MED2042 WEEK 3 - Laboratory Diagnosis of Infectious diseases

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In what circumstances do you use the lab in the diagnosis of infectious disease?: Suspect Infection
- clinical history
> patient characteristics
> special epideiology
> clinical syndrome
- examination
- (bloods/imaging)

Are Investigations required?
1. 'Sick' e.g. tachycardia, low blood pressure, shortness of breath
2. Immunocompromised patient e.g. febrile neutropaenia
3. Special Epidemiology
e.g. returned traveller
4. Significant effect on therapy
5. Failure of empiric therapy (also recurrent or relapsing)
6. Important public health implications e.g. measles

What Investigations
1. What site?
blood, urine, faeces, sputum, fluid, wound
2. What infectious agents are you looking for?
faces of returned traveller vs hospital diarrhoea
3. When to take?
- before antibiotics for bacterial culture
- Serology may be negative at disease onset
4. How to take?
Avoid contamination from non-sterile sites
urine, sputum
5. How to transport
- special transport conditions?
- time critical?
6. Which test
- accuracy
- cost
7. How urgent?
What techniques are used in diagnosis?: 1. Macroscopic Examination
2. Microscopy
3. Culture
4.Detection of Antigen
5. Detect immune response
- Antibody
- T-cell (gamma Interferon)
6. Detect nucleic acid (Nucleic Acid Testing - 'NAT')
- PCR (bDNA, LCR)

4,5 = serology
What is Macroscopic examination used for?: - Cloudy CSF
- Purulent sputum
- Purulent fluids (joint, pleural, pericardial, periotoneal)
Describe direct examination.: Microscopy
- Light microscopy - wet mount, staining, dark field
- Fluorescent - bacteria (pertussis), and viruses (respiratory, herpes)
- EM - viruses (nor, pox)

Histology
- viral inclusions (CMV)
- invasive fungal infections
- H.pylori
Describe wet prep examination (microscopy).: Direct examination under light microscope
- Cell counts - urine, CSF, other gluids
- Vaginal swabs - trichomonas, bacterial vaginosis, candida
- Faeces - white cells, Trophozoites, eggs, larvae
Describe staining microscopy.: - Gram's Stain
- Acid Fast
> auromine rhodamine
> modified acid fast
> fite stain
- Grocott's methenamine silver stain (fungi)
- PAS
- Wright-Giemsa (malaria, leishmania)
- Toluidine blue (pnemocystis)
- Trichrome
- India ink
Describe the Gram stain.: Procedure (5 mins)
Specimen or culture
--> crystal violet
--> iodine
--> acetone/alcohol

Gram positive
- retain crystal violet (cell wall impermeable to solvent)
- purple

Gram negative
- crystal violet lost (cell wall permeable to solvent)
- stain with counter stain - red
Describe bacterial culture.: - Highly sensitive
- Provides isolate for identification and antimicrobial sensitivity
- Different media
> solid (agar) vs liquid (blood)
> enrichment
> selective
- Different growth conditions
e.g. anaerobic vs aerobic
- Different time to grow
> Most pyogenic bacteria overnight
> Anaerobes 48 hours
> Mycobacteria 2-6 weeks
- Different appearance on media

The different media, growth conditions, time to grow and appearance on media assists identification.
Define contaminant: Normal flora or environmental flora containing clinical specimen during or after collection.
Describe the assessment of significance of a bacterial culture.: Gram stain result
- how much pus?
- were organissms seen microscopically?

Site of isolation
- sterile stie (CSF, joint, blood) more likely to be significant
- Is the isolate commonly a site coloniser?

