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Sonia Mara Raboni2005
Laboratório de Virologia – Hospital de Clínicas da UFPR
IBMP – Instituto de Biologia Molecular do Paraná
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Genus Genus Human diseaseHuman disease
BunyavirusBunyavirus LaCrosse encephalitis, LaCrosse encephalitis, othersothers
PhlebovirusPhlebovirus Rift Valley fever, sandfly Rift Valley fever, sandfly feverfever
NairovirusNairovirus Crimean-Congo Crimean-Congo hemorrhagic feverhemorrhagic fever
TospovirusTospovirus Plant virus, no known Plant virus, no known human diseasehuman disease
HantavirusHantavirus Hemorrhagic fever with Hemorrhagic fever with renal syndromerenal syndrome
Hantavirus pulmonary Hantavirus pulmonary syndromesyndrome
5 genera, 250 species5 genera, 250 species
Family Family BunyaviridaeBunyaviridae
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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
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No arthropod vector establishedNo arthropod vector establishedUnique among genera of BunyaviridaeUnique among genera of Bunyaviridae
Rodent hostsRodent hostsGenus and possibly species specificGenus and possibly species specific
TransmissionTransmissionAerosolization of rodent excretaAerosolization of rodent excreta
Characteristics of Characteristics of HantavirusesHantaviruses
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Chronically infected Chronically infected rodentrodent
Virus is present in Virus is present in aerosolized excreta, aerosolized excreta,
particularly urineparticularly urine
Horizontal transmission of Horizontal transmission of infection by intraspecific infection by intraspecific
aggressive behavioraggressive behavior
Virus also present in Virus also present in throat swab and fecesthroat swab and feces
Secondary aerosols, mucous Secondary aerosols, mucous membrane contact, and skin membrane contact, and skin
breaches are also a breaches are also a considerationconsideration
Transmission of HantavirusesTransmission of Hantaviruses
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Sin NombreSin NombrePeromyscus maniculatus
Rio SegundoRio SegundoReithrodontomys mexicanusReithrodontomys mexicanus
El Moro CanyonEl Moro CanyonReithrodontomys megalotisReithrodontomys megalotis
AndesAndesOligoryzomys longicaudatusOligoryzomys longicaudatus
BayouBayouOryzomys palustrisOryzomys palustris
Black Creek CanalBlack Creek CanalSigmodon hispidusSigmodon hispidus
Rio MamoreRio MamoreOligoryzomys microtisOligoryzomys microtis
Laguna NegraLaguna NegraCalomys lauchaCalomys laucha
MuleshoeMuleshoeSigmodon hispidus
New YorkNew YorkPeromyscus leucopusPeromyscus leucopus
JuquitibaJuquitibaUnknown HostUnknown Host MacielMaciel
Necromys benefactusNecromys benefactus
Hu39694Hu39694Unknown HostUnknown Host
LechiguanasLechiguanasOligoryzomys flavescensOligoryzomys flavescens
PergaminoPergaminoAkodon azaraeAkodon azarae
OrOránánOligoryzomys longicaudatusOligoryzomys longicaudatus
CCañaño Delgaditoo DelgaditoSigmodon alstoniSigmodon alstoni
Isla VistaIsla VistaMicrotus californicus
Bloodland LakeBloodland LakeMicrotus ochrogasterMicrotus ochrogaster
Prospect HillProspect HillMicrotus pennsylvanicusMicrotus pennsylvanicus
New World HantavirusesNew World Hantaviruses
BermejoBermejoOligoryzomys chacoensisOligoryzomys chacoensis
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Peromyscus maniculatusDeer mouse
Sigmodon hispidusCotton rat
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Hantavirus Pulmonary Hantavirus Pulmonary SyndromeSyndromePathogenesisPathogenesis
Funcional derangement of vascular endotheliumNo cytopathic effectsImmunopathologic
Mediated by cytokine responses Pulmonary and cardiac effects
Thrombocytopenia consumptionsequestration (RE system)
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Most FrequentMost Frequent OtherOther RareRare
FeverFever DizzinessDizziness RhinorrheaRhinorrhea
