Lab 5 Lower Respiratory Tract Infections

Question 1

Which organisms might cause lower resp. infections?

Answer 1

Most common causative bacteria for these infections (pathogens):
1. Streptococcus pneumoniae,
2. Haemophilus influenzae,
3. Moraxella catarrhalis as community acquired.
In the nosocomial infections,
4. Enterobacterales (Kleb. pneumoniae),
5. Pseudomonas aeruginosa,
6. S.aureus,
7. Candida spp..

Also see table in learn for the 3 groups of organisms considered.

Question 2

Which is the typical “normal flora”?

Answer 2

Resident Microbiota of the respiratory tract are a majority of anaerobic bacteria (but we will not see these in routine cultures) and then,
1. Viridans streptococci,
2. Neisseria spp.,
3. Enterococcus,
4. Corynebacterium,
5. Coag negative Staph and…
6. The organisms listed as possible pathogens too!

Question 3

What practices improve the diagnostic accuracy of analyzing sputum specimens?

Answer 3

The sputum specimens that improve diagnostic accuracy are:
1. Early morning samples taken before antimicrobial treatment, 2. With the aid of physiotherapy or supervised by the physician, and
3. processed promptly (within 1-2 hours of collection).

Question 4

What should the patient be told to do to reduce the amount of oral contamination?

Answer 4

Gargling with water or mouthwash may reduce the amount of oral contamination.

Question 5

How should the specimen be treated if immediate culturing cannot be done?

Answer 5

If immediate culturing cannot be done, the specimen should be refrigerated.

Transport < 2 hours at room temp.

Question 6

Describe how the patient is to produce a sputum specimen and the container?

Answer 6

Collected after rinsing with water, with instruction to the patient to cough deeply into a sterile, wide-mouthed, screw capped container.

Question 7

What is the minimum volume of an acceptable sputum specimen?

Answer 7

Minimum volume- > 1 ml.

Question 8

if the specimen can’t be processed right away what is the storage conditions?

Answer 8

Storage- < 24 hr., 4º C

Question 9

What can be done for pediatric patients unable to produce a specimen?

Answer 9

For pediatric patients unable to produce a specimen, a respiratory therapist should collect a specimen via suction.

Question 10

What is the screening criteria for sputum specimens and which samples do not require screening?

Answer 10

Acceptable screening (<10 SEC’s/LPF)

Unless:
- immunocompromised patients, - pediatric and
- induced sputa.
Then accept without screening.

Question 11

What are unacceptable sputum specimens (note two of these are not specific to sputums)?

Answer 11

1. ‘Sputum’ for anaerobic culture
2. Saliva for culture or Sputum with ≥10 epithelial cells/LPF (See reporting section for how to report)
3. Sputa collected over a 24 hr. period
4. Sputum > 24 hrs. without refrigeration
5. Sputum in preservative
6. Specimen leaking
7. Specimen not labeled or mislabeled

Question 12

What affects the gram staining of sputum specimens that causes lab variability in sensitivity and specificity?

Answer 12

Depends on the specimen itself and the skill and experience of the technologist or reader.

Question 13

What makes a sputum smear of value for screening?

Answer 13

Generally smears are only of value for screening when:

1. It is a good quality specimens.
2. Predominance of Gram-positive cocci in pairs end-to-end (suggestive of S. pneumoniae),

3 Gram-negative bacilli suggestive of Haemophilus (82% sensitivity) or

4. Gram-negative rods suggestive of Enterobacteriaceae or Non-fermenting bacilli.

Question 14

Why is the proper evaluation of gram stains critical?

Answer 14

To ensure that only appropriate specimens are processed and not specimens contaminated with normal flora.

Question 15

What portion of the sputum specimen is selected to prepare the smear?

Answer 15

The portion that looks purulent.

Question 16

What is the sputum gram stain smear examined for?

Answer 16

Examined for the presence of squamous epithelial cells (SEC’s) using low power (10 X objective).

