choosing the right microbial typing method: a quantitative approach
TRANSCRIPT
João André Carriço, PhDMicrobiology Institute/Institute for Molecular MedicineFaculty of Medicine, University of LisbonPortugal
Mayo Clinic, Rochester MS, 2 November 2015
CHOOSING THE RIGHT MICROBIAL TYPING METHOD: A QUANTITATIVE APPROACH
http://im.fm.ul.pthttp://imm.fm.ul.pthttp://www.joaocarrico.infoTwitter: @jacarrico
MICROBIAL TYPING
“Crude classifications and False generalizations are the curse of organized life”
George Bernard Shaw (1856 – 1950)
What is microbial typing: discriminating strains within a species/subspecies, for the identification of clones/lineages of interest
APPLICATIONS OF MICROBIAL TYPING
Bacterial PopulationGenetics
Pathogenesis and
NaturalHistory ofInfection
Surveillance ofInfectiousDiseases
Outbreak Investigation and Control
MICROBIAL TYPING METHODS
Genotypic – gel basedPulsed Field Gel Electrophoresis (PFGE )
Phenotypic : ATB Resistance profiles Serotyping Genotypic - Sequence based
Multilocus Sequence Typing (MLST)Multilocus VNTR Analysis (MLVA) Single Locus methods :
emm typing (Group B ,C & G Strep)
Spa typing (S aureus)
TRADITIONAL TYPING AND NGS
Chronicle of a Death Foretoldhttp://en.wikipedia.org/wiki/File:ChronicleOfADeathForetold.JPG
Next Generation Sequencing:
- Gene-by-gene: wgMLST, cgMLST,
MLST
- SNP approaches: comparison with reference strains
- Ability to recover most of the present sequence based typing information in a single experimental procedure
TYPE /CLONE/LINEAGE AND SUBTYPE CLASSIFICATIONS
Street market, Florence, Italy
Serotype :SerogroupSerotype
emm typing:emm type (95% similarity to prototype)emm subtypes (specific sequence)
Different typing method results are different partitions of a dataset
Spa typing:Spa typeBURP complex
PFGE :PFGE Type (cut-off 80% DICE/UPGMA)PFGE Subtype (cut-off 80% DICE/UPGMA)
MLST /MLVA/ cgMLST/SNP profile:Sequence TypeClonal Complex (eBURST/goeBURST) / cut-off on Minnimun Spanning Trees
TYPE /CLONE/LINEAGE AND SUBTYPE CLASSIFICATIONS
CHOOSING THE RIGHT METHOD
Struelens, M.J. et al, 1996. Clinical microbiology and infection, 2(1), pp.2–11.
Performance criteria:TypeabilityReproducibilityStabilityDiscriminatory powerEpidemiological concordanceTyping System concordance
Convinience Criteria:FlexibilitySpeedCost
Goal
COMPARING TYPING METHODS
Weissman S J et al. Appl. Environ. Microbiol. 2012;78:1353-1360
Conc
aten
ated
MLS
T lo
cus
flmH sequences
The Hard way….
NEED FOR QUANTIFICATION AND STATISTICS
When you can measure what you are talking about and express it in numbers you know something about it. When you cannot measure it, when you cannot express it, your knowledge is of a meagre and unsatisfactory kind.
- Lord Kelvin 1861
COMPARING PARTITIONS FRAMEWORK
Use of three Coefficients :1) Simpson’s Index of Diversity 2) Adjusted Rand3) Adjusted Wallace
And the respective 95% confidence intervals
COMPARINGPARTITIONS WEBSITE
http://www.comparingpartitions.info
Copy/Paste from Excel
COMPARINGPARTITIONS WEBSITE
MEASURING DIVERSITY: SID
Simpson’s Index of Diversity
This index indicates the probability of two strains sampled randomly from a population belonging to two different types
Since it is a probability varies between 0 – 1.
Highly discriminatory methods are desired…
..but are they always needed?
Confidence intervals were defined for SID and should be used.
Simpson, 1948Hunter and Gaston, 1988Grundmann et al ,2001
Comparing SID’s 95% CIs
Null Hypothesis: The values under comparison are the same
COMPARING METHODS RESULTS
PFG
E C
lust
ers1
s2
s3
s4
s5
s6
s7
Same Sequence Type?
Same PFGE cluster?
Y
N
Y N
aa b
c d
For each pair of isolates:
Seq
uenc
e Ty
pe
ADJUSTED RAND
Overall concordance of two methods taking into account that the agreement between results could arise by chance alone.
Bi-directional agreement measureConfidence intervals by jackknife pseudo-values method.
CHANCE AGREEMENT ILLUSTRATION
Two possible random rearrangements…
CHANCE AGREEMENT: RAND VS ADJUSTED RAND
Without correction by chance agreement
CHANCE AGREEMENT: RAND VS ADJUSTED RAND
With correction by chance agreement
ADJUSTED WALLACE
Probability that if two strains share the same classification by a Method A they also share the same classification by Method B, corrected by chance agreement
Analytical confidence intervals.Jackknife pseudo values confidence intervals
COMPARING AR AND AW 95% CI
Null Hypothesis: The values under comparison are the same
OTHER APPLICATIONS FOR SID,AR AND AW
• Determination of the best set of markers for typing purposes : given dozens to hundreds or thousands of possible loci or SNPs is there a subset with enough discrimination to produce the same results as other typing method?
http://www.cidmpublichealth.org/pages/ausetts.html.
OTHER APPLICATIONS FOR SID,AR AND AW
• Determination of the best set of markers /typing methods for typing purposes for predicting a specific outcome or any associated metadata. Examples:
• Using AW to determine the which typing method better predicts a clinical outcome or prognosis.
• Using AW to determine association between alleles and Clonal Complexes (Weissman S J et al. Appl. Environ. Microbiol. 2012;78:1353-1360)
• Determining association between alleles or types and geographical location of sampling
• Determining the best combination of locus to predict a clinical outcome in order to design a fast RT-PCR method
CONCLUSIONS•The larger the sample size the more accurate can be the conclusions. Usually >50 is enough but >100 is better to get statistically significant results. (Severiano PlosOne 2011)
•Always use SID, Adjusted Rand and Adjusted Wallace to have an overall idea how the methods relate
•Confidence intervals give more information than the point estimates because they intrinsically take the sample size into consideration
•Don’t use coefficients that not corrected by chance agreement when comparing typing methods
ACKNOWLEDGEMENTS
Mário RamirezFrancisco Pinto Ana Severiano
Mramirez Lab / UMMI Members
Funding from Fundação para a Ciência e TecnologiaEU 7th Framework programme
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TO KNOW MORE:
For examples of usage see the list of references in:http://www.comparingpartitions.info/index.php?link=References