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Systems: Resp. WK > 24. Microbiological Diagnosis of Chest Infection > Flashcards

24. Microbiological Diagnosis of Chest Infection Flashcards

1
Q

What are 4 of the most common diagnostic techniques used in diagnostic medicine of chest infections?

A
  1. microscopy and culture of sputum and blood
  2. antigen detection methods
  3. nucleic acid amplification (PCR)
  4. serology (antibody measurement)
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2
Q

What staining method is used for microscopy and culture of sputum?

A

Gram stain which tests for organisms and pus cells

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3
Q

What are 3 major respiratory pathogens which undergo overnight incubation for microscopy and culture of sputum in chest infections?

A
  1. strep pneumoniae
  2. haemophilus influenzae
  3. moraxella cararrhalis
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4
Q

What pathogen is found in the normal upper respiratory flora and is not usually significant?

A

Viridans strep ( nasopharyngeal contamination)

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5
Q

What therapy selects out organisms that are NOT respiratory pathogens?

A

Previous antibiotic therapy (selects out organisms e.g. E.coli or Staph aureus)

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6
Q

While interpreting sputum and culture results, how should the sample be interpreted in comparison to patient’s situation?

A

Interpret in light of clinical presentation

E.g. community pneumonia or aspiration pneumonia or ventilation patient? (selects out specific organisms)

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7
Q

What 2 stains can be done to diagnose TB (mycobacteria)?

A
  1. ZN stain

2. Auramine phenol stain

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8
Q

What type of mycobacteria is TB?

A

AAFB (acid and alcohol fast bacilli)

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9
Q

How long can it take for full identification of mycobacteria TB?

A

Several weeks for extended culture once the mycobacteria has grown

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10
Q

What can give a faster result for diagnosis of mycobacteria TB rather than Zn stain or Auramine phenol stain?

A

specialist molecular tests

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11
Q

What diagnostic method is useful for diagnosis of ventilator associated pneumonia patients (VAP)- patients on breathing machines?

A

BAL: Bronchio-alveolar lavage (fluid squirted into bronchi and sample collected at bronchoscopy)

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12
Q

What is the main advantage of BAL (bronchio-alveolar lavage)?

A

less liable to contamination and provides a more accurate diagnosis of LRTIs (more representative of LRTIs)

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13
Q

How is BAL (bronchio-alveolar lavage) sample collected in ventilated patients?

A

catheter aspirate collected from endotracheal tube

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14
Q

What form of culture is used for BAL ( bronchio-alveolar lavage)?

A
  • Quantitative culture (method of counting how many microbes in a particular sample)
  • colony forming units/ ml used
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15
Q

When are blood cultures taken from patients with chest infections?

A

When a patient presents with severe sepsis (when body’s own response to infection damages its own tissues and organs)

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16
Q

What blood condition do any patients with pneumonia develop?

A

Bacteraemia ( viable bacteria found in blood)

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17
Q

Why can’t microscopy be always used for blood cultures?

A

Too few organisms present in blood to see on microscopy

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18
Q

What do the 2 bottles where the blood is inoculated contain?

A

Culture media (and are incubated)

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19
Q

What 2 types of blood culture bottle are there?

A
  • aerobic

- anaerobic

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20
Q

How long does it approximately take for bacteria to be flagged up in a blood culture diagnosis?

A

Within 48 hours any significant bacteria should indicate a positive test

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21
Q

What happens on day 1 of obtaining blood culture results? (3)

A
  • microscopy positive in 6-48 hours (Gram stain)
  • results phoned to clinician and recommendations on antibiotics established
  • overnight subculture of sensitivity and identification tests
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22
Q

What happens on day 2 of obtaining blood culture results?

A
  • full identification and sensitivity tests obtained

- clinical significance of these results re-assessed

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23
Q

What are the 4 main organisms which are NOT easily cultured?

A
  1. Legionella pneumophila
  2. Mycoplasma pneumoniae
  3. Chlamydia psittaci (birds)
  4. Coxiella burnetti (query fever)
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24
Q

What is antigen detection?

A
  • non-cultural method of demonstrating the presence of an organism
  • detects specific antigen immunologically
  • every organisms has antigens so very useful method
25
Q

Which two bacterial antigens can be detected in urine and viruses in naso-pharyngeal secretions?

A
  1. Legionella antigen

2. Pneumococcal antigen

26
Q

What test uses the patient’s antigen-antibody immune response to detect a pathogen?

A

Latex agglutination

27
Q

Describe what happens in a latex agglutination.

