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Medical Microbiology and Virology > 32 Diagnosis of infection and assessment of host defense mechanisms > Flashcards

32 Diagnosis of infection and assessment of host defense mechanisms Flashcards

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1
Q

What is involved in pre-analytical stage of laboratory practice?

A

Appropriate selection of tests

Proper filling in request forms

Collection/ transport specimens

Check patient details on sample/ paperwork

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2
Q

What are routes of potential infection for lab staff in the lab?

A

Inhalation - aerosol

Ingestion - eating/ drinking in lab

Skin penetration/ uncovered wound

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3
Q

34 year old with two days fever, abdo pain, diarrhoea with blood, vomiting. Other people have similar symptoms.

What tests should be performed?

A

MC&S - like campylobacter or salmonella

No need viral PCR - not norovirus

No need oca/ cysts/ parasites

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4
Q

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%

A

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

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5
Q

What is positive predictive value?

What is negative predictive value?

A

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

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6
Q

What are main techniques when processing bacteriology specimens?

A

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

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7
Q

What are names of different types of agar used to culture bacteria?

Samples from faeces require selective agar, so that commensals do not grow

A

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

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8
Q

What organisms are these culture media used for?

Blood agar

Chocolate agar

MacConkey agar

A

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
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9
Q

What organisms are these culture media used for?

Sorbitol MacConkey agar

Xylose lysine deoxycholate (XLD) agar

A

Sorbitol MacConkey agar - E.coli O157

Xylose lysine deoxycholate (XLD) agar - salmonella/ shigella

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10
Q

What organisms are these culture media used for?

Chromogenic MRSA

Camyplobacter agar

Neomycin fastidious anaerobe agar

A

Chromogenic MRSA - MRSA

Camyplobacter agar - campylobacter. Seagull shaped gram-negative bacteria

Neomycin fastidious anaerobe agar - clostridium perfringens

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11
Q

What organisms are these culture media used for?

TCBS agar

Columbia CNA agar

A

TCBS agar - vibrio cholerae

Columbia CNA agar - gram positive bacteria e.g staphylococci, streptococci, enterococci

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12
Q

What is catalase test, and what is it used for?

A

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

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13
Q

What is oxidase test, and what is it used for?

A

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

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14
Q

What is urease test, and what is it used for?

A

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
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15
Q

What are steps of gram staining?

A
  1. Heat-fixed cells are stained with crystal violet stain, which colours the cells purple
  2. Iodine solution is added, which forms a complex with the crystal violet stain and traps it within the cell
  3. Cells are washed with ethanol solution (acetone), which decolorises Gram-negative bacteria (but not Gram-positive)
  4. 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

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16
Q

How are mycobacterium stained?

They have large lipid coat, so do not take up gram stain

A

Auramine stain is more sensitive/ less specific. So can use with microscopy as screening test

Ziehl Neelsen stain used as confirmatory test

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17
Q

How are cryptosporidium dtected?

A

Auramine stain

Examine under UV fluorescence microscope

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18
Q

What are tests to detect antimicrobial sensitivity?

A

Disk diffusion - compare zone of growth inhibiton around colony

E-strip - gradient to determine which concentration inhibits growth

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19
Q

If culture results are negative, what further molecular tests can be performed?

A

16S PCR is used to detect presence of organism using real-time PCR

Microorganisms have their own unique 16s rRNA gene

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20
Q

Patient with suspected bacterial meningitis, given benpen by GP.

What test of CSF will tell us causative organism?

A

CSF PCR

After given antibiotics, less likely blood/ CSF culture will yield result

21
Q

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
A

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

22
Q

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
A

All can cause wound infections.

All will grow on MacConkey agar, except pasteurella

23
Q

What is mainstain of fungal diagnostic testing?

A

Examination of rash

Microscopic characteristics

culture - can take up to four weeks

24
Q

There are many stains for fungal identification - spores/ hyphae/ fruiting bodies

What are these stains used for?

Gram stain

Fontana-Masson (MH)

A

Gram stain - yeast elements stain purple. Filamentous fungi do no take up stain

Fontana-Masson (MH) - stains cells wall of cryptococcus

25
Q

There are many stains for fungal identification.

