bio303 laboratory diagnosis of infection
DESCRIPTION
In this Bio303 module talk, I provide an overview of how infections are diagnosed in the clinical microbiology lab, focusing on technologies, old and new, and also on practical issues and workflows crucial to optimal use of the lab.TRANSCRIPT
Laboratory Diagnosis of Infectious Disease: From Gram-stain to Genomes
Professor Mark Pallen
Outline Laboratory Diagnosis of Infection
Appropriate use of the lab Microscopy Culture Sensitivities Immunoassays
Rapid Methods High-throughput sequencing
Clinical Diagnosis Non-microbiology
investigations Radiology Haematology Biochemistry
Diagnosis of Infection
Laboratory Diagnosis of Infection If physician suspects infection, samples of
tissue or fluid collected for Microbiological analyses Immunological analyses Molecular-biological analyses
Samples include blood urine faeces sputum cerebrospinal fluid pus
Diagnostic workflow Specimen collection Specimen receipt Specimen processing Testing Interpretation Reporting
A proper clinical assessment is essential for optimal use of laboratory services!
Garbage inGarbage outLaboratoryGarbage inGarbage outLaboratory
Is your investigation worthwhile?Do you know whatinformation you want?Does it affect patient
management?Is the informationalready available?
Contact the lab for info onBest test
Type of sampleTiming of sample
Transport of sampleInterpretation of results
Give the lab all relevant clinicalinformation
e. g. antibiotic treatmentrecent travel
special risks etc
stop!thinkagain
yesnoyesyesCan the lab provide thisinformation?
nononoyesnonoHappyclinician
Happymicrobiologist
Happypatient
Happymanager
Is your investigation worthwhile?Do you know whatinformation you want?Does it affect patient
management?Is the informationalready available?
Contact the lab for info onBest test
Type of sampleTiming of sample
Transport of sampleInterpretation of results
Give the lab all relevant clinicalinformation
e. g. antibiotic treatmentrecent travel
special risks etc
stop!thinkagain
yesnoyesyesCan the lab provide thisinformation?
nononoyesnonoHappyclinician
Happymicrobiologist
Happypatient
Happymanager
Factors limiting usefulness of mirobiology investigations Specimens must be
obtained and handled properly Specimen should be
obtained from site of infection
Sample must be taken aseptically
Sample size must be large enough
Metabolic requirements for the organism must be maintained during sampling, storage, and transport
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
Safety in the Microbiology Laboratory Clinical microbiology labs
present significant biological hazards for workers
Standard lab practices for handling clinical samples are in place to protect workers
Laboratories are classified according to their containment potential, or biosafety level (BSL), and are designated as BSL-1 through BSL-4
Laboratory Diagnosis of Infection
culture
on plates or in broth
identification by biochemical or serological tests on pure
growth from single colony
microscopy
Decolorise CounterstainStain
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
Stained preparations Gram-stain Acid-fast stain
Ziehl-Neelsen
Fluorescence Direct, e.g. auramine Immunofluorescence
Crystal violet
Gram's iodine
Decolorise with acetone
Counterstain withe.g. methyl red
Gram-positives appear purple
Gram-negatives appear pink
The Gram Stain
Gram-positive rods
Gram-negative rods
Gram-positive cocci
Gram-negative cocci
The Gram Stain
The Gram Stain The Gram stain can be applied
to pure cultures of bacteria or to clinical specimens
Gram-stain not useful for all bacteria Mycobacteria have very
thick walls and are best seen using an acid-fast staining procedure
Spirochaetes are long spiral bacteria that are too thin to be seen by Gram-stain
Specialised intra-cellular bacteria such as chlamydias and rickettsias cannot be seen by Gram stain
Pure culture of E. coli (Gram-negative rods)
Neisseria gonorrhoeae in a smear of urethral pus(Gram-negative cocci, with pus cells)
Culture of Pathogenic Microbes 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 E.g. blood cultures
Culture of Pathogenic Microbes Although most pathogenic microbes can be grown
after overnight culture in vitro, there are some important exceptions Anaerobes or fastidious bacteria may take several
days/weks Mycobacteria grow very slowly, if at all (M. leprae
uncultivable) Treponema pallidum cannot be cultured in vitro Obligate intracellular bacteria (e.g. Chlamydia,
Rickettsia) need to be grown in cell culture Diagnosis of infection with slow-growing or non-
culturable bacteria tends to rely on molecular methods (PCR) or serodiagnosis (antibody detection)
Culture of Pathogenic Microbes Most microbes of clinical importance can be grown, isolated,
and identified with specialised growth media General Purpose Media
Support growth of most aerobic and facultatively aerobic organisms (e.g., blood agar)
Enriched Media Contain specific growth factors that enhance growth of
certain fastidious pathogens Selective Media
Allow some organisms to grow while inhibiting others Differential Media
Allow identification of organisms based on their growth and appearance on the medium
Advantages of Solid Media tentative identification
of an isolate by colonial chararacteristics E.g. lactose-fermenter
on MacConkey isolation of single
clonal colonies get bacterium in pure
culture allows detailed tests for
definitive identification quantification by
colony-forming units
