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Molecular Epidemiology of infectious diseases
Gilman K.H. SiuAssistant Professor, The Hong Kong Polytechnic University
Ph.D, BSc, MSB, MB(ASCPi)
YOUR LOGOMolecular epidemiology of
infectious diseases
Microbiology
Epidemiology
Molecular Biology
General Concept
Integrates
practices and
principle of
molecular
biology and
microbiology
with those of
epidemiology
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Definition
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The study of the distribution and determinants of diseases
and injures in human populations
Epidemiology:
Molecular Epidemiology of Infectious Diseases:
The study of the distribution and determinants of infectious
diseases that utilizes molecular biology methods
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Purpose
To identify the microorganisms
responsible for infectious disease
To determine their physical sources
To identify the genetic factors that
determine and regulate an
organism’s specific pattern of
transmission
To identify the genes responsible for
their virulence, vaccine-relevant
antigens and drug resistance
─ Determinants
Distribution
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Epidemiology? Taxonomy? Phylogeny?
One third of them deal with infectious disease topic
More than half only focus on laboratory method, no discussion on
the use of these techniques to characterize disease occurrence,
distribution, or determinants of disease distribution.
NOT molecular epidemiology, but Molecular Taxonomy or
Molecular Phylogeny
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Epidemiology? Taxonomy? Phylogeny?
Science of classification of organisms into
naturally related groups based on genetic
factors common to each
Molecular Taxonomy:
Taxonomy uses strain-typing techniques to
describe properties and characteristics of
organisms
e.g. M. tuberculosis can be subdivided into East
Asia (Beijing) lineage, Africa lineage etc.
Epidemiology targets on organism itself
AND its interactions with the host and the
environment in which it resides.
e.g. M. tuberculosis Beijing lineage is more
virulent and associated with drug resistance
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Epidemiology? Taxonomy? Phylogeny?
Study of lines descent or evolutionary
development of an organism
Molecular Phylogeny:
Both epidemiology and phylogeny
describe the distribution of
particular genetic attributes of a
pathogen in a population over time
Phylogenetic analysis seeks to infer
past evolution event cannot be
empirically confirmed
Epidemiology focuses on events of the
present, uses analytical study designs
to predict or validate the relationship of
these attributes to disease distribution,
transmission and manifestation
Inferred evolution history of
H3N2 by genetic attributes
Determine the association of the genetic
attributes with disease severity
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Epidemiological problems addressed by strain-typing techniques
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Determining dynamics of disease transmission in geographically
widespread disease
Study how an organism or a strain introduce and spread into a
community
Identify reasons for changes in prevalence of infections or
syndromes
Study the factors (host, environment, organism) that contribute to
transmission
E.g. Strain typing techniques
changes our concept in transmission
dynamics of diarrheal disease
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Epidemiological problems addressed by strain-typing techniques
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Without the strain-typing
information, the cases of diarrheal
diseases may appear endemic
(no major fluctuation in
occurrence of disease over time)
The ability to better discriminate
between cases according to
strain types reveal outbreaks that
had not been previously detected.
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Epidemiological problems addressed by strain-typing techniques
Identifying risk of infectious diseases
A strain characterized by a typing
method that has previously been
shown to be linked to a particular
vehicle or reservoir may provide a clue
about the sources of infection
E.g. H9N2 is associated with poultry
Information can be used to generate a
hypothesis and to design cross-
sectional or prospective studies to
determine the proportion of infections
caused by such a strain.
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Epidemiological problems addressed by strain-typing techniques
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Stratifying data and refining epidemiologic study designs
Molecular strain typing methods can group data related to infectious
agents by subtypes a new discrete units.
Patients infected with these organisms can be reclassified according
to these isolates’ new groupings a new case definition.
E.g Patients from whom M. tuberculosis is isolated would all be
grouped as TB patient.
But if those M. tuberculosis strains were typed for drug resistance
Patients can be reclassified according to the drug resistance of
their isolates.
Redefine the cases (patients with DR-TB) and control (patients with
nonDR-TB) to identify risk factors for the infection (e.g risk factors for
drug resistant TB)
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Epidemiological problems addressed by strain-typing techniques
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Distinguishing pathovars from nonpathvars
A pathogenic variant of an organism is
referred as a pathovar
E.g. E. coli was harboured in every
healthy human host. Yet some strains
can cause hemolytic-uremic syndrome
and kill 53 people in Europe in 2011.