Culture results
- Is it a pure or mixed culture?
- Is organism the predominate isolate?
- What is the significance of other isolates?
Describe confirmatory testing.: Biochemcial reactions
- catalase or urease production
- sugar fermentation

Semi-automated or manual

API kits 24-48 hours
Describe antibiotic-sensitivity testing.: - Significant isolates tested for sensitivity to relevant antibiotics
- disc sensitivity reported as S/I/R
- minimum inhibitory conc (MIC) only done for some isolates - E-test, Broth microdilution
What are diagnostic methods in virology?: 1. Direct examination - detect virus or virus products (protein Ag, NA)
2. Indirect - detect immune response to virus (antibodies)
What are the direct examination methods in virology?: 1. Antigen detection - immunofluorescence, ELISA etc
2. Electron microscopy - morphology of virus particles immune electron microscopy
3. Light Microscopy
- histological appearance
- inclusion bodies
- highly specific
- requires biopsy tissue
4. Viral culture
- cell culture
5. Viral NA Detection
- hybridization with specific nucleic acid probes
- PCR
3. Light Microscopy
Describe Electron Microscopy.: 10^6 virus particles per ml required for visualisztion. x 50 000 - 60 000 magnification normally used

Faeces - Rotavirus, Adenovirus, Norwalk like viruses, Astrovirus, Calcivirus

Skin - orf. molluscum contagiosum

Unknown - SARS
What are the problems with electron microscopy?: Expensive equipment
Expensive maintenance
Require experienced observer
Sensitivity often low
Describe Antigen detection-methods: Immunohistochemistry
Immunoelectronmicroscopy
Immunofluorescence
- direct fluorescence assay (DFA)
- respiratory viruses - RSV, flu, paraflu, adeno
- skin - HSV, VZV
- Blood - CMV (pp65 antigenaemia test)

- Immunochromatographic assay (like pregnancy test)
> quick
> suitable for use in the field
- Agglutination
.. Latex aggluintation
> blood, faecies, CSF
> quick, easy
> subjective reading
> lower sensitivity
- ELISA
> blood, urine, faeces
> highly reproducible
> automatible
> objective readout
What are the advantages and disadvantages of Antigen-detection methods?: Advantages
- result available quickly, usually within a few hours

Potential problems
- often very much reduced sensitivity compared to cell culture, can be as low as 20%
- specifically can be low because interpretation sujective
- requires good specimens including cells.
- the procedures involved are often tedious and time-consuming and thus expensive in terms of laboratory time
Describe Virus Isolation (cell culture).: When - early in disease when virus present

Transport - swabs in viral transport medium (VTM)

- Detect growth by Cytopathic effect (CPE) or by haemadsorption
- Confirmation of the identity of the virus may be carried out using neutralization, haemadsorption-inhibition or immunogluorescence tests.
- Most frequently used for herpes virus, cytomegalovirus, enterovirus, respiratory viruses
What are some problems with cell culture?: - long period (up to 4 weeks) required for result
- often very poor sensitivity (sensitivity depends on the condition of the specimen which is affected by timing, quality of collection (cells present) and transport
- susceptible to bacterial contamination
- susceptible to toxic substances which may be present in the specimen
- many viruses will not grow in cell culture

NA based detection now replacing cell culture
What are the uses of polymerase chain reaction (PCR)?: Uses
- HSV encephalitis
- HIV viral load
- respiratory viruses
- herpes viruses
- hepatitis C
- some bacteria e.g. mycobacteria - tuberculosis, MRSA resistance, 16s ribosomal sequencing, fungi, protozoa
What are the advantages of PCR?: - Extremely high sensitivity and specificity
- quick
- multiplexing possible
- quantification possible
- may be positive post antibiotics when cultures negative e.g. meningococcus from CSF
What are the disadvantages of PCR?: - extremely liable to contamination
- expensive
- a positive result may be difficult to interpret, especially with latent viruses such as CMV, where any seropositive person will have virus present in their blood irrespective whether they have disease or not
- may detect non-viable organims (no use in monitoring therapy)
- no isolate for characterisation, susceptibility
Describe serology.: Detection of specific antibody responses to infectious agents.

