Laboratory diagnosis L7 L9 Flashcards
(118 cards)
1
Q
what are the functions of diagnostic microbiology labs
A
Detection, isolation, identification of disease causing microorganisms
Antibiotic sensitivity testing as a guide to therapy and monitoring resistance patterns
Serological analysis to detect antibody responses associated with infection or to detect antigens in clinical specimens
Information used locally to guide treatment/regionally if there is an outbreak
2
Q
what is serology
A
Look for evidence for immune response – detect antibody response
3
Q
what is surveillance for
A
emerging patterns of antibiotic resistance
4
Q
are bacteria always reported if found
A
Mandatory reporting e.g C.difficile, S.aureus bloodstream infection
5
Q
what types of research are used in diagnostic microbiology labs
A
molecular approaches to supplement or replace existing detection or identification methods to improve speed of diagnosis
molecular methods is quicker
6
Q
what is a request form for
A
completed request form is a request for consultation or referral for a specialist opinion
7
Q
what is analysed in a lab after form complete
A
Origin (site) of specimen determines likely pathogens
8
Q
what determines the procedures done in a lab after form complete
A
Depending on the type of specimen determines the procedure for processing
(microscopy, culture medium, identification, antibiotic sensitivity)
9
Q
what determines the urgency and priority of processing samples
A
clinical details
10
Q
what is a common infection
A
gastrointestinal
11
Q
what does gastrointestinal infection cause
A
usually get diarrhoea
12
Q
examples of bacteria that cause gastrointestinal infection
A
Campylobacter Salmonella Escherichia coli Shigella sp. Clostridium difficile
13
Q
examples of viral causes of gastrointestinal infection
A
Rotavirus adenovirus norovirus astro- calici-
14
Q
examples of parasites that cause gastrointestinal infection
A
Cryptosporidium sp.
Giardia lamblia
Entamoeba histolytica
15
Q
what specimen is used to study gastrointestinal infections
A
liquid stool
Generally when diarrhoea goes organisms tend to go
16
Q
why is vomit not usually used as a sample
A
Stomach and back of throat in vomit
17
Q
why are samples generally collected at the start of infection
A
organisms start to drop off when begins to clear – harder to find organism
18
Q
what are details of the patients history important
A
need to know if travelled acute chronic symptoms is caught in hospital etc
19
Q
how can stool samples be examined
A
Macroscopic appearance Selective Culture Microscopy PCR/ slide agglutination PCR/EIA
20
Q
what is Macroscopic appearance used for in stool examination
A
liquid, blood-stained, ‘rice-water’ etc
21
Q
what is selective culture used for in stool examination
A
only specific bacteria will survive
22
Q
what is microscopy used for in stool sample examination
A
for ova, cysts and parasites
23
Q
what is PCR /slide agglutination used for in stool sample examination
A
for viruses
24
Q
what is PCR/EIA used for in stool sample examination
A
C. difficile and its toxins
25
what does stool mainly contain, what problem does this cause
contains mostly commensal bacteria – detecting the pathogen is the challenge
26
how is salmonella / shigella examined
used DCA orXLD agar
Prior enrichment using Selenite broth – may also be used on food
Colonies identified using slide agglutination and biochemical tests
27
how is campylobacter examined
Agar containing vancomycin, polymyxin B & trimethoprim. Kill all except campylobacter
incubated 42 degrees Colonies identified by oxidase test
and Gram film appearance vibrio like shaped cells
28
why is campylobacter incubated at 42 degrees
body temp of chicken, where it is found
29
what agar is used to grow Vibrio cholerae
Thiosulphate-citrate-bile salt-sucrose (TCBS)
30
what is Vibrio cholerae enriched in
alkaline peptone water
31
what happens to Vibrio cholerae when grown on TCBS plate
ferments sucrose and produces yellow colonies
32
what are the two test stages in C. difficile detection in stool sample
Stage 1 - Screening (non specific)
a) Detection of toxin genes - PCR (or nucleic acid testing NAATs)
b) Detection of glutamate dehydrogenase enzyme – Enzyme immunoassay
Stage 2 - Confirmation
Confirmation of toxin production using an enzyme immunoassay
33
