Laboratory Diagnostics Lecture Overview 2024 PDF

Lecture Overview: Laboratory Diagnostics  Sensitivity vs Specificity  Norman-McKay assigned  Laboratory diagnostic readings: techniques  Chapter 14  1) Morphology  2) Biochemical tests...

Lecture Overview: Laboratory Diagnostics  Sensitivity vs Specificity  Norman-McKay assigned  Laboratory diagnostic readings: techniques  Chapter 14  1) Morphology  2) Biochemical tests  3) Immunological tests  Agglutination tests  EIA/ELISA  ICA/POCT  4) Nucleic acid tests  PCR/NAAT, DNA microarray 1 MMG1650 2024 Laboratory Diagnostics  Infectious disease can be diagnosed in several ways:  Growing pathogen in lab (culture) from patient samples  Identifying pathogen microscopically in patient samples  Detecting specific antibodies or antigens in patient samples  Detecting pathogen DNA or RNA in patient samples  Laboratory diagnostic methods include:  Culture methods, microscopy, immunologic methods, molecular methods  Each type of laboratory diagnostic method has pros and cons  Certain laboratory diagnostic methods are more appropriate for certain types of infections 2 MMG1650 2024 Diagnostic testing: sensitivity vs specificity  Sensitivity and specificity:  Both are measures of a test's ability to correctly classify a person as having a disease or not having a disease  Sensitivity  refers to test's ability to designate an individual with disease as positive  highly sensitive test = few false negative results; fewer cases of disease are missed  Specificity  refers to test’s ability to designate an individual who does not have a disease as negative  highly specific test = few false positive results; few incorrect diagnoses 3 MMG1650 2024 Methods of Identifying Microorganisms in patient samples  1) Morphology:  Microscopic morphology  Colony morphology (bacteria and fungi)  2) Biochemical Tests  Determine the presence of enzymes  3) Immunological tests  Utilize antibodies to identify specific proteins  4) Nucleic acid Tests  Detection of DNA/RNA 4 MMG1650 2024  Methods of Classifying and Identifying Microorganisms: Morphology Available from: https://www.researchgate.net/figure/Bacterial-colony-and-cell-morphology-of-sp- isolated-from-the-commercial-probiotic_fig1_333831275 [accessed 17 Sept 2024] Biotechnology & Biotechnological Equipment. 35. 264-275.2021 10.1080/13102818.2020.1868334. 5 MMG1650 2024 Methods of Classifying and Identifying Microorganisms: Biochemical tests Table 10.8 Microbiology: An Introduction, 14th ed. 6 MMG1650 2024 Methods of Classifying and Identifying Microorganisms: Immunological tests  Serology: the study of what is in a patient’s serum  The goal of serology is to determine if a patient has certain antigens or antibodies in their blood  Determined by immunological methods  Immunologic methods:  Laboratory techniques involving the interaction of antigens with specific antibodies  Requires:  Commercial kits, easy to use  Useful for:  Bacteria, Fungi, Protozoa, Viruses 7 MMG1650 2024 Immunologic Methods Very Common in infectious Less common in infectious disease diagnosis disease diagnosis  Agglutination reactions  Immunoprecipitations  EIA/ELISA  Not covered in lecture  Neutralization  Immunochromatography  Not covered in lecture assay (ICA)  Interferon gamma release  Point of care testing assays (IGRAs) (POCT)  Covered later  Western blotting and complement fixation  Not covered in lecture 8 MMG1650 2024 Lab Diagnostics: Immunologic methods  Less Sensitive than culture  Advantages  Less Specific than culture  Inexpensive  Ig cross-reactions  Low tech  Easy to use  Fast  Require:  Commercial kits  Useful for:  Disadvantages  Lower sensitivity/specificity  Bacteria, Fungi, Protozoa, than culture Viruses  Cannot always distinguish  TAT= 5 min – few hours between current and past infection  Measure IgM vs IgG 9 MMG1650 2024 Agglutination Reactions  Recall:  Antibodies have 2 antigen-binding sites  Can attach to more than one antigen  Agglutination reactions:  Occur when antibody bind antigens into a clump  Testing can identify either antibody or antigen in patient samples (e.g., serum, urine, CSF)  Can be visualized better by using tiny synthetic (latex) beads coated with antigens (latex agglutination) Figure 12.21, 12.20 Norman-McKay. Microbiology: Basic and Clinical Principles. 2019 10 MMG1650 2024 Agglutination tests: blood typing Figure 14.6 Norman-McKay. Microbiology: Basic and Clinical Principles. 2019 11 MMG1650 2024 Agglutination reactions  Latex agglutination for coagulase  Latex beads coated with antibody specific for coagulase  + result = Staphylococcus aureus  - result = other Staphylococcus species http://www.bacteriainphotos.com/clum ping_factor_(bound_coagulase).html 12 MMG1650 2024 Enzyme immunoassays (EIAs) or Enzyme-linked immunosorbent assay (ELISA)  Uses enzyme-bound antibody to detect the presence of antigens  General steps:  Enzyme-labeled antibody added to sample  Variable region bind its specific antigen  Addition of a substrate for the enzyme allows for detection  Common substrates:  chromogen, a colorless molecule that is converted into a colored end product  alkaline phosphatase  horseradish peroxidase 13 MMG1650 2024  Figure 18.12 Microbiology: An Introduction, 14th ed. 14 MMG1650 2024 Immunologic methods: ICA  