MyalgiaMyalgia ArthralgiaArthralgia Sore Sore ThroatThroat
Nausea/Nausea/VomitingVomiting
CoughCough
Shortness Shortness ofofBreath Breath (late (late in the course in the course
of disease)of disease)
Hantavirus Pulmonary Hantavirus Pulmonary SyndromeSyndromeClinical Clinical PresentationPresentation
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TachypneaTachypnea
TachycardiaTachycardia
HypotensionHypotension
Crackles or rales on lung Crackles or rales on lung examination examination
Hantavirus Pulmonary Hantavirus Pulmonary SyndromeSyndrome
Physical ExaminationPhysical Examination
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Bilateral interstitial Bilateral interstitial infiltratesinfiltrates
moderate to rapid progressionmoderate to rapid progression
Bilateral alveolar Bilateral alveolar infiltratesinfiltrates
Pleural effusionPleural effusion
Hantavirus Pulmonary Hantavirus Pulmonary SyndromeSyndrome
Radiographic FindingsRadiographic Findings
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May 27, 1993May 27, 1993
May 30, 1993May 30, 1993
May 31, 1993May 31, 1993Source: Dr. L. Ketai
Radiographic Progression of Radiographic Progression of HPS HPS
in the Lungsin the Lungs
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ChemistryChemistryLow albuminLow albumin
Elevated LDHElevated LDH
Elevated AST (SGOT)Elevated AST (SGOT)
Elevated ALT (SGPTElevated ALT (SGPT))
HematologyHematologyLow platelet count Low platelet count
Atypical lymphocytes Atypical lymphocytes
(immunoblasts)(immunoblasts)
Left shift on WBC differentialLeft shift on WBC differential
Elevated hematocritElevated hematocrit
Elevated WBCElevated WBC
Hantavirus Pulmonary Hantavirus Pulmonary SyndromeSyndromeCommon Laboratory FindingsCommon Laboratory Findings
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+++
FeverFeverPulmonaryPulmonaryedemaedemaShockShock
DiuresisDiuresis
ProdromeProdromeCardiorespiratoryCardiorespiratoryConvalescenceConvalescence
ImmunoblastsImmunoblasts
HCTHCTASTASTLDHLDH
3-6 days3-6 days 7-10 days7-10 days
PlateletsPlatelets +
+++
+
++
+
++
++
+
+
++
+
+
++
+
+
++
+
+
+++++++
++++
Clinical Progression of HPSClinical Progression of HPS
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Early aggressive intensive Early aggressive intensive carecare
Early use of inotropic agents Early use of inotropic agents (Dobutamine)(Dobutamine)
Early ventilationEarly ventilation Careful monitoringCareful monitoring
OxygenationOxygenation Fluid balanceFluid balance Blood pressureBlood pressure
HPS HPS ManagementManagement
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SerologySerologyIgMIgM
IgGIgG
ImmunohistochemistryImmunohistochemistry
Reverse transcription Reverse transcription and polymerase chain and polymerase chain reaction (RT-PCR)reaction (RT-PCR)
Hantavirus Pulmonary Hantavirus Pulmonary SyndromeSyndromeLaboratory-confirmed Laboratory-confirmed
DiagnosisDiagnosis
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Peridomestic exposurePeridomestic exposure
Peridomestic & occupational Peridomestic & occupational
exposureexposure
Peridomestic & recreational Peridomestic & recreational
exposureexposure
Occupational exposure Occupational exposure
Entering/cleaning rodent-infested Entering/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 Exposure70 confirmed HPS 70 confirmed HPS
casescases
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Risk groupRisk groupForest workersForest workers11
Health care workersHealth care workers22
Prodromal HPSProdromal HPS33
ContactsContacts44
Rural OCCRural OCC55
Rodent workersRodent workers66
TotalTotal
Location/timeLocation/timeSW US, 1993SW US, 1993SW US, 1993SW US, 1993SW US, 1993SW US, 1993SW US, 1993SW US, 1993SW US, 1994SW US, 1994US, 1994US, 1994
Positive/tested (%)Positive/tested (%)0/1430/1430/3960/3963/299 (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, 19954. 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 Prevalence of SNV IgG Antibodies in Select U.S. Antibodies in Select U.S.
