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CMOD Week 5 > Lab Diagnosis of Microbial Infections > Flashcards

Lab Diagnosis of Microbial Infections Flashcards

1
Q

What is Sensitivity?

A

the ability of a test to single out those with disease as positive

If high sensitivity- a negative result rules out disease

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2
Q

What is Specificity?

A

the ability of the test to classify people who do not
have illness as negative

If high specificity- a positive test result rules in disease

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3
Q

What are the three main methods of specimen collecting?

A
  • Direct Specimen - when the pathogen is located in an otherwise sterile site, e.g. deep abscess
  • Collect surgically
  • Needle aspiration
  • Indirect Sample – when the pathogen is located in an otherwise sterile site, but must pass through a site containing normal flora
  • Expectorated sputum
  • Voided urine
  • Sample from site with normal flora – sample collected is a mixture, then the normal flora are inhibited under growth conditions for analysis
  • Throat swab
  • Stool sample
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4
Q

The acid-fast stain is aka?

A

Ziehl-Neelsen

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5
Q

What is a negative stain sued for?

A

india ink is excluded from capsules to expose it

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6
Q

What is one of the main reasons for culturing?

A

to obtain purified colonies in case confirmative testing is needed

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7
Q

Are BAP differential or selective?

A

differential only

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8
Q

What are MacConkey agar plates selective for? differential?

A

selective- dyes that are selective for gram negative rods

differential- for lactose+ (bright color) vs. lactose- (same red color) like EMB

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9
Q

MacConkey plates that contain sorbitol instead of lactose are used for what?

A

selective for GNR and differential for E. Coli O157 (will be white while all other E. coli colonies will be red)

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10
Q

What are Thayer-Martin agar plates used for?

A

Chocolate agar + AGX (brownish color). Blood has been heated and lysed that release iron and other things bacteria can grow on, The addition of certain antibiotics make it SELECTIVE for Neisseria species because they kill any other bacteria there.

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11
Q

When are Thayer-Martin plates used?

A

Used in the isolation of N gonorrheae from genital secretions but only chocolate agar (without antibiotics) is used for isolation of N. meningitidis from CSF because the CSF is normally sterile so we don’t have to worry about other contaminants that might compete with the Neisseria

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12
Q

How should blood cultures be collected?

A
  • Obtain at least three 10-mL samples in a 24-hr period to increase chance of getting good sample and for replicability
  • Cleanse the site with 2% iodine before puncture to cut back on contamination (draw from different spots to get your samples)
  • Add to rich growth medium (brain-heart infusion broth) to capture anything thats on there
  • Need to consider anaerobic incubation in addition to aerobic
  • Check for turbidity or CO2 production daily for up to 7 days
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13
Q

What kinds of things would make you want to do a blood culture?

A 
Sepsis (Staph aureus)
Endocarditis (Strep pneumo)
Osteomyelitis (E. coli)
Meningitis (K. pneumoniae)
Pneumoni (Ps. aeruginosa)
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14
Q

How/When would you perform a throat culture?

A
  • Swab posterior pharynx and tonsils
  • Routine culture is on blood agar, but special media may be required
  • Confirm b-hemolytic colonies are GAS by sensitivity to Taxo A (bacitracin)
  • Note: Gram stain is of little use because of viridans Strep present
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15
Q

What kinds of bugs would you be looking for on a throat swap?

A

Diphtheria

Gonococcal pharyngitis Thrush (Candida)

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16
Q

How would you do a sputum culture?

A

Make sure sample is sputum, not saliva

• >25 leukocytes,

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17
Q

What kinds of bugs would you be looking for on a sputum culture?

A 
Strep pneumoniae 
Staph aureus
K pneumonia
Ps aeruginosa
M tuberculosis

thinking Pneumonia or TB

18
Q

How would you do a spinal fluid culture?

A

Lumbar puncture

  • Consider this a medical emergency, send to lab immediately (bacterial/viral meningitis)
  • Centrifuged samples are cultured but observed microscopically for the presence of bacteria
  • Rich medium to capture anything- Blood agar and chocolate agar
19
Q

What kinds of bugs would you be looking for on a spinal fluid culture?

A

Meningitis

S pneumoniae
N meningitidis
H influenzae

20
Q

How would you do a stool culture?

A
  • Direct examination of stool sample
  • Methylene blue staining of leukocytes would be seen in inflammatory diarrhea (opposite is watery diarrhea without leukocytes)
  • Culture on selective, differential media such as EMB or MacConkey because theres tons of normal flora there
  • Grossly bloody diarrhea should be grown on MacConkey Sorbitol to look for EHEC O157
  • Anaerobic cultures are NOT needed because of abundance of normal flora
21
Q

What kinds of bugs would you be looking for on a stool culture?

A

Enterocolitis

Salmonella
Campylobacter
Shigella
E coli O157

22
Q

How would you do a urine culture?

A
  • Urine is sterile (because bladder is), but will become contaminated as it is released
  • Capture first morning, clean-catch midstream because bacteria are concentrated then
  • Start cultures within 1 hr or refrigerate to avoid growth
  • Culture must be quantitative to confirm infection
  • Use a 0.001mL calibrated loop to streak a BAP and EMB plate
  • If no colonies on EMB plate, not a Gram negative infection. If colonies on both plates, likely Gram negative
  • Count colonies to determine whether >100,000/mL (count on late should be over 100 for diagnosis)
23
Q

When should a urine culture be performed?

