Week 13.3 Diagnostic Microbiology Flashcards
Diagnostic medicine progresses through 5 stages
- Patient history (What have they been exposed to?)
- Physical examination (Which organs are affected?)
- Evaluation of signs and symptoms (What are the disease characteristics?)
- Selection, collection, and transport of clinical specimens
- Conducting appropriate diagnostic tests
4 categories of diagnostic tests
- Microscopy
- In vivo culture
- Molecular
- Serological
Microscopy test purpose
initial detection and morphology
In vivo culture test purpose
grow a pathogen in a lab and test for signature metabolism
Molecular test purpose
genetic information (DNA, RNA, or protein)
Serological test purpose
detection of antigens and antibodies
-use antibodies and antigens to detect, identify, and quantify an infectious agent
Different types of microscopy tests
- Direct examination
- Differential stains
- Fluorescent stains
Direct examination test (microscopy)
-looking directly at a specimen (blood, urine, diarrhea)
-Ex: wet mount and simple stains
Differential stains test (microscopy)
-staining specific components of a cell
-Ex: gram stain, endospore stain, Giemsa-Wright stain
Fluorescent stains test (microscopy)
-can be used to stain cells and/or cellular components
-Ex: FISH, DAPI
In vivo culture (biochemical) test categories (5)
- Detecting changes in pH w/ a pH indicator
- Observing the production of gas bubbles
- Observing changes in transparency
- Formation of iron oxides (black precipitates)
- Miscellaneous chemical assays
Examples of pH tests (In vivo culture)
-MacConkey Agar
-Mannitol Salt Agar (MSA)
-Phenol Red Fermentation Broth
-Simmon’s Citrate Agar
-Urea broth
Examples of gas bubble production tests (In vivo culture)
-Catalase assay
-Phenol Red Fermentation broth
Examples of transparency tests (In vivo culture)
-Blood agar
-Coagulase assay
Examples of iron oxides tests (In vivo culture)
-Bile Esculin Agar (BEA)
-Sulfide Indole Motility (SIM)
Examples of miscellaneous chemical assays (In vivo culture)
-Methyl Red Voges Proskauer (MR-VP)
-Oxidase Pyrrolidonyl Aminopeptidase (PYR) test
-SIM
Molecular tests
- Non-amplified nucleic acid probes
- Nucleic acid amplification methods
- Nucleic acid sequencing
- Protein analysis
Non-amplified nucleic acid probes (molecular)
DNA or RNA oligonucleotides labeled w/ a reporter
-RARELY used on clinical samples but CAN be used on In vivo cultures
Nucleic acid amplification methods (molecular)
PCR amplification of specific genes
-use pathogen-specific primers to amplify and detect DNA
-still being developed but being used for gonorrhea, chlamydia, TB, and COVID-19
Nucleic acid sequencing (molecular)
16S/18S rRNA gene sequencing or whole genome sequencing
Protein analysis (molecular)
Western blot or Matrix-assisted laser desorption/ionization coupled with time-of-flight (MALDI-TOF)
Nucleic acid amplification test steps
- Sputum liquefaction and inactivation w/ 2:1 sample reagent
- Transfer of 2 mL material into test cartridge
- Cartridge inserted into MTB-RIF test platform
- Sample automatically filtered and washed
- Ultrasonic lysis of filter-captured organisms to release DNA
- DNA molecules mixed w/ dry PCR reagents
- Seminested real-time amplification and detection in integrated reaction tube
- Printable test result
Nucleic acid sequencing steps
- Sample
- Transfer to test tube
- Deliver to lab
- DNA extraction
- Sequencing and bioinformatics identification
- Comprehensive microbiome report
MALDI-TOF mass spectrometry
- Sample, applied to target, is overlaid w/ matrix solution and allowed to dry
- Spot on target is pulsed w/ laser
- Desorbed ionized molecules accelerated by a potential difference fly through a high-vacuum and field free flight tube
- Time of flight, based on mass of particles, is captured on detector
- Resulting spectrum is compared to library containing patterns of clinically relevant species
Serological tests
- Latex agglutination
- Enzyme-linked immunosorbent assay (ELISA)
Latex agglutination test (serological)
use known antibodies to detect their corresponding antigen
-Latex particles coated w/ pathogen-specific antibodies + patient serum or in vivo culture containing corresponding antigen = agglutination
ELISA test (serological)
detects antigen (or antibodies) in sera using conjugated antibodies (antibodies attached to an enzyme, typically horseradish peroxidase)
-Immobilized antigen > conjugated antibody > substrate addition (like TMB), signal detection, and quantification
-Horseradish peroxidase breaks down TMB to produce a blue color
