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2021-22 Clinical microbiology > W2 From patient to result > Flashcards

W2 From patient to result Flashcards

1
Q

State the diagnostic template

A
  1. Request form
  2. Sample collection
  3. Transport of samples
  4. Reception (macroscopic examination)
  5. Safety issues
  6. Non-culture techniques (indication at early stage/rapid diagnosis)
  7. Culture of clinical samples
  8. Identification and sensitivity testing
  9. Result
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2
Q

Name some sterile sites in the body

A
  • Blood/bone marrow
  • CSF
  • Tisssue
  • Lower respiratory tract
  • Bladder
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3
Q

Name some non-sterile sites

A
  • Upper respiratory tract (streptococci, anaerobes, Candida albicans)
  • Skin (coagulase negative staphylococcus eg S. epidermidis)
  • GI tract (‘coliforms’,anaerobes ‘faecal flora’)
  • Vagina (lactobacilli, anaerobes)
  • Urethra (skin and faecal flora)
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4
Q

Give examples of specimens received in clinical microbiology

A
  • Midstream Specimen of Urine (MSU)
  • Blood culture
  • Urethral swab
  • Faeces (stool)
  • Toe nail clippings
  • Sputum
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5
Q

What is the aim of the clinical microbiology laboratory?

A
  • To provide accurate information about the presence or absence of microorganisms from a clinical specimen
  • To provide antimicrobial susceptibility information on the microorganism(s) recovered
  • Accurate diagnosis and sensitivity testing:

(a) Successful treatment of infection
(b) Aids in preventing spread of infection
(c) Prevents emergence of antibiotic resistance

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6
Q

Describe sample collection: Aide-Memoire

A
  • Take appropriate specimen eg. sputum
  • Collect specimen before antibiotics are given (to avoid false negatives)
  • Avoid contamination from normal flora if possible and any contaminated equipment
  • Label specimens correctly and identify any known ‘High Risk’ eg HIV
  • Complete request form completely; sufficient clinical information
  • Transport sample to the lab as quickly as possible
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7
Q

What media is used to transport bacteria?

What does it contain?

What is it used for?

A

Stuarts transport media (STM)

Contains charcoal to inactivate any toxic bacterial bi-products. Used for swabs eg. W/S, HVS, ear, T/S (not fluids eg. sputum)

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8
Q

What media is used to transport bacteria?

What does it contain?

A

Viral transport media (VTM). Buffered salt solution containing serum. Contains antimicrobials to control overgrowth of contaminating bacteria and fungi

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9
Q

How is transport media stored?

A

Refrigeration at 4oC

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10
Q

What happens at the clinical microbiology reception?

A
  • Check specimen / form details
  • Allocation of unique laboratory number
  • Macroscopic appearance –discard unsuitable samples (cost issues; poor results)
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11
Q

Give an example of extra informtion hat may be included after reciept of the specimen

A

(a) Diarrhoea’: solid stool received
(b) ‘Chest infection’: saliva from the mouth
(c) Tissue samples received in formalin

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12
Q

What is it important to highlight on recept of the specimen?

A

Any safety issues

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13
Q

Give examples of potential safety issues

A

All clinical samples received MUST be regarded as high risk

  • Patient (eg. HIV, Hep B)
  • Potential pathogens within specimen / Advisory Committee on Dangerous Pathogens (ACDP) categorises microorganisms 1,2,3 or 4

eg. Mycobacterium tuberculosis (category 3 pathogen): cat 3 laboratory; class 1 safety cabinet

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14
Q

State the characteristics of a class I safety cabinets

A

(a) negative pressure, inward flow of air
(b) 0.74m3/sec air flow rate
(c) HEPA filter (high efficiency particle absorber)

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15
Q

Advisory Committee on Dangerous Pathogens (ACDP) categorisation of biological agents:

What is category 1?

Give examples

A

•A biological agent unlikely to cause human disease eg. saprophytic / soil organisms

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16
Q

ACDP categorisation of biological agents

What is category 2?

Give examples

A

•A biological agent that can cause human disease; hazardous to employees; unlikely to spread in the community; effective treatment/prophylaxis

eg. Staphylococcus aureus, Clostridium difficile, Streptococcus pneumoniae, Streptococcus pyogenes, Neisseria gonorrhoeae

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17
Q

ACDP categorisation of biological agents

Wha is category 3?

Give examples

A

•A biological agent that can cause severe human disease; serious hazard to employees; risk of spread in community; effective treatment / prophylaxis

eg. Mycobacterium tuberculosis

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18
Q

ACDP categorisation of biological agents

What is category 4?

Give examples

A

• A biological agent that causes severe human disease; serious hazard to employees; likely to spread in community; no effective treatment/ prophylaxis

eg. Ebola virus

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19
Q

Diagnostic Procedures

Give examples of non culture techniques

A

(A) Direct Microscopy

(B) Antigen Detection

(C) Molecular Microbiology (detection and identification of bacteria by molecular techniques eg. Nucleic acid amplification test-NAAT (PCR):

Used to detect a particular nucleic acid sequence and thus usually to detect and identify a particular species or subspecies of organism, often a virus or bacterium that acts as a pathogen in blood, tissue, urine, etc. Types of nucleic acid amplification tests

  • Polymerase chain reaction (PCR) – including nested (n), quantitative (q) or real-time reverse transcription (RT-PCR), loop mediated isothermal amplification (LAMP), and quantitative nucleic acid sequence-based amplification (QT-NASBA)

Serological Response

Serology tests in the laboratory include:

  • markers of Hepatitis B infection or past infection
  • Human Immunodeficiency Virus (HIV) status
  • Hepatitis C virus
  • Rubella immune status
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20
Q

Diagnostic procedures

State a culture technique

A

Solid agar

21
Q

Diagnostic Procedures: Non culture techniques

Direct microscopy

A

•Direct microscopy of clinical sample

(eg. light, phase contrast, fluorescence, EM)

•Light Microscopy widely used

(a) Wet preparations
eg. parasite ova and cysts (faeces)
(b) Stained preparations
eg. Gram stain: most bacteria and yeasts

•May be diagnostic eg. Gram-negative diplococci (Neisseria meningitidis) in CSF

22
Q

Diagnostic Procedures
Non-Culture Techniques: Antigen Detection

A
  1. Latex beads have specific antibodies attached that are specific to certain antigens
  2. Mix with CSF sample
  3. Agglutination =positive result

Useful even if the bacteria has been killed by antibiotics

23
Q

Culture of Microorganisms

What are the types of soilid agar?

