32 Diagnosis of infection and assessment of host defense mechanisms Flashcards
What is involved in pre-analytical stage of laboratory practice?
Appropriate selection of tests
Proper filling in request forms
Collection/ transport specimens
Check patient details on sample/ paperwork
What are routes of potential infection for lab staff in the lab?
Inhalation - aerosol
Ingestion - eating/ drinking in lab
Skin penetration/ uncovered wound
34 year old with two days fever, abdo pain, diarrhoea with blood, vomiting. Other people have similar symptoms.
What tests should be performed?
MC&S - like campylobacter or salmonella
No need viral PCR - not norovirus
No need oca/ cysts/ parasites
Asked to introduce new POC HIV test to A&E.
Which do you pick?
Sens 22% Spef 100%
Sens 50% Spef 50%
Sens 98% Spef 22%
Sens 98% Spef 22%
Sensitivity correctly identifies those with virus.
Specificity correctly identifies those without virus.
Tests with high sensitivity are used to rule in disease.
Tests with high specificity are used to rule out disease.
Screening test should be highly sensitive, and confirmatory test should be highly specific.
This way we will pick up 98% of all HIV coming to A&E. There will be some false positives, but this will be excluded on confirmatory test. Better to over-diagnose, than miss a case of HIV
What is positive predictive value?
What is negative predictive value?
PPV - reflects probability that HIV is present when test is positive
NPV - reflects probability that HIV is absent when test is negative
PPV is influeced by sensitivity/ specificity, and also the prevalence of a disease in a population
What are main techniques when processing bacteriology specimens?
Microscopy - use Neubauer champer to count WBC, gram stain
Culture - streak out on agar plate to obtain single colony
Sensitivity testing
MALDI-TOF - mass spectrometry
What are names of different types of agar used to culture bacteria?
Samples from faeces require selective agar, so that commensals do not grow
Blood agar
Chocolate agar
MacConkey agar
Sorbitol MacConkey agar
Xylose lysine deoxycholate (XLD) agar
Chromogenic MRSA
Camyplobacter agar
Neomycin fastidious anaerobe agar
TCBS agar
Columbia CNA agar
What organisms are these culture media used for?
Blood agar
Chocolate agar
MacConkey agar
Blood agar - most bacteria. Look for haemolysis if streptococci
Chocolate agar - fastidious H influenzae/ N meningitidis
MacConkey agar - to separate lactose fermenters
- lactose fermenter - E. coli/ klebsiella
- non-lactose fermenter - Salmonella/ shigella
What organisms are these culture media used for?
Sorbitol MacConkey agar
Xylose lysine deoxycholate (XLD) agar
Sorbitol MacConkey agar - E.coli O157
Xylose lysine deoxycholate (XLD) agar - salmonella/ shigella
What organisms are these culture media used for?
Chromogenic MRSA
Camyplobacter agar
Neomycin fastidious anaerobe agar
Chromogenic MRSA - MRSA
Camyplobacter agar - campylobacter. Seagull shaped gram-negative bacteria
Neomycin fastidious anaerobe agar - clostridium perfringens
What organisms are these culture media used for?
TCBS agar
Columbia CNA agar
TCBS agar - vibrio cholerae
Columbia CNA agar - gram positive bacteria e.g staphylococci, streptococci, enterococci
What is catalase test, and what is it used for?
Tests for presence of enzyme catalase, which is present in staphylococcal species (not streptococci)
Add hydrogen peroxide to colonies, and add cover slip. If catalase present, will convert hydrogen peroxide to water and oxygen and release bubbles
What is oxidase test, and what is it used for?
Used to test for pseudomonas species. Helps differentiate from other gram negative rods e.g E.coli, klebsiella
Add oxidase reagent to colony. If cyctochrome c oxidase enzyme present, will change to purple
What is urease test, and what is it used for?
Urea in culture medium is broken down by organisms possessing urease, into ammonia and carbon dioxide. Colour change from yellow to red-orange
Species with urease - proteus providencia klebsiella morganella ureplasma helicobacter pylori Nocardia cryptococcus
What are steps of gram staining?
- Heat-fixed cells are stained with crystal violet stain, which colours the cells purple
- Iodine solution is added, which forms a complex with the crystal violet stain and traps it within the cell
- Cells are washed with ethanol solution (acetone), which decolorises Gram-negative bacteria (but not Gram-positive)
- Gram-negative cells are visualised by applying safranin – which is a pink-coloured counterstain
Gram staining works as a result of the different effects of ethanol on the cell wall of the bacteria:
Gram-positive bacteria have a thick peptidoglycan layer which is dehydrated by ethanol, trapping the crystal violet stain - appearing purple.
Gram-negative bacteria have an outer lipid membrane which is dissolved by ethanol, releasing the crystal violet stain
Both cell types retain the counterstain, but the pink colour cannot be distinguished in the purple-stained Gram-positive cells
How are mycobacterium stained?