Immunochromatography assay (ICA)  simple device to detect the presence/absence antigen or antibody  combination of chromatography  separation of components of a sample based on differences in their movement through a sorbent  and immunologic reactions (antibody-antigen) http://www.prodivet.com/en-GB/Products/Diagnostic- tests/Immunochromatographic-Tests/Principles 15 MMG1650 2024 16 assay (ICA) MMG1650 2024 Immunological methods: Immunochromatography https://www.bio-rad.com/en-us/product/elisa-immuno-explorer-kit?ID=1e3f3100-99f6-49b3-b9a0-2c8aad9d9285  Results: Legionella pneumophila antigen detected or Legionella pneumophila antigen not detected https://www.mountainside- https://www.alere.com/en/home/produ medical.com/products/urine-specimen-cup-sterile ct-details/binaxnow-legionella.html Plasmodium spp. antigen detected or Plasmodium spp. antigen not detected https://www.123rf.com/photo_38663449_lavender-top-blood- https://www.cdc.gov/dpdx/diagnosticproced sample-collection-tubes-and-nitrile-gloves-.html ures/blood/antigendetection.html 17 MMG1650 2024 Point-of-care tests (POCTs)  Medical diagnostic testing  Examples: at or near the point of  Group A Streptococcus care (Strep throat)  Performed by clinicians  Trichomonas vaginalis (rather than laboratorians) (protozoan causing  POCTs for infection vaginitis) diagnosis often based ICAs  Urine pregnancy test https://www.ridacom.com/en/products/view/7075 18 MMG1650 2024 Point-of-care tests (POCTs) Pro Con  Efficiency  Cost?  Speed of diagnosis and  Processing errors treatment  Sample collection  Improper storage  Portable  Sensitivity  Relatively easy to use  Often less sensitive than lab methods  Reliability  Higher margin of error than lab methods 19 MMG1650 2024 Lab Diagnostics: Nucleic Acid Identification Tests  Highly sensitive  Advantages  Highly Specific  Very fast  Sensitive/ specific  Require:  Commercial kits  Disadvantages  High tech  Expensive (both equipment  Useful for: and test kits)  Bacteria, Fungi, Protozoa, Viruses  TAT= 30 min – few hours 20 MMG1650 2024 Laboratory Diagnostics: Nucleic Acid Identification Tests Very common in infectious Less common in infectious disease diagnosis disease diagnosis  PCR  CRISPR  DNA Microarray (Gene  Not covered microarray)  Genome maps  Not covered 21 MMG1650 2024 Laboratory Diagnostics: Nucleic Acid Identification Tests  Laboratory techniques involving the detection and/or amplification of nucleic acids (DNA or RNA)  AKA: Nucleic acid amplification tests (NAAT)  PCR = DNA template  RT (reverse transcriptase) PCR = RNA template  Requires:  Commercial kits  Technical expertise; expensive equipment  Useful for:  Bacteria, Fungi, Protozoa, Viruses 22 MMG1650 2024 Nucleic Acid Identification Tests : Polymerase chain reaction (PCR)  Polymerase chain reaction (PCR)  Sensitive enough to detect DNA of a single cell/virion in a sample  Creates billions of copies of a target gene in just a few hours  Highly specific: only DNA that is an exact match to primers will be amplified  Applications:  Facilitates gene sequencing for genetic disorders  Diagnosing infections 23 MMG1650 2024 PCR: amplification of DNA (or RNA) target sequences https://www.genome.gov/genetics-glossary/Polymerase-Chain-Reaction 24 MMG1650 2024 Nucleic Acid Identification Tests: DNA microarrays  DNA microarrays  Based on ability of ssDNA to hybridize (bind) to a complementary ssDNA  Short ssDNA molecules (oligos) bound to glass slide in a microscopic array  If complementary DNA molecules are in the sample, they will bind and can be visualized by detector (reader)  Requires:  Commercial kits, easy to use  Specialized equipment  Useful for:  Panel tests  Blood culture panels (specific for GP bacteria, GN bacteria or yeast species)  Respiratory panels (respiratory viruses) 25 MMG1650 2024 DNA microarray for detecting bacterial pathogens in blood: Verigene system https://scholar.dominican.edu/cgi/viewcontent.cgi?article=1337&context=masters-theses 26 MMG1650 2024  How can one microarray detect a variety of microbes? https://emcrit.org/wp-content/uploads/2019/03/027- 00039-01_c_ivd_bc-gn_package_insert.pdf https://www.luminexcorp.com/verigene-nanogrid-technology/ 27 MMG1650 2024 Nucleic Acid Identification Tests: DNA Microarray (DNA chip) Figure 10.18 Microbiology: An Introduction, 14th ed. 28 MMG1650 2024 Common diagnostic methods by microbe type Bacteria - Traditional culture - Microscopy - Immunological methods - Nucleic acid tests (Bacteremia, meningitis and species that are difficult to culture) Viruses - Nucleic acid tests - Serology (antibody detection) Yeasts/Molds - Traditional culture - Microscopy - Serology (antibody detection) Protozoa - Microscopy - Immunological methods are rare (ICA for malaria only) - Nucleic acid tests rare (enteric panel) Helminths - Microscopy 29 MMG1650 2024 Learning Objectives:  After attending the lecture and completing the assigned readings the student will:  Define sensitivity and specificity  Describe immunological methods including: agglutination tests, EIAs/ELISAs, ICAs  Define POCT and state the pro/cons  Describe the polymerase chain reaction (PCR)  Describe DNA microarrays and explain how they are used in clinical diagnostics  Compare and contrast common diagnostic methods for infectious disease 30 MMG1650 2024