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2.2 Microbiology > Celine - Diagnostic Microbiology > Flashcards

Celine - Diagnostic Microbiology Flashcards

1
Q

What are the three phases of microbiology testing

A

Pre-analytical
Analytical
Post-analytical

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2
Q

What are the three stages to the pre-analytical phase of micro

A

Patient assessment and test ordering
Specimen Collection
Specimen Transport

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3
Q

What are the three stages to the analytical phase of micro

A

Specimen evaluation
Specimen processing
Validation of results

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4
Q

What are the three stages to the post-analytical phase of micro

A

Reporting
Interpretation
Diagnosis and Treatment

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5
Q

What aspects might be evaluation in the patient evaluation stag of the pre-analytical phase?
(5)

A

Clinical history
Patient symptoms
Age of patient
History of travel
Patient occupation

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6
Q

Why are patient symptoms so important
(3)

A

cough may indicate respiratory tract infection
Fever may indicate bloodstream infection
Stiff neck and photophobia may indicate meningitis

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7
Q

Why is age of patient important

A

Can help predict the ID of pathogens

Pneumonia in children most likely viral but bacterial in adults

Meningitis in newborn most likely bacteria particularly Streptococcus agalactiae in babies but most likely N. meeningitidis

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8
Q

Why might history of travel be important

A

May indicate exotic/unusual organisms particularly parasites

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9
Q

Why might patient occupation be important?

A

May suggest exposure e.g. brucellosis in vets

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10
Q

What are the four things that need to be remembered when taking a clinical specimen?

A

Must be from the correct site

Must be taken at the correct time

Must be taken in the correct manner

Must be taken using the correct swab or specimen container

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11
Q

Why is it important to take specimens from the correct site?
(2)

A

Query abscess needs to be a specimen of pus not a skin swab

Query respiratory tract infection needs sputum not saliva

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12
Q

Why is it important to take a sample at the correct time

A

Perferably before the administration of antimicrobials

When pathogen number is maximal e.g. early morning urine or blood for blood culture sample before rigors (rigor mortis)

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13
Q

What are rigors/rigor mortis

A

Unexpected feeling of cold with shivering with a rise in body temperature

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14
Q

Why is it important to take samples in the correct manner

A

Specimen taken using an aseptic technique especially blood cultures as 5-30% positives are considered skin contaminants

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15
Q

Why is it important to use the correct swab or specimen container

A

Suitable sterile leak-proof container
Using correct swab with suitable transport media

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16
Q

Why is it so important to use the correct swab

A

Swab should allow optimal specimen collection, survival in transport and maximal recovery in the lab

Specialised transport medium required for collection and transport of specimens containing viruses and fastidious STI agents including chlamydiae

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17
Q

Comment on the transport system for conventional swabs today

A

Amies Transport Swab system

Cotton swab placed in Amies semi-solid transport media

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18
Q

What is amies + charcoal media used for?

A

Increased recovery of fastidious organisms e.g. Bordatella pertusis

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19
Q

What are four different types of swabs used and list what they are used for?

A

Regular single plastic applicator -> mouth, throat, vagina and wounds

Dual plastic screening swabs -> nasal, axilla, groin

Minitip narrow plastic shaft -> eye, ENT, nasopharynx, urogenital, pediatric

Flexible twisted aluminium wire -> nasopharynx

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20
Q

List the four main swabs + media used
(4)

A

Amies transwab for general purpose

Amies Transwab with aluminium wire for urethral and ear specimens

Amies Transwab + charcoal for anaerobes and fastidious organisms

Viral transport for liquid transport containing virus

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21
Q

What has been the newest development in swabs?

A

ESwab

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22
Q

What is the ESwab?

A

A Nylon Flocked Swab in Liquid Based Swab collection and transport systems

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23
Q

What are the main benefits of an ESwab?
(2)

A

Flocked bud allows for max collection, release and recovery of organism

1ml liquid Amies broth transport media improves viability of aerobic, anaerobic and fastidious bacteria for up to 48 hours

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24
Q

What does the lab do with ESwabs once taken in?

