Dịch tễ học phân tử: tổng quan và định nghĩa

“ Molecular epidemiology of cytomegalovirus infections in women and their infants”, New England J of Medicine, 1980 Abstract: We studied cytomegaloviruses CMV's isolated from mothers an

National Institute of Infectious Disease

Tokyo, Japan

January 16-20, 2017

Lee W Riley, MD

School of Public Health, University of California, Berkeley

Lecture 1: Molecular Epidemiology: overview and definitions

Principles of Molecular Epidemiology

What the course will cover

 Principles of evolutionary biology applied to infectious diseases

epidemiologic investigations

Paper discussion

What is molecular epidemiology?

 First paper to use the term “Molecular Epidemiology”:

Huang, E S., C A Alford, D W Reynolds, S Stagno, and R F Pass 1980

“ Molecular epidemiology of cytomegalovirus infections in women and their infants”, New England J of Medicine, 1980

Abstract: We studied cytomegaloviruses (CMV's) isolated from mothers and their

children to determine whether recurrent infections and transmission to the fetus in

immune women are due to reinfection or reactivation of endogenous virus …

Endogenous CMV appears to be most frequent source of recurrent infection and

intrauterine transmission in immune women; reinfection also occurs, but less commonly.

What is molecular epidemiology?

Definitions:

 Epidemiology: Study of the distribution and determinants of

distribution of diseases in human (and non-human animal) population

 Molecular epidemiology of infectious diseases

 Study of the distribution and determinants of distribution of infectious diseases using molecular techniques

 Study of the genetics of pathogens that determine disease transmission

 Next generation molecular epidemiology:

 Study of the distribution and determinants of distribution of infectious diseases using next-generation sequencing methods

Is this epidemiology?

 “Molecular epidemiology of the sil streptococcal invasive locus

in group A streptococci causing invasive infections in French children”

We found 31 different emm-toxin genotypes among 74 group A

streptococcal isolates causing invasive infections in French children The

predominant emm types were emm1 (25%), emm3 (8%), emm4 (8%),

emm6 (7%), and emm89 (9%) Sixteen percent of isolates harbored the streptococcal invasive locus, half of them belonging to emm4

Is this epidemiology?

 “ Molecular epidemiology of Mycobacterium tuberculosis in an urban

area in Japan, 2002-2006”

SETTING: Shinjuku City, Tokyo, Japan OBJECTIVE: To evaluate the status of transmission of Mycobacterium

tuberculosis in Shinjuku City to allocate resources efficiently and effectively for a successful tuberculosis (TB) control programme

DESIGN: Observational descriptive study combining the genotype data of M tuberculosis with TB patient profiles

RESULTS: The genotype clustering rate was significantly higher in males (adjusted odds ratio [aOR] 1.94, 95%CI

1.04-3.65, P = 0.038), patients aged <40 years (aOR 2.09, 95%CI 1.17-3.71, P = 0.012) and the homeless (aOR 2.72, 95%CI 1.42-5.20, P = 0.002), and was lower for the foreign-born (aOR 0.21, 95%CI 0.06-0.76, P

= 0.017) Among 45 genotype clusters containing 152 TB patients, 26 clusters containing 102 patients (67.1%) were composed of a mix of homeless and non-homeless patients

Definitions: cont.

development of an organism

natural, related groups based on a factor common to each

nucleic acid sequences to infer evolutionary relationships of organisms

studies relationship of organisms to each other

organisms to each other and to their hosts within an

environmental context

Components of epidemiology of infectious diseases

 People and nonhuman animals

 Pathogen

 Environment

 Hypothesis generation about risks and causes

 Identification of risks

 Suggestions for approaches to identify causes

 Devise appropriate intervention

Components of molecular epidemiology of infectious diseases

 People and nonhuman animals

 Pathogen characterized genetically

 Environment

 Hypothesis generation about risks and causes

 Identification of risks

 Suggestions for approaches to identify causes

 Devise appropriate intervention

Epidemiology vs phylogeny/taxonomy/molecular evolution

 Epidemiology

hypotheses can be generated and tested empirically

provides opportunity for intervention

Taxonomy example: Changes in the classification of Salmonella:

 Before 1960s: >1000 “species”, based on O, H, and Vi antigens

(Kauffman-White scheme)

 1960s-early 80s: 3 species (S typhi, S cholerasuis, S enteritidis), based

on biochemical reactions (Ewing’s classification)

 Current: 2 species (S enterica, S bongori), based on rRNA sequence

What is “species”?