PopulationsPopulations
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CountryCountry Positive/tested (%)Positive/tested (%) TimeTimeParaguayParaguay11 44/345 (12.8%)44/345 (12.8%) 19951995Western ParaguayWestern Paraguay2 2 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, 1996 2. Ferrer, 1998 3. Peters, 1998; Weissenbacher, 1996
Prevalence of SNV IgG Prevalence of SNV IgG Antibodies in Select South Antibodies in Select South
American PopulationsAmerican Populations
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Control Mice InsideControl Mice Inside
Control Mice OutsideControl Mice Outside
Use Safety PrecautionsUse Safety Precautions
HPS PreventionHPS Prevention
TRANSMISSÃO TRANSMISSÃO AÉREAAÉREA
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SafetySafety Label as infectious substance and/or Label as infectious substance and/or human human blood precautions blood precautions Double container with absorbent material Double container with absorbent material sufficient sufficient for volume being sentfor volume being sent Plastic tubes preferable over glassPlastic tubes preferable over glass
ConditionsConditions Sera -- room temperature or cold pack Sera -- 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 -- room Formalin-fixed tissue and blocks -- room temperature (don't freeze)temperature (don't freeze)
Safety PrecautionsSafety Precautions
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HANTAVIROSES NO BRASIL
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HPS
Síndrome Pulmonar por Hantavirus 1993 Vírus Sin Nombre
Brasil Novembro/93 – juquitiba(SP) Sazonalidade: roedores
Paraná Maior numero de casos identificados
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Hantavirose: Casos e Óbitos, Brasil
1993-2004*
8 2 10 8
166
83 8541
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
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Hantavirose: Série Histórica do Paraná,
1992-2004*
4 42 2
85
26
6
32
1315
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
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Map of Brazil showing the state of Parana where the HPS cases have occurred
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Hantavirose: Dados Demográficos, Paraná
1992-2004*Distribuição de Casos
por Sexo
M94%
F6%
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
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Hantavirose: Ambiente de Contaminação, Paraná, 1992-2004*
6% 3%
90%
1%
Domiciliar
Lazer
Trabalho
Ignorado
* Dados parciais. Fonte: SESA/CSA/DV zoonoses e intoxicações
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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
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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
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Hantavirose - Inquérito Soroepidemiológico
General CarneiroN = 14219,7%(+)
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HANTAVIROSESPropostas
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;
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Objetivos
Epidemiologia Molecular
Correlação Clínica x Molecular Produção de antígeno recombinante para diagnóstico
Desenvolvimento de métodos de diagnóstico laboratorial
Inicio 0ut/2002
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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
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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
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0
5
10
15
num
ber o
f cas
esFigure 2. Seasonal Distribution of
Hantavirus Pulmonary Syndrome, 1998-2004, Paraná State, Brazil
1998 1999 20022000 2001 2003 2004
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nRT-PCR S Segment
434 pb
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RT-PCR Nucleoproteína
434 pb
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Table 3: nRT/PCR for hantavirus detection from blood and clot fractions from patients with hantavirus pulmonary
syndrome
N Patient* nRT-PCRPrimers Johnson
et al.
nRT-PCRBrazil-specific Primers
N Patient* nRT-PCRPrimers 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%
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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, BrazilJ Med Virol, 2005, in press
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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
HantaanSeoulDobrava
PuumalaProspect Hill
PrgMac
BerLec
Andes
LNV
SNV
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Brazil Hantavirus Recombinant Nucleoprotein
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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
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-hantavirus -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
30oC
37oC
50kDa
40kDa
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Ensaio ImunoenzimáticoIgG
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EIE IgG: AMOSTRAS IgM POSITIVAS
5
4
5 5
10
9
0
2
4
6
8
10
12
PCR + PCR -
Positivo NegativoTotal
N = 19
47% de positividade
Soroteca: IBMP
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IgG: AMOSTRAS IgM POSITIVAS
PositivasColeta (dias)
Media: 4 dias
NegativasColeta (dias)
Media: 5,4 dias
ObservarAmostras 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
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IgG: AMOSTRAS IgM POSITIVAS
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IgG: Estudo Soroepidemiológicode General Carneiro
9
99
010
71
10
20
40
60
80
100
Kit IBMP Kit Focus
reagente não reagente indeterminado
N = 107
N = 82
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IgG: Estudo Soroepidemiológicode 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
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IgG: Estudo Soroepidemiológicode General Carneiro
11 amostras foram selecionadas para teste de imunoblotting Todos os
resultados foram concordantes com os testes IBMP e Pergamino
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IgG: Estudo Soroepidemiológico
de General Carneiro - Conclusão Gold Standard
EIEIBMP
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%
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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
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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
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IgG: Doadores de Sangueimunoblotting
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Ensaio ImunoenzimáticoIgM (em fase de padronização)
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IgM: Comparação de amostras da Soroteca do
IBMP25
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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