W2 From patient to result Flashcards
State the diagnostic template
- Request form
- Sample collection
- Transport of samples
- Reception (macroscopic examination)
- Safety issues
- Non-culture techniques (indication at early stage/rapid diagnosis)
- Culture of clinical samples
- Identification and sensitivity testing
- Result
Name some sterile sites in the body
- Blood/bone marrow
- CSF
- Tisssue
- Lower respiratory tract
- Bladder
Name some non-sterile sites
- Upper respiratory tract (streptococci, anaerobes, Candida albicans)
- Skin (coagulase negative staphylococcus eg S. epidermidis)
- GI tract (‘coliforms’,anaerobes ‘faecal flora’)
- Vagina (lactobacilli, anaerobes)
- Urethra (skin and faecal flora)
Give examples of specimens received in clinical microbiology
- Midstream Specimen of Urine (MSU)
- Blood culture
- Urethral swab
- Faeces (stool)
- Toe nail clippings
- Sputum
What is the aim of the clinical microbiology laboratory?
- To provide accurate information about the presence or absence of microorganisms from a clinical specimen
- To provide antimicrobial susceptibility information on the microorganism(s) recovered
- Accurate diagnosis and sensitivity testing:
(a) Successful treatment of infection
(b) Aids in preventing spread of infection
(c) Prevents emergence of antibiotic resistance
Describe sample collection: Aide-Memoire
- Take appropriate specimen eg. sputum
- Collect specimen before antibiotics are given (to avoid false negatives)
- Avoid contamination from normal flora if possible and any contaminated equipment
- Label specimens correctly and identify any known ‘High Risk’ eg HIV
- Complete request form completely; sufficient clinical information
- Transport sample to the lab as quickly as possible
What media is used to transport bacteria?
What does it contain?
What is it used for?
Stuarts transport media (STM)
Contains charcoal to inactivate any toxic bacterial bi-products. Used for swabs eg. W/S, HVS, ear, T/S (not fluids eg. sputum)
What media is used to transport bacteria?
What does it contain?
Viral transport media (VTM). Buffered salt solution containing serum. Contains antimicrobials to control overgrowth of contaminating bacteria and fungi
How is transport media stored?
Refrigeration at 4oC
What happens at the clinical microbiology reception?
- Check specimen / form details
- Allocation of unique laboratory number
- Macroscopic appearance –discard unsuitable samples (cost issues; poor results)
Give an example of extra informtion hat may be included after reciept of the specimen
(a) Diarrhoea’: solid stool received
(b) ‘Chest infection’: saliva from the mouth
(c) Tissue samples received in formalin
What is it important to highlight on recept of the specimen?
Any safety issues
Give examples of potential safety issues
All clinical samples received MUST be regarded as high risk
- Patient (eg. HIV, Hep B)
- Potential pathogens within specimen / Advisory Committee on Dangerous Pathogens (ACDP) categorises microorganisms 1,2,3 or 4
eg. Mycobacterium tuberculosis (category 3 pathogen): cat 3 laboratory; class 1 safety cabinet
State the characteristics of a class I safety cabinets
(a) negative pressure, inward flow of air
(b) 0.74m3/sec air flow rate
(c) HEPA filter (high efficiency particle absorber)
Advisory Committee on Dangerous Pathogens (ACDP) categorisation of biological agents:
What is category 1?
Give examples
•A biological agent unlikely to cause human disease eg. saprophytic / soil organisms
ACDP categorisation of biological agents
What is category 2?
Give examples
•A biological agent that can cause human disease; hazardous to employees; unlikely to spread in the community; effective treatment/prophylaxis
eg. Staphylococcus aureus, Clostridium difficile, Streptococcus pneumoniae, Streptococcus pyogenes, Neisseria gonorrhoeae
ACDP categorisation of biological agents
Wha is category 3?
Give examples
•A biological agent that can cause severe human disease; serious hazard to employees; risk of spread in community; effective treatment / prophylaxis
eg. Mycobacterium tuberculosis
ACDP categorisation of biological agents
What is category 4?
