Diagnostics PDF - LA TROBE University

DIAGNOSTICS Katelyn Mroczek [email protected] 1 Learning outcomes Understand the uses of diagnostic tests in microbiology How the test works Why it is useful How to use dichotomous key...

DIAGNOSTICS Katelyn Mroczek [email protected] 1 Learning outcomes Understand the uses of diagnostic tests in microbiology How the test works Why it is useful How to use dichotomous key 2 CHARACTERISING MICROORGANISMS 3 Collecting specimens Aseptic technique must be used Must have enough sample to culture Environmental conditions of possible pathogen should be considered Usually performed with sterile swab Blood sample Diagnostic tests: Specificity – Is the test specific for a certain pathogen? If so decreases chance of false positives Sensitivity – How small can the sample size be to be detected? High sensitively decreases chance of false negatives 4  Culture media Enrichment culture Original sample inoculated onto media that supports growth of many different organisms Selective media Uses chemicals and inhibitory compounds to select the growth of one organisms while inhibit the growth of others Differential media Distinguishes between different organism usually based on metabolism of chemicals and pH indicators Example: Mannitol salt agar Selective: Contains higher salt concentration (~7.5%) selecting for Gram positive bacteria Differential: Contains mannitol – distinguishes mannitol fermentation 5 https://medizzy.com/user/1604009 Example: Urinary tract infection Urine sample taken Direct microscopy will give idea of number of bacteria in sample Cell counting machines Known volume of sample plated onto media for viable count (usually 10µl) 108 cells per litre of urine = infection Can do gram strain directly from sample E.g. Gram negative rods = Enteric bacteria 6 Biochemical tests – Enteric bacteria MacConkey agar – Differentiates lactose fermenters from non-lactose fermenters Selective – Bile salts selects for enteric bacteria Differential – Lactose and pH indicator Can then use further biochemical media to identify causative organism TSIA – Triple sugar iron agar Urea agar Citrate Indole production 7  Shigella Can use dichotomous key H2S Salmonella Urease Proteus Interpretation of each test indicates next ONPG result to look at Lactose Yersinia The collective result fermenter of test will allow identification of an Enterobacter organism by giving a Indole biochemical profile Urease E.coli Negative Positive Klebsiella 8 Rapid testing – EnterotubeTM II Identification of gram negative rods All media is inoculated simultaneously After 24 hours can interpret the results If an organism indicates a positive result for the test the number is circled Each section is added up to a single digit number Final number or ‘code’ will reveal the identification of the organism 9  Microscopy – stains Stain Organism Gram stain Classifies gram negative and positive bacteria Acid-fast stain Mycobacterium, oocysts of some parasites Acirdine orange Bacteria and fungi (Fluorescent) Calcofluor white Mainly dermatophyte fungi (Fluorescent) Wet mount Fungi, parasites (helminths), motile (unstained) organisms Wright Parasites in blood, intracellular stain/Giemsa inclusions, stain India ink stain Cryptococcus neoformans Periodic acid- Fungi and Acanthamoeba Schiff (PAS) 10 SEROLOGICAL TESTING 11  Serology Look for antibodies in the patient serum Can indicates infection with certain pathogen Immune response produces antibodies against pathogen antigens Many different types of serological tests Precipitation Agglutination Immunofluorescence Immunoblot Enzyme immunoassays Rapid immunoassays https://bio.libretexts.org/Bookshelves/Microbiology/Book%3A_Microbiology_(Boundless)/12%3A_Immunology_Applications/12.2%3A_Im munoassays_for_Disease/12.2A%3A_Immunoassays_for_Disease https://www.researchgate.net/publication/320265684_Allergen Based_Diagnostic_Novel_and_Old_Methodologies_with_New_Approaches/download 12  Useful for fungal infections Precipitation Precipitation forms when soluble antibodies and antigens meet to form insoluble complex or precipitate Crosslinking of antibodies and antigens form when there are equal proportions of each Performed in semi-solid media either agar or agarose gel When antibodies and antigens meet form a line of precipitation http://www.biosciencenotes.com/precipitation-test/ 13  Useful to detect bacterial surface Agglutination antigens Usually antigens are coupled to latex beads This solution can