Laboratory Diagnosisof influenza a study

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Influenza Virus – General properties : 

Influenza Virus – General properties Orthomyxoviridae 3 Types: A,B and C Type A Virus: Epidemics Spherical in shape 80-120 nm size Genome: SS RNA Segmented: 8 units Lipid Envelope Haemagglutinin (HA) & Neuraminidase(NA) Proteins Subtypes based on HA & NA Antigens HA NA

Laboratory Diagnosis : 

Laboratory Diagnosis Specimen Collection, Storage & Transportation Lab. Diagnostic Methods

I. Specimen Collection, Storage & Transportation : 

I. Specimen Collection, Storage & Transportation

Slide 5: 

Classical Influenza

Slide 6: 

Influenza A & B viruses are primarily shed in the upper respiratory tract of humans Viral shedding occurs the day before illness onset Peak viral shedding on Day 1 of illness Duration Adults may shed viruses for 4-6 days Young children may shed for longer periods Immunocompromised can shed for months Influenza Viral Shedding

Specimen Collection : 

Specimen Collection

Sources of Clinical Specimen : 

Sources of Clinical Specimen Suspected cases Symptoms consistent with influenza Contacts Including people living or working with suspected cases in human and birds

Specimen Collection Kit : 

Specimen Collection Kit

Specimen Collection Kit : 

Specimen Collection Kit Collection vials with VTM Polyester fiber-tipped swabs Tongue depressors Sputum or mucus trap Specimen collection cups or Petri dishes Transfer pipettes Sterile saline (0.85% NaCl) Secondary container Ice Box Items for blood collection Personal Protective Equipment (PPE) Field collection forms Marker pen for labeling samples

Viral Transport Medium (VTM) : 

Viral Transport Medium (VTM) 2ml VTM in sterile vials Used for viral isolation and testing Contain Bovine Serum Albumin (BSA) or Gelatin 0.5% act as virus stabilizer Antibiotics – prevent bacterial growth Glycerol – Prevent drying of the specimens

Polyester Fiber-Tipped Swabs : 

Polyester Fiber-Tipped Swabs Should be drayon, rayon, or polyester-fiber swabs Do not use calcium alginated or cotton swabs with wooden sticks; they inhibit PCR

Tongue Depressors : 

Tongue Depressors

Ice Box : 

Ice Box

Field Data Collection Form : 

Field Data Collection Form Patient name Identification number Patient’s demographic information Patient’s Health Status

Type of Specimens : 

Type of Specimens Collect Specimen with in 6 days of illness Transport to the lab with in 24 hours Types of Specimens: Aspirate Nasopharyngeal Aspirate Swab Nasopharyngeal Swab Oropharyngeal Swab Throat Swab Blood for serum Other Specimens: Broncheoalveolar lavage Tracheal aspirate Sputum

Specimen Collection Procedures : 

Specimen Collection Procedures

Nasopharyngeal Swab : 

Nasopharyngeal Swab Insert dry swab into nostril and back to nasopharynx Leave the swab for a few seconds Slowly remove swab while slightly rotating it Use another swab for the other nostril Insert tip of swab into vial containing VTM, breaking applicator’s stick

Throat Swab : 

Throat Swab Ask the patient to open his or her mouth Depress the tongue Swab the posterior pharynx using a sterile swab Avoid touching the tonsils Insert tip of swab into vial containing VTM, breaking applicator’s stick

Blood : 

Blood 3-5 ml of blood collected Serum collected in a sterile vial Transport in cold chain at 2-8*C Paired serum samples to be collected Acute phase : with in 7 days of onset of illness Convalescent phase: after 14 days of onset of illness

Slide 22: 

Specimen Storage Specimens in VTM for viral isolation Keep at 4oC & transport quickly to lab Viability at 4oC up to 2 days Otherwise freeze below - 70oC until transport to lab. Repeated freezing & thawing must be avoided Serum samples Store at 4oC for up to 1 week Long term storage at - 20oC

Label Samples : 

Label Samples Use labels: On the specimen container On the Ice Box On the field data collection form On the log book Label each specimen with: Patient's name Patient's unique identification number

Packaging & Transportation(Triple Layer Pack) : 

Packaging & Transportation(Triple Layer Pack)

Transporting Specimens from Field to Lab : 

Transporting Specimens from Field to Lab

Where to send the samples? : 

Where to send the samples? National Reference Centre: National Institute of Virology (NIV), Pune Regional Reference Centres: NCDC, Delhi KIPM, Chennai Virology Division, AIIMS, New Delhi NICED, Kolkatta

II. Laboratory Diagnostic Methods : 

II. Laboratory Diagnostic Methods

Methods : 

Methods Direct Method Demonstration of Viruses Virus Isolation Viral Antigens Rapid tests IFA HA Test HAI Test Viral Genome PCR RT-PCR Indirect Method Demonstration of Antibodies Serological Tests Micro-neutralization Assay HAI Assay Western Blot ELISA – IgM & IgG

Slide 29: 

Direct Method Patient sample throat swab aspirate (NPA/BAL) Rapid test Directigen Flu A+B Binax Now A/B Capilia Flu A,B Lab assay Direct IFA RT-PCR HI assay Virus Isolation Grow virus Extensive HI Sequence HA & NA

Virus Isolation : 

