Role of lab

Question 1

Considerations for “getting it right first time” in infections

Answer 1

Question 2

Sample from sterile sites are plated onto…

Answer 2

Non-selective plates. as any growth is relevant

Question 3

Mcriscopy iscarreid out on day…

Answer 3

Day 0, only in important specimens

Pus is more sensitive

Question 4

Non culture techniques and future technologies

Answer 4

Serology – For antigens eg latex agglutination – For antibodies

Molecular methods eg PCR, sequencing

Other eg MALDI-TOF MS = Matrix-assisted laser desorption/ionisation-time of flight mass spectrometry

• A colony of the microbe in question is smeared directly on the sample target and overlayed with matrix. The mass spectra generated are analysed by dedicated software and compared with stored profiles •

Some tests performed in house; others sent away to reference laboratories or commercial diagnostic laboratories

Question 5

Question 6

Define MIC:

Answer 6

lowest concentration of an agent that inhibits growth (prevents development of visible growth) after overnight inoculation

Question 7

Define MBC

Answer 7

lowest concentration of an agent which is able to kill the bacterial strain (judged by a decrease in original inoculum by a factor of 1000)

Question 8

Even though molecular methods for antibiotic resistance show genotype for resistance, it may not be expressed in the phenotype

Question 9

How do Disc suceptibility tests work

Answer 9

• Inoculate test organism evenly across agar
• Apply antibiotic filter paper discs
• Incubate overnight
  
  • antibiotics diffuse through the agar
  • concentration of antibiotic deacreases as the distance from disc increases
• Circular zone of growth appears
• Size of zone of inhibition indicates susceptibility of organism

Question 10

Factors which affect zone size in diffusion test

Answer 10

• Bacterial inoculum (heavy or light) and growth rate
• Medium: Composition, osmolality, pH
• Antibiotic molecul: Sizem solubility, ionic charge i.e. diffusability
• Antiobiotic formulation
• Disc content, age, condition
• Enzymes secreted by bacteria into medium

Question 11

Tests for measuring MIC

Answer 11

• quantitative measure of antimicrobial activity
• Tube MIC – series of antibiotic doubling dilutions in tubes containing liquid media
• Etest (Gradient MIC)1 – exponential gradient of antimicrobial dried on one side of plastic strip
• 15x 2-fold dilutions

Question 12

Molecular methods for antibiotic resistnace testing

Answer 12

• Application of genotypic methods to allow rapid detection of resistance genes direct from the sample/culture
  
  • mecA gene detection by PCR denotes resistance to methicillin in S. aureus
  • rpoB gene detection by PCR followed by hybridisation using a DNA probe denotes rifampicin resistance in MDR Mycobacterium tuberculosis1
  • antiviral drug resistance due to genetic point mutations can be examined by PCR for HIV, CMV & HCV
• Single- and multiplex-PCR
• Real-time PCR
• DNA sequencing
• Hybridisation-based techniques

Question 13

How and why to use serum antibiotic assays

Answer 13

• To check for compliance (Rifampicin in TB)
• To demonstrate bactericidal activity endocarditis
• To manage therapy with antibiotics that have a narrow therapeutic range
  
  • aminoglycosides, e.g. gentamicin, tobramycin, amikacin, streptomycin
  • some glycopeptides, e.g. vancomycin

Therapeutic range = [toxic conc] – [therapeutic conc]

Microbiological assays on large plates

• concentration proportional to zone
• plot calibration curve with standards

Non-microbiological assays

• enzymatic
• Immunoassay
• high pressure liquid chromatography [HPLC]