitis or West Nile viruses
  49. 49. Laboratory Diagnosis of Dengue Fever: Serology: Haemagglutination-Inhibition test (HAI) The Interpretation of HAI resultsRef: Dengue haemorrhagic fever: diagnosis, treatment, prevention and control.2nd edition. Geneva : World Health Organization
  50. 50. Progress toward a Dengue Vaccine Control of dengue by widespread vaccination has been a priority of WHO for three decades (Ref:Brandt WE. J Infect Dis 1990) Background: Robust neutralising antibody responses develop after dengue infection and are believed to provide lifelong protection against reinfection with the same dengue serotype and short-lived protection, of several months, against a heterologous dengue serotype. This naturally acquired immunity provides optimism for the feasibility of a dengue vaccine.
  51. 51. Progress toward a Dengue Vaccine Vaccine development and the issue of ImmunopathogenesisThe fear:The pathogenesis of severe dengue results from a complex interaction between the virus, the host, and, at least in part, immune-mediated mechanisms. Vaccine development has been slowed by fears that immunisation might predispose individuals to the severe form of dengue infection.The assurance:Whatever the role of antibody-dependent enhancement, it seems that a vaccine inducing a long-lived neutralising antibody response against all four serotypes simultaneously should not induce any risk in this respect(Ref: 1. Guirakhoo F et al, Hum Vaccin 2006. 2. Sabchareon A et al, Am J Trop Med Hyg 2002. 3. Sabchareon A et al, Pediatr Infect Dis J 2004.)
  52. 52. Progress toward a Dengue Vaccine LEADING
  53. 53. Progress toward a Dengue Vaccine
  54. 54. Progress toward a Dengue Vaccine The leading candidate vaccine in clinical trials at present is the ChimeriVax dengue vaccine. Using a new technology, the premembrane and envelope genes of yellow fever 17D virus are replaced with those of each wild-type dengue virus serotype. ChimeriVax dengue vaccine viruses are then prepared by electroporation of Vero cells with RNA transcripts prepared from viral cDNA.(Ref: 1. Guirakhoo F, Kitchener S et al. Hum Vaccin 20062. Webster DP, Farra J. Lancet Infect Dis 2009.)
  55. 55. Kyasanur forest disease Kyasanur forest disease is a tick-borne viral hemorrhagic fever endemic to South Asia The disease was first reported from Kyasanur Forest of Karnataka in India The disease was first manifested as an epizootic outbreak among monkeys killing several of them in the year 1957. Hence the disease is also known as Monkey Disease.
  56. 56. Kyasanur forest disease (…contd) The reservoir hosts for the disease are porcupines, rats and mice. The vector for disease transmission is Haemaphysalis spinigera, a forest tick. Humans contract infection from the bite of nymphs of the tick The disease has a high mortality rate of 10% The clinical manifestations of the disease in humans are:  High fever  Headache  Hemorrhages from nasal cavity and throat  Vomiting
  57. 57. Crimean Congo Hemorrhagic Fever: India affected Index case: Ameena Momin (Case A), 32yr old woman from Korat village in Sanand, 20 kms from Ahmedabad was admitted to Sterling Hospital on December 29 and later shifted to Shalby Hospital on January 1, expired on January 3rd 2011. Secondary cases:  42 yr old Dr Gaganjeet Sharma (Case B) treating the index case at Shalby, died January 13th.  25 yr old nurse Asha John (Case C), attending the index case, died January 18th.  The husband of the index case (case D) also admitted to the same hospital on Jan 16, was positive for CCHF virus was treated with oral ribavarin and discharged after 10 days. (Ref: Mishra AC, Mehta M, Mourya DT, Gandhi S. Crimean-Congo haemorrhagic fever in India. Lancet July, 2011)
  58. 58. Crimean Congo Hemorrhagic Fever: India affected(Ref: Mishra AC, Mehta M, Mourya DT, Gandhi S. Crimean-Congo haemorrhagicfever in India. Lancet July, 2011)
  59. 59. Crimean Congo Hemorrhagic Fever: India affected DIAGNOSIS Only Nucleic acid Amplification Tests (eg. PCR, rt-PCR) are the most reliable method, along with RNA sequencing in case of outbreaks in previously unknown geographical areas High serum LDH, High serum Ferritin, and thrombocytopenia may lead to a strong suspicion Negative assays for locally prevalent diseases, that may lead to such fever (eg. Malaria, Leptospirosis, Dengue, Kyasanur Forest Disease for India) shall also lead to strong suspicion.
  60. 60. Crimean Congo Hemorrhagic Fever: India affected IMPORTANCE: Many severely ill patients with CCHF require admission to intensive care facilities. To avoid infections in hospital settings, stringent infection control practices, proper air handling in intensive-care units, isolation of patients, and correct handling of clinical specimens are essential. Tick bites and contact with infected animals are the main modes of infection to people. Disinfection of domestic animals and their accommodation can help reduce the risk of human infection. Febrile patients with haemorrhagic symptoms, who are negative for dengue virus, should be considered as possible cases of CCHF for the purpose of hospital infection control and isolation of patients in India
  61. 61. Novel Bunyavirus identified in China (SFTS) Why do we discuss it here in context of India? RNA from SFTS bunyavirus was detected in roughly 5 percent of ticks of the species Haemaphysalis longicornis recovered from animals in the area in which affected patients lived, and the authors propose that this tick may be a vector for SFTS bunyavirus. This tick is found throughout the Asia-Pacific region including India Considering globalization, population migration and lack of SFTS surveillance, INDIA is at a high risk.Ref: Yu XJ, Liang MF, Zhang SY, et al: Fever with thrombocytopenia associated with a novel bunyavirus in China. N Engl J Med 2011; 364(16):1523-1532.
  62. 62. Novel bunyavirus identified in China (…contd) Surveillance for infectious disease in China has advanced in recent years. In 2009 and 2010, surveillance detected the emergence of a new viral pathogen that causes a clinical syndrome including fever and thrombocytopenia that has been termed severe fever with thrombocytopenia syndrome (SFTS). SFTS is characterized by  Gastrointestinal symptoms  Leukopenia  Fever  Thrombocytopenia  30% mortality rate.
  63. 63. Novel bunyavirus identified in China(…contd) In June 2009, a patient in Xinyang City in Henan Province (central China) presented with SFTS Blood sample was obtained from this patient 1 week after onset of symptoms. Multiple cell lines that were susceptible to both viral and rickettsial agents were inoculated with the patient’s blood. During the period June 2009 to March 2010, additional cases of SFTS were identified in central and northeast China, and blood samples were obtained from these patients as well.
  64. 64. Novel bunyavirus identified in China(…contd) In these cases, serum or white blood cells were inoculated onto Vero cells. The virus isolated from the first patient was analyzed by RFLP assay, whereas samples from the second group of patients were analyzed using PCR. EM and neutralization assays were also performed.
  65. 65. Novel bunyavirus identified in China(…contd) The investigators isolated a novel pathogen, which they named SFTS bunyavirus. Analysis of viral RNA showed that the virus was a member of the Bunyaviridae family in the genus Phlebovirus. EM confirmed bunyavirus morphology. Based on identification of viral DNA or specific antibodies, or both, 171 patients with SFTS were shown to have infection with SFTS bunyavirus. Immune response specific to the virus was shown in 100 percent (35 of 35) of matched serum samples obtained during acute infection and convalescence.
  66. 66. All that are round and spiculated are not Dengue Thanks for your attention

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