LF
NLF
Identification of Bacteria Morphology Growth requirements Biochemistry Enzymes Antigens
Sensitivity tests on solid medium
disc diffusion technique E-test
in liquid medium minimum inhibitory concentration (MIC) test
Antimicrobial Susceptibility Testing Disk Diffusion
Test Standard
procedure for assessing antimicrobial activity
Inhibition Zones Used to
determine an organism’s susceptibility to an antimicrobial agent
The E Test
Antimicrobial Susceptibility Testing The MIC (minimum inhibitory
concentration) procedure is used to assess antibiotic susceptibility with regard to various concentrations
8mg/L 4mg/L 2mg/L 1mg/L 0.5mg/L 0.25mg/LAntibiotic concentration
Cloudiness represents growth after overnight incubationmeans bacteria can grow at that concentration of antibioticMIC=2mg/L
Diagnosis of Viral Infection Electron microscopy Antigen detection Antibody detection Virus culture
Detect cytopathic effect or antigen
Molecular methods Polymerase Chain
Reaction
Immunoassays for Infectious Disease Identify infection by measuring antibody titre
against antigen produced by pathogen Agglutination ELISA Radioimmunoassay
T Cell based tests Skin tests Interferon-gamma assays
Agglutination Passive Agglutination
The agglutination of soluble antigens or antibodies that have been adsorbed or chemically coupled to cells or insoluble particles (e.g., latex beads, charcoal)
Reactions can be up to five times more sensitive than direct agglutination tests
Latex Bead Agglutination Test for Staphylococcus aureus
Rapid Microbiological Methods Growth-Based
Technologies measurement of
biochemical or physiological parameters that reflect growth of microorganisms
include: ATP bioluminescence:
AKuScreen to screen for microbial contamination in pharmaceuticals
colorimetric detection of CO2 production: Bactec; BacT/Alert
Cellular Component-Based Technologies
detection of a specific cellular component
include: Fatty acid profiles mass spectrometry ELISA fluorescent probe detection
Rapid Microbiological Methods Nucleic Acid-Based
Technologies DNA probes: Gene-Trak;
Gene-Probe molecular typing polymerase chain reaction
(PCR) and other nucleic acid amplification technologies (NAATs)
sequencing
Automated Methods Simplest use classical
method for processing sample, then detect colorimetric change to spot growth earlier than visual detection
Replace human detection methods with machine detection; human judgment with machine intelligence
include: BacT/ALERT, VITEK
VITEK 2 fully automated system for
bacterial/fungal identification and antibiotic susceptibility testing
reduces set-up time and minimizes manual steps
Compact sealed ID/AST cards Rapid microorganism
identification Rapid, same-day antimicrobial
susceptibility testing Advanced Expert System
validates IDs Data management software
allows for generation of epidemiology reports and antibiograms
Xpert MTB/Rif Sealed cartridge Robust sonication/mechanical DNA extraction procedure Hemi-nested PCR targets rpoB gene associated with
rifampicin resistance 2 hour result
‘Next-generation’ High-throughput sequencing ~100x faster, ~100x cheaper than conventional
approaches Clonal template populations obtained by new
methods: amplification on solid phase to grow a ‘molecular colony’ Massive increase in number of ‘clones’ but shorter read
length New chemistries for sequence reading
Pyrophosphate detection (PPi release upon base addition): 454
Single (reversibly 3’-blocked) fluorescent base (quenchable) added per step: Solexa
Sequencing by Ligation (ABI SOLiD)
High-throughput Sequencing in Clinical Microbiology: Applications
Pathogen detection and discovery Clinical metagenomics
Polymorphism detection and discovery Genomic epidemiology Adaptive changes
Pathogen biology Gene detection and discovery
Sequencing Methodologies
Culture-dependent Culture-independent
Shotgun library of purified genomic DNA Delivers whole-
genome sequence
Phylogenetic profiling: PCR with 16S primers
Metagenomics: shotgun sequence community DNA
Culture-independent Pathogen Discovery
The Birth of Genomic Epidemiology for Bacteria
The Birth of Genomic Epidemiology for Bacteria
Case Study Acinetobacter baumannii Gram-negative bacillus Multi-drug resistant
colistin and tigecycline as reserve agents moving towards pan-resistance
Associated with wound infections and ventilator-associated pneumonia bloodstream infections returning military personnel from Iraq and Afghanistan transmission from military to civilian patients
Acinetobacter Genomic Epidemiology Outbreak in Birmingham Hospital in 2008 Isolates indistinguishable by current typing
methods
Acinetobacter Genomic Epidemiology 454 whole-genome sequencing of 6 isolates SNP detection by mapping reads against draft
reference assembly SNP filtering for false positives SNP validation with Sanger sequencing of PCR
amplicons
Results: Outbreak Isolates Are Distinguishable At Only Three Loci
SNP 1 SNP 2 SNP 3
AB0057 C A G
M1 C A G
M2 T A G
M3 T A T
M4 T A G
C1 T T G
C2 T A G
Take-away messages
Genome sequencing brings the advantages of open-endedness (revealing the “unknown
unknowns”), universal applicability ultimate in resolution
Bench-top sequencing platforms now generate data sufficiently quickly and cheaply to have an impact on real-world clinical and epidemiological problems
http://pathogenomics.bham.ac.uk/blog/2011/08/are-diagnostic-and-public-health-bacteriology-ready-to-become-branches-of-genomic-medicine/
19th Century 21st Century20th Century