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  • 1. Hantavirus pulmonary syndrome (HPS), caused by a previously unrecognized hantavirus, was first recognized in May 1993. The disease begins with non-specific symptoms which include fever, muscle aches, headache, abdominal pain, nausea, vomiting, and progresses to include coughing and shortness of breath. The symptoms usually occur between one and six weeks after exposure to virul ladent in rodent' excreta. The disease rapidly progresses to cardiac and respiratory failure, usually requiring intensive care management.
  • 2. Sin Nombre virus(formerly Muerto Canyon Virus (MCV))the most frequent virus recognized as a cause of the Hantavirus Pulmonary Syndrome(HPS) is a member of the family Bunyaviridae.
  • 3. Characteristics of Hantaviruses.
  • 10. Clinical presentation on admission.
  • 12.Radiographic progression of HPS in the Lung is shown in this slide; these chest X-rays of a HPS patient were taken on May 27, May 30, and May 31, 1999 (courtesy of L. Ketai). In HPS, the lung is the target organ, with a distinctive bilateral pulmonary edema due to increased vascular permeability. The abrupt onset follows a febrile prodrome of 1 to 10 days' duration (typically 3 - 5 days). The chest radiograph occasionally may be normal very early in the pulmonary phase of HPS, but it soon progresses to the more typical pattern of bilateral interstitial or alveolar pulmonary edema. The findings differ from those of adult respiratory distress syndrome in that indications of interstitial edema (Kerley B lines, peribronchial cuffing) are more common and the distribution is more central in HPS. *
  • 13. Clinical laboratory presentation.
  • 14.Clinical course of HPS: There is a febrile prodrome that progresses to cardiopulmonary insufficiency. The onset of the immune response precedes severe organ failure, and may mediate the vascular compromise. In HPS, severe hypotension is usually coincident with onset of respiratory failure, which reflects the severe physiologic impact of the non-cardiogenic lung edema. (AST--aspartate aminotransferase; HCT--hematocrit; LDH--lactate dehydrogenase; PTT-partial thromboplastin time.) *
  • 15. HPS patient management.
  • 18.Laboratory confirmation of HPS.
  • 26.Review of selected confirmed cases conducted to characterize rodent exposure.
  • 27.Seroprevalence of SNV IgG antibodies in select occupational groups.
  • 34.Shipping and labeling specimens.
  • Slide sem título

    1. 1. Sonia Mara Raboni 2005 Laboratório de Virologia – Hospital de Clínicas da UFPR IBMP – Instituto de Biologia Molecular do Paraná
    2. 2. GenusGenus Human diseaseHuman disease BunyavirusBunyavirus LaCrosse encephalitis, othersLaCrosse encephalitis, others PhlebovirusPhlebovirus Rift Valley fever, sandfly feverRift Valley fever, sandfly fever NairovirusNairovirus Crimean-Congo hemorrhagic feverCrimean-Congo hemorrhagic fever TospovirusTospovirus Plant virus, no known human diseasePlant virus, no known human disease HantavirusHantavirus Hemorrhagic fever with renalHemorrhagic fever with renal syndromesyndrome Hantavirus pulmonary syndromeHantavirus pulmonary syndrome 5 genera, 250 species5 genera, 250 species Family BunyaviridaeFamily Bunyaviridae
    3. 3. HANTAVIRUS  80 – 120 nm  Genoma s/s –  Capacidade de codificação  L – 8,5 kb / L, ?  M – 5,7 kb / G1, G2  S – 1,9 kb / N  Linear  RNA genômico não infecioso