Distinguishing between E. coli strains
associated with a particular type of
disease, and separating them from
non-pathogenic variants is an important
activity in epidemiological investigation
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Epidemiological problems addressed by strain-typing techniques
Distinguishing hospital and community infectious disease problems
Issues related to infectious disease problem in hospital settings
are often distinct from those related to field or community settings.
Strain-typing helps to identify the genetic determinants which are
specific to the strains circulating in hospital and community
respectively
CA-MRSA HA-MRSA
At-risk groups/conditions Children, athletes Long-term care facility
residents
SCCmec type IV, V I, II,and III
Antimicrobial resistance Usually β-lactam
resistance alone
Multidrug resistance
Panton Valentine leukocidin Frequent rare
Associated clinical syndromes Skin and soft tissue
infection
Sepsis, pneumonia, UTI
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Epidemiological problems addressed by strain-typing techniques
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Identifying genetic determinants of disease transmission
Infectious disease transmission is an interaction of
characteristic of the pathogen, the host, and the
environment
Molecular typing technique can help to identify:
Genetic determinants of human susceptibility to certain
infectious diseases
- People with single copy of sickle cell anaemia mutation are
naturally resistant to malaria.
Genetic determinants of microorganisms that facilitate
their transmission
- usually structural genes and regulatory genes
- Some strains of Clostridium difficile showed
overexpression of sporulation genes
hyperproduction of spore facilitate their transmission
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Conventional epidemiological typing methods
Antibiogram
Typing based on the susceptibility patterns of
the strains against different antibiotics
Serotyping
Identifying the distinct variations among the
strains based on the interactions between
antibodies and cell surface antigens
Phage typing
Differentiating bacterial strains based on the
specificities of different bacteriophages to
infect particular bacterial strains
Limitation
Low discrimination power – outbreak strains
may share the same antiobiogram with
sporadic strains
Fastidious bacteria / viruses cannot be grown
in culture media
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Molecular typing methods
Non-amplified typing methods
Restriction fragment length polymorphism (RFLP)
- with / without probe hybridization
Pulsed field gel electrophoresis (PFGE)
Involve analysis of the whole genome high discriminative power
Amplification-based typing methods
Involve analysis of single or multiple genetic regions variable
discriminative power
PCR-ribotyping
spa typing
Variable number of tandem repeat (VNTR)
Multiocus sequencing typing (MLST)
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Restriction fragment length polymorphism (RFLP)
Fragmentation of genomic DNA by restriction
enzymes
No. and size of fragments rely on the
frequency and distribution of the cutting sites
Fragments pattern can be simply visualized by
gel electrophoresis
Two clonally related strains contain almost
identical DNA sequences cutting site should
be conserved identical electrophoresis
Can be theoretically applied on all bacterial
species.
But some restriction sites may be commonly
found throughout bacterial genome large
number of DNA fragments or even smears
Principle
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Restriction fragment length polymorphism (RFLP)
The DNA fragments produced by RE is
separated by gel electrophoresis and
transferred to a membrane filter.
The filter is incubated with a probe that
hybridizes to a specific genes
Simplify the restriction fragments pattern by
selecting only those containing the specific
genes
Sequence differences in the regions
flanking the gene of interest lead to
variability in fragment patterns
discriminate between related strains
Insertion Sequence 6110 (IS6110) – RFLP High discriminative power
Still considered as gold standard for M.
