LABORATORY DIAGNOSIS OF SWINE FLU Dr Mohit Bhatia
LABORATORY DIAGNOSIS OF SWINE FLU Dr. Mohit Bhatia Assistant Professor Department of Microbiology AIIMS, Rishikesh
SPECIMENS v. Upper respiratory tract specimens: (1) Deep nostrils (Nasal swab) (2) Nasopharynx (Nasopharyngeal swab) (3) Nasopharyngeal aspirate (4) Bronchial aspirate (5) Throat swab v. Acute and convalescent serum specimens should be used for the detection of rising antibody titres
POSTMORTEM SAMPLING • Lung: all lobes, including affected areas • Proximal and distal trachea • Liver • Brain • Kidneys • Nasopharynx (this is best done from a supratentorial approach with removal en bloc).
DIAGNOSTIC TESTS
VIRAL CULTURE ØEmbryonated eggs ØMammalian cell lines: (1) Madin Darby canine kidney (MDCK) (2) A 549 (3) Mink lung epithelial cell line (Mv 1 Lu) (4) Rhesus monkey kidney (LLC MK 2) (5) Buffalo green monkey kidney (BGMK) (6) Primary cell lines: Rhesus monkey kidney (Rh. MK) or African green monkey kidney (AGMK)
SHELL VIAL CULTURE
DIRECT FLUORESCENT ANTIBODY TEST (DFA) • Mainly intended for the detection and differentiation of influenza A and B viruses. • Not useful for subtyping influenza A viruses.
SEROLOGICAL ASSAYS üHemagglutination inhibition assay (HAI) üMicroneutralization or virus neutralization assay (VN) üSingle radial hemolysis (SRH) üComplement fixation assay üEnzyme linked immunoabsorbant assay (ELISA) üWestern blotting.
HEMAGGLUTINATION INHIBITION ASSAY
VIRUS NEUTRALIZATION ASSAY
SINGLE RADIAL HEMOLYSIS • SRH is a technique commonly used to determine the induction of antibodies following natural infection or vaccination. • It is used to measure complement-mediated hemolysis induced by antigen-antibody complexes. • SRH is considered more sensitive than the HAI assay and does not require pretreatment of the serum to inactivate non-specific inhibitors.
COMPLEMENT FIXATION TEST
ELISA
RAPID INFLUENZA DIAGNOSTIC TESTS (RIDTs)
LAB ON A CHIP/MICROCHIP DEVICES
NUCLEIC ACID BASED TESTS (NATs) üReverse Transcriptase Polymerase Chain Reaction (RT-PCR) üLoop-Mediated Isothermal Amplification-Based Assay (LAMP) üSimple Amplification-Based Assay (SAMBA) üNucleic Acid Sequence-Based Amplification (NASBA) üMicroarray-Based Approaches
REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION (RTPCR)
LOOP-MEDIATED ISOTHERMAL AMPLIFICATIONBASED ASSAY (LAMP)
SIMPLE AMPLIFICATION-BASED ASSAY (SAMBA)
NUCLEIC ACID SEQUENCE-BASED AMPLIFICATION (NASBA)
MICROARRAY-BASED APPROACHES
NUCLEIC ACID SEQUENCING ü SANGER SEQUENCING üNEXT GENERATION SEQUENCING (NGS)
- Slides: 26