Lecture 5- Intro to clinical lab Flashcards
Role of the Clinical Microbiology Laboratory?
To examine and/or culture clinical specimens for microorganisms, to make accurate species identification of significant isolates and when indicated, perform antibiotic susceptibility tests.
Importance of obtaining an accurate identification?
Overt Pathogens always require accurate speciation
ID is important for ongoing and recurrent infections
Epidemiological accuracy – references would never get updated
Significance of antibiotic susceptibility results and ID?
- Many practitioners placeéemphasis on susceptibility profile over ID
- Some microorganisms are intrinsically resistant to certain antibiotics
- i.e. cannot use Augmentin to treat Pseudomonas – ID may limit therapy options
Decribe the diagnostic cycle?
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What is the turnaround time?
- The time elapsed from the time a specimen is collected until the time a final result is issued
Why is turnaround time so important for patients and doctors?
Patient – waiting to commence treatment - alleviate symptoms and/or anxiety e.g. consider possible diagnosis of UTI vs possible gonorrhea
Doctor – confirmation of correct treatment - empirical therapy is common - confirm diagnosis or exclude infection
What happen to a specimen quility over time?
It decreases with time. The longer it takes for a specimen to reach the laboratory, the more difficult it may be to isolate or identify a pathogen
Eg.
Overgrowth with normal flora
Non-survival of anaerobes or fastidious (fussy) organisms
what are the important factors that go into proper specimen collection?
Correct & clear labeling of all containers, swabs, smears AS SOON AS COLLECTED!
Collect material from the site of infection
Sufficient quantity of sample to perform all tests, esp. urine, aspirates
Sterile containers (except for faeces)
Use of transport media for swabs?
- preserves the viability of bacteria without multiplication
- the semisolid agar provides moisture to prevent drying out
- contains a reducing agent to improve recovery of anaerobes
- maintains pH
- use special tissue culture media for requests for viral studies
- charcoal containing media for gonorrhoeae (inhibits toxic fatty acids found in cotton fibers)
What is the aim of proper specimen transport?
The aim is to transport the specimen while maintaining the sample in as close to the original state as possible
This is achieved by;
Paperwork and specimen should be shipped together
Aviod extremes of tempuratures
Use leak proof containers
What is the ideal time to recieve a specimen in?
Ideally, specimens should be received within 2 hours
When would dry swab with no media be used?
Suitable for chlamydia and gonorrhoea PCR
Not suitable for MC+S (microscopy, culture & sensitivity)
When does Rejection of Specimens occour?
- Unlabeled specimens
- Samples with illegible labeling
- Mismatched details
- Insufficient material and/or leaking specimens
- Specimens for anaerobes that have been stored at 4 ̊C
- Inappropriate specimen collected
- Non-sterile container for a urine or aspirate
- Any specimen collected into formalin
What hapens in a hospital when a specimen is rejected?
- generally it should be easier to get another sample
- depends on the site of infection and the type of specimen
e.g. CSF (traumatic and difficult) vs. wound swab (easy) - patient must still be an ‘inpatient’
-patient must still be an ‘inpatient’
What happens when a specimen is rejected in a private lab?
- much more challenging to accomplish
- contact surgery, contact patient, recollect specimen, transport to lab., match to existing paperwork
- therefore, there is more pressure to accept and process an existing sample
What happens to specimens as they enter a lab?
- Small laboratories – likely to process individual specimens as they arrive
- Large laboratories – likely to process specimens in batches
What are classified as URGENT/High priority specimens?
CSF, tissue, blood cultures, sterile fluids
What are generally classed as lower priority specimens?
urine, swabs, sputum, faeces (unless marked URGENT)
What type of specimens are usually always batched?
Fungal, viral and acid fast specimens (for TB) usually batched
What type of specimens are examined microscopically?
Virtually all specimens are examined microscopically
What are items seen in wet preparations?
- Urine (WCC, RCC, bacteria, epithelial cells, casts, crystals i.e uric acid
- Effusions/aspirates (crystals, WBC, RBC)
- Genital swabs (yeast, parasites e.g.Trichomonas vaginalis)
- Faeces (WBC, RBC, OCP - ova, cysts and parasites)
What type of micriobes are usually stained prepartions and when are smears best?
- Gram stain of all smears and on spun deposits (done on all wound fluids, aspirates, some urines)
• WBC, bacteria, yeast, fungal elements, epithelial cells
- Smears are best if prepared at the time of specimen collection
What is generally a good indicator of infection
The presence of polymorphonuclear WBCs is a good indicator of infection
What does the presents of RBC’s in a sample usually indicate?
