This document provides an overview of molecular epidemiology, including definitions, applications, tools, and attributes. It defines molecular epidemiology as the combination of molecular biology and epidemiology to study disease distribution and determinants at the molecular level. The document outlines several molecular epidemiology techniques like fingerprinting pathogens using pulsed-field gel electrophoresis and multilocus sequence typing. It also discusses how molecular epidemiology can be used descriptively to examine disease distribution and analytically to evaluate associations with risk factors.

1. Molecular Epidemiology: An
Introduction
Dr. Bhoj R Singh
Act. Head, Division of Epidemiology
Indian Veterinary Research Institute, Izatnagar-243122, Bareilly, UP, India
Phone: +91-8449033222, e-mail: brs1762@ivri.res.in,
web: http://ivri.nic.in/

2. Definitions
• The term "molecular epidemiology" was first coined by Kilbourne in 1973 in his
article "The molecular epidemiology of influenza”.
• A science that deals with the contribution of genetic and environmental risk factors
identified at the molecular and biochemical level, to the etiology, distribution and
control of disease in families and populations (J. Dorman).
• It is “the study of the distribution and determinants of infectious diseases that
utilizes molecular biology methods” (Riley).
• The discipline of molecular epidemiology, in which patterns of disease transmission
are followed using selected markers that distinguish different populations of the
disease-causing agent, is now a well-established central theme in the study of
infectious diseases. (Nadin-Davis, 2013).
• Molecular epidemiology is a discipline that uses molecular or genetic markers to
trace the development of a disease in a population and to understand transmission,
as well as the population structure and evolution of bacterial pathogens (Wang and
Meyer, 2015).
• Molecular epidemiology is the discipline that combines molecular biology with
epidemiology; this means not merely using molecular techniques in epidemiology or
population approaches in molecular biology, but a marriage of the two disciplines
so that molecular techniques are taken into account during study design, conduct,
and analysis (Foxman, 2012).
• Molecular epidemiology has “come of age” and is now well placed to explore the
evolutionary history and global distribution of entire pathogens (Smith 2011).
• It is not just a term that describes adding new techniques to epidemiology. Rather,
it represents an opportunity to use new resolving power to develop theories of
disease causation that acknowledge complex interactions in the health process. (P
Schulte)

3. More Definitions
• Molecular epidemiology offers a powerful approach to the identification of genetic variants
that influence susceptibility to many common diseases (Yucesoy et al., 2015).
• A science that deals with etiology, distribution and control of disease in families and with
inherited causes of diseases in populations (N Morton).
• “the application of sophisticated techniques to the epidemiologic study of biological material”
(Higginson J )
• “molecular epidemiology is the use of biologic markers or biologic measurements in
epidemiologic research” (Schulte PA )
• “the application of molecular biology to the study of infectious disease epidemiology”
(Tompkins LS )
• “using molecular biomarkers in epidemiology” (McMichael AJ )
• “molecular epidemiologic research involves the identification of relations between previous
exposure to some putative causative agent and subsequent biological effects in a cluster of
individuals in populations” (Groopman JD, Kensler TW, Links JM)
• “the analysis of nucleic acids and proteins in the study of health and disease determinants in
human populations” (Hall A)
• “molecular epidemiology uses molecular techniques to define disease and its pre-clinical states,
to quantify exposure and its early biological effect, and to identify the presence of susceptibility
genes” (Shpilberg O, Dorman JS, Ferrell RE, et al.)
• “the practical goals of molecular epidemiology are to identify the micro-parasites responsible
for infectious diseases and determine their physical sources, their biological relationships, and
their route of transmission and those of the genes responsible for their virulence, vaccine
relevant antigens and drug resistance” (Levin BR, Lipsitch M, Bonhoeffer S)

4. Genetic Epidemiology versus Molecular
Epidemiology
• Molecular epidemiology evaluates the association of
variations in known genes with risk of the disease while
genetic epidemiology aims to identify the unknown genes
that influence risk of malignancies (Maria et al., 2007).
• Genetic Epidemiology:
– Is based on population genetics. Utilizes statistical
techniques to evaluate the genetic aspects of chronic
diseases. Little or no emphasis on environmental risk
factors.
• Molecular Epidemiology:
– Molecular surveillance of disease risk factors. Measuring
the geographical and temporal distribution of disease risk
factors. Characterizing the evolution of pathogens and
classifying new pathogen species.

5. Tenets of Molecular Epidemiology
• At the heart of molecular epidemiology lie the concepts of isolate, strain,
and clone. (Isolation, identification and fingerprinting techniques are
required).
• Isolate refers to “a population of microbial cells in pure culture derived
from a single colony on an isolation plate and identified to the species
level”.
• “[A] strain is made up of the descendants of a single isolation in pure culture
and usually made up of a succession of cultures ultimately derived from an
initial single colony”. Riley reserves the term strain for “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, is defined as “an organism or cell, or group of organisms or cells,
produced asexually from one ancestor or stock, to which they are genetically
identical” (Oxford Dictionary).
• Clones are identified using molecular methods to establish the identity. The
genome of a bacterial species fundamentally determines its identity. Thus, gel
electrophoresis techniques like pulsed-field gel electrophoresis can be used in
molecular epidemiology to comparatively analyze patterns of bacterial
chromosomal fragments and to elucidate the genomic content of bacterial cells
(Slattery, M. 2002).
• Requires consideration of standardization, analytical validity and clinical
validity of molecular tests.
• Utilizes family study designs, as well as case-control and cohort studies.