    4. 4. No arthropod vector establishedNo arthropod vector established Unique among genera of BunyaviridaeUnique among genera of Bunyaviridae Rodent hostsRodent hosts Genus and possibly species specificGenus and possibly species specific TransmissionTransmission Aerosolization of rodent excretaAerosolization of rodent excreta Characteristics of HantavirusesCharacteristics of Hantaviruses
    5. 5. Chronically infectedChronically infected rodentrodent Virus is present inVirus is present in aerosolized excreta,aerosolized excreta, particularly urineparticularly urine Horizontal transmission ofHorizontal transmission of infection by intraspecificinfection by intraspecific aggressive behavioraggressive behavior Virus also present inVirus also present in throat swab and fecesthroat swab and feces Secondary aerosols, mucousSecondary aerosols, mucous membrane contact, and skin breachesmembrane contact, and skin breaches are also a considerationare also a consideration Transmission of HantavirusesTransmission of Hantaviruses
    6. 6. Sin NombreSin Nombre Peromyscus maniculatus Rio SegundoRio Segundo Reithrodontomys mexicanusReithrodontomys mexicanus El Moro CanyonEl Moro Canyon Reithrodontomys megalotisReithrodontomys megalotis AndesAndes Oligoryzomys longicaudatusOligoryzomys longicaudatus BayouBayou Oryzomys palustrisOryzomys palustris Black Creek CanalBlack Creek Canal Sigmodon hispidusSigmodon hispidus Rio MamoreRio Mamore Oligoryzomys microtisOligoryzomys microtis Laguna NegraLaguna Negra Calomys lauchaCalomys laucha MuleshoeMuleshoe Sigmodon hispidus New YorkNew York Peromyscus leucopusPeromyscus leucopus JuquitibaJuquitiba Unknown HostUnknown Host MacielMaciel Necromys benefactusNecromys benefactus Hu39694Hu39694 Unknown HostUnknown Host LechiguanasLechiguanas Oligoryzomys flavescensOligoryzomys flavescens PergaminoPergamino Akodon azaraeAkodon azarae OrOránán Oligoryzomys longicaudatusOligoryzomys longicaudatus CCañaño Delgaditoo Delgadito Sigmodon alstoniSigmodon alstoni Isla VistaIsla Vista Microtus californicus Bloodland LakeBloodland Lake Microtus ochrogasterMicrotus ochrogaster Prospect HillProspect Hill Microtus pennsylvanicusMicrotus pennsylvanicus New World HantavirusesNew World Hantaviruses BermejoBermejo Oligoryzomys chacoensisOligoryzomys chacoensis
    7. 7. Peromyscus maniculatus Deer mouse Sigmodon hispidus Cotton rat
    8. 8. Hantavirus Pulmonary SyndromeHantavirus Pulmonary Syndrome PathogenesisPathogenesis Funcional derangement of vascular endothelium No cytopathic effects Immunopathologic  Mediated by cytokine responses  Pulmonary and cardiac effects Thrombocytopenia ↑ consumption sequestration (RE system)
    9. 9. Most FrequentMost Frequent OtherOther RareRare FeverFever DizzinessDizziness RhinorrheaRhinorrhea MyalgiaMyalgia ArthralgiaArthralgia Sore ThroatSore Throat Nausea/VomitingNausea/Vomiting CoughCough Shortness ofShortness of BreathBreath (late in(late in the course ofthe course of disease)disease) Hantavirus Pulmonary SyndromeHantavirus Pulmonary Syndrome Clinical PresentationClinical Presentation
    10. 10.  TachypneaTachypnea  TachycardiaTachycardia  HypotensionHypotension  Crackles or rales on lungCrackles or rales on lung examinationexamination Hantavirus Pulmonary SyndromeHantavirus Pulmonary Syndrome Physical ExaminationPhysical Examination