They have large lipid coat, so do not take up gram stain
Auramine stain is more sensitive/ less specific. So can use with microscopy as screening test
Ziehl Neelsen stain used as confirmatory test
How are cryptosporidium dtected?
Auramine stain
Examine under UV fluorescence microscope
What are tests to detect antimicrobial sensitivity?
Disk diffusion - compare zone of growth inhibiton around colony
E-strip - gradient to determine which concentration inhibits growth
If culture results are negative, what further molecular tests can be performed?
16S PCR is used to detect presence of organism using real-time PCR
Microorganisms have their own unique 16s rRNA gene
Patient with suspected bacterial meningitis, given benpen by GP.
What test of CSF will tell us causative organism?
CSF PCR
After given antibiotics, less likely blood/ CSF culture will yield result
Neonate develops symptoms of acute meningitis after birth. Gram stain of CSF shows gram-positive bacilli.
Which test will confirm identification of organism?
Catalase DNase plate Staphaurex latex agglutination Tube coagulase Tumbling motility
Tumbling motility - patient likely has listeria monocytogenes, obtained via birth canal. When grown at room temp displays tumbling motility
Catalase - used to differentiate staph/ strep
DNase plate - used to differentiate staph
Staphaurex latex agglutination- used to differentiate staph
Tube coagulase- used to differentiate staph
Gram negative bacillus isolated from pus drained from wound of farm-worker’s hand. It is oxidase positive and grew on blood agar, but not on MacConkey agar. What is most likely organism?
Aeromonas hydrophilia Escherichia coli Klebsiella pneumoniae Pasteurella multocide Pseudomonas aeruginosa
All can cause wound infections.
All will grow on MacConkey agar, except pasteurella
What is mainstain of fungal diagnostic testing?
Examination of rash
Microscopic characteristics
culture - can take up to four weeks
There are many stains for fungal identification - spores/ hyphae/ fruiting bodies
What are these stains used for?
Gram stain
Fontana-Masson (MH)
Gram stain - yeast elements stain purple. Filamentous fungi do no take up stain
Fontana-Masson (MH) - stains cells wall of cryptococcus
There are many stains for fungal identification.
What are these stains used for?
Giemsa
Potassium hydroxide (KOH)
Giemsa - histoplasma/ PCP
Potassium hydroxide (KOH) - identify fungal elements
There are many stains for fungal identification - spores/ hyphae/ fruiting bodies
What are these stains used for?
India ink
Periodic Acid Shift (PAS)
India ink - cryptococcus
Periodic Acid Shift (PAS) - identify fungal elements which stain pink
There are many stains for fungal identification - spores/ hyphae/ fruiting bodies
What are these stains used for?
Haematoxylin and Eosin (H&E)
Immunoflourescence
Haematoxylin and Eosin (H&E) - fungal elements
Immunoflourescence - PCP
Serology can be used with mycoses which are not easily cultured.
Which species might this be useful for?
Aspergillus - IgG particularly useful for bronchopulmonary aspergillosis
Blastomycosis
Coccidiodomycosis
Histoplasmosis
Complement fixation tests usually used
Beware antibody tests can give false negatives if immunocompromised
With fungal infection, which antigens can be detected by testing?
Beta-D-glucan polysaccharide present in majority of fungal cell walls. Useful for screening, and negative result has high negative predictive value. Ubiquitous in environment, so false positives can occur
Mannan - cell wall polysaccharide of yeasts is highly immunogenic, and more sensitive that BDG. Get rapid result before blood culture. Antigen rapidly cleared during disease, so negative value does not exclude invasive fungal infection.
Galactomannan is cell wall polysaccharide in most aspergillus species. Use EIA for early diagnosis of invasive aspergillosis e.g haematological malignancies, stem cell transplant
Which fungi do not have beta-D-glucan?
Which fungi do do have mannan?
Cryprococcus
Mucoraceous moulds
Cryptococcus
Radiological investigations are important in diagnosing invasive fungal infections.
What signs are seen on HR-CT chest?
Pulmonary nodule surrounded by ground-glass appearance, or crescent sign which appears later
Molecular techniques can be used to give rapid, quantitative diagnosis of fungal infection.
What is example of this?
PCR
Peptide nucleic acid fluoescence in-site hybridisation (PNA FISH). Rapidly identifies 5 most common candida species, which each emit different fluorescence
MALDI-TOF - compare results to spectral database.
Aspergillus antigen galactomannan can have false positives due to certain drugs which cause cross-reactivity. What are examples?
Co-amoxiclav
Tazocin
BAL sample from transplant recipient with suspected IA. Which is most appropriate test?
Galactomannan
IgM
Mannan
PCR
Galactomannan
Immunosuppressed so will not produce antibodies
Mannan only yeast species
How does MALDI-TOF identify yeasts/ fungi?
Examines spectra obtained from proteins of fungal cell wall. Compare to spectral database.
Does not detect DNA.
What is lab accreditation?