Question 17

Are polymorphonuclear leukocytes (PMN’s) counted in the gram stain smear and are these important to consider?

Answer 17

Yes, the higher the number of PMN’s and the lower the number of epithelial cells the more representative the specimen of the disease process.

However, the number of white blood cells may not be relevant, because many patients are severely neutropenic and specimens from these patients will not show white blood cells on Gram stain evaluation. But the presence of 25 or more PMN’s per 100x field, together with few squamous epithelial cells, implies an excellent specimen.

Question 18

What is considered an acceptable specimen based on the screening of the sputum gram stain smear?

Answer 18

An acceptable specimen yields less than 10 squamous epithelial cells per low-power field (10X eyepiece X 10X objective = 100 X).

Question 19

What is done if the gram stain smear screening results are unacceptable?

Answer 19

If the results are unacceptable, the physician or ward should be notified as soon as possible. The specimen will be rejected for culture and a new specimen requested if clinically relevant.

Question 20

What is the procedure for creating and evaluating a sputum gram stain smear?

Answer 20

1. Label slide.
2. In a BSC, select a purulent portion of sputum with a sterile swab and roll it over a generous area of the slide.
3. Air-dry the slide, fix and stain with Gram’s reagents.
4. Scan at least 20-40 fields under low power (10 X).
5. Count SEC’s and average.
6. If SEC’s <10/LPF= accept for culture and continue processing.

If SEC’s ≥10/LPF= reject specimen for culture (see exceptions)

SEC = squamous epithelial cells.

Question 21

What are the exceptions to the acceptability criteria for sputum gram stain smears?

Answer 21

1. If PMN’s are 10X more than SEC’s (in LPF) and only one morphotype of bacteria is observed at 3-4+ (in oil), specimen should be processed.
   E.G: >10 EPI’s, >100 PMN’s, 4+ GPC in pairs= do not reject.
2. No screening done for CF patients or pediatric patients.
3. No screening done for requests for Legionella, fungus or TB cultures

Question 22

What plates are being used in this lab to plant an acceptable sputum sample?

Answer 22

BA, Choc and a MAC plate

Question 23

How do you inoculate the plates for a sputum sample?

Answer 23

Using a new swab, the inoculum should be about the size of a quarter, going from the least inhibitory to the most inhibitory agar.

Question 24

Do you sterilize between streaks when streaking for isolation of a sputum sample?

Answer 24

Yes, flame between streaks.

Question 25

What are the incubating conditions for the BA, Choc and MAC plates.

Answer 25

Incubate the BA and Choc in CO2 at 35º C and the MAC in the 35º C incubator.

Question 26

How do you enumerate the gram stain smear?

Answer 26

Evaluate the smear with either 10X or oil and compare the count to the table provided. The table gives a semi-quantitative value as 1+, 2+, 3+ and 4+ based on the count per field within the range given in the table.

Question 27

What is the primary purpose of culturing for sputum specimens?

Answer 27

The primary purpose of culturing is to isolate, identify, and, when appropriate, determine resistance (the antimicrobial susceptibility - AST) of organisms which are significant arising from the lower respiratory tract.

Detailed descriptions of ‘normal’ flora is not required and a waste of resources.

Question 28

With the challenge of separating normal flora from a pathogen, what is the approach to identify a LRT pathogen?

Answer 28

1. Identify and report only the predominant aerobic and facultatively anaerobic bacteria present. Predominant growth could be defined as all organisms present in 3+ amounts or greater.
2. Correlate the clinical picture with the Gram-stained smear and the growth.

Question 29

What do you try to observe when comparing the BA to the Choc plates?

Answer 29

When observing the CA, try to see if there is anything growing on it that is not growing on BA.

Question 30

What are you considering on the MAC plates?

Answer 30

Mac, as usual, is to observe and differentiate LF or NLF, and mucoid colonies.

Question 31

What are you observing for and determining from the BA plate?