A

Inoculate culture plates using ESwab as applicator

Or vortex and pipette liquid suspension onto plates

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25
Q

What is the role of ESwabs in automation?

A

Sample inoculation and streaking can be done using the WASP robotic device

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26
Q

Write about the importance of specimen transport
(3)

A

Minimal delay to ensure the survival of fastidious organisms such as N. gonorrhoea which are very sensitive to heating or drying

Most hospitals use vacuum shoots

Specimens must be processed as soon as possible or stored appropriately to ensure survival of pathogens e.g. blood cultures must be incubated immediately and urine samples must be stored at 4 degrees celsius

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27
Q

What happens on day 1 of the lab
(2)

A

Microscopic visualisation of organism and or host cells in clinical specimen where appropriate

Isolation - culture of organism from clinical sample

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28
Q

What happens on day 2 of the lab?

A

Identification of the isolated organism

Determine antibiotic susceptibility

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29
Q

How is sputum or CSF investigated microscopically

A

Stained preparation
Sputum - gram stain
How many bacterial cells can you see

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30
Q

How is Urine or CSF microscopically investigated

A

Unstained prep - count host cells e.g. WCC and look for bacterial cells

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31
Q

How is faeces examined microscopically

A

Stain for parasites

But no microscope investigation for bacteria

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32
Q

What specimens are unsuitable for stained microscopic investigation?

A

Urine and faeces

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33
Q

Give some examples of samples which would undergo stained microscopic investigation

A

Sputum
CSF
Urethral swab
Joint fluid

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34
Q

How do you carry out a stained microscopic investigation?
(4)

A

Swabs -> rub swab on glass slide

Liquid specimen -> centrifuged to concentrate then prepare smear with swab of deposit

Gram stain carried out -> determine gram reaction, shape and formation of pathogen

Recognition of host cells -> white cells, epithelial cells

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35
Q

How do you rank the amount of bacterial cells?

A

+
++
+++

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36
Q

What would gram positive diplococci in a sputum sample indicate

A

Streptococcus pneumoniae

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37
Q

What would a gram negative intracellular diplococci in a urethral specimen indicate

A

Neisseria gonorrhoea

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38
Q

What would a gram negative intracellular diplococci in a urethral specimen indicate

A

Neisseria gonorrhoea

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39
Q

What are some applications of the gram stain
(2)

A

Provides rapid, invaluable therapeutic guide

May be invaluable in culture-negative samples

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40
Q

Gram stains of specimens provide rapid, invaluable therapeutic guide, comment on this
(4)

A

Differentiate gram-positive and gram-negative bacteria to guide empiric antibiotic treatment

Morphology can aid in preliminary pathogen identification e.g. GN intracellular diplococci in urethral pus provide a presumptive ID of N. gonorrhoeae

Presence of white cells in specimen indicate infection

Gram stain may be used as a rapid guide to initiate treatment without waiting for 24/48 hours for culture results until definitive identification is obtained

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41
Q

Gram stains of specimens may be invaluable in culture-negative samples, comment on this
(4)

A

Specimens may show organisms in gram stain but show no growth on culture plates

Fastidious organisms may be unable to grow on the culture media employed

Patient received antibiotics - damaged bacteria present and are unable to grow

In these cases the gram stain provides the only clue to the presence and identity of infecting organisms

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42
Q

What are three limitations of the gram stain?

A

Need a high number of organisms present to be seen in a gram stain

Visualisation with the gram stain requires greater than 10^4 organisms/mL

Microscopy is not a sensitive detection method

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43
Q

Give an example of another staining technique for clinical samples

A

Acid-fast stain such as Ziehl Neelson

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44
Q

What is the Ziehl Neelson stain?
(3)

A

The classic acid-fast stain

Used to stain organisms that have waxy material (mycolic acids) in their cell walls e.g. Mycobacterium tuberculosis

Acid fast staining is reserved for clinical samples from patients suspected of having mycobacterial infection

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45
Q

What is another stain used for Mycobacterium tuberculosis other than Ziehl-Neelson?

A

Auromine stain - fluorescent stain - more sensitive

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46
Q

How does mycobacterium appear on a Ziehl Neelson stain?

