Molecular epidemiology an introduction

Molecular epidemiology is an emerging branch of epidemiology developed by merging molecular biology into epidemiological studies. Molecular epidemiology studies identify infectious diseases’ causation and pathogenesis and unravel infectious agents’ sources, reservoirs, circulation pattern, transmission pattern, transmission probability, and transmission order. Molecular epidemiology, an area of epidemiology that is somewhat ambiguous, encompasses utilization of biomarkers and genetics as tools to define both exposures (factors that are inherited) and outcomes (factors that are acquired). In last few decades, it has grown extensively to help to understand of disease ecology, in planning disease control, and in drafting health policies.

1. Molecular Epidemiology : An Introduction Dr. Bhoj R Singh Act. Head, Division of Epidemiology Indian Veterinary Research Institute, Izatnagar-243122, Bareilly, UP, India   Phone: [phone], e-mail: [email protected] , 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.