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/

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