1. 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

2. 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

3. Influenza B Influenza C
Produces less serious disease than does Influenza type A
Not categorized as by H or N type as Influenza A is
Influenza C
First isolated in 1949
Not known to be responsible for epidemics

4. Influenza as a public health threat
Influenza Viruses are the respiratory viruses of greatest public health importance, particularly Influenza A

5. 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

6. 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

7. 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.

8. 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

9. 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

10. 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

13. PRUEBAS RÁPIDAS INV-416-110 BINAX 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
QDL-20183
Quidel 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
FluAlert1000
SAS FluAlert (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

14. Immunofluorescence Direct Y
Ab to tissue Ag is labeled with fluorochrome Ag Y
Fluorochrome
Labeled Ab
Tissue Section

15. Immunofluorescence Indirect Y Qualitative to Semi-Quantitative
Ab to tissue Ag is unlabeled
Fluorochrome-labeled anti-Ig is used to detect binding of the first Ab. Ag Y
Fluorochrome
Labeled Anti-Ig
Tissue Section
Unlabeled Ab
Qualitative to Semi-Quantitative

16. Figure A-17

17. PARTICIPANTES HA Y HI Anticuerpo Flu virus Eritrocitos NO AGLUTINA
AGLUTINACIÓN

18. Hemaglutinación (HA)
Flu virus
No virus
AGLUTINA
NO AGLUTINA

19. Hemagutinación para virus
Diluciones del Flu virus
No virus
advantages
sensitive and specific
simple to do, relatively fast compared to virus isolation
quantitative
disadvantages
requires laboratory facilities
NO AGLUTINAN
AGLUTINAN

20. Inhibición de la hemaglutinación (HI)
Virus es
bloqueado por Ac
Flu virus
NO AGLUTINAN
AGLUTINAN

21. HI para virus Antivirus específicoS Ac H5N1 Ac H3N2
advantages
sensitive and specific
simple to do, relatively fast compared to virus isolation
quantitative
disadvantages
requires laboratory facilities
HI POSITIVA hay inhibición por el Ac H3N2
HI NEGATIVA hay aglutinación de eritrocitos

22. Figure A-7
ELISA DE CAPTURA DE ANTÍGENO

23. Técnicas de amplificación de Ac. Nucleicos
1-PCR
2-LCR
3-NASBA, TMA
4-Amplificación de señal
5-Boomerang

24. PCR 1-Qué es? 2-En qué consiste? 3-Cómo? 4-Quién? 5-Ventajas
6-Desventajas

25. PCR
Amplifica en forma exponencial el DNA del microorganismo. Esto le confiere un enorme potencial para detectar pequeñas cantidades del material inicial
Tipos de PCR
Múltiple
RT-PCR
Anidada (nested-PCR)
En tiempo real
PCR-RFLP
PCR-ELISA

26. PCR

27. PCR
Termociclador

28. Detección del producto de PCR

29. DETECCIÓN COLORIMÉTRICA DE RT-PCR-ELISA PARA ENTEROVIRUS
Sustrato
Cromógeno
Color
Enzima
Amplificado con biotina
Sonda de captura
Estreptavidina

30. Sistema automatizado

31. PCR VARIEDADES PCR simple RT-PCR RAPD´s PCR semicuantitativa
PCR competitiva
Nested PCR
PCR múltiple
Otras: real time PCR: Higuchi et al.

32. RT-PCR