    11. 11.  Bilateral interstitialBilateral interstitial infiltratesinfiltrates moderate to rapid progressionmoderate to rapid progression  Bilateral alveolar infiltratesBilateral alveolar infiltrates  Pleural effusionPleural effusion Hantavirus Pulmonary SyndromeHantavirus Pulmonary Syndrome Radiographic FindingsRadiographic Findings
    12. 12. May 27, 1993May 27, 1993 May 30, 1993May 30, 1993 May 31, 1993May 31, 1993Source: Dr. L. Ketai Radiographic Progression of HPSRadiographic Progression of HPS in the Lungsin the Lungs
    13. 13. ChemistryChemistry Low albuminLow albumin Elevated LDHElevated LDH Elevated AST (SGOT)Elevated AST (SGOT) Elevated ALT (SGPTElevated ALT (SGPT)) HematologyHematology Low platelet countLow platelet count Atypical lymphocytesAtypical lymphocytes (immunoblasts)(immunoblasts) Left shift on WBC differentialLeft shift on WBC differential Elevated hematocritElevated hematocrit Elevated WBCElevated WBC Hantavirus Pulmonary SyndromeHantavirus Pulmonary Syndrome Common Laboratory FindingsCommon Laboratory Findings
    14. 14. + + + FeverFever PulmonaryPulmonary edemaedema ShockShock DiuresisDiuresis ProdromeProdrome CardiorespiratoryCardiorespiratory ConvalescenceConvalescence ImmunoblastsImmunoblasts HCTHCT ASTAST LDHLDH 3-6 days3-6 days 7-10 days7-10 days PlateletsPlatelets + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ++ + ++ + + Clinical Progression of HPSClinical Progression of HPS
    15. 15.  Early aggressive intensive careEarly aggressive intensive care  Early use of inotropic agentsEarly use of inotropic agents (Dobutamine)(Dobutamine)  Early ventilationEarly ventilation  Careful monitoringCareful monitoring  OxygenationOxygenation  Fluid balanceFluid balance  Blood pressureBlood pressure HPS ManagementHPS Management
    16. 16.  SerologySerology IgMIgM IgGIgG  ImmunohistochemistryImmunohistochemistry  Reverse transcriptionReverse transcription and polymerase chainand polymerase chain reaction (RT-PCR)reaction (RT-PCR) Hantavirus Pulmonary SyndromeHantavirus Pulmonary Syndrome Laboratory-confirmed DiagnosisLaboratory-confirmed Diagnosis
    17. 17. Peridomestic exposurePeridomestic exposure Peridomestic & occupational exposurePeridomestic & occupational exposure Peridomestic & recreational exposurePeridomestic & recreational exposure Occupational exposureOccupational exposure Entering/cleaning rodent-infestedEntering/cleaning rodent-infested structuresstructures Armstrong, L.R. et al., JID 1995; 172 (October)Armstrong, L.R. et al., JID 1995; 172 (October) 69% (48/70)69% (48/70) 19% (13/70)19% (13/70) 9% (6/70)9% (6/70) 4% (3/70)4% (3/70) 9% (6/70)9% (6/70) Rodent ExposureRodent Exposure 70 confirmed HPS cases70 confirmed HPS cases
    18. 18. Risk groupRisk group Forest workersForest workers11 Health care workersHealth care workers22 Prodromal HPSProdromal HPS33 ContactsContacts44 Rural OCCRural OCC55 Rodent workersRodent workers66 TotalTotal Location/timeLocation/time SW US, 1993SW US, 1993 SW US, 1993SW US, 1993 SW US, 1993SW US, 1993 SW US, 1993SW US, 1993 SW US, 1994SW US, 1994 US, 1994US, 1994 Positive/tested (%)Positive/tested (%) 0/1430/143 0/3960/396 3/299 (1.0%)3/299 (1.0%) 3/239 (1.3%)3/239 (1.3%) 1/522 (0.2%)1/522 (0.2%) 8/932 (0.9%)8/932 (0.9%) 15/2531 (0.6%)15/2531 (0.6%) 1. Vitek et al, 19961. Vitek et al, 1996 2. Vitek et al, 19962. Vitek et al, 1996 3. Simonsen et al, 19953. Simonsen et al, 1995 4. Zeitz et al, 19954. Zeitz et al, 1995 5. Zeitz et al, 19955. Zeitz et al, 1995 6. Armstrong et al, 19956. Armstrong et al, 1995 Prevalence of SNV IgG Antibodies inPrevalence of SNV IgG Antibodies in Select U.S. PopulationsSelect U.S. Populations
