Lecture 2 diagnostic clinical microbiology Flashcards
State the 9 step diagnostic template
1- request 2- sample collection 3- transport samples 4- reception of samples 5- safety issues 6- non culture methods 7-culture methods 8- ID and sensitivity 9- result to clinician
Name 5 sterile sites
Blood/bone marrow CSF Tissue Bladder Lower respiratory tract
Name 5 non sterile sites
Upper respiratory tract - c. Albicans, streptococci
Skin - s. Epidermidis
GI tract - coliforms, anaerobes, faecal flora
Vagina - lactobacilli, anaerobes
Urethra tip - skin and faecal flora
Name the main types of specimens received in an NHS micro lab
Mid stream urine Blood Urethral swab Faeces Toe nail clippings Sputum
What can toe nail clippings be used to identify
Fungal and dermatophyte (cause ringworm) infection
What can urethral swabs be used to identify
STI
What can faeces be used to identify
Diagnose for enteropathogenic bacteria,, campylobacter or parasites
What is the most common sample received
Mid stream urine
Why must specimen be collected before antibiotics are given
May killbacteria and give a false negative.
Do fastidious organisms need to be transported to the lab quickly
Nutrient demanding otherwise will die in transport
What is important to consider when viewing sputum samples
Sputum must travel through upper respiratory tract which isn’t sterile so must know normal flora
What is Stuart’s media used for
Bacteria swab transportation
What does Stuart’s medium contain and why
Nutrient gel
Charcoal to inactivate toxic byproducts from metabolism of nutrient gel.
On what circumstances will a swab need to be refrigerated during transportation?
If there is a delay but no more than 24 hrs
What is the transport media used to transport viral swabs
Viral transport media
What does viral transport media contain
Buffered salt solution containing serum and contain antimicorbials to control overgrowth to prevent contaminating bacteria and fungi
What differs chlamydial transport media from VTM
No antimicrobials which are toxic to chlamydia
What transport media is used for parasitic transportation
Merthiolate-iodine-formalin preserves ova and cysts but kills bacteria. Not suitable for protozoal trophozites
What are the main roles of reception
Check specimen and form details filled in properly
Allocate unique lab number
Macroscopic appearance - discard unsuitable ones (DIARRHOEA get solid stool
What are the key safety issues to consider with patient samples
Patient may have HIV or hep b
Category of organism 1,2,3,4.
How do lafu keep lab staff safe
Negative pressure
HEPA FILTER
What are the ACDP categorisation of biological agents
Cat 1 - saphrophytic
Cat 2- biological agent cause disease hazardous in lab but not likely to spread in community, effective treatment (s. Aureus, c. Diff, n. Gonorrhoeae, streptococcus pneumoniae)
Cat 3- biological agent cause severe human disease, serious hazard to employees , risk to spread in community, effective treatment
(Mycobacterium tuberculosis )
Cat 4- severe human disease and serious hazard to emploYees. Likely to spread in the community with no effective treatment.
(Ebola)
Name 4 non culture techniques
Direct microscopy
antigen detection
NAAT (molecular micro)
Serological response
What is the gold standard for diagnosis
Culture on solid agar
What is beneficial about antigen detection
Bacteria doesn’t need to be alive to detect antigen
Which common STI causing bacterium is commonly identified using serological response testing
Treponema pallidum cause syphilis Look for IgM and IgG response.
What are nucleic acid amplification tests for
Useful for slow growing or difficult microrogansims (hep b/c, HIV, mycobacteria)
Also for MRSA screening
Why are NAAT methods better than serological
Greater sensitivity
Name some examples of NAAT
Polymerase chain receptor (PCR)
Ligase chain reaction (LCR)
Nucleic acid sequence-based amplification (NASBA)
Strand displacement amplification (SDA)
What does ligase chain reaction involve
Hybridisation of two oligonucleotide probes at adjacent positions on a strand of target DNA which are then joined by thermostable ligase enzyme.
What does nucleic acid sequence based amplification involve
Use RNA as a target and utilise 3 enzymes simultaneously: reverse transcriptase, RNAase H and DNA-dependent RNA polymerase
What does strand displacement amplification involve
Utilise oligonucleotide primers containing a restriction enzyme site, DNA polymerase and a restriction enzyme at constant temp to produce exponential amplification of the target
When would you use a wet preparation
View parasite ova and cysts (faeces)
Name a common fluorescent stain used to detect mycobacterium tuberculosis in clinical specimens
Auramine
Name 3 organisms that can be identified using the non culture technique, antigen detection
Haemophilus influenzae
Streptococcus pneumoniae
Neisseria meningitidis
What is the benefit of antigen detection
Bacteria don’t need to be alive
What does antigen detection involve
Bacteria e.g n. Meningitidis in CSF
Latex beads have specific antibody for bacteria bound
Agglutination of carrier particles
Antigens can only be detected using co-agglutination. True or false?
FALSE.
Immunofluorescence for influenza virus or RSV respiratory sample
ELISA for chlamydia trachomatis for urogenital specimens
Enzyme immunoassay (EIA) for s.pnuemoniae for urogenital specimens/ C. difficile toxin in faeces
Name 4 different types of agar
Basic e.g nutrient
Enriched - take nutrient and add horse blood
Selective e.g contain antimicrobial
Differential e.g, MSA, s aureus yellow, s epidermidis white
What percentage of horse blood agar is horse blood
5-7%
How is chocolate agar made
Heat blood and add to agar so x and v factors are released.
H. Influenzae and n gonorrhoeae are grown on this
Name an example of selective media
CCFA (cycloserine, cefoxitin fructose agar) for C. difficile detection
Cycloserine and cefoxitin are antimicrobial and selective for c diff as kill normal faecal commensals.
Give an example of differential agar
CLED (cystine lactose electrolyte deficient agar)
Look for E. coli ferment lactose which produce acid, turn bromothymol blue to yellow.
Differentiation of enterobacteriaceae
State the 4 levels of ID
Stage 1 Basic microscopic e,g, gram stain (e,.g gram neg rod)
Stage 2 generic basic tests such as macroscopic and microscopic (e,g enterobacteriaceae/coliforms)
(Most clinical specimens) Stage 3 full - Basic ID plus additional tests e,g. API/MALDI-TOF (e.g. E.coli)
Stage 4 (for outbreaks) full plus genetic fingerprinting ( ecoli 0157)
What haemolysis does s pyogenes do on blood afar
Beta haemolytic (1mm, entire edge)
Benefit of MALDI TOFF (mass spec) over API
API takes 24 to 48 hrs
MALDI toff can take few mins
How can genetic fingerprinting/molecular typing be done
Pulsed field gel electrophoresis (PFGE)
Restriction enzymes chop up whole bacterial genome, and can compare the bands on a gel to identify strain
Which methodology is followed for sensitivity testing
EUCAST
EUROPEAN COMMITTEE ON ANTIMICROBIAL SUSCEPTIBILITY TESTING
What is sensitivity testing for
To identify sensitivity or resistance of clinical pathogens, provides national standardised sensitivity / resistance data, epidemiologically useful
What machines do labs use to perform antimicrobial sensitivity testing
VITEK 2. Strips contain antibitoics so put bacteria into the strip and put into a machine
Sensitivity patterns in 30 mins.
Can use disc susceptibility methodology using agar.
