RESUMEN

The coronavirus disease 2019 pandemic accelerated developments in biotechnology that underpin infection science. These advances present an opportunity to refresh the microbial forensic toolkit. Integration of novel analytical techniques with established forensic methods will speed up acquisition of evidence and better support lines of enquiry. A critical part of any such investigation is demonstration of a robust causal relationship and attribution of responsibility for an incident. In the wider context of a formal investigation into agency, motivation and intent, the quick and efficient assembly of microbiological evidence sets the tone and tempo of the entire investigation. Integration of established and novel analytical techniques from infection science into a systematic approach to microbial forensics will therefore ensure that major perspectives are correctly used to frame and shape the evidence into a clear narrative, while recognizing that forensic hypothesis generation, testing and refinement comprise an iterative process. Development of multidisciplinary training exercises that use this approach will enable translation into practice and efficient implementation when the need arises.

Asunto(s)

RESUMEN

The Anthrax mailings bioterrorism attack in 2001 revealed the need for universal and rapid microbial forensic analyses on unknown biological evidence. However, the gold standard for bacterial identification includes culturing isolates, which is laborious. Molecular approaches for bacterial identification revolve around 16S ribosomal gene sequencing using Sanger or next generation sequencing (NGS) platforms, but these techniques are laboratory-based and can also be time-consuming. The Oxford Nanopore Technologies (ONT) MinION sequencer can generate long read lengths that span the entire bacterial 16S rRNA gene and accurately identify the species level. This platform can be used in the field, allowing on-site evidence analysis. However, it requires higher quantities of pure DNA compared to other sequencing platforms; thus, the extraction method for bacterial DNA is critical for downstream analysis, which to date are tailored toward a priori knowledge of the species' taxonomic grouping. During an attack, the investigative team may not know what species they are handling; therefore, identifying an extraction method that can handle all bacterial groups and generate clean DNA for the MinION is useful for microbial forensic analysis. The purpose of this study was to identify a "universal" extraction method that can be coupled with ONT MinION sequencing for use in forensic situations for rapid identification. It also evaluated the cloud-based data analysis software provided by ONT, EPI2ME. No "universal" extraction method was identified as optimal for downstream MinION sequencing. However, the DNeasy PowerSoil Kit and Noda et al. Chelex-100 method gave comparable sequencing results and could be used as rapid extraction techniques. This study showed that the ONT 16S Barcoding Kit 1-24 coupled with the 16S FASTQ workflow might not be the best for use in forensic situations where species-level identification needs to be obtained, as most alignments were approximately 89% accurate. In all seven test organisms and nine extraction methods, accurate species identification was only obtained in 63% of the cases.

Asunto(s)

RESUMEN

Objects collect microorganisms from their surroundings and develop a microbial "fingerprint" that may be useful for determining an object's past location (provenance). It may be possible to use ubiquitous microorganisms for forensics or as environmental sensors. Here, we use microbial communities in the Chesapeake Bay region to demonstrate the use of natural microorganisms as biological sensors to determine the past location of boats. The microbiomes of two boats and of the open water were sampled as these vessels traveled from the Port of Baltimore to the Port of Norfolk, and back to Baltimore. 16S rRNA sequencing was performed to identify microorganisms. Differential abundance and machine learning analyses were utilized to identify microbial signatures and predicted probabilities which were used to determine the vessel's previous location. The work presented here provides a better understanding of how microbes in aquatic systems can be leveraged as utility for object biosensors.

Asunto(s)

RESUMEN

Diagnostic microbiology plays a vital role in managing infectious diseases, combating antimicrobial resistance, and containment of outbreaks. During the fourth industrial revolution, when artificial intelligence (AI) became an essential part of our day-to-day lives, its integration into healthcare would further revolutionize our knowledge and potential. Although in the budding stage, AI with machine learning is being increasingly utilized in various aspects of diagnostic microbiology. It can handle large datasets that are difficult to analyze manually. Researchers have developed and demonstrated several machine-learning algorithms for interpreting bacterial cultures, conducting image analysis for microbial detection, and predicting antimicrobial susceptibility patterns. Thus, AI may most likely be the ultimate solution to the ever-increasing demand for improved results with shorter turnaround times. AI can also assist forensic microbiologists in crime scene investigations, as it can guide individual identification, cause and time since death, and manner of death. This review summarizes the application of AI in diagnostic microbiology for performing diverse sets of microbial investigations and is an essential aid in forensic microbiology.

