Celine - Diagnostic Microbiology Flashcards
1
Q
What are the three phases of microbiology testing
A
Pre-analytical
Analytical
Post-analytical
2
Q
What are the three stages to the pre-analytical phase of micro
A
Patient assessment and test ordering
Specimen Collection
Specimen Transport
3
Q
What are the three stages to the analytical phase of micro
A
Specimen evaluation
Specimen processing
Validation of results
4
Q
What are the three stages to the post-analytical phase of micro
A
Reporting
Interpretation
Diagnosis and Treatment
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
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
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
8
Q
Why might history of travel be important
A
May indicate exotic/unusual organisms particularly parasites
9
Q
Why might patient occupation be important?
A
May suggest exposure e.g. brucellosis in vets
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
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
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)
13
Q
What are rigors/rigor mortis
A
Unexpected feeling of cold with shivering with a rise in body temperature
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
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
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
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
18
Q
What is amies + charcoal media used for?
A
Increased recovery of fastidious organisms e.g. Bordatella pertusis
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
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
21
Q
What has been the newest development in swabs?
A
ESwab
22
Q
What is the ESwab?
A
A Nylon Flocked Swab in Liquid Based Swab collection and transport systems
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
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
25
What is the role of ESwabs in automation?
Sample inoculation and streaking can be done using the WASP robotic device
26
Write about the importance of specimen transport
(3)
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
27
What happens on day 1 of the lab
(2)
Microscopic visualisation of organism and or host cells in clinical specimen where appropriate
Isolation - culture of organism from clinical sample
28
What happens on day 2 of the lab?
Identification of the isolated organism
Determine antibiotic susceptibility
29
How is sputum or CSF investigated microscopically
Stained preparation
Sputum - gram stain
How many bacterial cells can you see
30
How is Urine or CSF microscopically investigated
Unstained prep - count host cells e.g. WCC and look for bacterial cells
31
How is faeces examined microscopically
Stain for parasites
But no microscope investigation for bacteria
32
What specimens are unsuitable for stained microscopic investigation?
Urine and faeces
33
Give some examples of samples which would undergo stained microscopic investigation
Sputum
CSF
Urethral swab
Joint fluid
34
How do you carry out a stained microscopic investigation?
(4)
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
35
How do you rank the amount of bacterial cells?
+
++
+++
36
What would gram positive diplococci in a sputum sample indicate
Streptococcus pneumoniae
37
What would a gram negative intracellular diplococci in a urethral specimen indicate
Neisseria gonorrhoea
38
What would a gram negative intracellular diplococci in a urethral specimen indicate
Neisseria gonorrhoea
39
What are some applications of the gram stain
(2)
Provides rapid, invaluable therapeutic guide
May be invaluable in culture-negative samples
40
Gram stains of specimens provide rapid, invaluable therapeutic guide, comment on this
(4)
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
41
Gram stains of specimens may be invaluable in culture-negative samples, comment on this
(4)
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
42
What are three limitations of the gram stain?
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
43
Give an example of another staining technique for clinical samples
Acid-fast stain such as Ziehl Neelson
44
What is the Ziehl Neelson stain?
(3)
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
45
What is another stain used for Mycobacterium tuberculosis other than Ziehl-Neelson?
Auromine stain - fluorescent stain - more sensitive
46
How does mycobacterium appear on a Ziehl Neelson stain?
Pink, often beaded and slightly curved
47
How does Mycobacterium appear on an Auromine stain
Fluorescent
48
What specimens are microscopically examined as unstained preparations
Urines and CSFs
49
How are Urine and CSF samples examined microscopically
(2)
Specimens added to disposable counting chamber
Perform a WCC, RCC and observe presence of bacteria/yeasts
50
What is required for culture and isolation?
Requires that target pathogens be provided with their nutritional and environmental growth requirements
51
What are three examples of nutritional requirements?
Energy source
Nutrients
Growth factors
52
What are two examples of environmental requirements?
Temperature
Atmospheric Conditions - O2/CO2
53
What does success of culture depend on?
Selection of appropriate culture media
54
What do strategies to isolate pathogenic bacteria largely depend on?
Growth characteristics of suspected pathogens
Nature of the clinical sample (sterile vs contaminated)
55
What are the three types of conventional culture media?
General purpose
Enriched
Selective/differential
56
What does general purpose media do?
Supports the growth of many different species
57
What does enriched media do?
(3)
Fortified with lysed blood, yeast extracts etc
Useful in growing fastidious organisms
+- selective agents added to promote recovery from contaminated sites
58
What does selective/differential media do?
Promote the growth of pathogen whilst suppressing the growth of commensals and differentiate suspect pathogen by colonial morphology
59
What is chocolate agar and when is it used
Red cells heated to release nutrients
Supports growth of fastidious pathogens e.g. H. influenzae or N. meningitidis
60
What is NYC agar and when is it used?
Lysed blood + yeast extract + antibiotic
Supports growth of N. gonorrhoea from contaminated urethral site
61
What is Xylose lysine deoxycholate agar and when is it used
A selective and differential agar
Used in recovery of Salmonella from faeces
62
What is MSA agar and when is it used
Mannitol Salt agar
Used in the recovery of S. aureus from wound or from sputum in CF
63
What is chromogenic agar?
Selective and differential agar
64
What are chromogenic agar and what are they used for
Selective and differential agar used extensively in all micro labs for selection and preliminary identification of pathogens from clinical specimens or screening swabs
65
Chromogenic agar can be used for what screening swabs?