A
  • microbeads of latex are coated wtih monoclonal pathogen-specific antibodies (or antigens) which are specific for the organism in question
  • microbeads of latex are mixed with the clinical specimen; CSF, urine or serum
  • agglutination (clumping) visible by naked eye if organism present
28
Q

If the test supplied an antigen, what does clumping/ agglutination indicate? (positive test)

A

that the patient’s body has produced a pathogen-specific antibody for that antigen

29
Q

If the test supplied an antibody, what does clumping/ agglutination indicate? (positive test)

A

that the specimen contains the pathogen’s antigen (organism present)

30
Q

What is another methods for detecting specific antigens by using antibodies?

A

EIA/ELISA (same thing)

31
Q

What does EIA stand for?

A

Enzyme Immuno-Assay

32
Q

What does ELISA stand for?

A

Enzyme Linked Immunosorbent Assay

33
Q

What generates colour change in EIA/ELISA?

A

linked enzyme on 2nd antibody (2 levels of antibody)

34
Q

Describe the steps for EIA/ELISA.

A
  1. Monoclonal anti-Legionella antibody (product of single clone of plasma cells) coated on a well of microtitre plate
  2. Antibodies are incubated with patient’s urine and antigen binds if present to the antibodies
  3. Another layer of antibodies is added with enzyme linked monoclonal anti-Legionella
  4. Enzyme substrate is added which causes a COLOUR change which detects specific pathogen (because of its antigens)
35
Q

What diagnostic method is used for DNA detection?

A

PCR: Polymerase Chain Reaction

36
Q

Describe the process of PCR in DNA detection.

A
  1. Specific primers (short oligonucleotide sequences) made
  2. Specific primers bind to terget DNA sequence
  3. Multiple copies of target sequence produced
  4. Amplified copies of DNA easily detected
37
Q

Approximate how many nucleotides are present in the primer used in PCR?

A

approx. 20 nucleotides (very small chain)

38
Q

Is PCR method used for DNA detection a sensitive test?

A

Yes

39
Q

What can cause false positives in a PCR test?

A

contamination

40
Q

What substance helps with amplification of DNA in PCR?

A

specific enzymes which help “cut” specific DNA strands (once temperature reaches 70 degrees)

41
Q

What is a technical variation test of PCR?

A

NAAT; nucleic acid amplification test

42
Q

What does “real time” PCR enable as the reaction progresses?

A

Enables product of the reaction to be measured

43
Q

What sampling technique is used for respiratory diagnosis by PCR?

A

Throat swabs or other respiratory sample

44
Q

What type of throat swab is used for PCR diagnosis?

A

Use a flocked swab (different from charcoal swab for bacterial culture)

45
Q

What does PCR routinely screen for?

A

Mycoplasma pneumoniae (and a wide range of other respiratory viruses; multiplex assays)

46
Q

What are the main respiratory viruses? (7)

A
  1. Influenza A and B
  2. Adenovirus
  3. RSV
  4. Parainfluenza viruses
  5. Metapneumovirus
  6. Coronavirus
  7. Rhinovirus
47
Q

What is a serological diagnosis? (antibody detection)

A
  • measures host antibody response to organism
  • detects if patients have been exposed to infection before
  • indirect wat to detect infection which would be hard to diagnose otherwise
  • usually difficult to culture or detect the organism directly
48
Q

What is serological diagnosis now mostly replaced by?

A

PCR, but it was previously used for viral infections and atypical causes of pneumonia

49
Q

What marker indicates a CURRENT infection? ( infection ongoing NOW)

A

IgM

50
Q

What marker indicates a PREVIOUS infection? (infection in the PAST)

A

IgG

51
Q

If a total antibody number is measured, what needs to be shown?

A

You need to demonstrate a significant increase in levels of antibodies during the course of the illness ( “rising titre”)

52
Q

What is antibody measurement now mostly used to determine?

A

Immunity e.g. vaccine response

53
Q

What are the 4 main bacterial infections which are difficult to diagnose?

A

1, Legionella pneumophila (urinary antigen and sputum culture)

  1. Chlamydia psittaci
  2. C. pneumoniae
  3. Coxiella burnetti (Q fever)
54
Q

Is interpretation of sputum culture easy or difficult?

A

can be very difficult

55
Q

When is a blood culture used in diagnosis of pneumonia?

A

only in severe disease

56
Q

What 2 antigens are tested in urine?

A
  1. legionella antigen

2. pneumococcal antigen

57
Q

PCR is increasingly used for which 2 viruses?

A
  1. mycoplasma viruses

2. respiratory viruses

58
Q

What 2 things are measured in a serological diagnosis?

A
  1. rising titre

2. IgM and IgG

59
Q

In summary what are the main diagnostic methods in diagnosing pneumonia? (5)

A
  1. microscopy and culture of sputum or BAL
  2. Blood culture
  3. Legionella/pneumococcal antigen testing in urine
  4. PCR for resp.+ mycoplasma viruses
  5. serological diagnosis

Systems: Resp. WK (40 decks)

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