    19. 19. CountryCountry Positive/tested (%)Positive/tested (%) TimeTime ParaguayParaguay11 44/345 (12.8%)44/345 (12.8%) 19951995 Western ParaguayWestern Paraguay22 78/193 (40.4%)78/193 (40.4%) 19931993 (Indian Population)(Indian Population) ArgentinaArgentina33 <1%<1% 19961996 Salta ProvinceSalta Province22 38/222 (17.1%)38/222 (17.1%) 19931993 (Indian Population)(Indian Population) ChileChile33 2-13%2-13% 19971997 1. Williams, 19971. Williams, 1997 2. Ferrer, 1998 3. Peters, 1998; Weissenbacher, 19962. Ferrer, 1998 3. Peters, 1998; Weissenbacher, 1996 Prevalence of SNV IgG Antibodies inPrevalence of SNV IgG Antibodies in Select South American PopulationsSelect South American Populations
    20. 20. Control Mice InsideControl Mice Inside Control Mice OutsideControl Mice Outside Use Safety PrecautionsUse Safety Precautions HPS PreventionHPS Prevention TRANSMISSÃOTRANSMISSÃO AÉREAAÉREA
    21. 21. SafetySafety  Label as infectious substance and/or humanLabel as infectious substance and/or human blood precautionsblood precautions  Double container with absorbent materialDouble container with absorbent material sufficientsufficient for volume being sentfor volume being sent  Plastic tubes preferable over glassPlastic tubes preferable over glass ConditionsConditions  Sera -- room temperature or cold packSera -- room temperature or cold pack  Clot or buffy coat -- dry iceClot or buffy coat -- dry ice  Fresh tissues (1-cm cubes) -- dry iceFresh tissues (1-cm cubes) -- dry ice  Formalin-fixed tissue and blocks -- roomFormalin-fixed tissue and blocks -- room temperature (don't freeze)temperature (don't freeze) Safety PrecautionsSafety Precautions
    22. 22. HANTAVIROSES NO BRASIL
    23. 23. HPS  Síndrome Pulmonar por Hantavirus  1993  Vírus Sin Nombre  Brasil  Novembro/93 – juquitiba(SP)  Sazonalidade:  roedores  Paraná  Maior numero de casos identificados
    24. 24. Hantavirose: Casos e Óbitos, Brasil 1993-2004* 8 2 10 8 166 83 85 41 233 79 502 213 0 100 200 300 400 500 600 Norte Nordeste Sudeste Centro- Oeste Sul Total Casos Obitos *Dados parciais Dez/2004. Fonte: Ministério da Saúde
    25. 25. Hantavirose: Série Histórica do Paraná, 1992-2004* 4 4 2 2 8 5 26 6 32 13 15 6 14 6 10 5 0 5 10 15 20 25 30 35 1992 1998 1999 2000 2001 2002 2003 2004 Casos Obitos *Dados parciais. Fonte: SESA/CSA/DV zoonoses e intoxicações N=111
    26. 26. Map of Brazil showing the state of Parana where the HPS cases have occurred
    27. 27. Hantavirose: Dados Demográficos, Paraná 1992-2004* Distribuição de Casos por Sexo M 94% F 6% 0 5 10 15 20 25 30 35 0-9a 10- 19a 20- 29a 30- 39a 40- 49a 50- 59a 60- 69a Distribuição por Faixa Etária * Dados parciais. Fonte: SESA/CSA/DV zoonoses e intoxicações
    28. 28. Hantavirose: Ambiente de Contaminação, Paraná, 1992-2004* 6% 3% 90% 1% Domiciliar Lazer Trabalho Ignorado * Dados parciais. Fonte: SESA/CSA/DV zoonoses e intoxicações
    29. 29. Hantavirose: Evolução das Ocupações, Paraná, 1992-2004* 0 5 10 15 20 25 1992 1998 1999 2000 2001 2002 2003 2004 Agricultura Relacionado ao Pinus * Dados parciais. Fonte: SESA/CSA/DV zoonoses e intoxicações