Virus Isolation Gold standard for detection of Influenza Allows identification of virus and test for drug susceptibilities Technically difficult and requires BSL-3 laboratory Virus Isolation Methods: Embryonated Eggs Tissue Culture: Primary Monkey Kidney Cells Continuous Cell Lines – MDCK ( Madin Darby Canine Kidney Cell Line) LLC MK 2 ( Monkey Kidney Cell Line) Vero (Vervet Monkey Kidney Cell Line)

Slide 31: 

Sample collection Inoculation in Tissue Culture and Embryonated Eggs Virus Isolation Procedure

Slide 32: 

Harvesting of egg allantoic fluid Observation of tissue culture cells Harvesting of tissue culture supernatant

Detection of Virus Antigens 1. Rapid Tests Kits : 

Detection of Virus Antigens 1. Rapid Tests Kits Advantage Rapid results in 30 min Simple On-site testing Test clinical material or grown virus Useful for clinical settings and outbreak investigations

Slide 34: 

Directigen Fu A + B FLU OIA QuickVue ZstatFlu Rapid Diagnostic Test

Slide 35: 

Rapid Diagnostic Kits Disadvantages Less sensitive than viral culture or RT- PCR False negatives Some kits cannot type (A/B) Cannot subtype (H3, H1, H5?) Cost? ($12 - $25)

Slide 36: 

Direct testing of cell pellets from original clinical samples (BSL-2) Typing/sub typing (grown viruses – BSL-3+) WHO Influenza Reagent Kit Commercially available reagents 2. Immunofluorescence Assay - IFA

3. Haemagglutinin(HA) Test : 

3. Haemagglutinin(HA) Test Advantages: General sub typing to detailed antigenic characterization Disadvantage Cultured virus of high titers necessary Immune sera necessary Time consuming/labor intensive

Slide 38: 

4. Hemagglutination Inhibition (HAI) Test

Demonstration of Viral GenomePCR-based Molecular Diagnosis : 

Demonstration of Viral GenomePCR-based Molecular Diagnosis Reverse transcriptase PCR Real-time RT-PCR CDC-FDA Approved WHO Recommended Used Internationally for confirmation of H5N1/H1N1

Slide 40: 

The Influenza A Virus Genes (H,N & M) for molecular diagnosis PB1 PB2 PA HA NP NA M1 M2 NS1 NS2

Influenza-A Genome: 8 Segments : 

Influenza-A Genome: 8 Segments Seg Nt position Protein Function 1 (2341 bp) PB2 Polymerase component 2 (2341 bp) PB1 Polymerase component 3 (2233 bp) PA Polymerase component 4 (1778 bp) HA ►Hemagglutinin viral attachment protein: target of neutralizing antibody 5 (1565 bp) NP Nucleocapsid 6 (1413 bp) NA ►Neuraminidase (cleaves sialic acid+promotes viral release) 7 (1022 bp) M1 ►Matrix protein (promotes viral assembly). M2 Membrane protein (facilitates uncoating & HA production 8 (180 bp) NS1 Non structural protein (inhibits cellular messenger RNA translation) NS2 Non structural protein (important but unknown function)

Steps in Molecular Diagnosis : 

Steps in Molecular Diagnosis 1. Clinical Samples 2. RNA Extraction 3. RT-PCR amplification (Real Time) 4. PCR Product Purification 5. Sequencing-PCR Product 6. Automated Gene Sequencing 7. Detection of Mutation & Confirmation of Sub-type by Blast Search 8. Origin & Route of Transmission using Phylogenetic analysis

BSL 3/BSL 2 with PCR/other equipments : 

BSL 3/BSL 2 with PCR/other equipments 3 separate cubicles RNA Extraction/Master Mix Preparation/Sample Loading Separate class II biosafety cabinet RT-PCR Machine(20 lakhs) Ref.high speed centrifuge/vortexer/-20&-70 deep freezer PPE/Micropipettes/Tips Kits & Reagents

Step – 1 Sample Collection : 

Step – 1 Sample Collection Nasal/Throat Swab in VTM Transport in cold chain Submit case sheet Ensure personnel protection

Step 2 RNA Extraction : 

Step 2 RNA Extraction RNA Extraction Kit – Commercial Rs 300/sample(approx) Separate cubical with class II biosafety cabinet

Step-3 PCR Mix Preparation : 

Step-3 PCR Mix Preparation Fluorescent labeled nucleotides CDC recommended primer sequences Enzymes Expensive Reagents

Step-4 PCR Run : 

Step-4 PCR Run

Step-5 Reading : 

Step-5 Reading

Slide 50: 

Micro-neutralization Assay Seroconversion in paired sera ( 4 fold) Titer of 1:80 in 2 independent assay Confirmatory Assay: Western Blot ELISA: IgM IgG Indirect Method Serologic Methods to detect Antibodies

Summary : 

Summary Specimen Collection Kit Collect from suspected cases and contacts Specimens: Nasopharyngeal Aspirate/Swab/Throat Swab Storage: 4*C for short time & -20/-70*C long time Transportation: Triple Layer Packaging to Ref.Labs Methods: Direct Method: Virus Isolation – Embryo eggs & Tissue Culture Antigen Detection – Rapid / IFA / HA / HAI / Genome – RT-RCR / Sequencing of H,N & M genes Indirect Method: Antibody Detection



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