Give examples
• A biological agent that causes severe human disease; serious hazard to employees; likely to spread in community; no effective treatment/ prophylaxis
eg. Ebola virus
Diagnostic Procedures
Give examples of non culture techniques
(A) Direct Microscopy
(B) Antigen Detection
(C) Molecular Microbiology (detection and identification of bacteria by molecular techniques eg. Nucleic acid amplification test-NAAT (PCR):
Used to detect a particular nucleic acid sequence and thus usually to detect and identify a particular species or subspecies of organism, often a virus or bacterium that acts as a pathogen in blood, tissue, urine, etc. Types of nucleic acid amplification tests
- Polymerase chain reaction (PCR) – including nested (n), quantitative (q) or real-time reverse transcription (RT-PCR), loop mediated isothermal amplification (LAMP), and quantitative nucleic acid sequence-based amplification (QT-NASBA)
Serological Response
Serology tests in the laboratory include:
- markers of Hepatitis B infection or past infection
- Human Immunodeficiency Virus (HIV) status
- Hepatitis C virus
- Rubella immune status
Diagnostic Procedures: Non culture techniques
Direct microscopy
•Direct microscopy of clinical sample
(eg. light, phase contrast, fluorescence, EM)
•Light Microscopy widely used
(a) Wet preparations
eg. parasite ova and cysts (faeces)
(b) Stained preparations
eg. Gram stain: most bacteria and yeasts
•May be diagnostic eg. Gram-negative diplococci (Neisseria meningitidis) in CSF
Diagnostic Procedures
Non-Culture Techniques: Antigen Detection
- Latex beads have specific antibodies attached that are specific to certain antigens
- Mix with CSF sample
- Agglutination =positive result
Useful even if the bacteria has been killed by antibiotics
Culture of Microorganisms
What are the types of soilid agar?
(a) Basic eg. Nutrient agar (general purpose)
(b) Enriched agar (enriched general purpose)
(c) Selective agar (selects specific organisms from a sample)
(d) Differential agar (differentiates organisms within a sample)
What is horse blood agar and what is it useful for?
•Horse blood agar contains 5-7% horse blood – useful for demonstrating haemolysis of organisms such as streptococci
What are the characteristics of CCFA agar?
Grey (‘fried egg’) colonies of Clostridium difficile on CCFA agar (cycloserine, cefoxitin fructose agar)
•Cycloserine and Cefoxitin are both antimicrobials and ‘selective’ for C. difficile:
They inhibit the growth of normal commensals (sensitive) in faeces but allow growth of C. difficile (resistant)
•Many different types of selective agar utilised in microbiology; choice of selective agar will be influenced by a knowledge of the potential normal flora in a clinical sample
What is Cystine Lactose Electrolyte Deficient agar (CLED)?
used for the differentiation and enumeration of Enterobacteriaceae ‘coliforms’ (enteric Gram-negative rods) in urine/
ID: BASIC → FULL (example: Escherichia coli)
Give examples of basic identifiaction (stages 1 and 2)
a) Colonies of Staphylococcus aureus are golden on blood agar (1-2mm, entire edge, ‘buttery smell)
b) Colonies of Streptococcus pyogenes are beta- haemolytic on blood agar (1mm, entire edge)
BASIC IDENTIFICATION
microscopic appearance
Full ID (stage 3): Biochemical tests
Eg. API (Analytical Profile Index)
Commercially produced kits of 20 or more miniaturised biochemical tests are available. The results of the set of tests generate a ‘numerical profile’ which identifies the bacterium
API available for most organisms eg. enterobacteria, staph, strep, neisseria, haemophilus
NB. Many labs now use MALDI-TOF Mass Spectrometry for microbial identification
Give an example of Molecular Typing / Genetic Fingerprinting
•Pulsed field gel electrophoresis (PFGE) based on similarities between the whole genome of different strains; whole genome sequencing
DISC SUSCEPTIBILTY method