A

(a) Basic eg. Nutrient agar (general purpose)
(b) Enriched agar (enriched general purpose)
(c) Selective agar (selects specific organisms from a sample)
(d) Differential agar (differentiates organisms within a sample)

24
Q

What is enriched media?

A

A nutritious general purpose medium

25
Q

Give examples of enriched media

A
  • Horse blood agar
  • Chocolate agar
26
Q

What is horse blood agar and what is it useful for?

A

•Horse blood agar contains 5-7% horse blood – useful for demonstrating haemolysis of organisms such as streptococci

27
Q

Enriched media: What is chocolate agar?

A

Chocolate agar – made by heating the blood before it is added to the agar releases nutrients (x and v factors) from the red blood cells. This rich medium is useful for growing fastidious organisms such as Haemophilus influenzae and Neisseria gonorrhoeae

28
Q

What is selective media?

A

A medium to which a selective compound has been added

29
Q

Give examples of selective media

A

CCFA agar (Clostridium difficile)

30
Q

What are the characteristics of CCFA agar?

A

Grey (‘fried egg’) colonies of Clostridium difficile on CCFA agar (cycloserine, cefoxitin fructose agar)

•Cycloserine and Cefoxitin are both antimicrobials and ‘selective’ for C. difficile:

They inhibit the growth of normal commensals (sensitive) in faeces but allow growth of C. difficile (resistant)

•Many different types of selective agar utilised in microbiology; choice of selective agar will be influenced by a knowledge of the potential normal flora in a clinical sample

31
Q

What is differential media?

A

A medium to which an indicator eg.dye has been added

32
Q

Give examples of differential media

A
  • Bromothymol blue
  • Cystine Lactose Electrolyte Deficient agar (CLED)
33
Q

What is Cystine Lactose Electrolyte Deficient agar (CLED)?

A

used for the differentiation and enumeration of Enterobacteriaceae ‘coliforms’ (enteric Gram-negative rods) in urine/

34
Q

What is the differntial media bromothymol blue used for?

A

Bromothymol blue is a pH indicator

Bromothymol blue has a blue color when in basic conditions (pH over 7), a green color in neutral conditions (pH of 7), and a yellow color in acidic conditions (pH under 7)

35
Q

Level of identification of isolated bacterium depends upon:

A
  • Sample type
  • Clinical situation
  • Demand of clinician
36
Q

ID: BASIC → FULL (example: Escherichia coli)

37
Q

What is BASIC IDENTIFICATION (stages 1 and 2)?

A

macroscopic appearance

The appearance of bacterial colonies on agar plates can help in identification

38
Q

Give examples of basic identifiaction (stages 1 and 2)

A

a) Colonies of Staphylococcus aureus are golden on blood agar (1-2mm, entire edge, ‘buttery smell)
b) Colonies of Streptococcus pyogenes are beta- haemolytic on blood agar (1mm, entire edge)

39
Q

BASIC IDENTIFICATION
microscopic appearance

40
Q

Full ID (stage 3): Biochemical tests

A

Eg. API (Analytical Profile Index)

Commercially produced kits of 20 or more miniaturised biochemical tests are available. The results of the set of tests generate a ‘numerical profile’ which identifies the bacterium

API available for most organisms eg. enterobacteria, staph, strep, neisseria, haemophilus

NB. Many labs now use MALDI-TOF Mass Spectrometry for microbial identification

41
Q

Identification (Stage 4): Typing of Microorganis

Why are they typed?

A

•To determine genetic similarities between strains of a particular microorganism –useful in outbreak situations ie. the same microorganism is being spread from person to person

42
Q

Give an example of Molecular Typing / Genetic Fingerprinting

A

•Pulsed field gel electrophoresis (PFGE) based on similarities between the whole genome of different strains; whole genome sequencing

43
Q

What is EUCAST?

A

European Committee on Antimicrobial Susceptibility Testing

44
Q

What is the purpose of sensitivity testing?

A

Determine SENSITIVITY / RESISTANCE of clinical pathogens

45
Q

What is the European Standardised Method?

A

For determining sensitivity to antibiotics; provides national standardised sensitivity / resistance data; epidemiologically useful

46
Q

DISC SUSCEPTIBILTY method

47
Q

What do many labs use for antimicrobial sensitivity testing?

48
Q

What are the main roles of clinical microbiology?

A
  • Biological confirmation of a clinical diagnosis depends upon the collection of high quality specimens with detailed supporting information
  • Rapid and appropriate transport is essential
  • Non-culture and culture techniques may be employed by the laboratory to make the diagnosis
  • Level of bacterial identification: BASIC – FULL (situation dependant)
  • Sensitivity testing should be undertaken using a standardised method eg. EUCAST
  • Many labs now use semi automated technology for bacterial identification and sensitivity testing eg. MALDI –TOF (MS) and VITEK 2.

2021-22 Clinical microbiology (10 decks)

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