What are these stains used for?

Giemsa

Potassium hydroxide (KOH)

A

Giemsa - histoplasma/ PCP

Potassium hydroxide (KOH) - identify fungal elements

26
Q

There are many stains for fungal identification - spores/ hyphae/ fruiting bodies

What are these stains used for?

India ink

Periodic Acid Shift (PAS)

A

India ink - cryptococcus

Periodic Acid Shift (PAS) - identify fungal elements which stain pink

27
Q

There are many stains for fungal identification - spores/ hyphae/ fruiting bodies

What are these stains used for?

Haematoxylin and Eosin (H&E)

Immunoflourescence

A

Haematoxylin and Eosin (H&E) - fungal elements

Immunoflourescence - PCP

28
Q

Serology can be used with mycoses which are not easily cultured.

Which species might this be useful for?

A

Aspergillus - IgG particularly useful for bronchopulmonary aspergillosis

Blastomycosis

Coccidiodomycosis

Histoplasmosis

Complement fixation tests usually used

Beware antibody tests can give false negatives if immunocompromised

29
Q

With fungal infection, which antigens can be detected by testing?

A

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

30
Q

Which fungi do not have beta-D-glucan?

Which fungi do do have mannan?

A

Cryprococcus
Mucoraceous moulds

Cryptococcus

31
Q

Radiological investigations are important in diagnosing invasive fungal infections.

What signs are seen on HR-CT chest?

A

Pulmonary nodule surrounded by ground-glass appearance, or crescent sign which appears later

32
Q

Molecular techniques can be used to give rapid, quantitative diagnosis of fungal infection.

What is example of this?

A

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.

33
Q

Aspergillus antigen galactomannan can have false positives due to certain drugs which cause cross-reactivity. What are examples?

A

Co-amoxiclav

Tazocin

34
Q

BAL sample from transplant recipient with suspected IA. Which is most appropriate test?

Galactomannan
IgM
Mannan
PCR

A

Galactomannan

Immunosuppressed so will not produce antibodies

Mannan only yeast species

35
Q

How does MALDI-TOF identify yeasts/ fungi?

A

Examines spectra obtained from proteins of fungal cell wall. Compare to spectral database.

Does not detect DNA.

36
Q

What is lab accreditation?

A

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

37
Q

What is the role of these organisations?

Health and Safety Executive HSE

Human Tissue Authority

A

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

38
Q

What is the role of these organisations?

Medicines and Healthcare Products Regulatory Authority MHRA

European Federation for Immunogenetics EFI

A

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

39
Q

Lab quality control is performed daily to ensure test results are reliable e.g internal control samples.

What is quality assurance?

A

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

40
Q

What are Westgard rules used for, and what are examples of these?

A

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.

41
Q

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?

A

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

42
Q

What are examples of category 4 organisms?

Bacteria
Fungi
Helminths
Parasites
Prions
Viruses
A

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

43
Q

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

A

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
44
Q

What is role of biological safety cabinet? BSC

A

To protect worker from pathogen, and prevent spread to environment.

45
Q

What are uses for different class of BSC?

A

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

46
Q

Patient with suspect VHF can be classified into 4 categories.

  1. Unlikely to have VHF
  2. Low possibility of VHF
  3. High possibility of VHF
  4. Confirmed VHF

What action to we take for 1/2?

A
  1. Unlikely to have VHF - if not been exposed within 21 days. Check alternative diagnosis e.g malaria
  2. 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

47
Q

Patient with suspect VHF can be classified into 4 categories.

  1. Unlikely to have VHF
  2. Low possibility of VHF
  3. High possibility of VHF
  4. Confirmed VHF

What action to we take for 3/4?

A
  1. 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

  1. Confirmed VHF - diagnosis usually made by Rare and Imported Pathogens Lab (RIPL)

As above
Discuss with High Level Isolation Unit to arrange immediate transfer

48
Q

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
A

Class II BSC protects user and environment, including reagents

Medical Microbiology and Virology (60 decks)

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