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Influenza Virus review
• Family Orthomyxoviridae:
• three types– Influenza A– Influenza B– Influenza C (not considered of
critical importance)– Segmented (8) ssRNA genome with
lipid envelop
Influenza A
• Further classified by Hemagglutinin (H) and Neuraminidase (N) sub-types
• Current circulating strains are H1N1 and H3N2
• Human subtypes include H1N1, H3N2, H1N2, and H2N2
• Avian subtypes include H1 to H15 and N1 to N9
• Bird human H5N1, H9N2, H7N7, H7N2, H7N3
Influenza B• Produces less serious disease than does Influenza
type A
• Not categorized as by H or N type as Influenza A is
• First isolated in 1949
• Not known to be responsible for epidemics
Influenza C
Influenza as a public health threat
• Influenza Viruses are the respiratory viruses of greatest public health importance, particularly Influenza A
Epidemiology, Prevention, and Control of Influenza; esp. Influenza A
Why is Influenza A such a public health threat?
• Antigenic drift (variation within the HN sub-type) or Antigenic shift (variation between different HN sub-types) makes large portions of the population immunologically naïve on a regular basis
• Influenza epidemics can be characterized as inter-pandemic or pandemic
Global surveillance
– Avian influenza: H5N1: HIGHLY PATHOGENIC
• Present in Asia since 1996• Extent/distribution not firmly
established• Threat level is high• In 1997 there were 18 confirmed
cases of H5N1 with 6 deaths• Other avian strains being watched
include H7N7 and H9N2
Pandemic Influenza
– Recipe for a human pandemic• Emergence of a novel sub-type of
influenza to which the population is immunologically naïve
• Replication in humans disease• Efficient human-to-human transmission• Note: H5N1 has meet all criteria except the
third one.
Pandemic planning
– An influenza pandemic will be unlike other public health emergencies or common disasters.
• Inevitable• Will arrive with very little warning• Locally explosive epidemics• Widespread, not focused like a bio-terrorism
event• Will put an extraordinary strain on human and
material resources• Effect will be relatively prolonged –weeks to
months
Laboratory issues
• Laboratory safety• Tissue culture techniques• Rapid test kits• HA/HI sub-typing• Immuno-fluorescent testing• Real time PCR analysis
– Molecular typing and sub-typing
Previous testing algorithm
– Inoculation of specimens into cell culture; one diploid, one Hep-2, one Viromed Rhmk and two Diagnostic Hybrid Rhmk
– In the absence of CPE, “blind” Hemadsorption (HAD) at days 7 and 14.
– In the absence of CPE “blind” passage of Hep2 with “blind” FA for RSV at day 14 for all patients ≤5 years old
– Identification of Influenza isolates:• Immuno-fluorescent testing to identify type (A or B)
followed by Hemagglutination Inhibition (HI) testing to identify sub-type
Influenza Diagnostic Table
Procedure Influenza
Types Detected
Acceptable Specimens
Time for Results
Rapid result
available
Viral culture A and B NP swab2, throat swab, nasal wash, bronchial wash, nasal aspirate, sputum
3-10 days 3
No
Immunofluorescence DFA An tibody Staining
A and B NP swab2, nasal wash, bronchial wash, nasal aspirate, sputum
2-4 hours
No
RT-PCR5 A and B NP swab2, throat swab, nasal wash, bronchial wash, nasal aspirate, sputum
1-2 days No
Serology A and B paired acute and convalescent serum samples6
>2 weeks
No
Enzyme Immuno Assay (EIA)
A and B NP swab2 , throat swab, nasal wash, bronchial wash
2 hours No
Rapid Diagnost ic Tests
