Week 13.3 Diagnostic Microbiology

Question 1

Diagnostic medicine progresses through 5 stages

Answer 1

1. Patient history (What have they been exposed to?)
2. Physical examination (Which organs are affected?)
3. Evaluation of signs and symptoms (What are the disease characteristics?)
4. Selection, collection, and transport of clinical specimens
5. Conducting appropriate diagnostic tests

Question 2

4 categories of diagnostic tests

Answer 2

1. Microscopy
2. In vivo culture
3. Molecular
4. Serological

Question 3

Microscopy test purpose

Answer 3

initial detection and morphology

Question 4

In vivo culture test purpose

Answer 4

grow a pathogen in a lab and test for signature metabolism

Question 5

Molecular test purpose

Answer 5

genetic information (DNA, RNA, or protein)

Question 6

Serological test purpose

Answer 6

detection of antigens and antibodies
-use antibodies and antigens to detect, identify, and quantify an infectious agent

Question 7

Different types of microscopy tests

Answer 7

1. Direct examination
2. Differential stains
3. Fluorescent stains

Question 8

Direct examination test (microscopy)

Answer 8

-looking directly at a specimen (blood, urine, diarrhea)
-Ex: wet mount and simple stains

Question 9

Differential stains test (microscopy)

Answer 9

-staining specific components of a cell
-Ex: gram stain, endospore stain, Giemsa-Wright stain

Question 10

Fluorescent stains test (microscopy)

Answer 10

-can be used to stain cells and/or cellular components
-Ex: FISH, DAPI

Question 11

In vivo culture (biochemical) test categories (5)

Answer 11

1. Detecting changes in pH w/ a pH indicator
2. Observing the production of gas bubbles
3. Observing changes in transparency
4. Formation of iron oxides (black precipitates)
5. Miscellaneous chemical assays

Question 12

Examples of pH tests (In vivo culture)

Answer 12

-MacConkey Agar
-Mannitol Salt Agar (MSA)
-Phenol Red Fermentation Broth
-Simmon’s Citrate Agar
-Urea broth

Question 13

Examples of gas bubble production tests (In vivo culture)

Answer 13

-Catalase assay
-Phenol Red Fermentation broth

Question 14

Examples of transparency tests (In vivo culture)

Answer 14

-Blood agar
-Coagulase assay

Question 15

Examples of iron oxides tests (In vivo culture)

Answer 15

-Bile Esculin Agar (BEA)
-Sulfide Indole Motility (SIM)

Question 16

Examples of miscellaneous chemical assays (In vivo culture)

Answer 16

-Methyl Red Voges Proskauer (MR-VP)
-Oxidase Pyrrolidonyl Aminopeptidase (PYR) test
-SIM

Question 17

Molecular tests

Answer 17

1. Non-amplified nucleic acid probes
2. Nucleic acid amplification methods
3. Nucleic acid sequencing
4. Protein analysis

Question 18

Non-amplified nucleic acid probes (molecular)

Answer 18

DNA or RNA oligonucleotides labeled w/ a reporter
-RARELY used on clinical samples but CAN be used on In vivo cultures

Question 19

Nucleic acid amplification methods (molecular)

Answer 19

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

Question 20

Nucleic acid sequencing (molecular)

Answer 20

16S/18S rRNA gene sequencing or whole genome sequencing

Question 21

Protein analysis (molecular)

Answer 21

Western blot or Matrix-assisted laser desorption/ionization coupled with time-of-flight (MALDI-TOF)

Question 22

Nucleic acid amplification test steps

Answer 22

1. Sputum liquefaction and inactivation w/ 2:1 sample reagent
2. Transfer of 2 mL material into test cartridge
3. Cartridge inserted into MTB-RIF test platform
4. Sample automatically filtered and washed
5. Ultrasonic lysis of filter-captured organisms to release DNA
6. DNA molecules mixed w/ dry PCR reagents
7. Seminested real-time amplification and detection in integrated reaction tube
8. Printable test result

Question 23

Nucleic acid sequencing steps

Answer 23

1. Sample
2. Transfer to test tube
3. Deliver to lab
4. DNA extraction
5. Sequencing and bioinformatics identification
6. Comprehensive microbiome report

Question 24

MALDI-TOF mass spectrometry

Answer 24

1. Sample, applied to target, is overlaid w/ matrix solution and allowed to dry
2. Spot on target is pulsed w/ laser
3. Desorbed ionized molecules accelerated by a potential difference fly through a high-vacuum and field free flight tube
4. Time of flight, based on mass of particles, is captured on detector
5. Resulting spectrum is compared to library containing patterns of clinically relevant species

Question 25

Serological tests

Answer 25

1. Latex agglutination
2. Enzyme-linked immunosorbent assay (ELISA)

Question 26

Latex agglutination test (serological)

Answer 26

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

Question 27

ELISA test (serological)

Answer 27

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