Molecular Epidemiology of Infectious Diseases

Molecular Epidemiology of
infectious diseases
Gilman K.H. Siu

Assistant Professor, The Hong Kong Polytechnic University
Ph.D, BSc, MSB, MB(ASCPi)
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Principle and Approach
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General Concept
Molecular
Biology
Epidemiology
Microbiology
Molecular epidemiology of
infectious diseases
Integrates
practices and
principle of
molecular
biology and
microbiology
with those of
epidemiology
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Definition
Epidemiology:
The study of the distribution and determinants of diseases
and injures in human populations
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
Determinants
To determine their physical sources

To identify the genetic factors that
determine and regulate an
organisms specific pattern of
transmission
To identify the genes responsible for

their virulence, vaccine-relevant
antigens and drug resistance
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
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Molecular Phylogeny
Molecular Taxonomy:
Science of classification of organisms into
naturally related groups based on genetic
factors common to each
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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Molecular Phylogeny:
Study of lines descent or evolutionary
development of an organism
Both epidemiology and phylogeny
describe the distribution of
particular genetic attributes of a
pathogen in a population over time
Inferred evolution history of

H3N2 by genetic attributes
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
Determine the associationYOUR

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genetic
attributes with disease severity
Application of StrainTyping Techniques in

Epidemiology
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Epidemiological problems addressed by strain-typing

techniques
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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techniques
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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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 crosssectional or prospective studies to
determine the proportion of infections
caused by such a strain.
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techniques
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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techniques
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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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 -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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techniques
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
-
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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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Examples of Strain-typing
methods
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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

Involve analysis of the whole genome high discriminative power
Restriction fragment length polymorphism (RFLP)
- with / without probe hybridization
Pulsed field gel electrophoresis (PFGE)
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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Principle
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
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RFLP with DNA probe
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
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Application
IS6110-RFLP- Gold standard method for epidemiological investigation of

M. tuberculosis transmission
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Limitations
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
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Pulsed field gel electrophoresis

Principle
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
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PFGE protocol and rare cutting enzymes have been standardized for
various bacterial pathogen
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Application
Investigation of MRSA outbreak in Neonatal ICU
Incubator 1
Incubator 2
MRSA
MRSA
MRSA
MRSA
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Application
MRSA isolated from neonates who lived in incubator 1 had the same antibiogram
Cli-S/R; Ery-R; Fus-S/R; Gen-S/R; Mth-R; Rif-S/R; Van-S
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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Internal
air External
14
MRSA
in
neonates
control
sample control
Standardized
interpretation rule
Closely related
Difference in 2-3
fragments
Possibly related
Difference in 4-6
fragments
Unrelated
Difference in 7
fragments
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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)

Principle
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
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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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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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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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A web-based database for MIRU-VNTR typing - www.miru-vntrplus.org/
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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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Very High discriminative power !!
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
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Staphylococcus protein A gene (spa) typing

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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Step 1:
Obtain a sequence of spa using the PCR-sequencing protocol described by Harmsen D et
al. Harmsen D., Claus H., Witte W., Rothgnger 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
Step 2:
Identify the ends of the hypervariable region of the spa gene
TAAAAGCTAAAAGCTAAACGATGCTCAAGCACCAAAAGAGGAAGACAATAACAAGCCTGGTAAAGAAGACAACA
ACAAACCTGGCAAAGAAGACGGCAACAAGCCTGGCAAAGAAGACGGCAACAAGCCTGGTAAAGAAGATGGCAAC
AAACCTGGTAAAGAAGACAACAAAAAACCTGGTAAAGAAGACGGCAACGGAGTACATGTCGTTAAACCTGGTGA
TACAGTAAATGACATTGCAAAAGCAAACGGCACTACTGCTGA
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Step 3:
Find out the 24-bp repeating units
TAAAAGCTAAAAGCTAAACGATGCTCAAGCACCAAAA
GAGGAAGACAATAACAAGCCTGGT
AAAGAAGACAACAACAAACCTGGC
AAAGAAGACGGCAACAAGCCTGGC
6 x 24-bp
AAAGAAGACGGCAACAAGCCTGGT
repeating units
AAAGAAGATGGCAACAAACCTGGT
AAAGAAGACAACAAAAAACCTGGT
AAAGAAGACGGCAACGGAG
TACATGTCGTTAAACCTGGTGATACAGTAAATGACATT
GCAAAAGCAAACGGCACTACTGCTGA
Step 4:
Type the repeating units by input the
sequence one-by-one to the online database
- Ridom SpaServer
(http://spa.ridom.de/index.shtml)
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Step 5:
Identify the spa type of the strain by input the
repeat succession to the Ridom SpaServer
(http://spa.ridom.de/index.shtml)
r04
r20
r22
r17
r25
r34
04-20-22-17-25-34 (repeat succession)
Spa type: t4673
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Multilocus sequence typing (MLST)