Answer 31

Describe colonial morphology of suspicious pathogens:
- α (alpha) trans, dry gry, yellow-grey, small trans, dry grey β-haem, etc.
- describe the most numerous (heaviest) growth first eg. 4+ or 3+

• perform any Gram stains and spot tests necessary to ‘rule out’ possible pathogens
  e.g. wet α haem → smear, bile solubility to rule out S pneumoniae
  e.g. whop → smear, cat, coag to rule out Staph aureus
  e.g. gysm trans (CA only) → smear, BAss, Choc to rule out Haemophilus

Question 32

In general on the plates (BA, Choc and MAC) what are you looking for - these are 3 general groups to identify?

Answer 32

Organisms can be divided in 3 groups for this body site:

a. the ones that are always pathogens, no matter how much there is and what else is growing;
b. the “possible pathogens”: organisms that are considered pathogens if they are predominant (growing at 3-4+ and > than the normal flora), but if they are in small amounts or amounts < than the normal flora, they are considered part of the normal flora;
c. organisms that are usually normal flora

Question 33

What is important to remember if you think you have a potential pathogen on the plates?

Answer 33

If a potential pathogen has been identified, ensure that it is present in the Direct Gram-stained smear before reporting. E.g. If 4+ mucoid lactose fermenter isolated (potential Klebsiella pneumoniae) then the smear report should show a significant amount of GNB from the direct Gram: 4+ PMN; 3-4+ GNB; 1+ GPC

Question 34

How do you report sputum specimen results if you found a pathogen?

Answer 34

1. Report Gram-stained Direct smear.
2. Name of Pathogen (if present, with amount) and AST (if applicable) and the presence of normal respiratory flora and its quantity.

Positive:
* Report preliminary results. i.e. “4+ Streptococcus pneumoniae isolated. FRTF”
Final report include AST results.

Question 35

What do you report if the gram stain sputum smear is unsuitable for culture?

Answer 35

Report as:
“Smear contains ≥ squamous epithelial cells/ LPF, suggestive of poor quality; culture not performed. Please recollect if clinically indicated.”

Question 36

What do you report if found no pathogen or you get no growth on the plates?

Answer 36

Negative:
* If no growth report: “No growth in 48h”
* If only oropharyngeal microbiota, report: “Normal respiratory flora”

Question 37

What are the limitations that affect the lab results from sputum cultures (false-neg or pos, growth?)?

Answer 37

• Some agents do not grow in routine culture but can be a significant cause of disease.
• False-negative cultures can result from contamination of the specimen with normal oral microbiota and “hiding” the pathogen.
• False-positive results can be caused by over interpretation of the culture results.

Question 38

Who should sputum specimens be obtained from?

Answer 38

Patients with pneumonia who have a productive cough and are able to expectorate (spit deeply).

Question 39

What is a major source of contamination of sputum specimens?

Answer 39

The upper respiratory tract to the level of the larynx, (approximately 200 anaerobic, facultatively anaerobic and aerobic bacterial species – in concentrations from 10^8 - 10^12 /ml) including expectorated sputum, these specimens are NOT valid for anaerobic culture.

Question 40

How does a patient on antibiotic therapy affect the ability to produce a good sputum sample?

Answer 40

Antibiotic therapy markedly reduces the ability to isolate susceptible bacteria from any specimen source

Question 41

What organisms are pathogens no matter what the specimen source is?

Answer 41

1. Mycobacterium tuberculosis, 2. Mycoplasma pneumoniae,
2. Pathogenic fungi (Blastomyces, Coccidioides, Histoplasma),
3. Most respiratory tract viruses, and
4. Legionella spp.

Question 42

If a pathogen/infection is actually present in the LRT but does not get identified in the culture what could have happened?

Answer 42

1. The specimen could have been diluted,
2. The organism is highly fastidious,
3. There is recent antibiotic exposure or
4. The patient is immunocompromised

Note: Most bacterial pathogens responsible for infections of the lower airways are present in high concentrations- > 10^6 /ml of bronchial secretions. Thus semi-quantitative cultures usually yield these organisms in “heavy”(4th quadrant) or at least “moderate”(3rd quadrant) numbers on primary isolation plates unless for the above reasons.

