Laboratory diagnosis L7 L9 Flashcards
what are the functions of diagnostic microbiology labs
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
what is serology
Look for evidence for immune response – detect antibody response
what is surveillance for
emerging patterns of antibiotic resistance
are bacteria always reported if found
Mandatory reporting e.g C.difficile, S.aureus bloodstream infection
what types of research are used in diagnostic microbiology labs
molecular approaches to supplement or replace existing detection or identification methods to improve speed of diagnosis
molecular methods is quicker
what is a request form for
completed request form is a request for consultation or referral for a specialist opinion
what is analysed in a lab after form complete
Origin (site) of specimen determines likely pathogens
what determines the procedures done in a lab after form complete
Depending on the type of specimen determines the procedure for processing
(microscopy, culture medium, identification, antibiotic sensitivity)
what determines the urgency and priority of processing samples
clinical details
what is a common infection
gastrointestinal
what does gastrointestinal infection cause
usually get diarrhoea
examples of bacteria that cause gastrointestinal infection
Campylobacter Salmonella Escherichia coli Shigella sp. Clostridium difficile
examples of viral causes of gastrointestinal infection
Rotavirus adenovirus norovirus astro- calici-
examples of parasites that cause gastrointestinal infection
Cryptosporidium sp.
Giardia lamblia
Entamoeba histolytica
what specimen is used to study gastrointestinal infections
liquid stool
Generally when diarrhoea goes organisms tend to go
why is vomit not usually used as a sample
Stomach and back of throat in vomit
why are samples generally collected at the start of infection
organisms start to drop off when begins to clear – harder to find organism
what are details of the patients history important
need to know if travelled acute chronic symptoms is caught in hospital etc
how can stool samples be examined
Macroscopic appearance Selective Culture Microscopy PCR/ slide agglutination PCR/EIA
what is Macroscopic appearance used for in stool examination
liquid, blood-stained, ‘rice-water’ etc
what is selective culture used for in stool examination
only specific bacteria will survive
what is microscopy used for in stool sample examination
for ova, cysts and parasites
what is PCR /slide agglutination used for in stool sample examination
for viruses
what is PCR/EIA used for in stool sample examination
C. difficile and its toxins
what does stool mainly contain, what problem does this cause
contains mostly commensal bacteria – detecting the pathogen is the challenge
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
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
why is campylobacter incubated at 42 degrees
body temp of chicken, where it is found
what agar is used to grow Vibrio cholerae
Thiosulphate-citrate-bile salt-sucrose (TCBS)
what is Vibrio cholerae enriched in
alkaline peptone water
what happens to Vibrio cholerae when grown on TCBS plate
ferments sucrose and produces yellow colonies
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
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
what types of intestinal helminth ova are there
roundworm
whipworm
how are viruses usually examined in a stool sample
PCR based molecular methods
what is the old way that viruses were detected in stool samples
electron microscopy
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
what is slide agglutination
latex particles coated with rotavirus-specific monoclonal antibody)
used to detect viruses in stool samples
when are UTIs common
women and children
what causes UTIs
Cystitis or pyelonephritis
what can recurrent infections in children cause
renal damage
what are the symptoms of UTI
- dysuria (pain when go toilet)
- frequency (need to often go toilet)
- secondary enuresis (bladder leaking)
what is the most common cause of UTIs
E.coli
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)
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
what can be used to test urine
Dipstick for blood, nitrite, leukocyte esterase
put stick into urine
wait for colour changes
what is the downside of using dipstick method for urine examination
not always accurate
what does a high number of RBC indicate
haematuria
what does a high number of WBC indicate
infection
what does a high number of epithelial cells indicate
contamination
how are bacteria isolated for urine example
Semi-quantitative culture using CLED agar or Chromogenic media
antibiotic sensitivity testing
what is Sterile pyuria
presence of high numbers of WBCs
WBCs suggest infection but no bacterial growth obtained
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)
what is the problem of using antibiotic treatment before collect sample
already killed the organism
why may catheterisation cause ‘infection’
it causesinflammation not infection
why might tumour think is infection
WBC in urine
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
what are the specimens collected for wound infections
pus
tissue
body fluid
all superior to swabs
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’
how should the specimen be cultured
aerobically an anaerobically
what should be done to identify bacteria in infections
test antibiotic sensitivity coagulase Lancfield etc
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
what is URTI
upper respiratory tract infection
examples of URTIs
Otitis media
sinusitis
pharyngitis
tracheitis
how common are URTIs
Very common; often viral in aetiology
what is URTIs severity
Relatively minor, but if get into lung is very serious
what is LRTI
Lower respiratory tract infection
examples of LRTIs
Bronchitis
bronchiolitis
pneumonia
what may cause LRTIs
May be bacterial, viral or fungal in aetiology
what is the history of the patient important for in LRTIs
to determine likely pathogens
examples of community acquired pneumonia
Streptococcus pneumoniae Haemophilus influenzae Staphylococcus aureus (post ‘flu) Mycoplasma pneumoniae Legionella pneumophila
examples of hospital acquired pneumonia
Coliforms (e.g. E. coli, Klebsiella sp., Enterobacter sp.)
