diagnostic priniciples

Question 1

Assessing the performance of laboratory tests

Answer 1

specifity and sensitivity

Question 2

sensitivity formula

Answer 2

Question 3

specifity formula

Answer 3

Question 4

positive predictive value

Answer 4

higher value at higher prevalences, determines amount of real positive results

Question 5

For ruling out syphilis

Answer 5

• Rapid plasma reagin (RPR) test
• Venereal Diseases Reference Laboratory(VDRL) test
• These tests have a low false negative rate (very sensitive)

Follow up a positive RPR or VDRL test to confirm syphilis (need a veryspecific test)

Question 6

Diagnosing infections by microscopy

Answer 6

Direct microscopic examination of clinical specimen

Question 7

Syphilis microscopy

Answer 7

Syphilis - spirochetes observed in scrapings of lesions

Question 8

use of staining

Answer 8

1. Bacteria in normally sterile body fluid (CSF, pleural fluid, urine)
2. Staining properties part of larger effort at diagnosis
3. Actual diagnosis (sometimes)
   typically will not reveal genus and spp

Question 9

Gram stain

Answer 9

• Apply the primary stain (crystal violet) to a heat-fixed smear of bacteria.
• Add a trapping agent (or mordant) (Gramsiodine).
• Decolorize with acetone or alcohol. Thick peptidoglycan layer of Gram positives keeps crystal violet-iodine complex trapped within cells. This step must be done quickly (seconds); if too long the crystal violet will be washed from both Gram-negatives and Gram positives).
• Counterstain with safranin to stain decolorized cells.

Question 10

acid-fast stain (Ziehl-Neelsen method)

Answer 10

• Apply the primary stain (fuschin) + mordant (carbolic acid) (combination is called carbolfuchsin) to a heat-fixed smear of bacteria. Place piece of absorbant paper soaked with carbolfuchsin over the smear and heat the slide to drive the stain+mordant into cells.
• Decolorize with dilute acid in alcohol. The carbolfuchsin will wash out of most cells, but not those with high levels of mycolic acid in their membranes (e.g. mycobacteria)
• Counterstain with methylene blue to stain decolorized cells.

Question 11

Antibody-based identification

Answer 11

Direct Fluorescent Antibody (DFA) test

Question 12

Ab compostions and their specifities

Answer 12

monoclonal Ab to epitope would be most specific

Question 13

bacterial diagnosis with culture

most bacteria vs. some special ones

Answer 13

most bacteria:
culturing involves use of numerous kinds of growth media
can provide preliminary information about biochemical nature of bacterium
additional biochemical tests used following isolation

some bacteria are not routinely cultured:
rickettsias, chlamydiae, and mycoplasmas
identified with special stains, immunologic tests, or molecular methods

Question 14

approaches to culturing bacteria

Answer 14

Open-ended sampling versus looking for a particular pathogen

Question 15

types of media

Answer 15

selective, differential, enrichment, and characteristic

Question 16

selective media

Answer 16

prepared to facilitate the growth of some spp and inhibit others
ex: salmonella-shigella

Question 17

differential media

Answer 17

incorporation of certain chemicals into the media for diagnostically useful growth or visible change
ex; eosin methylene blue agar

Question 18

enrichment media

Answer 18

additon of blood/ serum to support fastidious bacteria, use mainly for the isolation of bacteria from body fluids

Question 19

characteristic media

Answer 19

test bacteria for certain activities, products or requirements
ex; urea broth
choice of media depends on pathogen suspected to be in specimen

Question 20

Bacteriophage Typing

Answer 20

• only done by CDC and certain labs
• based on specificity of phage surface molecules for host cell surface molecules
• phagovar– bacterium with sensitivity to certain collection of phage types

Question 21

Measuring the antibody response to infection methods

Answer 21

ELISA and western blot

Question 22

direct ELISA steps

Answer 22

Question 23

western blot

Answer 23

Question 24

Diagnosing infection by detecting microbial macromolecules methods

Answer 24

Detecting microbial antigens, Nucleic acid-based diagnosis of infection, PCR, Microarrays, Next generation sequencing

Question 25

Latex agglutination test:

Answer 25

latex bead coated with antibody to particular pathogen material (capsular material or surface protein), will agglutinate when exposed to Ag can have false negatives with no agglutination due to Ag surrounding the beads= prozone, corrected with dilutions

Question 26

. Enzyme immunoassays for microbial antigen detection.

Answer 26

ELISA for Ag detection

Question 27

Nucleic acid amplification (polymerase chain reaction (PCR)), basic components?

Answer 27

pathogen DNA, pair of pathogen-specific primers, dNTPs, stable DNA poly

Question 28

real time PCRs

Answer 28

same as regular PCR but use a probe that is displaced with DNA synthesis causing activation of the fluorescent portion that can be detected

Question 29

Microarrays

Answer 29

Question 30

Tests for SARS-CoV-2 Virus (COVID-19)

Answer 30

Real-time reverse transcription-polymerase chain reaction (rRT-PCR) or sequencing of reverse transcribed SARS-CoV-2 RNA genome regions Use nasal swab or saliva Sensitive and specific

Question 31

Diagnosing HIV infection

Answer 31

Standard tests detect HIV antigen and anti-HIV antibodies

Question 32

Tests for Zika Virus, positive and negative results?

Answer 32

For symptomatic persons suspected for Zika virus infection 1. Real-time reverse transcription-polymerase chain reaction (rRT-PCR) Use serum or urine collected within 2 weeks after symptom onset. A positive rRT-PCR result confirms Zika virus infection and no additional testing is indicated. Sensitive and specific but subject to limited time window 2. A negative rRT-PCR result does not exclude Zika virus infection and serum should be analyzed by IgM antibody (serological) testing. Sensitive with longer time window but not as specific cross-reactivity with other flaviviruses