Infection or physical trauma
E.g.
- In urine may be due to kidney stones, tumours (bladder, kidney, prostate)
- In faeces may be due to dysentery or tumours
- In sputum with some infections or with tumours
What type of diagnoses does microscopy deliver and example?
Microscopy may provide a presumptive diagnosis
Eg.
- Neisseria gonorrhoeae - urogenital sites – GNIDC
- Neisseria meningitidis – Upper RT - GNIDC
What is the Importance of Microscopy?
May provide a definitive diagnosis for some microbes/infections (wet preparations and stains)
Microscopy can influence if additional culture media is used
Provides information about the quality of a specimen
Microscopy results should correlate with the subsequent culture results
What does epithelial cells in septum and urine sample indicate?
indicate likely contamination with normal flora
What is the best way to correlate swap cultures?
Correlation for swab cultures is best with a separate smear from the time of collection
What determines the media a culture is prepared in?
-depending on the type of specimen and/or the site of infection (varies between labs. due to individual preferences)
What type of media is Blood agar? (BA)
- enriched media, grows most pathogens
- Assess haemolysis around single colonies (? a differential feature)
- Separate BA plate incubated for anaerobes (AnO2)
What type of media is chocolate agar? (CHOC)
– heated BA plate, enriched media
- ALWAYS incubated in CO2 to accommodate Haemophilus and Neisseria
What type of media is MacConkey (MAC)?
- selective and differential (different formulations available)
- bile salts (some also contain crystal violet – 2nd year Curtin) – selective for GNB - inhibits gram positives (only in 2nd year at Curtin)
- neutral red pH indicator, lactose fermenters = pink (differential)
What type of media is Colistin/ Nalidixic Acid Agar (CNA)
– selective for GPC
- Blood agar base (enriched)
- Antibiotics inhibit GPB and Gram negatives – selective for GPC
What type of media is Sabouraud Dextrose Agar (SAB)
-Used with antibiotics
– selective for fungi
- used to culture yeast (colonies) and molds (hairy)
- antibiotics inhibit bacteria
- Chloramphenicol is a common antibacterial additive
What is the incubation time for most agars?
- Most agar plates are incubated in air and CO2
- Inspected after 18-24 hrs and reincubated for a further 24 hrs, reinspected
After how long are agar plates that a incubated anaerobically checked?
Agar plates that are incubated anaerobically - inspected x1 at 36-48 hours
How long is selective media for fungi incubated?
Incubated at 28 ̊C for up to 28 days
What tempurature are most bacterial culture incubated at?
Most bacterial cultures incubated at 35-37 ̊C, Campylobacter plates at 42 ̊C
How can colony mophology be interpretated?
- provides a major cue to the identity of many organisms
- Size, shape, colour, surface texture, haemolysis if on BA
- Appearance & growth or no growth on different agars
i. e. Haemophilus sp. on CHOC (flat grey colonies), NG on BA - Pseudomonas aeruginosa – green pigment
What are the rapid confirmatory tests?
- Oxidase POS (Pseudomonas, Campylobacter, Vibrio, Neisseria, Moraxella)
- Spot indole POS (Escherichia coli, Proteus vulgaris, Klebsiella oxytoca, Vibrio, Aeromonas)
- Catalase (staphylococci vs. streptococci)
- Tributyrin (positive for Moraxella/Branhamella catarrhalis)
- Bile solubility for Streptococcus pneumoniae
- Latex agglutination (coagulase) for Staphylococcus aureus
- Co-agglutination (Phadebact kit for Streptococci)
What is done if further biochemical tests are required for ID?
commercial ID kits can be used Examples of the range of products available
Some ID methods being replaced by?
by PCR technology as more PCR kits become available (stringent QA) ***
MALDI TOF - Matrix-assisted laser desorption/ionization (MALDI)
What is a MALDI – TOF (Mass spectrometry) used for?
- Bacterial identification results in < 1 hour
- Still requires initial culture of all microbial specimens
- Scientist still required to examine plates and identify potential pathogens for ID
- Desorption triggered by UV laser beam (controlled fragmentation of biomolecules)
What can be some issues in reporting results of a specimen?
- Doctor doesn t understand or can t interpret result!
- Patients ringing for results (no proof of ID over the phone = no result)
How are results of a specimen usually reported?
Telephone
Facsimile
Hardcopy (by courier or post)
Electronic (email, doctors can be given direct web access to patient database)