Methods used in microbial forensics and epidemiological investigations for stronger health systems

Figures & data

Table 1. Common steps of workflow and interaction between microbial forensics and molecular epidemiology during initial investigations.

Steps	Microbial forensics	Molecular epidemiology
1. Collection of specimen	Enforcement of chain of custody Follows standard operating procedures (SOPs) for sampling Lawful and accredited personnel are involved in specimen collection	More research-oriented Utilises active or passive surveillance samples
2. Recognition that an attack or outbreak/disease is happening/ spreading	Requires inputs from healthcare professionals for case definition Answers questions pertinent to: Pathogen’s route of transmission and survivability (airborne, waterborne, vector-borne, persistence on surfaces, etc.) Pathogen’s virulence (comparison with control specimens and detection of virulence factors/enhanced phenotypes/strains) Appropriate control strategy (is there enough time to quarantine or eradicate)
3. Validation for quality assurance and control	Establishes limits and sensitivity of methods and data collected to ensure replicability
4. Analysis of collected specimen	Application of established workflows to collect relevant data (genetic, physical, and chemical) Performed in dedicated laboratories that follow ISO-accredited operating protocols with established chain of custody	Application of established workflows to collect relevant data (genetic, physical, and chemical) Performed in research/academic laboratories that do not necessarily follow stringent SOPs/accreditation

Table 2. Salient features of epidemiological investigations and microbial forensics.

Points of comparison	Epidemiological investigations	Microbial forensic investigations
Primary goal	Source-tracking of the pathogen to ensure prevention of the transmission and spread of disease [5, 11]	Source-tracking of the pathogen to accomplish attribution [5, 6]
Use of molecular methodologies	Present, molecular biotechniques that derive information from nucleic acids, proteins, etc. [5, 12]	Present, molecular biotechniques that derive information from nucleic acids, proteins, etc. [5, 12]
Use of traditional forensic methods	Absent [15]	Present, especially in cases of covert biocrime or bioterrorism (fingerprinting, trace material analysis, handwriting analysis) [15]
Standard of evidence production	Techniques used should be validated, peer-reviewed, and accepted by the relevant scientific community [7, 10, 11]	Techniques used should be validated, peer-reviewed, and accepted by the relevant scientific community; Chain of custody is strictly observed [7, 10, 11]
Operates within a legal framework?	No [7, 10, 11]	Yes, country-specific and international (at the level of the United Nations Security Council if multiple geographical locations are involved) [7, 10, 11, 23]

Table 3. Comparison among molecular techniques employed in microbial forensics and molecular epidemiology.

Molecular techniques	Advantages/strengths	Disadvantages/weaknesses
Nucleic acid sequence analyses and fingerprinting	Uses a stable biomolecule as the data source, i.e. DNA Analyses can be carried out without direct manipulation of DNA, at the minimum DNA extraction and shearing in DNA fingerprinting	Analyses dependent on quantity and quality of extracted DNA Database-dependent—analyses involving novel sequences/fingerprints may be inconclusive
Molecular phylogeny	Applicable to present and historical (banked) samples Could be used in deriving supplementary information in support of taxonomy assignment	Interpretation of resulting outputs will require advanced technical expertise Database-dependent—if reference sequences are limited, analyses and potential inferences are affected
Gene expression	Complements/supplements genotype, biochemical, and chemotaxonomic data Helpful in understanding the physiology of pathogens	Costly and may not be suitable in resource-limited settings, i.e. LMICs Needs comprehensive understanding of the role of the gene of interest in microbial metabolism
Microbe-derived biomolecule analyses	Could serve as direct evidence, i.e. bacteria-derived toxins Could be used in deriving supplementary information in support of taxonomy assignment	Costly and may not be suitable in resource-limited settings, i.e. LMICs Database-dependent—limited analyses if the database is not all-encompassing or is not regularly updated

LMICs: lower- and middle-income countries.

Figure 1. Key activities in establishing a national microbial forensic system.

Table 4. Challenges in establishing national microbial forensic systems.

Limitations	Potential problems/challenges
Financial resources	Expensive for countries with no resources and no pre-systems/early adopters for forensic analysis
Natural ubiquity, high genetic diversity, and rapid evolutionary adaptability of microbes	Sufficient biobanking facility to maintain and preserve all microbial strains encountered during investigations Continuous evolution and adaptation of microbes resulting in emerging and re-emerging pathogens
Methodological and analytical requirements	Absence of robust molecular typing systems Choice of informative analytical tools
Infrastructure and human resource requirements	Low number of trained scientists and technicians Absence of microbial forensics-ready laboratories

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