![Page 1: Choosing the Right Microbial Typing Method: A Quantitative Approach](https://reader035.vdocuments.net/reader035/viewer/2022081513/5879a61b1a28ab082c8b6f7f/html5/thumbnails/1.jpg)
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
![Page 2: Choosing the Right Microbial Typing Method: A Quantitative Approach](https://reader035.vdocuments.net/reader035/viewer/2022081513/5879a61b1a28ab082c8b6f7f/html5/thumbnails/2.jpg)
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
![Page 3: Choosing the Right Microbial Typing Method: A Quantitative Approach](https://reader035.vdocuments.net/reader035/viewer/2022081513/5879a61b1a28ab082c8b6f7f/html5/thumbnails/3.jpg)
APPLICATIONS OF MICROBIAL TYPING
Bacterial PopulationGenetics
Pathogenesis and
NaturalHistory ofInfection
Surveillance ofInfectiousDiseases
Outbreak Investigation and Control
![Page 4: Choosing the Right Microbial Typing Method: A Quantitative Approach](https://reader035.vdocuments.net/reader035/viewer/2022081513/5879a61b1a28ab082c8b6f7f/html5/thumbnails/4.jpg)
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)
![Page 5: Choosing the Right Microbial Typing Method: A Quantitative Approach](https://reader035.vdocuments.net/reader035/viewer/2022081513/5879a61b1a28ab082c8b6f7f/html5/thumbnails/5.jpg)
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
![Page 6: Choosing the Right Microbial Typing Method: A Quantitative Approach](https://reader035.vdocuments.net/reader035/viewer/2022081513/5879a61b1a28ab082c8b6f7f/html5/thumbnails/6.jpg)
TYPE /CLONE/LINEAGE AND SUBTYPE CLASSIFICATIONS
Street market, Florence, Italy
![Page 7: Choosing the Right Microbial Typing Method: A Quantitative Approach](https://reader035.vdocuments.net/reader035/viewer/2022081513/5879a61b1a28ab082c8b6f7f/html5/thumbnails/7.jpg)
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
![Page 8: Choosing the Right Microbial Typing Method: A Quantitative Approach](https://reader035.vdocuments.net/reader035/viewer/2022081513/5879a61b1a28ab082c8b6f7f/html5/thumbnails/8.jpg)
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
![Page 9: Choosing the Right Microbial Typing Method: A Quantitative Approach](https://reader035.vdocuments.net/reader035/viewer/2022081513/5879a61b1a28ab082c8b6f7f/html5/thumbnails/9.jpg)
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….
![Page 10: Choosing the Right Microbial Typing Method: A Quantitative Approach](https://reader035.vdocuments.net/reader035/viewer/2022081513/5879a61b1a28ab082c8b6f7f/html5/thumbnails/10.jpg)
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
![Page 11: Choosing the Right Microbial Typing Method: A Quantitative Approach](https://reader035.vdocuments.net/reader035/viewer/2022081513/5879a61b1a28ab082c8b6f7f/html5/thumbnails/11.jpg)
COMPARING PARTITIONS FRAMEWORK
Use of three Coefficients :1) Simpson’s Index of Diversity 2) Adjusted Rand3) Adjusted Wallace
And the respective 95% confidence intervals
![Page 12: Choosing the Right Microbial Typing Method: A Quantitative Approach](https://reader035.vdocuments.net/reader035/viewer/2022081513/5879a61b1a28ab082c8b6f7f/html5/thumbnails/12.jpg)
COMPARINGPARTITIONS WEBSITE
http://www.comparingpartitions.info
![Page 13: Choosing the Right Microbial Typing Method: A Quantitative Approach](https://reader035.vdocuments.net/reader035/viewer/2022081513/5879a61b1a28ab082c8b6f7f/html5/thumbnails/13.jpg)
Copy/Paste from Excel
COMPARINGPARTITIONS WEBSITE
![Page 14: Choosing the Right Microbial Typing Method: A Quantitative Approach](https://reader035.vdocuments.net/reader035/viewer/2022081513/5879a61b1a28ab082c8b6f7f/html5/thumbnails/14.jpg)
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
![Page 15: Choosing the Right Microbial Typing Method: A Quantitative Approach](https://reader035.vdocuments.net/reader035/viewer/2022081513/5879a61b1a28ab082c8b6f7f/html5/thumbnails/15.jpg)
Comparing SID’s 95% CIs
Null Hypothesis: The values under comparison are the same
![Page 16: Choosing the Right Microbial Typing Method: A Quantitative Approach](https://reader035.vdocuments.net/reader035/viewer/2022081513/5879a61b1a28ab082c8b6f7f/html5/thumbnails/16.jpg)
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
![Page 17: Choosing the Right Microbial Typing Method: A Quantitative Approach](https://reader035.vdocuments.net/reader035/viewer/2022081513/5879a61b1a28ab082c8b6f7f/html5/thumbnails/17.jpg)
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.
