Transcript North America

‫تركيب فيروس األنفلونزا‬
‫• اسم العائلة الفيروسية ‪:‬‬
‫‪Orthomyxoviridae‬‬
‫•‬
‫•‬
‫•‬
‫•‬
‫مغلف‬
‫القطر ‪ 120-80 :‬نانومتر‬
‫الشكل ‪ :‬دائري أو بيضوي‬
‫الحمض النووي ‪RNA :‬‬
‫‪-‬ثالثة أنواع ‪A, B, C :‬‬
‫المستضدات السطحية ‪:‬‬
‫• )‪HA : (haemaglutinin‬‬
‫• )‪NA : (neuraminidase‬‬
‫الثالثاء‪ 11 ،‬نيسان‪2017 ،‬‬
‫‪1‬‬
Influenza Virus
2
2017 ،‫ نيسان‬11 ،‫الثالثاء‬
Types of Influenza Virus


3
Three types: A, B, C
Influenza Type A can infect: People, birds, pigs, horses,
seals, whales and others
 Classified into subtypes

Influenza Type B: Human virus
 Not classified according to Subtype
 Cause human epidemics but not pandemics

Influenza Type C cause mild illness in humans
 Not classified according to subtype
 Do not cause epidemics or pandemics
2017 ،‫ نيسان‬11 ،‫الثالثاء‬
Influenza A Virus Subtyping
■ Influenza A subtypes are
determined by two
surface glycoproteins:
Hemagglutinin (HA)
Neuraminidase (NA)
■ 16 HA’s and 9 NA’s
4
2017 ،‫ نيسان‬11 ،‫الثالثاء‬
Species Infected by Influenza A, HA
and NA Subtypes
H1
H2
H3
H4
H5
H6
H7
H8
H9
H10
H11
H12
H13
H14
H15,16
5
N1
N2
N3
N4
N5
N6
N7
N8
N9
2017 ،‫ نيسان‬11 ،‫الثالثاء‬
History
6
Epidemic
data
Year
Influenza A
Virus
subtype
Deaths
Spanish flu
1918-1919
H1N1
50 millions
Asian flu
1956-1958
H2N2
2 millions
Hong kong flu
1968-1969
H3N2
1 million
2017 ،‫ نيسان‬11 ،‫الثالثاء‬
Influenza Pandemics 20th Century
Credit: US National Museum of Health and
Medicine
1918: “Spanish Flu”
1957: “Asian Flu”
50 million deaths
2 million deaths
A(H1N1)
A(H2N2)
7
1968: “Hong Kong
Flu”
1 million deaths
A(H3N2)
2017 ،‫ نيسان‬11 ،‫الثالثاء‬
p(H1N1) 2009
The CDC determined that the
strain contained genes from four
different flu viruses:
–North
American
swine
influenza,
-North
American
avian
influenza,
- Human influenza, and
- Swine influenza virus typically
found in Asia and Europe .
This new strain appears to be a
result of the reassortment of two
swine influenza viruses, one
from North America and one
from Europe
HA
Hemagglu
tinin
swine
(H1)
North
America
NA
Neuramini
dase
swine (N1)
Europe
PA
RNA
polymeras
e subunit
PA
avian
North
America
RNA
polymeras
e subunit
PB1
human
1993
H3N2
strain
RNA
polymeras
e subunit
PB2
avian
North
America
NP
Nucleopro
tein
swine
North
America
M
Matrix
protein
M1, M2
swine
Eurasia
Nonstructural
proteins
NS1
swine
North
America
PB1
PB2
NS
Specimen Collection
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Specimen Collection Kit
• Personal protective
equipment
• Collection vials with
VTM
• Polyester fiber-tipped
applicators
• Tongue depressors
• Items for blood
collection
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• Secondary container/
cooler
• Ice packs
• Suspect case forms
• A pen or marker for
labeling samples
• Labels
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Personal Protective Equipment
•
•
•
•
•
•
14
Masks (N-95)
Gloves
Protective eyewear (goggles)
Hair covers
Boot or shoe covers
Protective clothing (gown or apron)
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Safety Glasses
How to wear a Particulate
Respirator?
How to wear a Particulate
Respirator?
Types of Personal Protective
Equipments
- Cap
- Plastic apron (splashing of
blood, secretions, excretions)
gloves
• Different kinds of gloves
• Avoid to touch
– Eyes, nose, mouth
– Surfaces , if not necessary
• Change gloves between
patients
Gowns
An ideal gown must:
• Fully cover the body
• Have long sleeves
• Fit snuggly at the wrists & neck
Creative alternatives for Gown:
laboratory coat
Boots or Shoe covers
Creative alternatives for
boots:
plastic bags cover for regular shoes
Eye protection
• Eye Protection
1. Face shields
2. Goggles
Viral Transport Media
Viral Transport Media
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What is Viral Transport Medium?
• Used in the collection of samples for viral isolation
and testing
• Prevents specimen from drying out
• Prevents bacteria and fungi growth
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Storing VTM
• Sterile collection vials containing
1 ml of VTM
• Vials can be stored in a freezer
at -20 ºC until use
• Vials can be stored for short
periods of time at :
4 - 8 ºC
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Phlebotomy Supplies
•
•
•
•
•
•
•
•
Tourniquet
Disposable needles
Vacuum tubes with EDTA
Plastic needle holder
Alcohol and iodine swabs
Gauze
Band-aids
Biohazard sharps
container
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Polyester Fiber-Tipped Applicator
• Should be drayon,
rayon, or polyester fiber
swabs
• individually wrapped to
ensure they are sterile
• Do not use calcium
alginated or cotton
swabs ; they inhibit PCR
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How to Manage Kits
• Store specimen collection kits “except VTM” in
a dry, clean place
• Store specimen collection kit where it will be
accessible after hours and on weekends
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Methods of Collection
• Oropharyngeal (Throat) swab
• Nasopharyngeal swab
• Nasopharyngeal aspirate
• Blood sample
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Nasopharyngeal Swab
1.
Insert dry swab into nostril