    30. 30. Hantavirose – Coleta de Roedores N = 181 (+ 13,2%) Oligoryzomys sp (19 – 79,1%) Akodon sp (4 – 16,6%) Trichomys sp (1 – 4,1%)Fonte SESA;CSA;DV zoonoses e intoxicações
    31. 31. Hantavirose - Inquérito Soroepidemiológico General Carneiro N = 142 19,7%(+)
    32. 32. HANTAVIROSES Propostas Investigar sorologias de contatos com pacientes soropositivos para analisar transmissão inter-humana; Caracterização genômica dos hantavirus detectados em pacientes e camundongos; Produção de antígeno N-recombinante para o desenvolvimento de ensaio para detecção sorológica desta infecção;
    33. 33. Objetivos Epidemiologia Molecular Correlação Clínica x Molecular Produção de antígeno recombinante para diagnóstico Desenvolvimento de métodos de diagnóstico laboratorial Inicio 0ut/2002
    34. 34. Clinical Survey of Hantavirus in Southern Brazil and the Development of an Specific Molecular Diagnosis Tools Sonia M Raboni1 , Gisélia Rubio2 , Luana de Borba1 , Aurélio Zeferino1 , Irene Skraba3 , Samuel Goldenberg1 and Claudia N Duarte dos Santos1 1 Instituto de Biologia Molecular do Paraná, IBMP /Fiocruz/Brazil 2 Secretaria Estadual de Saúde do Paraná, Brazil 3 Laboratório Central do Estado do Paraná, Brazil Am J Trop Med Hyg, 2005, in press
    35. 35. Table 1. Clinical and Laboratory Records from Brazilian HPS Patients Clinical or Laboratory Data Frequency n/N % Fever 72/82 87.8 Headache 70/82 85.3 Cough 67/81 82.7 Myalgia 66/81 81.4 Thoracic pain 49/81 60.4 Dyspnea 54/82 65.8 Vomiting 39/82 47.5 Hypotension (SP <100 mm Hg) 19/38 50 Interstitial infiltrate 38/47 80.8 Pleural effusions 1/38 2,6 Hematocrit >45% 52/71 73.2 Thrombocytopenia (<150,000/mm3 ) 45/63 71.4 Leukocytosis (>10,000 cells/mm3 ) 22/54 40.7 Creatinine > 2,0mg/dl 19/29 65.5
    36. 36. 0 5 10 15 Sep Dec Mar Jun Sep Dec Mar Jun Sep Dec Mar Jun Sep Dec Mar Jun Sep Dec Mar Jun Sep Dec numberofcases Figure 2. Seasonal Distribution of Hantavirus Pulmonary Syndrome, 1998-2004, Paraná State, Brazil 1998 1999 20022000 2001 2003 2004
    37. 37. nRT-PCR S Segment 434 pb
    38. 38. RT-PCR Nucleoproteína 434 pb
    39. 39. Table 3: nRT/PCR for hantavirus detection from blood and clot fractions from patients with hantavirus pulmonary syndrome N Patient* nRT-PCR Primers Johnson et al. nRT-PCR Brazil- specific Primers N Patient* nRT-PCR Primers Johnson et al. nRT-PCR Brazil- specific Primers 1 BR/01-50 - + 12 BR/02-85 - + 2 BR/01-51 - - 13 BR/02-86 NT - 3 BR/01-52 - - 14 BR/03-91 NT - 4 BR/01-55 - + 15 BR/03-92 NT - 5 BR/01-60 - - 16 BR/03-95 NT + 6 BR/02-67 - - 17 BR/03-97 NT + 7 BR/01-69 - - 18 BR/03-98 NT + 8 BR/02-71 - + 19 BR/03-99 NT + 9 BR/02-72 - + 20 BR/03-100 NT + 10 BR/02-73 - + 21 BR/03-101 NT + 11 BR/02-74 + - 22 BR/04-102 NT + 60%
    40. 40. Hantaviruses in Central South America: Insights from the Phylogenetic Analysis of the S Segment from HPS Cases in Paraná, Brazil Sonia M Raboni, MD, MSc;* Christian M.Probst, MD,MSc.*; Juliano Bordignon, MSc;* Aurélio Zeferino;* Claudia N. Duarte dos Santos, PhD*# . *Instituto de Biologia Molecular do Paraná, IBMP /Fiocruz/Brazil  Universidade Federal do Paraná, Brazil J Med Virol, 2005, in press
    41. 41. 0.1 SEO DOB ILV1 MO46 PHV TUL PUU TOP KHU BAY MUL BCC PRG MAC OWR ACRF ACB LPM JM AS LNA CAN DA GFE APM VS BER LEC Hu39694 ORN AND Nort 9717869 9718133 CHI 7913 AH 1 LN OM 556 RM 97 RMx 1 CC107 CC074 SN 77734 NM H10 NM R11 MON H NY1 RI 1 NYa Paraná Hantaan Seoul Dobrava Puumala Prospect Hill Prg Mac Ber Lec Andes LNV SNV
