Laboratory Diagnosis of Infection

Laboratory Diagnosis
of Infection
Professor Mark Pallen
We ask the lab for a diagnosis, expecting a yes or no, but often end up with just a maybe…
Outline
Clinical assessment
Collecting and transporting specimens
Microscopy
Culture
Sensitivity
Non-cultural diagnostic methods
Virological diagnosis
Interactions between humans and microbes
Normal flora
Diagnosis of Bacterial Infection
Patient
Clinical
diagnosis
Haematology
Biochemistry
Non-microbiological investigations
Radiology
Sample
Take the correct specimen
Take the specimen correctly
Label & package the specimen up correctly
Appropriate transport & storage of specimen
A proper clinical assessment is essential for optimal use of laboratory services!
Collecting the correct specimen
Endocervical swabs for GC
Pernasal swabs for pertussis
whole EMU for TB
Sputum , not saliva
Blood culture bottles, not clotted blood
Correctly timed Gentamicin assays
Pus, not swabs
Getting the specimen to the lab
Problems in delay or inappropriate storage• delay in dignosis & treatment
pathogens die
contaminants overgrow
Blood cultures directly into incubator
not refigerator!
CSF straight to lab
Don`t put an entire surgical specimen into formalin!
Send a portion to microbiology in a sterile container
Collecting the specimen correctly
Take an mid-stream urine
avoids contamination with perineal flora
CSF
Avoid contamination
Avoid bloody tap
Throat swab
Make the patient gag!
Blood cultures
Avoid contamination with skin organisms
Specimens & Infection Control
Please be considerate to lab staff!!
Label hazardous specimens
Don`t send specimens to the lab without proper packing
Leaking or blood-stained specimens are not acceptable!!!
Factors limiting usefulness of bacteriological investigations
wrong sample
e.g. saliva instead of sputum
delay in transport / inappropriate storage
e.g. CSF
overgrowth by contaminants
e.g. blood cultures
insufficient sample / sampling error
e.g.in mycobacterial disease
patient has received antibiotics
Diagnosis of Bacterial Infection
culture
on plates or in broth
identification by biochemical or serological tests on pure growth from single colony
microscopy
Decolorise
Counterstain
Stain
unstained or stained with e.g. Gram stain
sensitivities
Serodiagnosis
DNA technologies
by disc diffusion methods, breakpoints or MICs
Microscopy
Unstained preparations
“Wet prep”
Dark-ground illumination for syphilis
Microscopy
Stained preparations
Gram-stain
Acid-fast stain
Ziehl-Neelsen
Fluorescence
Direct, e.g. auramine
Immunofluorescence
Culture of Bacteria
Solid media
Agar plates
For Identification
For Enumeration
Slopes
For safe long-term culture, e.g. Lowenstein-Jensen media for TB
Liquid media (broth)
For enrichment or maximum sensitivity
Advantages of Solid Media
isolation of single clonal colonies
get bacterium in pure culture
identify by colonial morphology
quantification by colony-forming units
Identification of Bacteria
Morphology
Growth requirements
Biochemistry
Enzymes
Antigens
Non-cultural diagnostic methods
Antigen detection
e.g. latex agglutination
Antibody detection
e. g. agglutination tests, complement fixation tests, indirect immunofluorescence
Molecular methods
Polymerase Chain Reaction
Sensitivity tests
on solid media
disc diffusion technique
in liquid media
minimum inhibitory concentration (MIC) test
Breakpoint methods
E-test
Diagnosis of Viral Infection
Electron microscopy
Antigen detection
Antibody detection
Virus culture
Detect cytopathic effect or antigen
Molecular methods
Polymerase Chain Reaction
Sequencing (e.g. for sensitivities)
Microbes and humans
Very few microbes are
always pathogenic
Many microbes are
potentially pathogenic
Most microbes are
never pathogenic
Microbes and humans
Disease can come about in several overlapping ways
1. Some bacteria are entirely adapted to the pathogenic way of life in humans. They are never part of the normal flora but may cause subclinical infection, e.g. M . tuberculosis
2. Some bacteria which are part of the normal flora acquire extra virulence factors making them pathogenic, e.g. E. coli
3. Some bacteria which are part of the normal flora can cause disease if they gain access to deep tissues by trauma, surgery, lines, e.g. S. epidermidis
4. In immunocompromised patients many free-living bacteria and components of the normal flora can cause disease, especially if introduced into deep tissues, e.g. Acinetobacter
How do we know that a given pathogen causes a specific disease?
Koch`s postulates
the pathogen must be present in every case of the disease
the pathogen must be isolated from the diseased host & grown in pure culture
the specific disease must be reproduced when a pure culture of the pathogen is inoculated into a healthy susceptible host
the pathogen must be recoverable from the experimentally infected host
Spectrum of virulence
poliomyelitis in a child
0.1-1% of infections are
clinically apparent
rubella
50% of infections are
clinically apparent
rabies
100% of infections
are clinically apparent
The iceberg concept of infectious disease
asymptomatic infection
classical
clinical disease
less severe
disease
potential pathogen isolated from or detected in clinical samples
Recognised syndromes

patient`s clinical condition

e.g.
septicaemia, endocarditis,
osteomyelitis meningitis,
UTI, pneumonia
pharyngitis
How do we know that a given pathogen causes a specific disease?
Diagnosis and effective treatment of infection depends not just on isolating an organism, but in establishing a plausible link between the laboratory findings, recognised syndromes and the patient`s clinical condition
Microbes and humans
Evidence for a potential pathogen being clinical significant (particularly for bacteria)
Isolated in abundance
Isolated in pure culture
Isolated on more than one occasion
Isolated from deep tissues
Evidence of local inflammation
Evidence of immune response to pathogen
Fits with clinical picture
Normal flora
All body surfaces possess a rich normal bacterial flora, especially the mouth, nose, gingival crevice, large bowel, skin
This can be a nuisance in that
it can contaminate specimens
it can cause disease
This is beneficial in that
it can protect against infection by preventing pathogens colonising epithelial surfaces (colonisation resistance)
removal of the normal flora with antibiotics can cause superinfection, usually with resistant microbes
Endogenous viruses reside in the human genome
worries about similar pig viruses in xenografts
Outline
Clinical assessment
Collecting and transporting specimens
Microscopy
Culture
Sensitivity
Non-cultural diagnostic methods
Virological diagnosis
Interactions between humans and microbes
Normal flora