RESUMEN

Microbial forensics is a rapidly evolving discipline that has gained significant momentum in recent years. The study evaluated relevant results over the last four decades from 1984 to 2022 all over the world, aiming to analyze the growing trends and research orientations of microbial forensics. Using "microbial forensics" as the search topic in the Web of Science Core Collection, the systematic retrieval identified 579 documents relevant to the field and draw many statistical tables and maps to make the retrieval results visible. According to further bibliometric analysis, there are an increasing number of publications related to microbial forensics from the overall trend, with the highest number of publications recorded in 2021. In terms of the total number of articles, the USA and China were both the leading contributors to the field among 40 countries. The field has developed rapidly in recent years based on the development of next-generation sequencing. Over the course of its development, there are rich keywords in the research of scholars, which focus on diversity and identification. Moreover, despite the early hot topic being PCR (the use of PCR to probe microorganisms), in recent years, the topics, markers, and the potential application of microorganisms in forensic practice have become hot, which also indicates the future research directions of microbial forensic.

RESUMEN

With the adoption of infection science as an umbrella term for the disciplines that inform our ideas of infection, there is a need for a common language that links infection's constituent parts. This paper develops a conceptual framework for infection science from the major themes used to understand causal relationships in infectious diseases. The paper proposes using the four main themes from the Principia Aetiologica to classify infection knowledge into four corresponding domains: Clinical microbiology, Public health microbiology, Mechanisms of microbial disease and Antimicrobial countermeasures. This epistemology of infection gives form and process to a revised infection ontology and an infectious disease heuristic. Application of the proposed epistemology has immediate practical implications for organization of journal content, promotion of inter-disciplinary collaboration, identification of emerging priority themes, and integration of cross-disciplinary areas such as One Health topics and antimicrobial resistance. Starting with these foundations, we can build a coherent narrative around the idea of infection that shapes the practice of infection science.

Asunto(s)

RESUMEN

Microorganisms, which are widely distributed in nature and human body, show unique application value in forensic identification. Recent advances in high-throughput sequencing technology and significant reductions in analysis costs have markedly promoted the development of forensic microbiology and metagenomics. The rapid progression of artificial intelligence (AI) methods and computational approaches has shown their unique application value in forensics and their potential to address relevant forensic questions. Here, we summarize the current status of microbial metagenomics and AI analysis in forensic microbiology, including postmortem interval inference, individual identification, geolocation, and tissue/fluid identification.

RESUMEN

The recent increase in publicly available metagenomic datasets with geospatial metadata has made it possible to determine location-specific, microbial fingerprints from around the world. Such fingerprints can be useful for comparing microbial niches for environmental research, as well as for applications within forensic science and public health. To determine the regional specificity for environmental metagenomes, we examined 4305 shotgun-sequenced samples from the MetaSUB Consortium dataset-the most extensive public collection of urban microbiomes, spanning 60 different cities, 30 countries, and 6 continents. We were able to identify city-specific microbial fingerprints using supervised machine learning (SML) on the taxonomic classifications, and we also compared the performance of ten SML classifiers. We then further evaluated the five algorithms with the highest accuracy, with the city and continental accuracy ranging from 85-89% to 90-94%, respectively. Thereafter, we used these results to develop Cassandra, a random-forest-based classifier that identifies bioindicator species to aid in fingerprinting and can infer higher-order microbial interactions at each site. We further tested the Cassandra algorithm on the Tara Oceans dataset, the largest collection of marine-based microbial genomes, where it classified the oceanic sample locations with 83% accuracy. These results and code show the utility of SML methods and Cassandra to identify bioindicator species across both oceanic and urban environments, which can help guide ongoing efforts in biotracing, environmental monitoring, and microbial forensics (MF).

Asunto(s)

RESUMEN

Microbial diversity's potential has been investigated in medical and therapeutic studies throughout the last few decades. However, its usage in forensics is increasing due to its effectiveness in circumstances when traditional approaches fail to provide a decisive opinion or are insufficient in forming a concrete opinion. The application of human microbiome may serve in detecting the type of stains of saliva and vaginal fluid, as well as in attributing the stains to the individual. Similarly, the microbiome makeup of a soil sample may be utilised to establish geographic origin or to associate humans, animals, or things with a specific area, additionally microorganisms influence the decay process which may be used in depicting the Time Since death. Further in detecting the traces of the amount and concentration of alcohol, narcotics, and other forensically relevant compounds in human body or visceral tissues as they also affect the microbial community within human body. Beside these, there is much more scope of microbiomes to be explored in terms of forensic investigation, this review focuses on multidimensional approaches to human microbiomes from a forensic standpoint, implying the potential of microbiomes as an emerging tool for forensic investigations such as individual variability via skin microbiomes, reconstructing crime scene, and linking evidence to individual.