A

Pink, often beaded and slightly curved

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47
Q

How does Mycobacterium appear on an Auromine stain

A

Fluorescent

48
Q

What specimens are microscopically examined as unstained preparations

A

Urines and CSFs

49
Q

How are Urine and CSF samples examined microscopically
(2)

A

Specimens added to disposable counting chamber

Perform a WCC, RCC and observe presence of bacteria/yeasts

50
Q

What is required for culture and isolation?

A

Requires that target pathogens be provided with their nutritional and environmental growth requirements

51
Q

What are three examples of nutritional requirements?

A

Energy source
Nutrients
Growth factors

52
Q

What are two examples of environmental requirements?

A

Temperature
Atmospheric Conditions - O2/CO2

53
Q

What does success of culture depend on?

A

Selection of appropriate culture media

54
Q

What do strategies to isolate pathogenic bacteria largely depend on?

A

Growth characteristics of suspected pathogens
Nature of the clinical sample (sterile vs contaminated)

55
Q

What are the three types of conventional culture media?

A

General purpose
Enriched
Selective/differential

56
Q

What does general purpose media do?

A

Supports the growth of many different species

57
Q

What does enriched media do?
(3)

A

Fortified with lysed blood, yeast extracts etc

Useful in growing fastidious organisms

+- selective agents added to promote recovery from contaminated sites

58
Q

What does selective/differential media do?

A

Promote the growth of pathogen whilst suppressing the growth of commensals and differentiate suspect pathogen by colonial morphology

59
Q

What is chocolate agar and when is it used

A

Red cells heated to release nutrients

Supports growth of fastidious pathogens e.g. H. influenzae or N. meningitidis

60
Q

What is NYC agar and when is it used?

A

Lysed blood + yeast extract + antibiotic

Supports growth of N. gonorrhoea from contaminated urethral site

61
Q

What is Xylose lysine deoxycholate agar and when is it used

A

A selective and differential agar
Used in recovery of Salmonella from faeces

62
Q

What is MSA agar and when is it used

A

Mannitol Salt agar

Used in the recovery of S. aureus from wound or from sputum in CF

63
Q

What is chromogenic agar?

A

Selective and differential agar

64
Q

What are chromogenic agar and what are they used for

A

Selective and differential agar used extensively in all micro labs for selection and preliminary identification of pathogens from clinical specimens or screening swabs

65
Q

Chromogenic agar can be used for what screening swabs?

A

Antibiotic resistant HCAI pathogens e.g.
MRSA
VRE
ESBL
CRE

66
Q

What are the selective agents of chromogenic agar

A

Antibiotics

67
Q

What are the differential agents in chromogenic agar

A

Contain colourless chromogen substrates which target specific bacteria enzymes - plus chromophore

When the enzyme from the target organism cleaves chromogen, coloured chromophore is released

Target colony exhibits distinctive colour -> can do a preliminary ID from this

68
Q

Give two Chromogenic medias for samples

A

Chromogenic GIT media (E. coli = pink, salmonella enterica = teal with black centres, shigella sonnei = teal, proteus mirabilis = pale pink with tan centres)

Chromogenic UTI media

69
Q

Give two chromogenic agars for AST

A

Oxoid chromogenic MRSA agar (MRSA = denim blue colonies)

Oxoid chromogenic VRE agar (VRE = pink colonies)

70
Q

What atmospheric conditions do most organisms grow in?

A

Incubation in air overnight at 35 degrees Celsius

71
Q

What incubation is required for H. influenzae and N. gonorrhoea?

A

24-48 hours in 5-10% CO2 for growth

72
Q

Comment on the incubation requirements for obligate anaerobes such as Bacteroides fragilis

A

These only grow in absence of O2

They are killed by small amounts of oxygen

73
Q

What is the point of basic characterisation tests?

A

Narrow down identification

To distinguish bacterial families/genera not species level

74
Q

What should you not about the colonial morphology?