tuberculosis typing
RFLP with DNA probe
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Restriction fragment length polymorphism (RFLP)
Application
IS6110-RFLP- Gold standard method for epidemiological investigation of
M. tuberculosis transmission
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Restriction fragment length polymorphism (RFLP)
Involves tedious and time consuming
experimental procedure Gel electrophoresis > 24 hours
Not amplification steps require very
heavy bacterial inocula for DNA extraction
particularly dangerous for MTB
Require intact DNA extract (no DNA
shearing is allowed )
Low throughput
Poor interlaboratory portability
getting improved by image capturing
software which converts analog band
pattern into digital pattern
Limitations
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Pulsed field gel electrophoresis
Bacterial chromosome is digested with
rare cutting enzyme (6-8 bases)
Protect chromosomal DNA from
mechanical damage by immobilizing the
bacteria into agarose block during lysis
Fragments are separated by gel
electrophoresis subjected to an
alternating voltage gradient in which the
orientation of electric field switches
direction periodically
Can resolve DNA fragment up to 800kb
Point mutations, deletions/ insertion, lost
or gain of plasmids difference in
profile among epidemiologically related
strains
Very high discriminative power good
for outbreak investigation
Principle
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Pulsed field gel electrophoresis
PFGE protocol and rare cutting enzymes have been standardized for
various bacterial pathogen
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Pulsed field gel electrophoresis
Incubator 2
MRSA
MRSAMRSAMRSA
Application
Investigation of MRSA outbreak in Neonatal ICU
Incubator 1
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Pulsed field gel electrophoresis
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Cli-S/R; Ery-R; Fus-S/R; Gen-S/R; Mth-R; Rif-S/R; Van-S
Application
MRSA isolated from neonates who lived in incubator 1 had the same antibiogram
PFGE after SmaI digestion were performed for the following samples
14 MRSA from new born
1 MRSA from air sample
2 MRSA from baby with no contact to the incubator
(internal control group)
12 MRSA from a small epidemic in another hospitals
(external control group)
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Pulsed field gel electrophoresis
Internal
control 14 MRSA in neonates External
control
air
sample
Closely related Difference in 2-3
fragments
Possibly related Difference in 4-6
fragments
Unrelated Difference in ≥ 7
fragments
Standardized
interpretation rule
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Pulsed field gel electrophoresis
Limitations
Labour intensive, requiring multiple
days to perform experimental work
Poor portability
Low intra- or inter-laboratory
reproducibility
- The intensity of banding patterns can
be affected by many factors, e.g.
i. Cell concentration
ii. Incomplete digestion of DNA
iii. Faulty electrodes
iv. Uneven gel thickness
v. Buffer height due to uneven
surfaces used for electrophoresis
Some bacterial species, such as
Streptococci spp. and Pseudomonas,
are difficult to be typed due to DNA
degradation by their DNAse production
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Repetitive Sequence-Based PCR (Rep-PCR)
PCR amplification of spacer fragments
lying between repeat motifs of the using
primers that bind to the repeat motifs.
Amplicons are separated by
electrophoresis to generate banding
patterns
Banding patterns differ as a result of the
number of repetitive elements and their
relative position within the bacterial
genome.
Moderate discriminatory power - depends
on the method used and the number of
repetitive sequences present in strains
Also affect by PCR reagents, thermal
cycling and gel electrophoresis
Principle
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Repetitive Sequence-Based PCR (Rep-PCR)
Application
PCR-ribotyping for Clostridium difficile
Uses specific primers
complementary to the 3’ end
of the 16S rRNA gene and
to the 5’ end of the 23S
rRNA gene to amplify the
variable-length intergenic
spacer region.
The most common method
for C. difficile typing
Ribotype 027 considered as
hypervirulent strain highly
associated with severe form
of infection
(Pseudomembranous colitis)
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Repetitive Sequence-Based PCR (Rep-PCR)
In Hong Kong, The most
predominant C. difficile strains is
PCR ribotype 002
Significantly higher sporulation
frequency
(20.2% [002] vs 3.7% [other ribo])
Warning on potential outbreak in
Hong Kong hospitals
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Variable number of tandam repeat (VNTR) analysis
Principle
Repetitive sequence DNA motifs ranging
from a few bases to > 100 bp
Tandem: copies of repeat motifs
clustered together and oriented in the
same direction
No. of repeats in a tandem highly
variable due to DNA strand slippage
during replication
Primers anneal non-repetitive sequences
just outside the repeat region
amplicon size determines no. of repeat
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Variable number of tandam repeat (VNTR) analysis
Application
Mycobacterial Interspersed repeat units – VNTR (MIRU-VNTR)
Short sequence repeats in
M. tuberculosis genome
Tandemly repeated
sequences of 40-100 bp
Location and number of
repeats varies in different M.