Directigen Flu A7 (Becton-Dickinson)
A NP wash and aspirate <30 minutes
Yes
Directigen Flu A+B7,9 (Becton-Dickinson)
A and B NP swab2,aspirate, wash; lower nasal swab; throat swab; bronchioalveolar lavage
<30 minutes
Yes
Directigen EZ Flu A+B7,9 (Becton-Dickinson)
A and B NP swab2, aspirate, wash; lower nasal swab; throat swab; bronchioalveolar lavage
<30 minutes
Yes
FLU OIA4,7 (Biostar)
A and B NP swab2 , throat swab, nasal aspirate, sputum
<30 minutes
Yes
FLU OIA A/B 7, 9 (Biostar)
A and B NP swab2 , throat swab, nasal aspirate,
<30 minutes
Yes
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FluAlert1000SAS FluAlert-046230 (15 tests)The SAS Influenza A and B tests are visual and rapid assays for presumptive qualitative detection of Influenza A and B antigens from nasal washes and aspirates, respectively. This test is FDA Cleared and CLIAwaived. Use CPT CODE: 87804QW$250.00
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QDL-20183Quidel QuickVue Influenza A/B-(25 tests)The QuickVue® Influenza A+B test (dipstick format)allows for the rapid, qualitative detection of influenza type A and type B antigens directly from nasal swab, nasopharyngeal swab,nasal wash and/or nasal aspirate specimens. FDA Cleared and CLIAwaived$449.00
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INV-416-110BINAX NOW Influenza A/B (10 per kit)BINAX NOW Influenza A & B is an in vitro immunochromatographic assay to aid in the rapid differential diagnosis of influenza type A and B viral infections. 15-minute test for Influenza Types A& B w/ excellent sensitivity. FDA Cleared & CLIAwaived$175.00
PRUEBAS RÁPIDAS
ImmunofluorescenceImmunofluorescence
• Direct– Ab to tissue Ag is labeled with fluorochrome
Ag
FluorochromeLabeled Ab
Tissue Section
ImmunofluorescenceImmunofluorescence
• Indirect– Ab to tissue Ag is
unlabeled– Fluorochrome-labeled
anti-Ig is used to detect binding of the first Ab.
Ag
FluorochromeLabeled Anti-Ig
Tissue Section
UnlabeledAb
• Qualitative to Semi-Quantitative
HI para virus
Antivirus específicoS
Ac H5N1 Ac H3N2
HI NEGATIVA hay aglutinación de eritrocitos
HI POSITIVA hay inhibición por el Ac H3N2
Técnicas de amplificación Técnicas de amplificación
de Ac. Nucleicosde Ac. Nucleicos
• 1-PCR1-PCR
• 2-LCR2-LCR
• 3-NASBA, TMA3-NASBA, TMA
• 4-Amplificación de señal4-Amplificación de señal
• 5-Boomerang 5-Boomerang
PCRPCR
• 1-Qué es?1-Qué es?
• 2-En qué consiste?2-En qué consiste?
• 3-Cómo?3-Cómo?
• 4-Quién?4-Quién?
• 5-Ventajas5-Ventajas
• 6-Desventajas6-Desventajas
PCRPCR• Amplifica en forma exponencial el DNA del Amplifica en forma exponencial el DNA del
microorganismo. Esto le confiere un enorme microorganismo. Esto le confiere un enorme potencial para detectar pequeñas cantidades del potencial para detectar pequeñas cantidades del material inicialmaterial inicial
• Tipos de PCRTipos de PCR– MúltipleMúltiple– RT-PCRRT-PCR– Anidada (nested-PCR)Anidada (nested-PCR)– En tiempo realEn tiempo real– PCR-RFLPPCR-RFLP– PCR-ELISAPCR-ELISA
Enzima
SustratoSustrato
CromógenoCromógeno ColorColor
DETECCIÓN COLORIMÉTRICA DE RT-PCR-DETECCIÓN COLORIMÉTRICA DE RT-PCR-ELISA PARA ENTEROVIRUSELISA PARA ENTEROVIRUS
Amplificado con biotina
Sonda de captura
Estreptavidina
PCRPCR
• VARIEDADESVARIEDADES– PCR simplePCR simple– RT-PCRRT-PCR– RAPD´sRAPD´s– PCR semicuantitativaPCR semicuantitativa– PCR competitivaPCR competitiva– Nested PCRNested PCR– PCR múltiplePCR múltiple– Otras: real time PCR: Otras: real time PCR: Higuchi et al.Higuchi et al.