Lecture 1: Molecular Epidemiology: overview and definitions

Principles of Molecular Epidemiology

Lecture 1: Molecular Epidemiology: overview and
definitions
National Institute of Infectious Disease
Tokyo, Japan
January 16-20, 2017

Lee W. Riley, MD
School of Public Health, University of California, Berkeley

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What the course will cover
 Basic epidemiology concepts
 Laboratory methods used to conduct epidemiologic investigations
 Principles of molecular epidemiology
 Principles of evolutionary biology applied to infectious diseases
 Practices of molecular epidemiology: examples
 Modular exercises illustrating practical approaches to molecular

epidemiologic investigations
 Next generation molecular epidemiology
 Paper discussion

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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.

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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
 Study of complex microbial niches that determine infectious and noninfectious disease occurrence.

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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.

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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.
CONCLUSION: The study revealed that M. tuberculosis transmission occurred more frequently among the
homeless than in non-homeless persons. However, transmission by casual contact between the homeless and the
general population was also shown to occur.

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Definitions: cont.
 Phylogenetics: study of lines of descent or evolutionary

development of an organism

 Taxonomy: the science of classification of organisms into

natural, related groups based on a factor common to each

 Molecular evolution: phylogenetics based on analyses of

nucleic acid sequences to infer evolutionary relationships of
organisms

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Definitions-cont.
 Taxonomy/phylogenetics/molecular evolution:
studies relationship of organisms to each other
 Molecular epidemiology: studies relationship of

organisms to each other and to their hosts within an
environmental context

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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

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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

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Epidemiology vs phylogeny/taxonomy/molecular evolution
 Epidemiology
 hypotheses can be generated and tested empirically
 provides opportunity for intervention
 not technique-dependent
 Phylogenetics/taxonomy/molecular evolution
 descriptive
 information is inferred
 intervention not implied
 technique-dependent

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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
 S. enterica
 6 subspecies (I, II, IIIa, IIIb, IV, VI)
 2501 serotypes
 S. bongori (formerly subspecies V)

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What is “species”?

(Janda & Abbott; J Clin Microbiol. 2007)

 Number of bacteria ranked at the level of species:
 1980: 1,791
 2012: 9,620

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“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

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“Species” (Janda & Abbott; J Clin Microbiol, 2007)
 16S rRNA sequences:
 Species definition: strains with <97% similarity score

belong to new species
 Similarity score >97%--unclear; no general agreement
 September 30, 2016: 3,356,809 rRNA sequences

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

Genus
Aeromonas

Species

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
Edwardsiella

Non-jejuni-coli group

Enterobacter
Neisseria
Pseudomonas

Streptococcus

E. cloacae

A. veronii

E. tarda, E. hoshinae, E. ictaluri

N. coinerea, N. meningitidis
P. fluorescens, P. jessenii
S. mitis, S. oralis, S. pneumoniae

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Pan-genome
 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
unique

core
dispensable

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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%

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Phylogenetic tree of E. coli O157:H7 by their core genes
(Kaas RS et al, 2012)

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Phylogenetic tree based on 1278 core genes of 186 E. coli strains
(Kaas et al, 2012)

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

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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
 etiologic role of a microbe for a newly-recognized disease or a disease previously not
recognized to be associated with an infectious agent

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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 noncommunicable disease outcomes
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Infectious disease epidemiological problems
addressed by molecular biology techniques (2009)
 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
 Studying microorganisms associated with hospital or
institutional infections
 Identifying genetic basis for disease transmission

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Infectious disease epidemiological problems addressed by
molecular biology techniques (2016)
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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
NGS
Identifying direction and chain of transmission
Identifying hidden social networks and transmission links
Analyzing microbiomes to study non-infectious disease epidemiology

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