tuberculosis strains
Based on 15 to 24 loci
Better predictive value than
IS6110-RFLP
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Variable number of tandam repeat (VNTR) analysis
A web-based database for MIRU-VNTR typing - www.miru-vntrplus.org/
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Variable number of tandam repeat (VNTR) analysis
Application
Typing for C.difficile outbreak investigation
Mini-outbreak in elderly home of Kowloon Hospital
Collected 13 C.difficile isolates from patients with
severe diarrhoeae
PCR-ribotyping reveals all belong to R-002 (cannot
differentiate from sporadic cases)
Investigate 7-loci VNTR
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Variable number of tandam repeat (VNTR) analysis
13 suspected outbreak
isolates from Kowloon
Hospital
3 isolates from other wards
in Kowloon Hospital
Sporadic R002 isolates
from other hospitals
Isolates with a summed
tandem repeat difference
of ≤2 are genetically
related
Very High discriminative power !!
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Staphylococcus protein A gene (spa) typing
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The emergence of MRSA has
become a major concern,
especially in the hospital
environment, because of the
high mortality of the infections
caused by these strains
There is evidence for
recombination in S. aureus
Single locus DNA-sequencing of
repeat regions of the
Staphylococcus protein A gene
(spa) could be used for reliable
and accurate typing of MRSA
Identify the number and the
sequence of repeat unit (24-bp)
in the spa gene
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Staphylococcus protein A gene (spa) typing
Step 1:
Obtain a sequence of spa using the PCR-sequencing protocol described by Harmsen D etal. Harmsen D., Claus H., Witte W., Rothgänger J., Claus H., Turnwald D., & Vogel U. (2003). Typing of methicillin-resistant Staphylococcus
aureus in a university hospital setting using a novel software for spa-repeat determination and database management. J. Clin. Microbiol.41:5442-5448
TAAAAGCTAAAAGCTAAACGATGCTCAAGCACCAAAAGAGGAAGACAATAACAAGCCTGGTAAAGAAGACAACAA
CAAACCTGGCAAAGAAGACGGCAACAAGCCTGGCAAAGAAGACGGCAACAAGCCTGGTAAAGAAGATGGCAACAA
ACCTGGTAAAGAAGACAACAAAAAACCTGGTAAAGAAGACGGCAACGGAGTACATGTCGTTAAACCTGGTGATAC
AGTAAATGACATTGCAAAAGCAAACGGCACTACTGCTGA
TAAAAGCTAAAAGCTAAACGATGCTCAAGCACCAAAAGAGGAAGACAATAACAAGCCTGGTAAAGAAGACAACA
ACAAACCTGGCAAAGAAGACGGCAACAAGCCTGGCAAAGAAGACGGCAACAAGCCTGGTAAAGAAGATGGCAAC
AAACCTGGTAAAGAAGACAACAAAAAACCTGGTAAAGAAGACGGCAACGGAGTACATGTCGTTAAACCTGGTGA
TACAGTAAATGACATTGCAAAAGCAAACGGCACTACTGCTGA
Step 2:
Identify the ends of the hypervariable region of the spa gene
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Staphylococcus protein A gene (spa) typing
TAAAAGCTAAAAGCTAAACGATGCTCAAGCACCAAAA
GAGGAAGACAATAACAAGCCTGGT
AAAGAAGACAACAACAAACCTGGC
AAAGAAGACGGCAACAAGCCTGGC
AAAGAAGACGGCAACAAGCCTGGT
AAAGAAGATGGCAACAAACCTGGT
AAAGAAGACAACAAAAAACCTGGT
AAAGAAGACGGCAACGGAG
TACATGTCGTTAAACCTGGTGATACAGTAAATGACATT
GCAAAAGCAAACGGCACTACTGCTGA
Step 3:
Find out the 24-bp repeating units
6 x 24-bp repeating units
Type the repeating units by input the sequence one-by-one to the online database - Ridom SpaServer (http://spa.ridom.de/index.shtml)
GAGGAAGACAATAACAAGCCTGGT
AAAGAAGACAACAACAAACCTGGC
AAAGAAGACGGCAACAAGCCTGGC
AAAGAAGACGGCAACAAGCCTGGT
AAAGAAGATGGCAACAAACCTGGT
AAAGAAGACAACAAAAAACCTGGT
Step 4:
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Staphylococcus protein A gene (spa) typing
GAGGAAGACAATAACAAGCCTGGT r04
AAAGAAGACAACAACAAACCTGGC r20
AAAGAAGACGGCAACAAGCCTGGC r22
AAAGAAGACGGCAACAAGCCTGGT r17
AAAGAAGATGGCAACAAACCTGGT r25
AAAGAAGACAACAAAAAACCTGGT r34
Step 5:
Identify the spa type of the strain by input the repeat succession to the Ridom SpaServer (http://spa.ridom.de/index.shtml)
04-20-22-17-25-34 (repeat succession)
Spa type: t4673
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Multilocus sequence typing (MLST)
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Characterize isolates of microbial species
using the DNA sequences of internal
fragments of multiple housekeeping genes
(present in all isolates)
Approximately 450-500 bp internal
fragments of each gene
For each housekeeping gene, Sequences
that differ at even a single nucleotide are
assigned as different alleles
About seven to eight house-keeping
genes are commonly used in the
laboratories
For each isolate, the alleles at each of the
loci define sequence type (ST).
