Lab 4 Upper Respiratory Tract Infections

Question 1

What are the only routine pathogens looked at in a throat swab culture?

Answer 1

Throat swab culture:
- Beta hemolytic Streptococci Group A, C, G, (or Streptococcus pyogenes and Strep dysgalactiae)

but primarily Strep Group A.

Question 2

What media and incubation environment are typically used? Is that the ideal conditions?

Answer 2

Throat swabs are planted on BA and usually incubated in CO2 even though ideal conditions would be anaerobic.

Question 3

Can other pathogens (other than Strep Group A, C and G be cultured? Why?

Answer 3

Yes on request because they need specific media.

Neisseria gonorrhoeae needs Thayer Martin to inhibit normal flora.

Corynebacterium diphtheria needs Tinsdale agar.

Question 4

Why is routine AST not needed for beta hemolytic Strep?

Answer 4

Penicillin is the recommended treatment and no resistance has been found to it.

If a patient is allergic to penicillin then AST could be performed on MH blood following CLSI method.

Question 5

What are the symptoms of pharyngitis?

Answer 5

Fever, extremely red and perhaps swollen throat, and tonsillar exudate (pus) typically characterize pharyngitis.

Question 6

Is it easy to diagnose pharyngitis from clinical appearance alone?

Answer 6

It is very difficult to make a diagnosis from clinical appearance alone; therefore a swab must be taken.

Question 7

What secondary infection/conditions are some people at risk of from Gr. A Strep?

Answer 7

Group A strep may SECONDARILY infect wounds from trauma and produce scarlet fever.

Other major concerns (non-pus forming conditions) are:
- acute rheumatic fever
- acute glomerulonephritis, also
- streptococcal toxic-shock like syndrome

Question 8

Why may Group A Strep (GAS) cause scarlet fever?

Answer 8

GAS elaborate an erythrogenic toxin that may produce scarlet fever, which is characterized by a scarlatina-like rash that affects the trunk, neck and extremities.

Question 9

Why is it important to select a method to diagnose Group A Strep with optimal sensitivity?

Answer 9

It is important to select methods of optimal sensitivity, since a low number of GAS in a specimen does not necessarily indicate colonization and may represent infection. If the method is not too sensitive, it might be missed.

Question 10

What symptoms can Group C and G beta-haemolytic Strep cause?

Answer 10

Group C & G beta-haemolytic streptococci can cause pharyngitis and fever.

Question 11

What secondary condition are patients not at risk from a Group C and G Strep infection that they are with Group A?

Answer 11

Group C & G Strep do not place patients at risk of acute rheumatic fever.

Question 12

What is Diphtheria and what bacteria causes it? What is its classic symptom?

Answer 12

Diphtheria is an acute infectious disease primarily of the upper respiratory tract but occasionally of the skin. It is caused by toxigenic strains of Corynebacterium diphtheria.

The disease is shown by a classic pseudomembrane of the pharynx; however, this symptom may be lacking in mild cases.

Question 13

What test is done and media used to confirm a diagnosis of diphtheria?

Answer 13

To confirm a clinical diagnosis of diphtheria, the strain isolated must be shown to produce toxin= Elek test (done in reference labs).

Tinsdale medium is a selective and differential medium to isolate and presumptively identify C. diphtheria strains.

Question 14

Which organisms cause upper respiratory infections?

Answer 14

The most common cause of bacterial pharyngitis (bacterial sore throat, not viral), in North America is Streptococcus pyogenes- Group A streptococci (GAS). Other bacterial causes of pharyngitis include beta hemolytic Group C and G streptococci, Neisseria gonorrhoeae, Corynebacterium diphtheria, and Arcanobacterium haemolyticum.

Question 15

Which are the usual “normal flora” organisms?

Answer 15

Resident Microbiota of the URT: Coag neg Staph, Viridans Strep, Corynebacterium, non pathogenic Neisseria spp.

Question 16

What are the steps for taking an acceptable throat swab specimen from the tonsillar area and/or posterior pharynx?

Answer 16

1. Depress tongue gently with tongue depressor.
2. Extend sterile Dacron-tip swab between the tonsillar pillars and behind the uvula. (avoid touching the cheeks, tongue, uvula, or lips)
3. Sweep the swab back and forth across the posterior pharynx, tonsillar areas, and any inflamed or ulcerated areas to obtain sample.

Question 17

What should be avoided when taking a throat swab from the tonsillar area and/or posterior pharynx?

Answer 17

Avoid the tongue and uvula, is recommended.
Do not obtain throat samples if epiglottis is inflamed, as sampling may cause serious respiratory obstruction.

Question 18

Do Streptococci survive well in a dry environment?

Answer 18

Yes, Streptococci survive well in a dry environment, and the original numbers present in the throat are preserved.

Question 19

When is a transport medium needed for a throat swab specimen?

Answer 19

If the specimen is transported over a distance or not being processed until the next day or is to be tested for other pathogens, then a swab in transport medium should be used.

Question 20

Is a nasopharyngeal specimen also acceptable and how is it obtained? What other types of nasopharyngeal specimens could be acceptable?

Answer 20

A nasopharyngeal specimen is also acceptable and is obtained using a flexible-wire calcium alginate-tipped swab through the nose or up the back of the throat into the posterior nasopharynx.

Question 21

What other types of nasopharyngeal specimens could be acceptable?

Answer 21

Nasopharyngeal suction or sinus aspirates are also acceptable, provided they are obtained aseptically and kept moist.

Question 22

When is a ‘dry’ throat swab not acceptable?