Question 43

What affects the choice of lab work done for a LRT?

Answer 43

The choice of procedures should be influenced by the:
1.Suspected pathogen (clinical history),
2. by prior therapy,
3. by the invasiveness of the technique used to collect the specimen and
4. the lab resources.

Question 44

What communication is important to obtain optimal results in the lab?

Answer 44

Optimal results are obtained when there is good communication between the clinician and the lab/microbiologist.

Question 45

What bacteria are associated with CF lung disease?

Answer 45

The number of microbial species associated with CF lung disease is relatively limited. They include:

1. Mucoid and nonmucoid Pseudomonas aeruginosa,
2. Stenotrophomonas maltophilia,
3. Burkholderia cepacia complex and
4. Other non-glucose fermenting gram negative rods;
5. Other organisms occurring are
   a) H. influenzae,
   b) S.aureus, and
   c) S. pneumoniae.

Question 46

What is the oxidase result, and morphology on BA, MAC and CA for B. cepacia that can be seen on CF patients?

Answer 46

B. cepacia is weakly oxidase POSITIVE.
Morphology on BA, smooth, slightly raised, dirt-like odor, NLF on MAC but might look pink because of oxidation of lactose after a few days
CA shows a green pigment (with time)

Question 47

What environment does Legionella pneumophila serogroup 1 like and what enhances their multiplication?

Answer 47

The source of these organisms is warm moist environments, both natural and artificial, and their multiplication is enhanced by the presence of free-living amoebae, in which the organisms multiply.

Question 48

Despite that culture of respiratory secretions for a Legionella infection is the most sensitive method for diagnosis, why might the bacteria not be seen in sputum samples?

Answer 48

Because they are intracellular pathogens, residing primarily in macrophages, they may not be present in sputum samples and, when present, their numbers may be low. Since many patients with legionellosis do not produce sputum, collection of multiple respiratory specimens may increase chance of isolation of organisms.

Question 49

What tests are done on patients suspected of having a Legionella infection? What is not done and why?

Answer 49

Ideally, culture + DFA (direct fluorescent antibody) or antigen in urine + serology should be all tested to get the greatest combined sensitivity.

Sputum for Legionella should not be screened for acceptance by Gram stain. These patients typically produce thin, watery sputum with few PMNs.

Question 50

What media (plates) are recommended to be used for culturing Legionella spp.?

Answer 50

• Buffered charcoal-yeast extract agar (BCYE) – contains ferric pyrophosphate, cysteine, α-ketoglutarate, and charcoal – nonselective
• BCYE with polymyxin, anisomycin, cefamandole, and α-ketoglutarate – selective - although it is useful to inhibit other microorganisms, this media may be inhibitory to some Legionella species.

The use of these two media simultaneously is recommended.

Question 51

How does Legionella look like on a gram stain?

Answer 51

Gram stains: Thin and faint gram negative bacilli (thin cell wall)

Question 52

How do process and evaluate the two types of BCYE plates?

Answer 52

BCYE (buffered charcoal-yeast-extract agar)
o 24-48 hours looks speckled green/blue colonies with pinkish/purple iridescence
o After 3-4 days colonies appear 3-4mm diameter, entire, convex with frosted glass internal appearance with a stereoscopic microscope
o After 7days colonies appear grayish with white centers and lose their iridescence
o With the use of a long-wave UV lamp, auto fluorescence can be demonstrated

Because they are L-cysteine dependent, that is the first step for ID: prove it by growing it in media with and without L-cysteine. After that: serology is needed.

Question 53

What is the DFA result for Legionella?

Answer 53

DFA (Direct Immunofluorescent Antibody): Positive
o Polyclonal antisera positive then monoclonal

Question 54

What immunoassay is useful for Legionella?

Answer 54

Enzyme immunoassays (EIA) are useful and easier than cultures