Methods of measuring:
- ELISA, western blot
- CFT
- HAI - inhibition of haemagglutination by specific Ab
- Immunofluorescence
- Immunoelectrophoresis

- Particularly useful in detecting viral infections
- But also some bacteria, protozoa, helminths
How to interpret serology?: - Criteria for diagnosing Primary Infection
> presence of IgM
> seroconversion
> 4 fold or more increase in titire of IgG or total antibody between acute and convalescent sera (>2 weeks)
- negative serology at presentation doesn't necessarily exclude infection
> lag to develop Ab reponses (e.g. HIV)
- Positive IgG can indicate past or current infection
> a single high titre of IgG (or total antibody) - very unreliable indicator of recent infection
What are the problems with serology?: - Time delay till confirmation
- Extensive antigenic cross-reactivity between related viruses e.g. Japanese B encephalitis and Dengue, may lead to false positive reults
- Immunocompromised patients often give a reduced or absent humoral immune response
- False positive result
e.g. postivie HIV ELISA, negative Western Blot
- Patients given blood or blood products may give a false positive result due to the transfer of antibody.
Possible HIV infection.: Are investigations required?
- yes
- important treatment and prognostic information
- important public health

Which organisms are likely
- HIV
- Also EBV, CMV, toxoplasmosis

How do we diagnose HIV in the lab?
- Blood
> Microscopy - NO
> Culture - NO (research)
> Serology - YES
> NAA - YES

Is it urgent?
- Yes/no depending on clinical context

Communication with lab
- Has the patient had HIV test before?
- Positive EIA will be phoned through to you
How to diagnose HIV?: Microscopy - NO
Cutlure - NO (research)
Serology - YES
> ELISA (Antibody to HIV-1, HIV-2)
> Western Blot
> P24antigen (ELISA)
NAT = HIV viral load
> RT-PCR
> bDNA
> resistance typing
-> RT-PCR sequence
-> RT-PCR hybridisation
What are the advantages of ELISA for HIV antibody?: Easy, automatable
High reproducibility
Antigen or Antibody or both (4th gen HIV EIA)
What is the Western Blot test?: HIV-1 Western Blot
Lane1: postive control
Lane2: negative control
Sample A: Negative
Sample B: Indeterminate
Sample C: Positive
Febrile Returned traveller: Are investigations required?
- yes, special epidemiology

Which organisms are likely?
- Malaria
- Dengue, typhoid, meningitis, bacterial enteritis, pneumonia, Rickettsioses

How do we diagnose malaria in the lab?
Blood examination
- Microscopy (thick and thin) - YES
- Culture - NO
- Serology (ICT) - YES
- NAA - (YES)

Is it urgent?
- yes - falciparum malaria can kill

Communication with lab
- you want result asap
What does ICT stand for?: Immunochromatographic test
What are the causes of community acquired pneumonia?: Strep pneumoniae - 25% bacteraemic
H-influenze
Legionella
Chalmydia, Mycoplasma
Respiratory Viruses
What is the significance of a clinical isolate? - interpreting reports: Sputum
- can be unreliable
- oropharyngeal colonisation
- gram stain very important in interpretation
> significant isolate - seen in gram stain, PMN's
> 10 epithelial cells/HPF = oral contamination (some labs reject/don't report these)
- "amount of growth" of potential pathogen

Blood culture
- bacteraemia is potentially life threatening
- All positive BC's should be reported and acted upon as a matter of urgency
- one blood culture set = 2 bottles
- always significant (any positive bottle) - S.aureus, S.pneumoniae, P. aeruginosa, GNB, Fungus
- possibly significant - Coagulase negative staphylococci (S.epidermidis), Corynebacterium spp
What are the general principles of laboratory investigations: 1. Don't do investigations for an infection unless you first clinically suspect infection
2. The syndrome and likely causes dictate the site and type of specimen
3. Do the right test for what you suspect the infecting agent to be
4. Communication with the lab important (right site, right amount, right transport, right test!)
5. You may have to select treament before test results are available (empirical therapy)
6. Not all bacteria are bad
- normal flora
- always look at the microscopy report first
Rapid - likely to be the first clue that there is definitely infection
Cell counts - PMNs
Gram stain
Assis you to determine the significance of subsequent culture results
7. Serology requires careful interpretation. The lag period may mean serology needs to be repeated
9. Usefulness of tests for types of infectious agents
- Bacteria - micro, culture > antigen detection > serology, PCR
- Viruses - Antigen detection > serology, PCR > micro, culture
- Fungi - Micro, culture, antigen detection >> PCR, serology
- Protozoa - Micro, Antigen detection > serology

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