(Janda & Abbott; J Clin Microbiol 2007)

 Number of bacteria ranked at the level of species:

1980: 1,791

2012: 9,620

(http://www.bacterio.cict.fr/number.html#total)

http://explorebio.wikispaces.com/The+Art+of+Phylogeny

“Species” (Janda & Abbott; J Clin Microbiol 2007)

 DNA-DNA hybridization (“gold standard”):

Species definition:

 >70% DNA-DNA relatedness and

 5°C or less TM for the stability of heteroduplex molecules

“Species” (Janda & Abbott; J Clin Microbiol, 2007)

Species definition: strains with <97% similarity score belong to new species

Similarity score >97% unclear; no general agreement

catalogued (http://rdp.cme.msu.edu/)

Bacteria that cannot be classified accurately by 16S rRNA sequencing (Janda & Abbot, JCM, 2007)

Aeromonas A veronii

Bacillus B anthracis,B cereus, B globisporus, B psychrophilus

Bordetella B bronchideptica, B parapertussus, B pertussus

Burkholderia B cocovenenans, B gladioli, B pseudomallei, B thailandensis

Campylobacter Non-jejuni-coli group

Edwardsiella E tarda, E hoshinae, E ictaluri

Enterobacter E cloacae

Neisseria N coinerea, N meningitidis

 Set of all the genes within a species

 Core genome : genes found in all strains in a species

 Dispensable genome : genes found in 2 or more strains of a species

 Unique genes : genes specific to one strain

core

dispensable

unique

E coli pan-genome (Kars RS et al, BMC Genomics, 2012)

• 186 E coli isolates

• 945,211 genes

• 16,373 gene clusters

• 3051 “soft core” genes

• 1702 “strict core” genes

“soft core” –found in 95%

“strict core”—found in 100%

Phylogenetic tree of E coli O157:H7 by their core genes

(Kaas RS et al, 2012)

Phylogenetic tree based on 1278 core genes of 186 E coli strains

(Kaas et al, 2012)

Core/pangenome ratio (Raouli et al, New Microbes and New Infections,2015)

Scope of investigation covered by epidemiology

Identifying…

 disease occurrence and distribution in time and place

 reservoir of infectious agents

 modes and pattern of disease transmission

 setting of disease transmission

 pathogen-related biologic factors that influence transmission

 host-related (demographic, behavioral, clinical, genetic) factors that influence

transmission

 environmental factors (socioeconomic, anthropologic, ecologic) that influence

transmission

Scope of investigations covered by next generation molecular epidemiology

Identifying …

 risk factors that could not be identified by conventional or early-generation molecular biology laboratory methods

 new or hidden transmission pathways

 direction of transmission of an infectious agent

 endogenous reactivation vs exogenous reinfection

 ecological niche from which clonal pathogenic strains are selected and

disseminate

 pathogen microbial population structures associated with a syndrome

 host commensal microbial population structures that determine

non-communicable disease outcomes

Infectious disease epidemiological problems

addressed by molecular biology techniques (2009)

occurrence

saprophytes

institutional infections

Infectious disease epidemiological problems addressed by

molecular biology techniques (2016)

 Tracking strains across time and geography

 Distinguishing endemic from epidemic disease occurrence

 Stratification of data to refine study designs

 Distinguishing pathovars vs commensal flora or saprophytes

 Identifying new modes of transmission

 Studying microorganisms associated with healthcare or institutional infections

 Surveillance and monitoring response to intervention

 Characterizing population distribution and determinants of distribution of parasitic organisms

 Identifying genetic basis for disease transmission

 Validating microdiversity genotyping methods applied to epidemiology

 Virus quasispecies population structure analysis

 Identifying direction and chain of transmission

 Identifying hidden social networks and transmission links

 Analyzing microbiomes to study non-infectious disease epidemiology

NGS

Definitions: cont

 Isolate: a population of microbial cells in pure culture derived from a single colony on an isolation plate and identified to the species level

 Strain: an isolate or group of isolates exhibiting phenotypic and/or

genotypic traits belonging to the same lineage, distinct from those of

other isolates of the same species

 Clone :: an isolate or group of isolates descending from a common

precursor strain by non-sexual reproduction exhibiting phenotypic or genotypic traits characterized by a strain typing method to belong to the same group

 Genotype: a strain belonging to a group of strains shown to be

Definitions: cont.

a specific and discrete unit of information belonging to a strain displayed upon application of a strain typing

procedure (Examples: antibiotic resistance pattern,

serotype, electrophoretic banding pattern, nucleic acid

sequence)

When to use molecular biology techniques to

address an epidemiological problem

 Simplicity (not necessarily speed)

 High throughput: capacity to process a large number of samples simultaneously

Criteria for evaluating performance of strain typing techniques ( Maslow

“What we observe is not nature itself, but  nature exposed to our method of 

questioning.”

–Werner Heisenberg, 1963.

Ask a question before you start a study!!!!

Questions before you start a study:

1 Which molecular typing method should I use?

2 How should I analyze the PFGE patterns that I generated from my collection of

E coli isolates?

3 What kind of information should I get to compare the strain typing data I have?

4 What is the best genotyping method for Salmonella?

Questions must be based on a

hypothesis or a set of hypotheses—not

 Maslow JN, Mulligan ME, Albeit RD Molecular epidemiology: application of

contemporary techniques to the typing of microorganisms Clin Infect Dis 1993; 17:153-64.

 Streulens M and Members of ESGEM Consensus guidelines for appropriate use and evaluation of microbial epidemiologic typing systems Clin Microbiol Infect 1996;2:2-11.