how are intestinal helminth ova detected in stool sample microscopy
Microscopy to detect helminths and protozoa– stool concentrated and examined mixed with Lugols iodine or stained
34
what types of intestinal helminth ova are there
roundworm
| whipworm
35
how are viruses usually examined in a stool sample
PCR based molecular methods
36
what is the old way that viruses were detected in stool samples
electron microscopy
37
what is the downside of electron microscopy
expensive, time-consuming, insensitive only looking at a tiny amount under microscope, if there is not a lot of the virus may miss it and misdiagnose
38
what is slide agglutination
latex particles coated with rotavirus-specific monoclonal antibody)
used to detect viruses in stool samples
39
when are UTIs common
women and children
40
what causes UTIs
Cystitis or pyelonephritis
41
what can recurrent infections in children cause
renal damage
42
what are the symptoms of UTI
- dysuria (pain when go toilet)
- frequency (need to often go toilet)
- secondary enuresis (bladder leaking)
43
what is the most common cause of UTIs
E.coli
44
when are microbiological diagnosis' required
- in children (risk of renal damage)
- recurrent UTI (resistant organisms likely)
- in males (UTI much less common & often -indicates other pathology)
- if pyelonephritis is suspected (fever/loin pain)
45
what are the ways urine is sampled
Midstream urine (MSU) not the first bit of urine the bit after
‘Clean- catch’ or ‘bag’ urine young children can't always produce urine on demand
Catheter urine (CSU)
Suprapubic aspirate (SPA)
Nephrostomy / Ureteric urine
46
what can be used to test urine
Dipstick for blood, nitrite, leukocyte esterase
put stick into urine
wait for colour changes
47
what is the downside of using dipstick method for urine examination
not always accurate
48
what does a high number of RBC indicate
haematuria
49
what does a high number of WBC indicate
infection
50
what does a high number of epithelial cells indicate
contamination
51
how are bacteria isolated for urine example
Semi-quantitative culture using CLED agar or Chromogenic media
antibiotic sensitivity testing
52
what is Sterile pyuria
presence of high numbers of WBCs
| WBCs suggest infection but no bacterial growth obtained
53
what causes sterile pyuria
Antibiotic treatment prior to specimen collection
Catheterisation
Tumour
Urine infection with fastidious
Vaginal discharge / urethritis (e.g. Chlamydia) (wouldn’t detect on agar plate)
Renal tuberculosis (wont grow under normal conditions used)
54
what is the problem of using antibiotic treatment before collect sample
already killed the organism
55
why may catheterisation cause 'infection'
it causesinflammation not infection
56
why might tumour think is infection
WBC in urine
57
what is fastidious and the problem it causes
wouldn’t normally grow under media used, would need special conditions to grow it organism e.g. mycoplasma
58
what are the specimens collected for wound infections
pus
tissue
body fluid
all superior to swabs
59
how should swabs be taken
taken from as deep as practicable within the substance of the lesion. Send to the laboratory in special 'transport medium'
60
how should the specimen be cultured
aerobically an anaerobically
61
what should be done to identify bacteria in infections
test antibiotic sensitivity coagulase Lancfield etc
62
what type os agars are used to streak bacteria to identify from wounds
Use fairly rich agars (non-selective media) which will allow most bacteria to grow, so have a change to isolate bacteria
63
what is URTI
upper respiratory tract infection
64
examples of URTIs
Otitis media
sinusitis
pharyngitis
tracheitis
65
how common are URTIs
Very common; often viral in aetiology
66
what is URTIs severity
Relatively minor, but if get into lung is very serious
67
what is LRTI
Lower respiratory tract infection
68
examples of LRTIs
Bronchitis
bronchiolitis
pneumonia
69
what may cause LRTIs
May be bacterial, viral or fungal in aetiology
70
what is the history of the patient important for in LRTIs
to determine likely pathogens
71
examples of community acquired pneumonia
```
Streptococcus pneumoniae
Haemophilus influenzae
Staphylococcus aureus (post ‘flu)
Mycoplasma pneumoniae
Legionella pneumophila
```
72
examples of hospital acquired pneumonia
Coliforms (e.g. E. coli, Klebsiella sp., Enterobacter sp.)
