1. Dr. Halesh.L.H. Professor and Head of the department, Microbiology SIMS,Shimoga

4. * Causative agent of dengue fever, belongs to family flaviviridae, genus flavivirus. * It is a spherical enveloped virus * Genomic material – single stranded RNA * There are presently 5 serotypes identified

5. * Fifth serotype, identified in 2013, october follows sylvatic cycle,and is found only in Sarawak forest, Malaysia * Each serotype provides specific lifetime immunity, and short-term cross-immunity * All serotypes can cause severe and fatal disease

6. * Genetic variation within serotypes * Some genetic variants within each serotype appear to be more virulent or have greater epidemic potential

7. o The first record of dengue fever is in chinese medical encyclopedia referred as water poison caused by flying insects o Reports of epidemics – 1779-80 o Then until 1940, epidemics were infrequent o Then there was marked spread of dengue during and after second world war

8. * The incidence is dramatically increasing * 390 million dengue cases per year * Infections are acquired in urban environment * Rate of dengue has increased 10folds between 1960-2010

10. 1. Virus is transmitted to human in mosquito saliva 2. Virus replicates in target organs 3. Virus infects white blood cells and lymphatic tissues 4. Virus released and circulates in blood

11. 5. Second mosquito ingests virus with blood 6. Virus replicates in mosquito midgut and other organs, infects salivary glands 7. Virus replicates in salivary glands

13. * Dengue transmitted by infected female mosquito * Primarily a daytime feeder * Lives around human habitation * Lays eggs and produces larvae preferentially in artificial containers

16. * Undifferentiated fever * Classic dengue fever * Dengue hemorrhagic fever * Dengue shock syndrome

17. * May be the most common manifestation of dengue * Prospective study found that 87% of students infected were either asymptomatic or only mildly symptomatic * Other prospective studies including all age- groups also demonstrate silent transmission

19. * Decreased level of consciousness: lethargy, confusion, coma * Seizures * Nuchal rigidity * Paresis

20. 4 Necessary Criteria: 1.Fever, or recent history of acute fever 2.Hemorrhagic manifestations 3.Low platelet count (100,000/mm 3 or less) 4.Objective evidence of “leaky capillaries:” * elevated hematocrit (20% or more over baseline) * low albumin * pleural or other effusions

21. criteria for DHF 1.Evidence of circulatory failure manifested indirectly by all of the following: * Rapid and weak pulse * Narrow pulse pressure (  20 mm Hg) OR hypotension for age * Cold, clammy skin and altered mental status 2.Frank shock is direct evidence of circulatory failure

22.  Grade 1 * Fever and nonspecific constitutional symptoms * Positive tourniquet test is only hemorrhagic Manifestation  Grade 2 * Grade 1 manifestations + spontaneous bleeding

23.  Grade 3 * Signs of circulatory failure (rapid/weak pulse, narrow pulse pressure, hypotension, cold/clammy skin)  Grade 4 * Profound shock (undetectable pulse and BP)

24. * Abdominal pain - intense and sustained * Persistent vomiting * Abrupt change from fever to hypothermia, with sweating and prostration * Restlessness or somnolence

25. When Patients Develop DSS: 3 to 6 days after onset of symptoms When Patients Develop DSS: 3 to 6 days after onset of symptoms Initial Warning Signals: Disappearance of fever Drop in platelets Increase in hematocrit Initial Warning Signals: Disappearance of fever Drop in platelets Increase in hematocrit Alarm Signals: Severe abdominal pain Prolonged vomiting Abrupt change from fever to hypothermia Change in level of consciousness (irritability or somnolence) Alarm Signals: Severe abdominal pain Prolonged vomiting Abrupt change from fever to hypothermia Change in level of consciousness (irritability or somnolence) Four Criteria for DHF: Fever Hemorrhagic manifestations Excessive capillary permeability  100,000/mm 3 platelets Four Criteria for DHF: Fever Hemorrhagic manifestations Excessive capillary permeability  100,000/mm 3 platelets

26. * Encephalopathy * Hepatic damage * Cardiomyopathy * Severe gastrointestinal hemorrhage

28. * Virus strain * Pre-existing anti-dengue antibody * previous infection * maternal antibodies in infants * Host genetics * Age

29. * Higher risk in secondary infections * Higher risk in locations with two or more serotypes circulating simultaneously at high levels (hyperendemic transmission)

30. Hyperendemicity Increased circulation of viruses Increased probability of secondary infection Increased probability of occurrence of virulent strains Increased probability of immune enhancement Increased probability of DHF

31. * Persons who have experienced a dengue infection develop serum antibodies that can neutralize the dengue virus of that same (homologous) serotype

32. Neutralizing antibody to Dengue 1 virus 1 1 Dengue 1 virus 1 Homologous Antibodies Form Non-infectious Complexes Non-neutralizing antibody 1 1 Complex formed by neutralizing antibody and virus

33. * In a subsequent infection, the pre-existing heterologous antibodies form complexes with the new infecting virus serotype, but do not neutralize the new virus

34. Non-neutralizing antibody to Dengue 1 virus Dengue 2 virus 2 2 2 2 2 Heterologous Antibodies Form Infectious Complexes Complex formed by non-neutralizing antibody and virus 2

35. * Antibody-dependent enhancement is the process in which certain strains of dengue virus, complexed with non- neutralizing antibodies, can enter a greater proportion of cells of the mononuclear lineage, thus increasing virus production

36. 2 2 2 2 2 2 2 2 2 2 Non-neutralizing antibody Dengue 2 virus 2 Complex formed by non- neutralizing antibody and Dengue 2 virus 2