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Multilocus sequence typing (MLST)
MLST databases contain the reference allele sequences and
sequence types for various organisms - http://www.mlst.net/
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Multilocus sequence typing (MLST)
A commercial CE-IVD
MRSA real time PCR
diagnostic assay
showed >95%
sensitivities and
specificities in US and
Europe
Characterize these strains with MLST
Application
Identification of a sequence type of S. aureus that are failed to be
detected by a commercial real time PCR assay
But more than 50% MRSA samples in Hong
Kong yield negative results
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Multilocus sequence typing (MLST)
Most of these strains belong to ST45 highly prevalent MRSA strains in
Hong Kong
carbamate kinase (arcC), shikimate dehydrogenase (aroE), glycerol kinase (glpF),
guanylate kinase (gmk), phosphate acetyltransferase (pta), triosephosphate
isomerase (tpi) and acetyl coenzyme A acetyltransferase (yqiL)
Seven housekeeping genes were sequenced
AGCC
ACC
C
Fluorescent signal produced
Other MLST type
No fluorescent signal produced
ST45
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How to choose appropriate molecular typing method ???
Simplicity– ability of the method to be handled in non-
specialized and non research laboratories. Molecular
tests require less previous training time than culture-
based tests do.
Turnaround time – may be important if the investigation
involves clinical management of patients infected with a
pathogen under study, or if the etiologic agent in an
outbreak is unknown, e.g. SARS
High throughout – ability to process a large number of
strains in a reasonable interval of time. In epidemiology,
this may affect the power (due to sample size) of a
statistical test to assess risk and association.
Cost and affordability
Convenience-related features of the methods
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How to choose appropriate molecular typing method ???
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Typeability – ability of the technique to generate an
unambiguous result for an isolate tested. i.e. how many strains
are untypeable? E.g. <1% M. tuberculosis strains do not
harbour IS6110 cannot be typed by IS6110 RFLP.
Discriminatory power – ability to generate distinct units of
information from epidemiologically unrelated isolates
Reproducibility - ability to generate identical results when a
strain is tested repeatedly. It depends on the natural stability of
a test strain’s genetic marker used as the basis for typing.
Portability – ability to share interpretable format of the results
among laboratories
Performance-related features of the methods
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Molecular strain typing methods for outbreak investigation
An outbreak is defined as the acute appearance of a cluster of
disease caused by a single pathogen that occurs in numbers in
excess of what is expected for that time and place.
Strain typing technique must have the ability to distinguish strains
that are epidemiologically related from those that are not by
minimizing misclassification and confounding variables
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Molecular strain typing methods for outbreak investigation
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How do we validate the performance of molecular
strain typing methods for outbreak investigation?
No gold standard method
Most of the time, in an outbreak setting, the study
subjects are already recognized to have characteristics
that define them as being related, e.g. infected with the
same pathogens in a setting or period of time where
such cluster of the disease is not expected.
No strain typing information is needed to define the
disease cluster as an outbreak.
