Microbiological Testing

Question 1

Describe the 6 key requirements for collecting a quality microbiology specimen

Answer 1

1. Adequately represents the diseased area for direct detection of the pathogen
   (unless blood [for antibody detection] or urine [for circulating antigen detection])
2. Sufficient quality and quantity
3. Avoid preventable contamination from the normal microbiota and environment
4. Collected in appropriate containers, kept at an appropriate temperature and forwarded promptly for testing
5. Obtained before antimicrobials have been given (where possible)
6. Sent to the laboratory with detailed clinical information, allowing the laboratory to do appropriate testing and interpretation

Question 2

Describe a typical clinical microbiology workflow

Answer 2

In a typical clinical microbiology workflow:

There is syndrome and disease based sampling

At the hospital, there may be point-of-care testing

Then the specimen is sent to the diagnostic lab where there is phenotypic identification and antibiotic susceptibility testing

If there is an unidentified or unusual bacterium then there is molecular detection & identification by sequencing the organism

And if it is a particularly new/atypical bacterium, then you do genomic sequencing

Question 3

Understand the differences between differential and selective media

Answer 3

Non-selective media is used when we want everything to grow

Selective media selects the growth of the desired organism, inhibiting the growth of undesired organisms like the flora

Differential media takes advantage of the biochemical properties of target organism, leading to a visible change when target organisms are present

Question 4

Describe different methods for identification of bacterial or fungal isolates

Answer 4

Biochemical testing relies on biochemical changes, and colour changes in the media, due to the growth of the organism (slow process)

MALDI TOF MS

Mass spectrometry and the molecules are separated based on their mass:charge ratio

Question 5

Describe different methods for antimicrobial susceptibility testing

Answer 5

Disk diffusion, which gives a categorical result (susceptible/intermediate/resistant) based on the zone of inhibition

MIC (min inhibitory concentration) testing gives a quantitative result (min conc of antibiotic needed to inhibit the growth of the microorganism)
Broth or agar dilutions

Question 6

With respect to diagnosis of pneumonia, describe:

Appropriate specimen types

Answer 6

Appropriate specimen types:
1) Lung Aspirate (best quality) High specificity; no contamination from upper airway flora… but rarely done in practice

2) Sputum (specimen from the lower respiratory tract)
But can be contaminated by upper respiratory flora, nasopharynx & mouth
Is the sample actually sputum or saliva
Good quality samples have lots of neutrophils & inflammatory cells, can see some gram + cocci (suggests that the sample is from the lower respiratory tract, not saliva)
Poor quality samples mean lots of squamous epithelial cells

3) Nasopharyngeal swab (many bacteria & viruses infect the upper respiratory tract first, before they cause disease in the lungs and the lower respiratory tract)

blood, urine, pleural fluid is useful in some cases

Question 7

With respect to diagnosis of pneumonia, describe:

Examples of antigen, antibody and nucleic acid detection

Answer 7

Can do microscopy & culture of the respiratory tract secretions

1) gram stain, specialised staining
2) routine cultures (standard agar plates), but special media for fastidious bacteria (bacteria that has complex or particular nutritional requirements)

Can detect the antigen in urinary & respiratory samples:

RAT tests:-
S. pneumonia:
A big polysaccharide capsule that is shed, comes out into the bloodstream, is filtered through the glomerulus and can be detected in the urine
But positive in children who carry S. pneumonia in their nasopharynx

Can go through nucleic acid (PCR) detection on respiratory samples
Is sensitive - detects very low levels of NA
More rapid than culture
Less affected by antibiotic therapy
Can detect viruses as well (DNA & RNA)
Multiplex PCR is useful in syndromic panel

Blood culture (only seldom positive)

Serology (blood tests looking for antibodies in the blood)
this signifies whether the patient has recently seen bacteria
Need acute & convalescent serum (4 weeks after)
If there is a 4-fold rise in antibody titre, implies recent infection

Question 8

Understand and calculate sensitivity, specificity, positive and negative predictive values for diagnostic tests

Answer 8

Sensitivity is the proportion of those who have the disease, who are correctly identified
(TP/ TP + FN)

Specificity is the proportion of those without the disease who are correctly identified by the test
(TN/ TN + FP)

The Positive Predictive Value is the proportion of those with a positive test, who have the disease
(TP/ TP + FP)

The Negative Predictive Value is the proportion of those with a negative test, who don’t have the disease
(TN / TN + FN)

Question 9

Understand how prevalence of a condition impacts on the predictive value of a test result

Answer 9

With decreasing prevalence of a disease, the PPV also decreases

NPV increases as prevalence decreases

Question 10

80% sensitivity means

Answer 10

80% TP, and 20% FN

Question 11

90% specificity means

Answer 11

90% TN

10% FP

Question 12

PPV of 89 % means

Answer 12

89% are TP

11% are FP

Question 13

NPV is 82%, meaning

Answer 13

82% are TN

18% are FN