and back to nasopharynx
2.
Leave in place for a few
seconds
3.
Slowly remove swab while
slightly rotating it
4.
Use a different swab for the
other nostril
5.
Put tip of swab into vial
containing VTM, breaking
applicator’s stick
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Nasopharyngeal Aspirate
Collection Process
1.
2.
3.
4.
5.
6.
38
Attach mucus trap to vacuum source
Place catheter into nostril parallel to palate
Apply vacuum
Slowly remove catheter while slightly rotating it
Repeat with other nostril using the same catheter
After collection, flush catheter with 3 ml VTM and
return VTM to a plastic vial
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Oropharyngeal (Throat) Swab
• Easy to do
• Highest yield in detecting avian influenza in
suspected cases
• Have the patient open his/her mouth wide
open.
• The patient should try to resist gagging and
closing the mouth while the swab touches the
back of the throat near the tonsils.
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Oropharyngeal Swab
(preferred specimens)
1. Ask the subject to open
his or her mouth
2. Depress the tongue
3. Swab the posterior
pharynx behind the
tonsils
4. Avoid the tonsils
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When to Collect
Respiratory Specimens
• As soon as possible after symptoms begin
• before antiviral medications are
administered
• Even if symptoms began more than one
week ago and antivirals administered but
record both the time from symptom onset
and the type and dosage of antivirals
treatment
• Multiple specimens on multiple days could
be collected if you have access to patient
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Serological Samples
Paired serum samples are most useful
Acute sample :
Within 7 days after symptom onset
Convalescent sample :
2 to 4 weeks after acute sample
Serology is useful for epidemiologic investigations, but it
is rarely helpful for acute clinical care of a patient. Keep
in mind that because there is no immunity to avian
influenza A (H5N1) in the population (i.e. there should
be no past exposure), any positive result for antibodies
should raise the index of suspicion.
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How to Label Samples
Use pre-printed barcode*
labels:
– On the specimen container
– On the field data collection
form
– On the log book
* If barcode labels are not available use
Marker.
Label each specimen with:
– Subject’s name
– Subject’s unique identification
number
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How to Store Respiratory Specimens:
For specimens in VTM:
• Transport to laboratory as soon as possible
• Store specimens at 4 °C before and during transportation
within 48 hours
• Store specimens at -20 °C beyond 48 hours
• Avoid freeze-thaw cycles as this will destroy the virus
– Better to keep on ice for a week than to have repeat freeze and thaw
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How to Store serum Specimens:
• Store specimen at 4 °C within 48 hours
• Store specimens at -20 °C beyond 48 hours
• Avoid repeated freeze-thaw cycles For both
VTM specimens and sera
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Suspect Case Form
•
•
•
•
•
•
•
47
Patient name
Unique identification number
Patient symptoms and date of onset
Specimen type and collection date
Whether or not patient is hospitalized
Patient contact information
Patient demographic information
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Handling Infectious Materials in
the Field
• Always wear personal protective equipment
• Be careful with sharp objects
• Treat all clinical samples as potentially infected
with influenza
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Packing Specimens for Transportation
• Use three
packaging layers
• First layer should
be water tight
• Use absorbent
material in all
layers
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Packing Specimens for
Transportation
• Keep specimens at 4 ºC
– Fill a cooler with ice packs or coolant packs but do
not use dry ice unless the specimens are doublebagged and airtight; carbon dioxide from the dry
ice can inactivate the virus.
• Coordinate with the laboratory
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Summary
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Summary
• Oropharyngeal swabs and lower respiratory
specimens are the best specimens to collect for
avian influenza A (H5N1).
• Nasopharyngeal swabs and nasal washes (for
young children) are better for seasonal influenza
and other pathogens.
• Collect multiple specimens (respiratory and
blood) on multiple days.
• Properly dispose of any infectious material.
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‫أهم الطرق المخبرية للتشخيص المخبري‬
‫تقنية اإلليزا‬
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‫الزرع الفيروسي‬
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‫دور مخابر الصحة العامة‬
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‫‪CPE‬‬
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PCR
Exponential Amplification of template DNA
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Visualizing the DNA
DNA ladder

