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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 02 ،أيار2017 ، 1 Influenza Virus 2 2017 ، أيار02 ،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 ، أيار02 ،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 ، أيار02 ،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 ، أيار02 ،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 ، أيار02 ،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 ، أيار02 ،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 11 م09:55 02/05/2017 09:55 02/05/2017م 12 Specimen Collection Kit • Personal protective equipment • Collection vials with VTM • Polyester fiber-tipped applicators • Tongue depressors • Items for blood collection 13 • Secondary container/ cooler • Ice packs • Suspect case forms • A pen or marker for labeling samples • Labels م09:55 02/05/2017 Personal Protective Equipment • • • • • • 14 Masks (N-95) Gloves Protective eyewear (goggles) Hair covers Boot or shoe covers Protective clothing (gown or apron) م09:55 02/05/2017 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 24 م09:55 02/05/2017 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 25 م09:55 02/05/2017 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 26 م09:55 02/05/2017 Phlebotomy Supplies • • • • • • • • Tourniquet Disposable needles Vacuum tubes with EDTA Plastic needle holder Alcohol and iodine swabs Gauze Band-aids Biohazard sharps container 27 م09:55 02/05/2017 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 28 م09:55 02/05/2017 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 29 م09:55 02/05/2017 Methods of Collection • Oropharyngeal (Throat) swab • Nasopharyngeal swab • Nasopharyngeal aspirate • Blood sample 36 م09:55 02/05/2017 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 37 م09:55 02/05/2017 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 م09:55 02/05/2017 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. 39 م09:55 02/05/2017 09:55 02/05/2017م 40 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 41 م09:55 02/05/2017 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 42 م09:55 02/05/2017 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. 43 م09:55 02/05/2017 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 44 م09:55 02/05/2017 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 45 م09:55 02/05/2017 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 46 م09:55 02/05/2017 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 م09:55 02/05/2017 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 48 م09:55 02/05/2017 Packing Specimens for Transportation • Use three packaging layers • First layer should be water tight • Use absorbent material in all layers 49 م09:55 02/05/2017 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 50 م09:55 02/05/2017 Summary 51 م09:55 02/05/2017 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. 52 م09:55 02/05/2017 أهم الطرق المخبرية للتشخيص المخبري تقنية اإلليزا 5/2/2017 9:55 PM دور مخابر الصحة العامة 55 الزرع الفيروسي 5/2/2017 9:55 PM دور مخابر الصحة العامة 56 CPE 5/2/2017 9:55 PM دور مخابر الصحة العامة 57 5/2/2017 9:55 PM دور مخابر الصحة العامة 58 PCR Exponential Amplification of template DNA 59 دور مخابر الصحة العامة 5/2/2017 9:55 PM 5/2/2017 9:55 PM دور مخابر الصحة العامة 60 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 61 دور مخابر الصحة العامة 5/2/2017 9:55 PM March 12, 2006 REAL TIME PCR 62 دور مخابر الصحة العامة www.biorad.com 5/2/2017 9:55 PM 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 ? 86 دور مخابر الصحة العامة 5/2/2017 9:55 PM