Answer 22

1. If > 2 hrs has passed after collection. A ‘dry’ swab is sufficient if no more than 2 hours is expected to elapse between the time the swab specimen is collected and the time it is examined in the laboratory.
2. Demographic mismatches between requisition and sample and lack of basic patient information
3. Frozen sample or leaking specimen

Question 23

What are the requirements for media to help isolate Streptococci?

Answer 23

Care must be taken in choosing an appropriate medium for isolation.

1. Free of reducing sugars
2. pH of media between 7.3 and 7.4

Note re #1: Streptococci require media free of reducing sugars i.e. substances that influence the expression of beta-hemolysis by strep-for example glucose. (eg. enriched infusion agar-such as tryptic soy, heart infusion, Todd-Hewitt- or protease peptone are free from reducing sugars)

Question 24

What media type is recommended to help isolate Streptococcus and why?

Answer 24

Sheep blood is recommended to prevent the growth of Haemophilus haemolyticus, which, if present in a throat specimen, has a colonial morphology indistinguishable from that of beta-hemolytic streptococci. This blood lacks sufficient amounts of pyridine nucleotides ( V factor) to support the growth of H. haemolyticus.

Question 25

What factor affects the size of the beta-hemolytic zone?

Answer 25

Different concentrations of blood affect the size of the area of erythrocyte (RBC) destruction (hemolysis zone size).

Question 26

What percentage of blood and how deep of agar is recommended for primary isolation of Streptococcus (defined hemolysis)? What also enhances hemolysis?

Answer 26

The best blood agar for primary isolation contain 5% defibrinated blood in agar approximately 4 mm deep.

Haemolysis is also enhanced by using ‘split bilayer BA plates’ where the bottom portion is clear nutrient agar and the top half is 5% sheep’s blood.

Question 27

What is the method to plant and incubate the throat swab specimen?

Answer 27

1. Inoculate a routine throat swab onto BA, making sure the swab is rolled on the plate in a way that all the sides touch the agar.
2. Streak for isolation and
3. Incubate in CO2 at 35° C for 18-24 hours.

Question 28

What are the alternate and/or ideal incubation methods for Strep?

Answer 28

Alternate incubation methods include ambient air with agar stabs or anaerobic (ideal but more complicated for lab)

Question 29

What morphology are you looking for after 18-24 hours of incubation in sheep blood agar?

Answer 29

1. Observe for presence of β- hemolytic strep-looking colonies. 2. Streptococci typically are about 0.5 mm in diameter, transparent or translucent, and domed; they have a smooth or semi-matte surface and an entire edge.
2. Group C & G differ very little in morphology.

Question 30

What do you do if beta-hemolytic colonies are present?

Answer 30

Gram stain, Catalase then PYR (see algorithm).

If there are not enough isolated colonies, then subculture to another BA plate.

Question 31

For review describe what alpha, beta, and gamma hemolysis looks like on a BA plate?

Answer 31

1. Alpha-hemolysis - An indistinct zone of partial destruction of RBC’s around the colony, seen as greenish discoloration of the medium
2. Beta-hemolysis - A clear, colorless zone around the colonies in which the RBC’s have undergone complete destruction.
3. Gamma-hemolysis - No apparent hemolytic activity or discoloration produced by the colony.

Question 32

What is alpha-prime hemolysis?

Answer 32

Alpha-prime- or wide-zone alpha-hemolysis:

A small halo or envelope of intact or partially lysed RBC’s lying adjacent to the bacterial colony, with a zone of complete hemolysis extending farther into the medium.

Caution: When examined macroscopically, alpha-prime hemolysis can be confused with beta-hemolysis.

Question 33

What serological test is recommended to ID beta-hemolytic streptococcus?

Answer 33

Serological grouping: Beta-hemolytic streptococci isolated from human infections possess specific carbohydrate antigens- called streptococcal group antigens (Lancefield grouping). Use of group A, B, C, and G antisera is recommended for identification of beta-hemolytic streptococcus.

Review and learn lab diagnosis algorithm.

Question 34

What do you put on the final report if no beta-hemolytic colonies are found?

Answer 34

If there are no β-haemolytic colonies identified, then report as
“No Beta-haemolytic strep Group A, C or G isolated.”

Question 35

If there are beta-hemolytic colonies present what different statements could be reported depending on subsequent test results?

Answer 35

1. If there are Beta-haemolytic colonies identified as Group A, then “Beta-haemolytic strep Group A isolated.” This should include enumeration e.g. 4+, 3+ etc. (semi-quantitative).
2. If there are Beta-haemolytic strep identified as group C or G, report as “Beta-haemolytic strep Group C (or G) isolated.”
3. “Beta-haemolytic strep, not Group A, isolated.” if Lancefield typing is not available.
   This should include enumeration e.g. 4+, 3+ etc.

Question 36

What does a positive Group A Strep ID in the micro lab not distinguish between?

Answer 36

Even if the culture is positive for Group A strep, it does not distinguish between infection and colonization.

Question 37

What is the next antibiotic of choice if the patient is allergic to penicillin?

Answer 37

For penicillin-allergic patients, the antibiotic of choice is usually erythromycin, but doctors might request AST testing in these cases.

Question 38

What can cause a false-negative culture for a URT?

Answer 38

False-negative cultures can be caused by overgrowth of cultures by normal flora (streaking?) or from lack of detection of Beta-haemolysis in cultures incubated aerobically.

Question 39

What can cause a false-positive culture for a URT?

Answer 39

False-positive pharyngeal culture reports can result from mis-identification tests. E.g. PYR + colonies which are enterococci.

(Note: I think you would have to miss the beta-hemolysis too in that case).