6. Applications of Molecular Epidemiology
• Molecular epidemiology allows for an understanding of the
molecular outcomes and implications of diet, lifestyle, and
environmental exposure, particularly how these choices and
exposures result in acquired genetic mutations and how these
mutations are distributed throughout selected populations
through the use of biomarkers and genetic information.
Molecular epidemiological studies are able to provide additional
understanding of previously-identified. risk factors and disease
mechanisms.
• Specifically it-
– Will facilitate the ability of scientists to conduct etiologic research.
– Will increase our knowledge about the determinants of disease.
– Will contribute to the development of approaches for disease
prevention.
– Will improve public health.
– Provides personalized estimates of risk may empower susceptible
individuals to intervene on: - Diet, lifestyle, Environmental
exposures.
– Helps to build Targeted approaches may be more effective in
preventing disease.

7. Weaknesses & Challenges
• Miquel Porta identified several challenges that the field of molecular epidemiology faces, particularly
selecting and incorporating requisite applicable data in an unbiased manner (Porta, M., 2002)
• Limitations (Slattery, M. 2002) of molecular epidemiological studies are similar in nature to those of
generic epidemiological studies, that is, samples of convenience - both of the target population and genetic
information, small sample sizes, inappropriate statistical methods, poor quality control, and poor definition
of target populations.
• Requires more elaborate team work: Collaboration among-
- Epidemiologists
- Geneticists
- Environmental health scientists
- Health professionals
- Biostatisticians
- Basic scientists
• Challenges: Develop and sustain collaboration among individuals with different
- Backgrounds
- Training
- Experience
- Goals
- Language &
• Foster links with:
- Members of the community
- Policy makers
- Educators
- General public

8. Objectives of Molecular Epidemiology
• Conduct descriptive and analytical studies to
evaluate gene / environment interactions in
disease etiology.
• Provide risk factor-specific morbidity rates for
purposes of education and intervention.

9. Molecular epidemiology tools
Identification of Aetiological Agent(s)
• Conventional Methods
– Culture
– Enzyme-linked immunosorbent assay (ELISA)
– Enzyme immunosorbent assay (EIA)
– Antibodies & Monoclonal antibodies based assays, LFA, agglutination etc.
• Nucleic acid based Methods
– DNA hybridization for known genes
– Direct sequencing of one or more regions
– Multilocus sequence typing (MLST)
• PCR (nucleic acid amplification) based Methods
– Amplification of a single target specific to a pathogen
– Ligase chain reaction (LCR)
– Loop mediated isothermal amplification (LAMP)
• Protein based Methods
– Western blot or immunoblotting.

10. Molecular epidemiology tools
Fingerprinting
• Conventional
– Serotyping, Antibiotic susceptibilities (Antibiogram), Toxinotyping, Biotyping, Phenotyping
• Nucleic acid based
– Plasmid profiles, Plasmid replicon typing
– Restriction fragment length polymorphism (RFLP)
– Pulsed field gel electrophoresis (PFGE)
– Segmented RNA gel electrophoresis, Ribosomal RNA gel electrophoresis
– Direct sequencing of one or more regions and now NGS
– Multilocus sequence typing (MLST)
• PCR Based
– Amplification of a single target specific to a pathogen
– Targeting known repetitive sequences (enterobacterial repetitive intergenic consensus sequences (ERIC),
repetitive extragenic palindromic sequences (REP), double repetitive element (DRE), BOX, insertional
sequence (IS), polymorphic guanine/cytosine-rich repetitive sequences (PGRS))
– Random primers (randomly amplified polymorphic DNA (RAPD), arbitrary primed PCR (AP-PCR)
– Restriction endonuclease of a single amplified product
– Amplified fragment length polymorphism (AFLP)
• Protein based
– Multilocus enzyme electrophoresis (MLEE)
– Enzyme-linked immunosorbent assay (ELISA)
• Gene expression
– Reverse transcriptase PCR
– Microarray technologies

11. Comparative and library typing systems.
• Pathogen typing methods can be described as comparative or library typing
systems:
– The comparative typing system is mainly used for outbreak investigation, a set
of outbreak-related and unrelated isolates are tested to identify outbreak-related
strains and to distinguish epidemic from endemic or sporadic isolates. Generally,
comparative systems produce significant results only in a local context for
delineation of isolates closely related from those significantly different in genomic
backgrounds.
– Library typing methods (or definitive typing), that use more stable genotypic
markers, are useful to compare strains from a current outbreak with previous
circulating strains in order to monitor clonal spread and distribution in different
populations over extended periods of time. Library typing methods can be used in
different laboratories at various time intervals, in order to generate data to be
aggregated in a single database for comparative assessment in great detail, at any
time, in long-term retrospective and prospective multicenter studies, as well as
epidemiological surveillance studies. The methods should be robust and sufficiently
standardized, thus various international networks developed databases on the basis of
molecular typing data in order to standardize library typing methods.
• Comparative or library epidemiological typing systems are not to be considered as
intrinsic characters of each method but an alternative way of use of it, for example, PFGE
may be used as comparative typing in outbreak investigations and as library typing in
surveillance of infectious diseases.