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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MLST databases contain the reference allele sequences and

sequence types for various organisms - http://www.mlst.net/
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Application
Identification of a sequence type of S. aureus that are failed to be

detected by a commercial real time PCR assay
A commercial CE-IVD
MRSA real time PCR
diagnostic assay
showed >95%
sensitivities and
specificities in US and
Europe
But more than 50% MRSA samples in Hong
Kong yield negative results
Characterize these strains with MLST
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Seven housekeeping genes were sequenced

carbamate kinase (arcC), shikimate dehydrogenase (aroE), glycerol kinase (glpF),
guanylate kinase (gmk), phosphate acetyltransferase (pta), triosephosphate
isomerase (tpi) and acetyl coenzyme A acetyltransferase (yqiL)
Most of these strains belong to ST45 highly prevalent MRSA strains in

Hong Kong
C
C
AGC
Other MLST type
Fluorescent signal produced
ACC
ST45
No fluorescent signal produced
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How to choose appropriate molecular typing method ???
Convenience-related features of the methods

Simplicity ability of the method to be handled in nonspecialized and non research laboratories. Molecular
tests require less previous training time than culturebased 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
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How to choose appropriate molecular typing method ???
Performance-related features of the methods

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 strains genetic marker used as the basis for typing.
Portability ability to share interpretable format of the results
among laboratories
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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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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.
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But it provides a good opportunity to validate a

molecular typing technique for epidemiological
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application.
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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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 doesYOUR

not LOGO
necessarily correlate with high discriminatory power of that technique.
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
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epidemiological tool.
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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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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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.
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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 days 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
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Any Questions?
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Molecular Epidemiology of Infectious Diseases

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Molecular Epidemiology of

Gilman K.H. Siu

Principle and Approach

Molecular Epidemiology of Infectious Diseases:

To identify the microorganisms

To determine their physical sources

To identify the genes responsible for

Epidemiology? Taxonomy? Phylogeny?

One third of them deal with infectious disease topic

Epidemiology? Taxonomy? Phylogeny?

Epidemiology targets on organism itself

Epidemiology? Taxonomy? Phylogeny?

Inferred evolution history of

Phylogenetic analysis seeks to infer

Determine the associationYOUR

Application of StrainTyping Techniques in

Epidemiological problems addressed by strain-typing

Determining dynamics of disease transmission in geographically

E.g. Strain typing techniques

Epidemiological problems addressed by strain-typing

Without the strain-typing

The ability to better discriminate

Epidemiological problems addressed by strain-typing

Identifying risk of infectious diseases

A strain characterized by a typing

Information can be used to generate a

Epidemiological problems addressed by strain-typing

Stratifying data and refining epidemiologic study designs

Epidemiological problems addressed by strain-typing

Distinguishing pathovars from nonpathvars

Epidemiological problems addressed by strain-typing

Distinguishing hospital and community infectious disease problems

Long-term care facility

Panton Valentine leukocidin

Associated clinical syndromes

Skin and soft tissue

Sepsis, pneumonia, UTI

Epidemiological problems addressed by strain-typing

Identifying genetic determinants of disease transmission

People with single copy of sickle cell anaemia mutation are

Genetic determinants of microorganisms that facilitate

usually structural genes and regulatory genes

Conventional epidemiological typing methods

Molecular typing methods

Non-amplified typing methods

Amplification-based typing methods

Restriction fragment length polymorphism (RFLP)

Fragmentation of genomic DNA by restriction

No. and size of fragments rely on the

Fragments pattern can be simply visualized by

Two clonally related strains contain almost

Can be theoretically applied on all bacterial

But some restriction sites may be commonly

Restriction fragment length polymorphism (RFLP)

The DNA fragments produced by RE is

The filter is incubated with a probe that

Simplify the restriction fragments pattern by

Sequence differences in the regions

Insertion Sequence 6110 (IS6110) RFLP

High discriminative power

Restriction fragment length polymorphism (RFLP)

IS6110-RFLP- Gold standard method for epidemiological investigation of

Restriction fragment length polymorphism (RFLP)

Involves tedious and time consuming