S. aureus (MRSA)
Pseudomonas aeruginosa
73
examples of respiratory tract infection specimens
```
throat swab
sputum
tracheal aspirate
pernasal swab
urine antigens
acute and convalescent serum
```
74
where must a throat swab be from
pharynx
75
where must a sputum swab be from
NOT Saliva only from mouth not from lung)
| May be obtained after chest physiotherapy as some cant cough up well e.g. cystic fibrosis
76
how must a tracheal aspirate be taken
taken from ventilated patients in Intensive care
77
what is a pernasal swab for
Designed to obtain optimum sample for culture of Bordetella pertussis (Whooping cough)
78
what does an acute and convalescent serum detect
When first have disease then later on too see change in antibodies
Detection of pathogen-specific antibodies
it is slow
79
if cannot cough how are respiratory tract infections done
may need a more invasive method
80
what precautions must be taken when performing respiratory sample examination
All processing performed in a high level - Category 3 containment lab (due to TB risk
Samples handled in a Class 1 safety cabinet (delivers a negative pressure environment)
Gown & gloves are worn
81
how is sputum examined
Sputum microscopy
Gram stain: pus cells, organisms (historical)
Ziehl-Neelsen (ZN) stain for Mycobacteria
82
what must be added to sputum and why
```
Sputum very sticky so add something to dilute it down and remove other organisms present
Sample diluted (reduces growth of commensal oropharyngeal organisms)
```
83
what contaminates sputum
Sputum gets contaminated by things in the mouth when coughed up too
84
how are respiratory samples cultured
Cultured on selective and non-selective agar plates to maximise the chances of finding organisms
85
what stain is first used in TB microscopy
```
Auramine staining (UV microscopy)
Detect with microscope
```
86
why is a second stain required in TB microscopy
auramine stain sensitive but not very specific for mycobacteria so do the ZN stain to confirm it
ZN stain always used to confirm auramine positive samples will be red
87
how is TB culture decontaminated
using sodium hydroxide
treat sputum with NaOH will kill most bacteria
Mycobacterium Tb has a thick waxy coat so survives the NaOH
Is then plated onto special media
88
how long does it take for TB plated cultures to grow
Solid agar slopes (Lowenstein-Jenson media): 6-8 weeks to grow
89
how long does it take for TB liquid cultures to grow
Liquid culture using Mycobacteria Growth Indicator Tube (MGIT): 1-2 weeks or less
90
what is immunofluorescence
using organism-specific monoclonal antibodies linked to a fluorescent dye
When antibodies bind to bacteria it makes them fluoresce
91
what colour is lactose fermenting bacteria and which plate is this on
pink
| MacConkey agar
92
what is bacteraemia
Positive blood culture (blood is normally sterile)
93
what can cause bacteraemia
infected organs, abscesses, catheters etc
| may have transient bacteraemia from tooth brushing
94
what is sepsis
blood poisoning
| bacteria is growing in the blood
95
what is the most common cause of sepsis
E.coli
96
what is infective endocarditis
Infection of heart valves – affects function and may be lethal
97
what happens to heart valves in infective endocarditis
Heart valves are colonised by bacteria circulating
in the blood, if previous damage to the heart valve makes hem sticky and bacteria can stick, forms “vegetations” on the heart valves
98
what happens once infective endocarditis occurs
Once infection is established, bacteria or parts of the vegetation may break away from the valve and enter the bloodstream
99
how is infective endocarditis diagnosed
So analysis of blood cultures is also used in diagnosis of infective endocarditis
100
how is the specimen obtained for analysis of blood cultures
up to 10 ml venous blood from patient using aseptic techniques to not contaminate the blood/bottle; 2-3 separate sets taken at different times during the day, bacteria numbers may change during the day; should collect before starting antimicrobial therapy; transport immediately.
101
once the blood cultures are obtained what happens to the bottles
Bottle contains growth medium add the blood
Incubate bacterial bottles at 37 degrees
Detects metabolites, incubator will signal result
102
what tests are done on blood cultures to analyse them
subculture onto various agar media under different conditions
antibiotic sensitivity
gram stains
identify bacteria using e.g. AP1 test
103
what is CSF
Cerebrospinal fluid
104
what does CSF look like
Clear, cloudy, bloody, purulent
105
how is a CSF sample taken
collected in a sterile bottle under aseptic conditions
| Taken immediately to the laboratory, day or night. (Blood cultures must be taken)
106
how is CSF measured
Total cell count, different cell types (e.g. neutrophils, lymphocytes), proteins, glucose – differ for bacterial and viral meningitis
107
what is done to CSF to make a gram film
CSF is centrifuged and the deposit stained. It may or may not be positive
108
what type of cultures are CSFs grown on
eposit on various rich agars (e.g. blood and chocolate agar) and incubate under different conditions (CO2, aerobically, anaerobically)
109
how is viral meningitis diagnosed
PCR of CSF, stool & throat swabs
110
how are rarer forms of TB meningitis diagnosed
TB meningitis - Ziehl-Neelsen film and special culture methods
Cryptococcal meningitis - India ink 'wet' film to demonstrate capsule
111
what will the CSF be like if infected with bacteria
clear/turbid
many neutrophils (more WBC)
more protein
less or no glucose as transporters interfered with
112
what will the CSF be like if infected with virus
clear
increased lymphocytes
normal or more proteins
normal glucose
113
what causes meningitis
different bacteria depending on age
114
what can still affect meningitis infected people when recover
Even if recover can cause long lasting problems e.g. losing limbs due to coagulation neurological, hearing problems
Not just the initial infection that is the problem
115
what still requires a bacteria culture
antibiotic sensitivity testing
116
what will PCR detect even if dead
PCR will detect bacterial DNA even if the bacteria have already been killed by antibiotic treatment
117
how is bacteria identified in sterile sites
Sometimes although infection is suspected, causative organism cannot be grown in culture e.g joint fluids
PCR is used to amplify 16S Ribosomal RNA genes, these contain conserved regions and sequences unique to individual bacterial species
DNA sequence analysis of PCR amplified DNA allows identification of the specific bacterium causing infection
118
how is Maldi-Tof identified
Each organism has a unique pattern made in mass spectrometer
| compare against a database