12. Molecular typing methods
Method Principle Advantages Limits
PFGE Whole genome restriction
polymorphism
Excellent discriminatory power,
high intra- and inter-laboratory
reproducibility, high
epidemiological concordance,
moderate cost
Limited ease of use, not rapid,
limited portability, moderate
interpretation, low resolution
for similar fragments size.
AFLP Selective PCR amplification
of a subset of restriction
fragments
Excellent discriminatory power,
high reproducibility
Limited ease of use, not rapid,
high cost
RAPD PCR amplification of
random segments of
genomic DNA with single
primer of arbitrary
nucleotide Sequence
High rapidity, ease of use, low
cost
Low discriminatory power, low
intra-laboratory
reproducibility
rep-PCR PCR amplification of non
coding intergenic
Repetitive Sequences
High rapidity, high
discriminatory power,
ease of use low cost
Low inter-laboratory
reproducibility (improved by
semi-automated commercial
systems)
Variable-
Number
Tandem Repeat
(VNTR) typing
and Multilocus
VNTR analysis
(MLVA)
PCR amplification of
polymorphisms of genomic
variable number tandem
repeat Elements
Excellent reproducibility, high
discriminatory power, ease of
use, accessibility, high rapidity,
moderate cost
Moderate inter-laboratory
reproducibility

13. Method Principle Advantages Limits
Single locus
sequence typing
(SLST)
Sequencing of single
target gene
High discriminatory
power for some species
(e.g. spa-typing for s.
aureus), ease of use, high
rapidity, moderate cost
Potential misclassification of
particular types due to
recombination and/or homoplasy.
Multilocus
sequence typing
(MLST)
Sequencing of allelic
variants of 7
housekeeping genes.
Excellent reproducibility,
portability, standard
nomenclature, high
discriminatory power (not
for all species)
Limited ease of use, not rapid,
limited accessibility, high cost
Comparative
genomic
hybridisation
(CGH):
microarrays
Labelled cDNA/RNA,
hybridized with
specific probes
High throughput ,
simultaneous genotyping
and profiling
Poor accessibility, the intra- and
inter-laboratory reproducibility of
microarray data needs to be
established prior to the application,
high cost
Whole genome -
Next generation
sequencing
(WG-NGS)
Sequencing of
multiple, overlapped
regions
High throughput
technique
Limited ease of use, limited
accessibility
The strong advantage of NGS over traditional Sanger sequencing is its ability to generate
millions of reads in a single run at comparatively low costs. However, WGS-NGS is still too
laborious and time-consuming to obtain useful data in routine surveillance and in small research
and clinical laboratories.

14. Attributes of Molecular Epidemiology
• Descriptive Epidemiology
– Examines the distribution of disease by person, place and
time, consequences to population.
– Rates are expressed as incidence and prevalence (i.e.,
morbidity rates, mortality rates etc.)
• Descriptive Molecular Epidemiology
– Assesses effects and / or outcomes early in the disease
process.
– Reduces heterogeneity in disease classification.
– Examines the distribution of markers of susceptibility or
exposure and aetiologic agent(s).

15. • Analytical Epidemiology
– Evaluates associations with potential risk factors
• Host & Parasite (agent) characteristics.
• Environmental exposures.
– Associations are expressed as relative risks or
odds ratios.
• Analytical Molecular Epidemiology
– Utilizes biological markers to replace surrogate
measures that have been typically employed for
traditional epidemiologic studies
• Genetic susceptibility.
• Environmental exposures or effects.

16. Books in Molecular Epidemiology
• Molecular Epidemiology: Principles and Practices. 2012. Paul A.
Schulte, Frederica P. Perera. Academic Press. pp.588.
• Molecular Epidemiology of Microorganisms: Methods and Protocols
(Methods in Molecular Biology). 2009. Dominique A. Caugant.
• New Frontiers of Molecular Epidemiology of Infectious Diseases. 2012.
Morand, Serge, Beaudeau, François, Cabaret, Jacque. pp. 216.
• The Molecular Epidemiology of Human Viruses. 2002. Leitner,
Thomas. Springer US. pp. 444.
• Molecular Epidemiology of Chronic Diseases. 2008. Chris Wild, Paolo
Vineis, Seymour Garte. John Wiley & Sons, Inc. Wiley Online Library.
• Genetic and Molecular Epidemiology of Multiple Myeloma.
2013. Lentzsch, Suzanne. Springer-Verlag New York. pp. 125.
• Molecular Epidemiology of Infectious Diseases: Principles and
Practices. 2004. Lee W. Riley. ASM Press. pp. 348.
• Molecular Tools and Infectious Disease Epidemiology. 2018. Betsy
Foxman. Academic Press . pp. 240.