Asunto(s)

RESUMEN

The skin microbiome is a highly abundant and relatively stable source of DNA that may be utilized for human identification (HID). In this study, a set of single nucleotide polymorphisms (SNPs) with a high mean estimated Wright's fixation index (FST) (>0.1) and widespread abundance (found in ≥75% of samples compared) were selected from a diverse set of markers in the hidSkinPlex panel. The least absolute shrinkage and selection operator (LASSO) was used in a novel machine learning framework to generate a SNP panel and predict the human host from skin microbiome samples collected from the hand, manubrium, and foot. The framework was devised to emulate a new unknown person introduced to the algorithm and to match samples from that person against a population database. Unknown samples were classified with 96% accuracy (Matthews correlation coefficient [MCC], 0.954) in the test (n = 225 samples) data set. A final panel of informative SNPs was determined for HID (hidSkinPlex+) using all 51 individuals sampled at three body sites in triplicate. The hidSkinPlex+ panel comprises 365 SNPs and yielded prediction accuracy for the correct host of 95% (MCC = 0.949). The accuracy of the hidSkinPlex+ panel may be somewhat overestimated due to using 26 individuals from the training data set for the selection of the final panel. However, this accuracy still provides an indication of performance when tested on new samples. IMPORTANCE One of the fundamental goals in forensic genetics is to identify the source of biological evidence. Methods for detecting human DNA have advanced and can be quite sensitive, but not all DNA samples are amenable to current methods. However, the human skin microbiome is a source of DNA with high copy numbers, and it has the potential for high discriminatory power. The hidSkinPlex panel has been used for HID; however, some aspects of it could be improved. Missing information is ambiguous, as it is unclear if marker drop-out is a by-product of a low-template sample or if the reasons for not observing a marker are biological. Such ambiguity may confound methods for HID, and as such, an improved marker set (hidSkinPlex+) was designed that is considerably smaller and more robust to drop-out (365 SNPs contained in 135 markers) yet still can be used to accurately predict the human host.

Asunto(s)

RESUMEN

For many years, scientists have aspired to edit any DNA segment of living organisms precisely, and with the discovery of CRISPR/Cas9, they have moved closer to achieving their objective. Using this tool, researchers can knock out or knock-in a gene into or from any part of the genome. The CRISPR-Cas9 system is a highly effective and accurate tool and has replaced previous genome editing technologies such as ZFN and TALEN having lesser precision and accuracy. Advances in CRISPR/Cas technology have been remarkable in recent years, with significant applications in various fields of life sciences research. Despite the diverse applications of the CRISPR system, this tool also poses a threat to living organisms and the environment. The intentional misuse of CRISPR could be a disaster to society and the environment. Different countries have imposed laws and guidelines to regulate genome editing through multiple agencies. However, in some countries, these rules and regulations are still under development or don't exist, leading to higher chances of the technology getting misused. Dual-use of this technology could be a threat to biosecurity and biodefense. This article summarizes the potential applications of the CRISPR system in human and plants, and also evaluate the laws and regulations imposed by different countries to keep genome editing technology under check.

Asunto(s)

RESUMEN

This review discusses microbial forensics as an emerging science that finds application in protecting human health. It is important to distinguish naturally acquired infections from those caused by the intentional release of microorganisms to the environment. This information is crucial in formulating procedures against the spread of infectious diseases and prosecuting persons who may be involved in acts of biocrime, bioterrorism, or biowarfare. A comparison between epidemiological investigations and microbial forensic investigations is provided. In addition, a discussion on how microbial forensics strengthens health systems is included in this review. Microbial forensic investigations and epidemiologic examinations employ similar concepts and involve identifying and characterising the microbe of interest. Both fields require formulating an appropriate case definition, determining a pathogen's mode of transmission, and identifying the source(s) of infection. However, the two subdisciplines differ in their objectives. An epidemiological investigation aims to identify the pathogen's source to prevent the spread of the disease. Microbial forensics focuses on source-tracking to facilitate the prosecution of persons responsible for the spread of a pathogen. Both fields use molecular techniques in analysing and comparing DNA, gene products, and biomolecules to identify and characterise the microorganisms of interest. We included case studies to show methods used in microbial forensic investigations, a brief discussion of the public significance of microbial forensic systems, and a roadmap for establishing a system at a national level. This system is expected to strengthen a country's capacity to respond to public health emergencies. Several factors must be considered in establishing national microbial forensic systems. First is the inherent ubiquity, diversity, and adaptability of microorganisms that warrants the use of robust and accurate molecular typing systems. Second, the availability of facilities and scientists who have been trained in epidemiology, molecular biology, bioinformatics, and data analytics. Human resources and infrastructure are critical requirements because formulating strategies and allocating resources in times of infectious disease outbreaks must be data-driven. Establishing and maintaining a national microbial forensic system to strengthen capacities in conducting forensic and epidemiological investigations should be prioritised by all countries, accompanied by a national policy that sets the legislative framework and provides for the system's financial requirements.