S. aureus (MRSA)
Pseudomonas aeruginosa
examples of respiratory tract infection specimens
throat swab sputum tracheal aspirate pernasal swab urine antigens acute and convalescent serum
where must a throat swab be from
pharynx
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
how must a tracheal aspirate be taken
taken from ventilated patients in Intensive care
what is a pernasal swab for
Designed to obtain optimum sample for culture of Bordetella pertussis (Whooping cough)
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
if cannot cough how are respiratory tract infections done
may need a more invasive method
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
how is sputum examined
Sputum microscopy
Gram stain: pus cells, organisms (historical)
Ziehl-Neelsen (ZN) stain for Mycobacteria
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)
what contaminates sputum
Sputum gets contaminated by things in the mouth when coughed up too
how are respiratory samples cultured
Cultured on selective and non-selective agar plates to maximise the chances of finding organisms
what stain is first used in TB microscopy
Auramine staining (UV microscopy) Detect with microscope
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
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
how long does it take for TB plated cultures to grow
Solid agar slopes (Lowenstein-Jenson media): 6-8 weeks to grow
how long does it take for TB liquid cultures to grow
Liquid culture using Mycobacteria Growth Indicator Tube (MGIT): 1-2 weeks or less
what is immunofluorescence
using organism-specific monoclonal antibodies linked to a fluorescent dye
When antibodies bind to bacteria it makes them fluoresce
what colour is lactose fermenting bacteria and which plate is this on
pink
MacConkey agar
what is bacteraemia
Positive blood culture (blood is normally sterile)
what can cause bacteraemia
infected organs, abscesses, catheters etc
may have transient bacteraemia from tooth brushing
what is sepsis
blood poisoning
bacteria is growing in the blood
what is the most common cause of sepsis
E.coli
what is infective endocarditis
Infection of heart valves – affects function and may be lethal
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
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
how is infective endocarditis diagnosed
So analysis of blood cultures is also used in diagnosis of infective endocarditis
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.
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
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
what is CSF
Cerebrospinal fluid
what does CSF look like
Clear, cloudy, bloody, purulent
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)
how is CSF measured
Total cell count, different cell types (e.g. neutrophils, lymphocytes), proteins, glucose – differ for bacterial and viral meningitis
what is done to CSF to make a gram film
CSF is centrifuged and the deposit stained. It may or may not be positive
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)
how is viral meningitis diagnosed
PCR of CSF, stool & throat swabs
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
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
what will the CSF be like if infected with virus
clear
increased lymphocytes
normal or more proteins
normal glucose
what causes meningitis
different bacteria depending on age
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
what still requires a bacteria culture
antibiotic sensitivity testing
what will PCR detect even if dead
PCR will detect bacterial DNA even if the bacteria have already been killed by antibiotic treatment
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
how is Maldi-Tof identified
Each organism has a unique pattern made in mass spectrometer
compare against a database
