M22: Diagnostic Microbiology

Question 1

The Clinical Microbiology Laboratory

1. Priorities
   a. Accurately diagnose pathogens: _ + _.
   b. Sufficient _ to aid in patient care.
2. Challenges
   a. Manage _ per day.
   b. Manage data from _.
   c. Maintain _ with quality control.
3. Rapid Diagnostic Test
   a. High _, high _ predictive value, _.

Answer 1

a. detection + identification.
b. speed

a. large numbers of samples
b. individual samples
c. accuracy

a. High sensitivity/specificity, high positive/negative predictive value, reproducible.

Question 2

Traditional Technologies:
Staining

a. e.g. (3)
b. Advantages: (2)
c. Disadvantages: (2)

Answer 2

Gram stain
acid-fast stain
India ink preparation

Rapid
very inexpensive detection.

Lower sensitivity for detection
not usually specific identification

Question 3

Traditional Technologies:
Culture-based (bacteria)

a. e.g. (2)
b. Advantages: (3)
c. Disadvantages: (2)

Answer 3

MacConkey agar
eosin methylene blue (EMB)

high detection sensitivity
more information on identification
relatively inexpensive.

overnight culture required
still not species-specific information.

Question 4

Traditional Technologies:
Culture-based (virus)

a. e.g. (2)
b. Advantages: (1)
c. Disadvantages: (3)

Answer 4

cytopathic effect
immunofluorescent staining

specific identification (especially immunofluorescent staining)

Slow
expensive
difficult to grow.

Question 5

Traditional Technologies:
Biochemicals

a. e.g. _ (catalase, oxidase, PYR test), _ (tubes, API strips, Vitek cards)
b. Advantages: (1)
c. Disadvantages: (2)

Answer 5

tests on colonies
culture tests

species-specific identification

Sometimes ambiguous results or strain has atypical behavior,
expense with more sophisticated automation.

Question 6

Newer Technologies:
Antigen testing

a. e.g. (2)
b. Advantages: (1)
c. Disadvantages: (1)

Answer 6

PBP2a agglutination (MRSA)
influenza and RSV rapid antigen tests 

extremely fast (compare with viral culture)

for viral antigens - low sensitivity varying by specimen collection methodology and duration of illness

Question 7

Newer Technologies:
Nucleic acid amplification testing (NAAT)

a. e.g. (5)
b. Advantages: (3)
c. Disadvantages: (6)

Answer 7

type IV SCCmec (MRSA)
Neisseria gonorrhoeae
Chlamydia trachomatis
HSV
endpoint PCR or real time PCR 

more rapid than culture
very specific
quantitative (e.g. viral load)

cost
labor intensive
technically challenging
concerns over contamination
slow if sample sent to reference lab and/or if samples batched for a run
not necessarily as sensitive as culture.

Question 8

Newer Technologies:
Nucleic acid amplification testing (NAAT)

Rapid TB diagnostic

i. Meant to be deployed in _.
ii. High sensitivity of detecting _ cases = earlier than weeks of culture

Multiplex PCR

i. e.g. _: 12 viruses
ii. e.g. _: 22 common bloodstream pathogens (bacteria, fungi), results 6 hours, 300 colony forming units/ml
iii. e.g. _: 24 pathogens and 3 antimicrobial resistance genes identified after blood cultures turn positive. FDA approved June 2013.

16S rRNA sequencing:

i. Uses _ against conserved sequences flanking variable segments.
ii. Sequence PCR product.
iii. Most commonly identification of pathogens with isolates that have _ biochemical phenotypes.
iv. Capable of detecting _ or _ pathogens, especially if unable to culture.

Answer 8

developing world

AFB stain negative

Luminex xTAG Respiratory Virus Panel (UPMC)

SeptiFast real time PCR

BioFire Blood Culture Identification Panel

primers

atypical

poorly described or unknown

Question 9

Newer Technologies:
Mass Spectrometry

3 components:

i. _ (ion source)
ii. measure _ (mass analyzer)
iii. abundance of _ (mass detector)
e. g.
i. _: measure PCR products; distinguish microorganisms by abundance of each nucleotide (not sequence).
ii. _: measure most abundant proteins from colonies; distinguish isolates by pattern of peaks.

Answer 9

vaporization

mass-to-charge ratio

molecule

PLEX-ID

Vitek MS

Question 10

Newer Technologies:
Mass Spectrometry

Advantages: (3)

Disadvantages: (4)

Answer 10

rapid analysis
very general (any pathogen?)
direct from patient sample (PLEX-ID)

requires overnight culture (Vitek MS)
sample prep (PLEX-ID)
setup expense
sensitivity?