Clinical Lab Assays 1

Question 1

Specimen collection - best specimens, what determines significance?

Answer 1

 For culture, specimen site determines significance
 GI and respiratory tracts are colonized with bacteria and fungi
 Best specimens: Bronchoalveolar lavage, Sterile fluids, Tissue or aspirate
NOT swabs, stool, sputum
 No formalin!

Question 2

What is the goal of a direct exam?

Answer 2

Get maximum information from direct exam of specimen:
 Provides a rapid report for initial treatment
 Presumptive identification of organism
- Gram negative vs Gram positive bacteria
- Yeast versus mould
Evidence of infection in spite of a negative culture
Direct exam should be done for all specimens,
Not highly sensitive so a negative result does not rule out
mycotic infection

Question 3

Explain Gram Stain

Answer 3

Apply crystal violet, apply iodine, alcohol wash, apply safranin

Fix specimen, apply cyrstal violent, ioding which bionds crystal violet to organisim
Decolourizeing step

Counter stain safranin

Question 4

What organisms dont stain with gram?

Answer 4

Organisms without a cell wall
 i.e. Mycoplasma species
Acid Fast Bacteria
 e.g. Mycobacterium species, Nocardia species
Fungal organisms
 i.e. Candida species, `Aspergillus species

Question 5

What is Inoculation of Media for Culture, and the 2 types of media

Answer 5

Nutrient rich media are used to isolate bacteria and fungi from clinical specimens.
 Selective: contain specific compounds to select for specific organisms
 Differential: allows for pigment formation for specific species of organisms

Question 6

Ex of Inoculation of Media for Culture, purpose and what type?

Answer 6

Blood Agar; General purpose; Non-selective, hemolysis
Chocolate Agar: Enriched, general purpose; Non-selective, Supports growth of fastidious organisms
MacConkey Agar; negative; Selective, Differential

Question 7

What is blood culture, what is it used for?

Answer 7

A laboratory test used to determine if microorganisms have invaded a patient’s bloodstream.
 Organisms in the blood are at a quantity so low, they cannot be recovered without a liquid culture step first.
 Specimens are collected into blood culture bottles containing nutrient rich media and incubated in automated detection systems

Question 8

What are some identification ways? (5)

Answer 8

Growth on each inoculated medium?
 CHOC&raquo_space; BA  suggests fastidious organism
 No growth on MAC suggests a particular group of organisms
 Obligate or facultative anaerobe?
Gram-stain
Colonial morphology
Spot tests, limited tube/strip biochemicals
Automated identification systems, MALDI-TOF MS,
Molecular testing

Question 9

Biochemical Identification Tests (5 of them) and how its used

Answer 9

Catalase, Coagulase, Pyrase, Oxidase, Indole
Miniaturized biochemicals often used in system-dependent approach
 (+) and (-) reaction patterns emerge
 “Metabolic profile”  compare to database

Question 10

MALDI-TOF-MS stand for?

Answer 10

Matrix-
Assisted
Laser
Desorption/
Ionization –
Time
Of
Flight
Mass
Spectrometry

Question 11

Advantages of MALDI-TOF

Answer 11

Turnaround time 
Low consumable costs 
High-throughput 
Reliable and reproducible 
Easy to use 
Minimal sample required 
Potential to ID organisms that cannot be identified by conventional methods

Question 12

disadvantages of MALDI-TOF

Answer 12

Upfront instrumentation cost
Closely related organisms are difficult to separate
Limited by database size and quality
Some organisms require extraction prior to analysis
Cannot perform susceptibility testing
Unable to perform quantitation

Question 13

Serology Testing - what is it? used for?

Answer 13

Used for detection of antigen or antibody in the blood
in response to infection with a given organism
 Measure Ag or Ab using several techniques
 agglutination
 immunochromatography
 immunofluorescence
 enzyme linked immunosorbent assay (ELISA)
Can detect IgG or IgM

Question 14

Serology Testing utility - antibody and antigen

Answer 14

ANTIBODY
 Used to stage the infection (acute vs past):
 IgM vs IgG
 Increasing antibody titers
 Avidity testing
 Titers can be monitored for treatment success
 Surveillance

ANTIGEN
 Provides evidence of current infection
 Titers/quantities can be monitored for treatment
success

Question 15

When is serologic testing used in

infectious diseases?

Answer 15

Some examples:
 HIV (antigen and antibody)
 Hepatitis (antibody)
 COVID (antigen and antibody)
 Treponema pallidum (antibody)
 Borrelia burgdorferi (antibody)
 Coxiella (antibody)
 Aspergillus galactomannan (antigen)
 Histoplasma (antigen)
 Strongyloides (antibody)
 And many many more...

Question 16

Molecular Testing Refers to ? 4 purposes?

Answer 16

Detection of nucleic acids:
Molecular assays have variable purposes:
1. Presence or absence of bacteria or virus
e.g. B. anthracis, C. trachomatis, N. gonorrhea, HIV
2. Presence or absence of a particular gene
e.g. PBP2’ gene of MRSA
3. Measuring amount of bacteria or virus
e.g. HIV viral load
4. Determining what type of a particular virus or bacteria is present.
e.g. Hepatitis C genotyping
e.g. Clusters of enteric bacteria ie: Salmonella - are they all the same strain of Salmonella?

Question 17

Most common assay for molecular testing employed in the laboratory today?

Answer 17

DNA amplification assays:
most common is polymerase chain reaction or PCR.
Amplification of a single strand of DNA into millions of
copies. Basis for a large number of molecular diagnostic assays

Question 18

Explain PCR testing

Answer 18

Slide 39 in Assay 1 ppt. pictorials

Question 19

Examples of DNA amplification assays

currently used in the lab:

Answer 19

SARS-CoV2
M. tuberculosis
C. difficile toxin
MRSA and VRE gene detection
16S rRNA PCR followed by sequencing
HIV viral load assays
Hepatitis C and CMV viral load assays
Norovirus
Malaria
Respiratory viruses
And many others!

Question 20

Sensitivity VS Specificity

Answer 20

SENSITIVITY: If a person has a disease, how often will the test be positive (true positive rate)?
Put another way, if the test is highly sensitive and the test result is negative you can be nearly certain that they don’t have disease.
SPECIFICITY: If a person does not have the disease how often will the test be negative (true negative rate)?
Put another way, if the test result for a highly specific test is positive you can be nearly certain that they actually have the disease.
High sensitivity- then negative results suggest absence of disease
High specificity - a positive result from the assay means a high probablity of the presence of disease.

Question 21

Screening vs Confirmation Assays

Answer 21

Screening assays have HIGH sensitivity, LOW specificity.

Confirmation assays have HIGH specificity, LOW sensitivity.