37. * Infected monocytes release vasoactive mediators, resulting in increased vascular permeability & hemorrhagic manifestations that characterize DHF and DSS

38.  Virus serotype * DHF risk is greatest for DEN-2, followed by DEN-3, DEN-4 & DEN-1

40. * Influenza * Measles * Rubella * Malaria * Typhoid fever * Leptospirosis * Meningococcemia * Rickettsial infections * Bacterial sepsis * Other viral hemorrhagic fevers

41. * Blood pressure * Evidence of bleeding in skin or other sites * Hydration status * Evidence of increased vascular permeability- pleural effusions, ascites

43. A B PEI = A/B x 100 Pleural Effusion Index

44. * Inflate blood pressure cuff to a point midway between systolic and diastolic pressure for 5 minutes * Positive test: 20 or more petechiae per 1 inch 2 (6.25 cm 2 )

47. * Clinical laboratory tests * CBC--WBC, platelets, hematocrit * Albumin * Liver function tests * Urine--check for microscopic hematuria VIRUS SPECIFIC TEST * Virus isolation * Serology

48. * Mosquito cell line * Mammalian cell lines : Vero cells, BHK21, Primary Chick & duck embryo cells * Sero type Identified by specific mouse monoclonal antibodies. * PCR for Sero type is also available.

50. Virus Isolation: Mosquito Inoculation

52. * Serological tests * Two types of serum response * Primary Immunological response * Secondary Immunological response

53. * HI test Anti body response S1-S2 interval Convalescent titre Interpretation > 4 x rise > 7 days < 1: 1280 Def. Inf. Primary > 4 x rise Any specimen > 1: 2560 Def. Inf. Secondary > 4 x rise < 7 days < 1: 1280 Def. Inf. Possibly primary or secondary No change Any Specimen > 1: 2560 Presumed Infection secondary No Change > 7 days < 1: 1280 Not dengue Interpretation of the test Antibodies can persist for decades. CFT Test is less sensitive but more specific Positive after 6-8 weeks of illness, 1/2 life three years.

54. * Plaque reduction Neutralization Test Most specific, time consuming, expensive, special lab facility required.

56. * MAC - ELISA * Detects IgM, indicates active or recent infection. Both in primary and secondary infection. * IgM ELISA – Primary infection 95% sensitive after 7 –10 days. * Secondary infection IgM + IgG 100% sensitive after 4-5 days.

57. * Simple * No expertise required for interpretation * False positive results occur * Should be confirmed by MAC-ELISA

59. * No hemorrhagic manifestations and patient is well-hydrated: home treatment * Hemorrhagic manifestations or hydration borderline: outpatient observation center or hospitalization * Warning signs (even without profound shock) or DSS: hospitalize

60. * Patients treated at home * Instruction regarding danger signs * Consider repeat clinical evaluation * Patients with bleeding manifestations * Serial hematocrits and platelets at least daily until temperature normal for 1 to 2 days

61. * All patients * If blood sample taken in first 5 days after onset, need convalescent sample between days 6 - 30 * All hospitalized patients need samples on admission and at discharge or death

62. * Fluids * Rest * Antipyretics (avoid aspirin & NSAIDs) * Monitor blood pressure, hematocrit, platelet count, level of consciousness

63. * Only needed until fever subsides, to prevent Aedes aegypti mosquitoes from biting patients and acquiring virus * Keep patient in screened sick room or under a mosquito net

64. * Continue monitoring after defervescence * If in doubt, provide intravenous fluids, guided by serial hematocrits, blood pressure, and urine output * The volume of fluid needed is similar to the treatment of diarrhea with mild to moderate isotonic dehydration (5%-8% deficit)

65. weight in kg ml /kg/ day < 7 220 7 - 11 165 12 - 18 132 19 - 40 88

66. * Volume required for rehydration is twice the recommended maintenance requirement * Formula for calculating maintenance volume: 1500 + 20 x (weight in kg - 20) * For example, maintenance volume for 55 kg patient is: 1500 + 20 x (55-20) = 2200 ml * For this patient, the rehydration volume would be 2 x 2200, or 4400 ml

67. * Avoid invasive procedures when possible * Effectiveness of steroids, IV immuno-globulin, or platelet transfusions to shorten the duration or decrease the severity of thrombocytopenia is not known * Patients in shock may require treatment in an intensive care unit

68. * Absence of fever for 24 hours (without anti-fever therapy) and return of appetite * Visible improvement in clinical picture * Stable hematocrit * 3 days after recovery from shock * Platelets  50,000 / mm 3 * No respiratory distress from pleural effusions / ascites

69. 8 Dengue + bleeding = DHF 4 Need 4 WHO criteria, capillary permeability 8 DHF kills only by hemorrhage 4 Patient dies as a result of shock 8 Poor management turns dengue into DHF 4 Poorly managed dengue can be more severe, but DHF is a distinct condition, which even well-treated patients may develop 8 Positive tourniquet test = DHF 4 Tourniquet test is a nonspecific indicator of capillary fragility

70. 8 DHF is a pediatric disease 4 All age groups are involved 8 DHF is a problem of low income families 4 All socioeconomic groups are affected

71. * No licensed vaccine at present * Effective vaccine must be tetravalent * Field testing of an attenuated tetravalent vaccine currently underway * Effective, safe and affordable vaccine is awaited

73. * Larvicides may be used to kill immature aquatic stages * Ultra-low volume fumigation ineffective against adult mosquitoes * Mosquitoes may have resistance to commercial aerosol sprays

74.  Biological control * Largely experimental * Option: place fish in containers to eat larvae  Environmental control * Elimination of larval habitats * Most likely method to be effective in the long term