But it provides a good opportunity to validate a
molecular typing technique for epidemiological
application.
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Molecular strain typing methods for outbreak investigation
Demonstrating that a pathogen is clonal in a recognized outbreak
setting Which of the following typing methods have the higher discriminatory power?
What of the following typing methods satisfies the criterion for outbreak
investigation given that FF, F, G, C, CC, J and JJ were isolated from an
outbreak?
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Molecular strain typing methods for outbreak investigation
Demonstrating that a pathogen is clonal in a recognized outbreak
setting
The higher the no. of discrete units of typing information generated by a typing
method from a set of isolates, the higher the discriminatory power of that test.
Method A generate discrete units of information for almost every isolates.
It may be a mistake to abandon the conclusion that an outbreak had occurred
based on information generated by Method A
But Method A is quite useful for taxonomic purpose many subtypes
identified.
Method B is epidemiologically suitable since the relatedness of the strains
according to this method matches with the previous knowledge of outbreak
The utility of a new typing method for epidemiologic purposes does not
necessarily correlate with high discriminatory power of that technique.
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Molecular strain typing methods for outbreak investigation
Selecting appropriate comparison isolates
The validity of a typing method should NOT be evaluated a collection
of isolates from only one outbreak.
The test should be simultaneously applied to appropriate comparison
isolates
The comparison isolates can be :
collected from geographic sources distinct from the setting of
outbreak,
a collection of isolates obtained during some period before or well
after the outbreak
If the method is able to classify the comparison isolates into many
distinct taxonomic units satisfies another criterion for its utility as an
epidemiological tool.
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Molecular strain typing methods for outbreak investigation
Ascertaining fidelity of the typing information
In response to changing selective
pressure, bacteria undergo mutations
during replications or acquire external
genetic materials by transduction or
conjugations
Strain typing method must take into
account the intrinsic “ biological clock” and
evolutionary relationships of the genetic
markers.
Some pathogens predominantly
propagate clonally (like TB) whereas
some undergo frequent genetic
recombination (like E.coli)
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Molecular strain typing methods for outbreak investigation
Ascertaining fidelity of the typing information
Even within a single organism, there are differences in the type, number, and the
rate at which specific genes undergo mutations.
Gene encode outer membrane proteins show greater sequence diversity than
those that encode “housekeeping” genes ( probably because outer membrane
proteins are under selective pressure to undergo more frequent changes)
E.g. the “clock speed” of a single spa gene is equal to, or even greater than the
combination of 7 housekeeping genes used in MLST.
Even the strains have the same
MLST, their spa types are always
different
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Molecular strain typing methods for outbreak investigation
Ascertaining fidelity of the typing information
Typing method needs to be validated epidemiologically or experimentally to
assess the temporal stability of the typing information generated by such a
method.
Subject an organism to multiple passages in vitro and analyze the passaged
strain by the typing method.
If stable typing information obtained after 6 month to a year of multiple passage
may be suitable for future outbreak investigations or short-term investigation
If the typing information remains unchanged for more than 2 years, the method is
useful for surveillance purpose or for multiregional comparison of strains.
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Concluding remark
Molecular epidemiology is not just a technique or a tool
All the laboratory-based information have to be associated with
epidemiological analysis, either analytical or descriptive studies
The ultimate goal of the strain typing method is to reveal a particular
subtype to be associated with a specific risk factor.
E.g Clostridium difficile ribotype 002 has significantly higher sporulation rate
The reliability of a typing test must be assessed in multiple systemically
designed epidemiological studies.
The validity of the typing method is ultimately affirmed by demonstration
of the effectiveness of an intervention based on the epidemiological
observations made possible by the use of the typing method.
E.g. Using more effective disinfectants to eliminate the spores of C. difficile in
the environment. Page 56
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Whole Genome Sequencing (WGS) for microbial typing
Potential
Take all genetic information into account highest discriminatory power
Replace all other sequencing-based method for outbreak investigation
Lower economic costs and turnaround time (US$100 in one day’s time)
Questions?
Any good bioinformatics tools for
translation of results into a format
that can be understood by health
professional
Too discriminative
overestimate the amount of recent
transmission of the disease.
Expensive for large-scale
investigation