Procedure which an authoritative body gives formal recognition that an organisation is competent to carry out specific tasks
Based on set of defined standards
UK Accreditation Service has annual assessments. Based on four-year cycle which covers all of lab tests. responsible for certification, testing, inspection, calibration of services
Each lab must have a Quality Manager, to ensure processes meet standard operating procedure (SOP).
If problem with accreditation of certain test, then recommendations will be made, and lab given 1-3 months to improve these
What is the role of these organisations?
Health and Safety Executive HSE
Human Tissue Authority
Health and Safety Executive HSE - responsible for inspection/ licensing of microbiological containment level 3/4 facilities
Human Tissue Authority - responsible for legal registration of labs, that process and store human tissue e.g histology
What is the role of these organisations?
Medicines and Healthcare Products Regulatory Authority MHRA
European Federation for Immunogenetics EFI
Medicines and Healthcare Products Regulatory Authority MHRA - provides guidelines on good laboratory practice, regulation of medical devices such as implants/ reagents/ analytical platforms
European Federation for Immunogenetics EFI - provides standards for transplantation and tissue typing laboratories across Europe
Lab quality control is performed daily to ensure test results are reliable e.g internal control samples.
What is quality assurance?
Has broader scope, and its activities are less frequent.
Quality assurance is used to ensure that quality control is working.
Important elements of a quality management system include:
Documentation
Standard Operating Procedures (SOP’s)
Quality Control samples
External Quality Assessment Scheme
What are Westgard rules used for, and what are examples of these?
Westgard Rules are multirule QC rules to help analyze whether or not an analytical run is in-control or out-of-control. They should trigger assessment of assay reagents and calibration of equipment.
It uses a combination of decision criteria, usually to judge the acceptability of an analytical run.
A run is rejected when a single control measurement exceeds the mean +3SD control limits.
A run is rejected with 2 consecutive control measurements 2 standard deviations of control limits on the same side of mean with this rule.
A run is rejected when there are 10 consecutive controls on the same side of the mean.
Control of Substances Hazardous to Health Regulations 2002 (COSHH) classifies biological agents into four categories.
Advisory committee on Dangerous Pathogens (ACDP) advises about classification. What is this based on?
Likelihood that it will cause disease
Likelihood disease would spread to community
Availability of any prophylaxis or treatment
Group 1 - unlikely to cause human disease
Group 2 - can cause disease, but treatment available
Group 3 - can cause severe disease, may spread in community, usually treatment available
Group 4 - causes severe disease, spread in community, no effective treatment available
Does not take into account individuals health e.g pregnancy, immunodeficiency
What are examples of category 4 organisms?
Bacteria Fungi Helminths Parasites Prions Viruses
Only viruses -
Ebola Marburg Hendra Nipah Lassa Junin Chapare Guanarito Sabia Crimean-congo TBE Kyasanur Forest disease Omsk haemorrhagic feber Variola major/ minor - smallpox
VHF typically has up to 21 day incubation from initial exposure
COSHH stipulates different rules for managing different pathogens at different containment levels.
Containment level 1 is basic lab
What is unique about these containment levels?
Containment level 2
Containment level 3
Containment level 4
Containment level 2 -
- biological safety cabinets
- autoclave
- immunised staff
Containment level 3 -
- higher security measures
- negative air flow through HEPA filter
Containment level 4 -
- high level security
- body suits with air supply
What is role of biological safety cabinet? BSC
To protect worker from pathogen, and prevent spread to environment.
What are uses for different class of BSC?
Class I BSC - protects worker/ environment, but not samples
Class II BSC - protects worker/ environment, also protects samples
Class III - in category 4 laboratories providing air tight protection
fume cupboard is for handling volatile chemicals, and is different
Patient with suspect VHF can be classified into 4 categories.
- Unlikely to have VHF
- Low possibility of VHF
- High possibility of VHF
- Confirmed VHF
What action to we take for 1/2?
- Unlikely to have VHF - if not been exposed within 21 days. Check alternative diagnosis e.g malaria
- Low possibility of VHF - typically fever within 21 days of leaving VHF-endemic country, but no high-risk contact.
Isolate in side room. Standard precautions - gloves/ aprons. Treat samples as normal
Patient with suspect VHF can be classified into 4 categories.
- Unlikely to have VHF
- Low possibility of VHF
- High possibility of VHF
- Confirmed VHF
What action to we take for 3/4?
- High possibility of VHF - typically fever within 21 days of leaving VHF-endemic country, with high-risk contact. Clinically bleeding/ bruising
Isolate in side room
Droplet precatuion
Discuss with ID/ Imported Fever service regarding VHF test
Notify Health Protection Team
- Confirmed VHF - diagnosis usually made by Rare and Imported Pathogens Lab (RIPL)
As above
Discuss with High Level Isolation Unit to arrange immediate transfer
Clean PCR reagents are being prepared in the lab.
Which is most appropriate to prevent cross-contamination of the reagents?
Class I BSC Class II BSC Class III BSC FFP3 respirator Fume Cupboard
Class II BSC protects user and environment, including reagents