wells
PCR
Product
+ - - - - + + - - + - +
 2,000 bp
 1,500
 1,000
 750
 500
 250
Samples # 1, 6, 7, 10 & 12 were positive for Wolbachia DNA
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‫دور مخابر الصحة العامة‬
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March 12, 2006
REAL TIME PCR
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‫دور مخابر الصحة العامة‬
www.biorad.com
4/11/2017 3:26 AM
CDC Realtime RTPCR protocol for Detection
of Influenza A (H1N1) of swine origin
qRT-PCR
• Used to determine whether sample is
Influenza A
– M gene primers and probes (segment 7)
• Tested for Influenza B
– B NS (segment 8)
• Tested for H5, H1, H3 (others if thought
likely) on segment 4 primers and probes
• Tested for RNA integrity based on human
ribonuclease P sequence.
RNA Extraction Layout
Sample A
Sample B
Sample C
Sample D
Step
Reagent Volume/Rx
Total number of Rxs
Total Volume
of Reagent
1
Nuclease free Water
N X 5.5 ul
N = n+1 = 6 + 1 = 7
38.5 ul
2
Forward Primer
N X 0.5 ul
N = n+1 = 6 + 1 = 7
3.5 ul
3
Reverse Primer
N X 0.5 ul
N = n+1 = 6 + 1 = 7
3.5 ul
4
Probe
N X 0.5 ul
N = n+1 = 6 + 1 = 7
3.5 ul
5
SuperScript III
RT/Platinum Taq Mix
N X 0.5 ul
N = n+1 = 6 + 1 = 7
3.5 ul
2X PCR Master Mix N X 12.5 ul
N = n+1 = 6 + 1 = 7
87.5 ul
N = n+1 = 6 + 1 = 7
140 ul
6
Total Volume
N X 20 ul
InfB
S1
S1
S1
S1
S1
S2
S2
S2
S2
S2
S3
S3
S3
S3
S3
S4
S4
S4
S4
S4
empty
empty
empty
empty
NC
NC
NC
NC
empty
empty
empty
empty
PC
PC
Z4
PC
PC
empty
NC
empty
PC
RP control (human Rnase P gene)
• Each specimen should be tested for RNP gene.
This reaction serves as an internal pos control
for the presence of human nucleic acid in the
extract as well as a control for inhibition of the
reaction.
RP interpretation
• Must be pos for each specimen
– Failure to detect RP may indicate improper extraction resulting in loss
of nucleic acid or carryover of PCR inhibitors, absence of sufficient
human cells in the sample, improper assay set up and execution and
reagent/equipment malfunction.
• IF RP is negative
– Repeat sample for all targets
– HOWEVER, if for a specific sample, RP is negative but the pathogenspecific target is positive, consider it a true positive. (Note: inhibition
in the reaction could cause the extraction control, as well as certain
targets, to fail although other targets amplify; it is advised to re-extract
and repeat this sample for all targets to detect potential co-infections)
96 wells plate
Novel influenza A (H1N1) Diagnostic Flowchart
Specimen: swabs in VTM
Preserve remnant at -70C (if
not available, then -20C)
Nucleic Acid Extraction:
140 ul specimen
PCR Universal Flu A
Negative:
Univ. Flu A
Stop:
Non-Flu A virus
Positive: Univ. Flu A (continue with
SwH1N1 primers)
PCR set up:
Univ Flu A
Sw Flu A
SW H1
RP
Univ. Flu A = Pos
Sw Flu A = Pos
Sw H1 = Pos
Sw H1N1
Univ. Flu A = Pos
Sw Flu A = Pos
Sw H1 = Neg
Univ. Flu A = Pos
Sw Flu A = Neg
Sw H1 = Neg
RP = Pos
STOP:
Flu A virus
Univ. Flu A = Pos
Sw Flu A = Neg
Sw H1 = Pos
Repeat reaction from extraction
Contact NAMRU-3 or CDC
Seasonal Flu
Subtype specimen
Thank you
Questions ?
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