A

Pyelonephritis or Cystitis

E coli (gram-)
Enterobacter (gram-)
Proteus (gram-)
Enterococcus (gram positive)
Staph saprophyticus (gram positive)
24
Q

What are some things to consider on wound and abscess cultures?

A

Frequently involve aerobic and anaerobic mixed infections

• Culture aerobically and anaerobically on a variety of media

25
Q

Infection of a brain, lung, or abdominal abscess would probably be caused by which bacteria

A

bacteriodes, Staph aureus, Strep pyogenes (GAS)

26
Q

Infection of a surgical wound would probably be caused by which bacteria?

A

Staph aureus

27
Q

Infection of a traumatic open wound would probably be caused by which bacteria?

A

C. perfringens

28
Q

Infection of a cat or dog bite would probably be caused by which bacteria?

A

Pasteurella multocida

29
Q

How is viral culturing performed?

A

Start with a specimen in a transport medium, then do a low speed centrifugation to pellet large things (our cells, bacterial cells, fungal cells) to the bottom. Small viruses will remain in the supernatant. Then add antibiotics to the supernatant and then inoculate a cell monolayer grown under culture. The virus will cause cytopathic effect and the appearance of the monolayer changes

30
Q

What is the gold standard for bacterial infection diagnosis?

A

Four-fold rise in specific convalescent vs acute titer

31
Q

How is syphillis testing performed?

A
  • Non-specific testing
  • Rapid plasma reagin (RPR)
  • Venereal Disease Research Lab (VDRL)
  • Beef heart cardiolipin (diphosphatidylglycerol) is the antigen, conjugated to carbon particles, which clumps in the presence of antibody to T. pallidum

if positive, follow up with:

  • Specific testing
  • Fluorescent treponemal antibody absorption (FTA-ABS)
  • Patient serum is absorbed with non-Tp treponemes, then reacted with fixed T pallidum on a slide
  • Fluorescent anti-human IgG is added to visualize the bound bacteria
32
Q

Why is the FTA-ABS expensive?

A

T. palladium cannot be grown in culture (just rabbit testicles)

33
Q

What is the advantage of antigen detection over antibody detection when possible?

A

Antigen may be present at higher levels earlier in an infection than antibodies while the immune response is being formed

34
Q

How are viral neutralization assays performed?

A

detecting antigen

have source with virus in it and add it to a series of tubes with commercial neutralizing antibodies against suspected causative antigens and then look on a monolayer plate for inhibition of the virus. Need a control to make sure its working properly.

35
Q

How are Rapid Antigen Detection tested (RADT) performed?

A

often used for strep throat and for flu infections

Called lateral flow immunoassays and can typically read in about 15 minutes and they are accurate

Take collected samples (throat swab) and put it in a medium that breaks up the bacteria to release the antigens and then we add a droplet of sample onto a well on a wickable paper so that the sample will flow. It will encounter antibody to antigens present in that organism to make sure the organism is present. Once they bind they are concentrated and trapped to a line via another antibody fixed to the slide. The second fixed line binds the antibody to make sure its present as a control.

Doesn’t tell you which antigen you are binding, you assume it is the right antigen

36
Q

How is HIV testing done?

A

• ELISA (antibody testing)

• Murex Single Use Diagnostic System (SUDS)
-Assays antibodies and presence of p24

• OraQuick (home testing)
-Saliva antibodies

• Western blot (confirmatory test)
-Positive: p24, gp41 and/or gp120/160
-Indeterminate: some bands, but not those above
-Negative: no bands

37
Q

How is direct probing done?

A

denature nucleic acid of target organism and then bring primers to amplify the DNA via PCR

38
Q

When are Nucleic Acid Amplification Tests (NAT or NAAT used)?

A
  • Particularly useful when organism is difficult to routinely grow and isolate
  • Often becoming the “gold standard” of diagnosis
  • Fairly rapid, and because of amplification, very sensitive
  • Can be used to detect pathogen before significant immune response
  • Can be used to determine pathogen load
• Uses (there are plenty others)
• HIV
• HBV, HCV
• Chlamydia, GC

39
Q

Why is probe hybridization preferred over an NAAT in BV?

A

PCR could be too specific and would give false positives if other flora were introduced

40
Q

Metabolite Analysis

A

useful in identification of gram- rods

uses enterotubes with agar ‘cubes’ that can change color depending on whats in it. Use an inoculating rod with organism for each rod and can see each the change in each cube. Uses a scoring system to identify

41
Q

Urine Dipstick

A

common UTi-causing bacteria can reduce nitrate to nitrite. Can be assayed using the dipstick format

42
Q

Mass Spec

A

Matrix-Assisted Laser Desorption Ionization Time-of-Flight

add bacteria in a sample and use a laser to ionize the proteins and then line them up through an electrostatic field. They are then sprayed and they “fly” through an analyzer and the small proteins fly faster than the heavier ones. Read on computer.

CMOD Week 5 (16 decks)

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