A

Size
Wet/dry
Colour
Odour
Haem

75
Q

What is the point of a gram stain

A

Reaction and microscopic arrangement such as a coccus, rod, coccobacili or diplococcus may be very useful in preliminary recognition of pathogen

76
Q

What is the point in a catalase test

A

Key in differentiating gram-positive cocci

77
Q

What is the point in an oxidase test

A

Key in differentiating gram-negative bacteria
Enterobacterales are oxidase negative
Pseudomonas is oxidase-positive

78
Q

How do confirmatory tests work
(4)

A

They detect antigenic determinant that is unique to the pathogen

Differential identification

Detection based on the use of an antibody that is specific for microbial antigen

Microbial antigen detection methods are rapid and specific

79
Q

What are the two types of agglutination tests

A

Side agglutination tests

Latex agglutination test

80
Q

What is a side agglutination test?

A

Observe agglutination (clumping) of suspension of suspect bacterial colonies when mixed with specific antibody on a slide e.g. Salmonella and Shigella species

81
Q

What is a latex agglutination test?

A

Latex particle coated with antibody specific for antigen-agglutination readily observed

e.g. Staphylococcus aureus (Staphaurex latex beads)
e.g. Streptococcus species (Streptex kit)

82
Q

How does detection of biochemical properties work

A

Detection of bacterial biochemical properties can be used in the differential identification of the organism

83
Q

The biochemical test works be identifying what?
(2)

A

Single biochemical reaction (enzyme) specific to the suspect pathogen - species-specific reaction

Differential biochemical profile from a range of reactions

84
Q

What are the two ways of detecting biochemical properties?

A

Species-specific reaction for identification
Differential biochemical profile

85
Q

How does species-specific reaction for identification?

A

Coagulase-tube coagulase test-used to identify Staphylococcus aureus

Butyrate esterase-used to identify Moraxella catarrhalis

86
Q

Write about the differential biochemical profile
(4)

A

Detection of a combination of enzymes generates a differential profile for species level ID

Enzymes function in bacterial metabolic pathway in the:
- fermentation of different carbohydrates
- Degradation of amino acids
- Utilisation of specific substrates

e.g. manual test - peptone water sugars (gives an ID on day 3)

Automated - Vitek - 64 substrate microwells - differnetial ID of all common pathogens - 8 hours with ID on day 2/3

87
Q

Write about the MALDI-TOF
(6)

A

Revolutionary - widely used for ID of bacteria and fungi

Analysis very short turn around time of 6 minutes/ day 2

Simple sample prep and result acquisition

Cost - 5 times cheaper than conventional ID

In contrast to manual and VITEK approach which rely on growth with subsequent biochemical control

MALDI - analyse the protein composition organism - no need to wait for growth test directly on colony

88
Q

What is the point of AST
(5)

A

The sensitivity of a pathogen to specific antibiotics serves as guide for antibiotic therapy

Accurate AST of isolated pathogens is essential function of Diagnostic Micro Lab

AST set up on Day 2 in parallel with ID test

Qualitative AST Disk-diffusion

Quantitative AST Micro-broth dilution

89
Q

What is qualitative AST-Disk-Diffusion?

A

Test bacteria exposed to fixed antibiotic concentration

90
Q

What is quantitative AST Micro-broth dilution

A

Test bacteria exposed to range of antibiotic concentrations

Can be done on the VITEK

91
Q

What are some advantages of molecular detection

A

Very sensitive methods of detection
Very specific methods of detection
Viable cells not required
Rapid TAT - Day 1 result - only takes 3 hours

92
Q

What is molecular detection considered the gold standard for?
(5)

A

Non-culturable organisms - viruses, chylamdiae
Fastidious organism - N. gonorrhoeae
Slow growing organisms - Mycobacteria
Important HCAI pathogens - MRSA, VRE
Multiple suspect pathogens - GIT, RTI - labour saving

93
Q

How do molecular methods work?