    42. 42. Brazil Hantavirus Recombinant Nucleoprotein
    43. 43. Objectives  Produce a recombinant antigen to detect IgM antibodies against hantavirus by immunoblot and/or capture ELISA  The development of region-specific antigens should be undertaken to improve serological reactivity
    44. 44. α-hantavirus α-histidina BM 1 2 3 4 5 6 48kDa 6 5 4 3 2 1 BM 6 5 4 3 2 1 BM 50kDa 40kDa 1. NI 2. 0,5mM IPTG 3. 1mM IPTG 4. NI 5. 0,5mM IPTG 6. 1mM IPTG 30o C 37o C 50kDa 40kDa
    45. 45. Ensaio Imunoenzimático IgG
    46. 46. EIE IgG: AMOSTRAS IgM POSITIVAS 5 4 5 5 10 9 0 2 4 6 8 10 12 PCR + PCR - Positivo Negativo Total N = 19 47% de positividade Soroteca: IBMP
    47. 47. IgG: AMOSTRAS IgM POSITIVAS Positivas Coleta (dias) Media: 4 dias Negativas Coleta (dias) Media: 5,4 dias Observar Amostras com resultados de IgG desconhecidos Períodos de coleta variados Sem coleta de segunda amostra para avaliar soroconversão Maior parte das amostras: coágulos ou material hemolisado
    48. 48. IgG: AMOSTRAS IgM POSITIVAS
    49. 49. IgG: Estudo Soroepidemiológico de General Carneiro 9 99 0 10 71 1 0 20 40 60 80 100 Kit IBMP Kit Focus reagente não reagente indeterminado N = 107 N = 82
    50. 50. IgG: Estudo Soroepidemiológico de General Carneiro  82 Amostras testadas pelos dois kits  14 amostras tiveram resultados discordantes  Repetido estes testes pelo kit IBMP e pelo kit Pergamino (Argentina) 7 7 0 7 7 00 2 4 6 8 kit IBMP kit PERG reagente não reagente indetem N = 14
    51. 51. IgG: Estudo Soroepidemiológico de General Carneiro  11 amostras foram selecionadas para teste de imunoblotting  Todos os resultados foram concordantes com os testes IBMP e Pergamino
    52. 52. IgG: Estudo Soroepidemiológico de General Carneiro - Conclusão Gold Standard EIE IBMP Positivo Negativo Total Positivo 5 0 5 Negativo 0 77 77 Total 82 PELO TESTE IBMP: Prevalência de 8,4% (9/107) de anticorpos IgG anti- hantavirus na população de General Carneiro. CONSIDERANDO COMO VERDADEIRO POSITIVO OS RESULTADOS CONCORDANTES EM 2 TESTES E O WESTERN BLOT PARA OS RESULTADOS DISCORDANTES TEM-SE: (Em 82 amostras analisadas por pelo menos 2 testes) Neste estudo: S = 100% e E = 100%
    53. 53. IgG: Doadores de Sangue 1 2 21 2 3 81 98 115 83 102 120 0 20 40 60 80 100 120 data 23/nov data 26/nov data 29/nov reagente indeterminado não reagente total Soroteca Hospital de Clínicas - UFPR Cut off: Média DO + 3x Desvio padrão Indeterminado: 10% acima ou abaixo do Cut off. N = 305
    54. 54. IgG: Doadores de Sangue  30 soros que tiveram absorbância acima de 0,2 foram repetidas com o kit IBMP  18 mantiveram absorbância acima de 0,2 na repetição.  17 destas foram avaliadas pelo teste de imunoblotting  3 amostras foram positivas
    55. 55. IgG: Doadores de Sangue imunoblotting
    56. 56. Ensaio Imunoenzimático IgM (em fase de padronização)
    57. 57. IgM: Comparação de amostras da Soroteca do IBMP 25 13 0 5 10 15 20 25 kit IBMP reagente não reagente Das 13 amostras negativas:  5 amostras eram de casos negativos  As outras 8 amostras negativas foram repetidas com kit IBMP, Pergamino e Focus:  6 foram negativas  1 negativa IBMP e positiva pelos outros 2 kits  1 negativa IBMP e Pergamino, mas indeterminada com kit Focus N = 38

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