molecular epidemiology of infectious diseases

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Molecular Epidemiology of infectious diseases Gilman K.H. Siu Assistant Professor, The Hong Kong Polytechnic University Ph.D, BSc, MSB, MB(ASCP i )

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Molecular Epidemiology of Infectious Diseases

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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)

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Principle and Approach

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

Page 4

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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Application of Strain-Typing Techniques in Epidemiology

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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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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

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

Page 38

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)

Page 42

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

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Do You Have

Any Questions?