Antibiotic resistant HCAI pathogens e.g.
MRSA
VRE
ESBL
CRE
66
What are the selective agents of chromogenic agar
Antibiotics
67
What are the differential agents in chromogenic agar
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
Give two Chromogenic medias for samples
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
Give two chromogenic agars for AST
Oxoid chromogenic MRSA agar (MRSA = denim blue colonies)
Oxoid chromogenic VRE agar (VRE = pink colonies)
70
What atmospheric conditions do most organisms grow in?
Incubation in air overnight at 35 degrees Celsius
71
What incubation is required for H. influenzae and N. gonorrhoea?
24-48 hours in 5-10% CO2 for growth
72
Comment on the incubation requirements for obligate anaerobes such as Bacteroides fragilis
These only grow in absence of O2
They are killed by small amounts of oxygen
73
What is the point of basic characterisation tests?
Narrow down identification
To distinguish bacterial families/genera not species level
74
What should you not about the colonial morphology?
Size
Wet/dry
Colour
Odour
Haem
75
What is the point of a gram stain
Reaction and microscopic arrangement such as a coccus, rod, coccobacili or diplococcus may be very useful in preliminary recognition of pathogen
76
What is the point in a catalase test
Key in differentiating gram-positive cocci
77
What is the point in an oxidase test
Key in differentiating gram-negative bacteria
Enterobacterales are oxidase negative
Pseudomonas is oxidase-positive
78
How do confirmatory tests work
(4)
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
What are the two types of agglutination tests
Side agglutination tests
Latex agglutination test
80
What is a side agglutination test?
Observe agglutination (clumping) of suspension of suspect bacterial colonies when mixed with specific antibody on a slide e.g. Salmonella and Shigella species
81
What is a latex agglutination test?
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
How does detection of biochemical properties work
Detection of bacterial biochemical properties can be used in the differential identification of the organism
83
The biochemical test works be identifying what?
(2)
Single biochemical reaction (enzyme) specific to the suspect pathogen - species-specific reaction
Differential biochemical profile from a range of reactions
84
What are the two ways of detecting biochemical properties?
Species-specific reaction for identification
Differential biochemical profile
85
How does species-specific reaction for identification?
Coagulase-tube coagulase test-used to identify Staphylococcus aureus
Butyrate esterase-used to identify Moraxella catarrhalis
86
Write about the differential biochemical profile
(4)
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
Write about the MALDI-TOF
(6)
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
What is the point of AST
(5)
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
What is qualitative AST-Disk-Diffusion?
Test bacteria exposed to fixed antibiotic concentration
90
What is quantitative AST Micro-broth dilution
Test bacteria exposed to range of antibiotic concentrations
Can be done on the VITEK
91
What are some advantages of molecular detection
Very sensitive methods of detection
Very specific methods of detection
Viable cells not required
Rapid TAT - Day 1 result - only takes 3 hours
92
What is molecular detection considered the gold standard for?
(5)
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
How do molecular methods work?
Detect a short sequence of nucleotide bases (target sequence) unique to pathogen
Most common molecular methods amplification-based -> Polymerase Chain Reaction
94
How does PCR work
PCR allows target sequences of pathogen to be amplified million of times - very sensitive detection
95
How can PCR be used in the lab
(2)
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
What is end point PCR?
(7)
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
What is real-time PCR?
(6)
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
What are the three phases of PCR
Initiation phase
Exponential phase
Plateau
99
What is the Ct (cross threshold) / Cp (crossing point) value?
PCR cycle at which fluorescence from PCR product in sample crosses the threshold
100
What are four methods of direct antigen/antibody detection?
1. Latex agglutination methods
2. Immuno-chromatographic methods
3. Direct Fluorescence Antibody Technique (DFA)
4. Enzyme-linked immunosorbant assay (ELISA)
101
Give an example of latex agglutination methods
Latex side agglutination kit for detect of antigen to N. meningitidis in CSF
102
Give an example of immuno-chromatographic methods
BinaxNOW S. pneumoniae
Antigen Card detection for S. pneumoniae antigen in CSF
103
What is Direct Fluorescence Antibody Technique
(4)
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
What is an ELISA?
(3)
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
How does the ELISA work?
(4)
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
How does serology work
(4)
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
What are the two serology methods used in the lab
Immuno-chromatographic methods
ELISA
108
What are immuno-chromatographic methods
Antibody detection card
e.g. HIV antibody in blood qualitative only
109
What are ELISA test?
(3)
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
What are the advantages of conventional culture
Isolate pathogen
Identify and sensitivity
111
What are the disadvantages of conventional culture
(4)
Needs 48 to 72 hours
Laborious
Expensive
No growth for nonviable organism
112
What are the advantages of molecular diagnostics
(3)
Detect pathogen 2/3 hours
Very sensitive - can detect low pathogen numbers
Detect non-viable organisms
113
What are the limitations of molecular diagnostics?
(3)
No pathogen isolated
No susceptibility profile
Expensive equipment/kits
114
What are the advantages of serology/detecting antigens/antibodies
(2)
Detecting antigens are rapid and specific
Detecting antibodies are rapid, automated and low cost
115
What are the limitations of detection of antibodies/antigen ?
Detect antigen - not sensitive
Detect antibody - indicates infection
Ab 2/3 week response - retrospective