![Page 18: Choosing the Right Microbial Typing Method: A Quantitative Approach](https://reader035.vdocuments.net/reader035/viewer/2022081513/5879a61b1a28ab082c8b6f7f/html5/thumbnails/18.jpg)
CHANCE AGREEMENT ILLUSTRATION
Two possible random rearrangements…
![Page 19: Choosing the Right Microbial Typing Method: A Quantitative Approach](https://reader035.vdocuments.net/reader035/viewer/2022081513/5879a61b1a28ab082c8b6f7f/html5/thumbnails/19.jpg)
CHANCE AGREEMENT: RAND VS ADJUSTED RAND
Without correction by chance agreement
![Page 20: Choosing the Right Microbial Typing Method: A Quantitative Approach](https://reader035.vdocuments.net/reader035/viewer/2022081513/5879a61b1a28ab082c8b6f7f/html5/thumbnails/20.jpg)
CHANCE AGREEMENT: RAND VS ADJUSTED RAND
With correction by chance agreement
![Page 21: Choosing the Right Microbial Typing Method: A Quantitative Approach](https://reader035.vdocuments.net/reader035/viewer/2022081513/5879a61b1a28ab082c8b6f7f/html5/thumbnails/21.jpg)
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
![Page 22: Choosing the Right Microbial Typing Method: A Quantitative Approach](https://reader035.vdocuments.net/reader035/viewer/2022081513/5879a61b1a28ab082c8b6f7f/html5/thumbnails/22.jpg)
COMPARING AR AND AW 95% CI
Null Hypothesis: The values under comparison are the same
![Page 23: Choosing the Right Microbial Typing Method: A Quantitative Approach](https://reader035.vdocuments.net/reader035/viewer/2022081513/5879a61b1a28ab082c8b6f7f/html5/thumbnails/23.jpg)
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.
![Page 24: Choosing the Right Microbial Typing Method: A Quantitative Approach](https://reader035.vdocuments.net/reader035/viewer/2022081513/5879a61b1a28ab082c8b6f7f/html5/thumbnails/24.jpg)
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
![Page 25: Choosing the Right Microbial Typing Method: A Quantitative Approach](https://reader035.vdocuments.net/reader035/viewer/2022081513/5879a61b1a28ab082c8b6f7f/html5/thumbnails/25.jpg)
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
![Page 26: Choosing the Right Microbial Typing Method: A Quantitative Approach](https://reader035.vdocuments.net/reader035/viewer/2022081513/5879a61b1a28ab082c8b6f7f/html5/thumbnails/26.jpg)
ACKNOWLEDGEMENTS
Mário RamirezFrancisco Pinto Ana Severiano
Mramirez Lab / UMMI Members
Funding from Fundação para a Ciência e TecnologiaEU 7th Framework programme
www.comparingpartitions.info
![Page 27: Choosing the Right Microbial Typing Method: A Quantitative Approach](https://reader035.vdocuments.net/reader035/viewer/2022081513/5879a61b1a28ab082c8b6f7f/html5/thumbnails/27.jpg)
Save the date !http://www.escmid.org/immem11
Registration and Abstract submission open at :
Plenary Sessions•Small scale microbial epidemiology - outbreaks•Large scale microbial epidemiology - surveillance•Bioinformatics for genome-based microbial epidemiology•Population genetics: pathogen emergence•Population dynamics: transmission networks and surveillance•Molecular epidemiology for global health and One Health
Parallel Sessions•Food and environmental pathogens•Microbial forensics•Viral evolution and surveillance•Fungal and yeast pathogens•Novel diagnostics and typing methodologies•Antimicrobial resistance: from mechanisms to impact in healthcare-associated infections•Microbial phylogenetic inference•Making sense of genomic data: bioinformatics tools
![Page 28: Choosing the Right Microbial Typing Method: A Quantitative Approach](https://reader035.vdocuments.net/reader035/viewer/2022081513/5879a61b1a28ab082c8b6f7f/html5/thumbnails/28.jpg)
TO KNOW MORE:
For examples of usage see the list of references in:http://www.comparingpartitions.info/index.php?link=References