be mixed with patient serum (on a slide) The antibodies in the sample bind to antigen This is visualised as visible clumping on the slide If no antibodies present for the specified antigen = uniform suspension no clumping Can be performed in many ways In test tubes Microtiter plates Glass slides/coated paper slides 14 Staphytect test Antibodies can also be coupled to latex beads This can be useful in detecting bacterial surface antigens Staphytect test uses antibodies to Protein A and clumping factor These are found on the cell wall of S. aureus only Specific for identification of S. aureus A colony is mixed into the latex bead solution Clumping = Positive result 15  Immunofluorescence Using fluorescent antibodies to detect antigens in cells This technique is readily used for diagnostic and research purposes Direct staining method The antibody itself is tagged with fluorescent dye (fluorophore) E.g. Fluorescein isothiocyanate (FITC) Indirect staining method Primary antibody to target the antigen (non fluorescent) Secondary antibody to target primary antibody (fluorescent) https://www.biocompare.com/Antibody-Manufacturing/354958-CST- Immunofluorescence-Validated-Antibodies/ 16  Useful to identify microorganism in patient sample Disease diagnosis Viral pathogens Madigan, Michael T., et al. Brock Biology of Microorganisms, Global Edition, Pearson Education Limited, 2017. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/latrobe/detail.action?docID=5203167. Visualised using a fluorescent microscope Fluorophore excited with light at different wavelengths E.g. Green fluorescent protein (GFP) can be excited by 488 nm laser line Can also be applied directly to host tissue May be able to detect disease before culturing test results come back https://www.researchgate.net/figure/Green-fluorescent-protein-GFP-tagged-microtubules-GFP-MAP4-in-Arabidopsis-hypocotyl_fig2_280918641 17  Enzyme immunoassays Involves an enzyme bound to constant end of antibody Variable end of antibody free to bind to antigen Enzyme will react with certain substrates to produce a colour reaction Chromogen = colourless chemical compound that can be converted by chemical reactions into a coloured compound Or link antibody to fluorophore Direct EIA – Detects antigens (can have low detection) Indirect EIA – Detects antibodies Sandwich EIA – Quantifies antigens (very fast https://www.memorangapp.com/flashcards/28659/Antibody+Structure/ and accurate) Antibodies must bind to different sites of target antigen 18  Highly sensitive to Useful to detect antibodies antibodies to to pathogens in pathogens in body body fluids Useful to detect fluids antigens in a samples E.g. viral particles 19 Allergen-Based Diagnostic: Novel and Old Methodologies with New Approaches http://dx.doi.org/10.5772/intechopen.69276  Useful for rapid Rapid tests – lateral diagnosis of infectious diseases flow immunoassay Rapid identification of diseases Uses capillary action to pull sample through a matrix Moves through line of beads (usually latex (blue) or gold particles (red)) bound with antibodies Antigen binds with antibody-coated beads This complex then flows over ‘test’ line with fixed antibodies against antigen Retains the beads that have antigen bound Third line contains fixed antibodies to pick up remaining antibody bound beads - not antigen 20  Positive result Negative result 21 MOLECULAR METHODS 22 PCR Primers to target DNA Cycles make many copies of DNA PCR product run using agarose gel electrophoresis to separate bands 23  Useful to assess the abundance of Quantitative PCR (qPCR) pathogen in a sample Thermocycler includes fluorometer to detect fluorescence in real time Fluorescent probe can be specific for target DNA or non-specific Specific fluorescent probes Fluorescent dye linked to DNA probe for specific target Only fluoresces when double stranded DNA of correct sequence accumulates Non-specific fluorescent probes Fluoresce only when bound to dsDNA Fluorescence increases as more DNA is amplified Can quantify the amount of target DNA in a sample Real time fluorescence so no need for gel electrophoresis 24  Useful for the RT-qPCR detection of RNA viruses RNA initial template to create cDNA RNA degraded PCR to amplify cDNA Can use qPCR Madigan, Michael T., et al. Brock Biology of Microorganisms, Global Edition, Pearson Education Limited, 2017. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/latrobe/detail.action?docID=5203167. 25 Laboratory identification of pathogens 26