A

Detect a short sequence of nucleotide bases (target sequence) unique to pathogen

Most common molecular methods amplification-based -> Polymerase Chain Reaction

94
Q

How does PCR work

A

PCR allows target sequences of pathogen to be amplified million of times - very sensitive detection

95
Q

How can PCR be used in the lab
(2)

A

Used to confirm ID suspect microbial colonies on day 2 such as important HAI pathogens - MRSA, VRE

More commonly PCR used for direct investigation of clinical specimen on day 1 -> no need to isolate pathogen just detect target DNA from pathogen in specimen

96
Q

What is end point PCR?
(7)

A

1 full day

Non automated systems

Manual DNA/RNA extraction

Run for 40 cycles on PCR machine

Prepare agarose gel

Electrophoresis gel

Stain gel to see DNA bands

97
Q

What is real-time PCR?
(6)

A

Takes 2/3 hours on day 1

Fully automated systems

Automated/rapid DNA/RNA extraction

Run on real-time PCR platform

Combines amplification and detection with fluorescent probes

Observe amplification plot

98
Q

What are the three phases of PCR

A

Initiation phase
Exponential phase
Plateau

99
Q

What is the Ct (cross threshold) / Cp (crossing point) value?

A

PCR cycle at which fluorescence from PCR product in sample crosses the threshold

100
Q

What are four methods of direct antigen/antibody detection?

A
  1. Latex agglutination methods
  2. Immuno-chromatographic methods
  3. Direct Fluorescence Antibody Technique (DFA)
  4. Enzyme-linked immunosorbant assay (ELISA)
101
Q

Give an example of latex agglutination methods

A

Latex side agglutination kit for detect of antigen to N. meningitidis in CSF

102
Q

Give an example of immuno-chromatographic methods

A

BinaxNOW S. pneumoniae

Antigen Card detection for S. pneumoniae antigen in CSF

103
Q

What is Direct Fluorescence Antibody Technique
(4)

A

Sample smear incubated with a fluorescein-labelled AB directed against a specific microbial antigen

Labelled-antibody binds microbial antigen within the clinical specimen and emits visible fluorescence that can be detected using a fluorescence microscope

e.g. Detection of Syphilis

Smear from syphilis lesion is stained with specific Fluorescent Ab

104
Q

What is an ELISA?
(3)

A

Enzyme-linked immunosorbent assay

Used for direct identification of microbial antigens from clinical specimens

ELISA commonly used for the detection of viral (e.g. Hepatitis B) and fungal (e.g. Aspergillus) antigens in patient samples

105
Q

How does the ELISA work?
(4)

A

Antibody specific for an antigen of interest is bound to the walls of a plastic microtitre well

Patient serum is incubated in the well. Any antigen in the serum is bound by the antibody on the well walls

Enzyme-labelled antibody is added to the well and binds to the antigen

Enzyme makes coloured product from added substrate. Intensity of colour produced is proportional to the amount of bound antigen

106
Q

How does serology work
(4)

A

Detect antibodies in pt. serum directed against microbial antigen

Provides evidence infection with a pathogen

IgG antibody response may indicate a current infection

Serology used extensively in the identification of viral and fungal infection

107
Q

What are the two serology methods used in the lab

A

Immuno-chromatographic methods

ELISA

108
Q

What are immuno-chromatographic methods

A

Antibody detection card
e.g. HIV antibody in blood qualitative only

109
Q

What are ELISA test?
(3)

A

ELISA wells are coated with Ag

e.g. HIV, Hepatitis B etc. Aspergillus

Measure rise in the amount of antibody (IgG titre) over a 7-10 day period - Quantitative

110
Q

What are the advantages of conventional culture

A

Isolate pathogen
Identify and sensitivity

111
Q

What are the disadvantages of conventional culture
(4)

A

Needs 48 to 72 hours
Laborious
Expensive
No growth for nonviable organism

112
Q

What are the advantages of molecular diagnostics
(3)

A

Detect pathogen 2/3 hours
Very sensitive - can detect low pathogen numbers
Detect non-viable organisms

113
Q

What are the limitations of molecular diagnostics?
(3)

A

No pathogen isolated

No susceptibility profile

Expensive equipment/kits

114
Q

What are the advantages of serology/detecting antigens/antibodies
(2)

A

Detecting antigens are rapid and specific

Detecting antibodies are rapid, automated and low cost

115
Q

What are the limitations of detection of antibodies/antigen ?

A

Detect antigen - not sensitive
Detect antibody - indicates infection
Ab 2/3 week response - retrospective

2.2 Microbiology (60 decks)

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