RESUMEN

Over the last few years, advances in massively parallel sequencing technologies (also referred to next generation sequencing) and bioinformatics analysis tools have boosted our knowledge on the human microbiome. Such insights have brought new perspectives and possibilities to apply human microbiome analysis in many areas, particularly in medicine. In the forensic field, the use of microbial DNA obtained from human materials is still in its infancy but has been suggested as a potential alternative in situations when other human (non-microbial) approaches present limitations. More specifically, DNA analysis of a wide variety of microorganisms that live in and on the human body offers promises to answer various forensically relevant questions, such as post-mortem interval estimation, individual identification, and tissue/body fluid identification, among others. However, human microbiome analysis currently faces significant challenges that need to be considered and overcome via future forensically oriented human microbiome research to provide the necessary solutions. In this perspective article, we discuss the most relevant biological, technical and data-related issues and propose future solutions that will pave the way towards the integration of human microbiome analysis in the forensic toolkit.

Asunto(s)

RESUMEN

The human microbiome has begun to emerge as a potential forensic tool, with varied applications ranging from unique identification to investigative leads that link individuals and/or locations. The relative abundance of the combined DNA of the microbiome, compared to human nuclear DNA, may expand potential sources of biological evidence, especially in cases with transfer or low-copy number DNA samples. This work sought to determine the optimal swab type for the collection and analysis of microorganisms. A bacterium (Proteus mirabilis) was deposited by pipette onto four swab types (cotton, flocked, dental applicators, and dissolvable), and extraction and real-time PCR quantitation of the bacterial DNA were performed, which allowed for absolute microbial DNA recovery and comparison of yields across the four sampling substrates. Flocked swabs had the highest yield (~1240 ng) compared to the cotton swabs (~184 ng), dental applicators (~533 ng), and dissolvable swabs (~430 ng). The collection efficiency was further evaluated for cotton and flocked swabs using dried microbial samples spotted onto non-porous surfaces (treated wood, glass, plastic, and tile). Flocked swabs performed consistently better across wood, glass, and tile, but showed decreased recovery from plastic. The cotton swabs failed in the recovery of P. mirabilis DNA across all surfaces. Knowing the appropriate sampling substrate will be useful as others continue to investigate the use of the microbiome as a forensics tool.

Asunto(s)

RESUMEN

Microbial forensics represents a promising tool to strengthen traditional forensic investigative methods and fill related knowledge gaps. Large-scale microbiome studies indicate that microbial fingerprinting can assist forensics in areas such as trace evidence, source tracking, geolocation, and circumstances of death. Nevertheless, the majority of forensic microbiome studies focus on soil and internal organ samples, whereas the microbiome of skin, mouth, and especially vaginal samples that are routinely collected in sexual assault and femicide cases remain underexplored. This review discusses the current and emerging insights into vaginal, skin, and salivary microbiome-modulating factors during life (e.g., lifestyle and health status) and after death (e.g., environmental influences and post-mortem interval) based on next-generation sequencing. We specifically highlight the key aspects of female reproductive tract, skin, and mouth microbiome samples relevant in forensics. To fill the current knowledge gaps, future research should focus on the degree to which the post-mortem succession rate and profiles of vaginal, skin, and saliva microbiota are sensitive to abiotic and biotic factors, presence or absence of oxygen and other gases, and the nutrient richness of the environment. Application of this microbiome-related knowledge could provide valuable complementary data to strengthen forensic cases, for example, to shed light on the circumstances surrounding death with (post-mortem) microbial fingerprinting. Overall, this review synthesizes the present knowledge and aims to provide a framework to adequately comprehend the hurdles and potential application of vaginal, skin, and salivary post-mortem microbiomes in forensic investigations.

RESUMEN

Microbial DNA, shed from human skin, can be distinctive to its host and, thus, help individualize donors of forensic biological evidence. Previous studies have utilized single-locus microbial DNA markers (e.g., 16S rRNA) to assess the presence/absence of personal microbiota to profile human hosts. However, since the taxonomic composition of the microbiome is in constant fluctuation, this approach may not be sufficiently robust for human identification (HID). Multimarker approaches may be more powerful. Additionally, genetic differentiation, rather than taxonomic distinction, may be more individualizing. To this end, the nondominant hands of 51 individuals were sampled in triplicate (n = 153). They were analyzed for markers in the hidSkinPlex, a multiplex panel comprising candidate markers for skin microbiome profiling. Single-nucleotide polymorphisms (SNPs) with the highest Wright's fixation index (FST) estimates were then selected for predicting donor identity using a support vector machine (SVM) learning model. FST is an estimate of the genetic differences within and between populations. Three different SNP selection criteria were employed: SNPs with the highest-ranking FST estimates (i) common between any two samples regardless of markers present (termed overall); (ii) each marker common between samples (termed per marker); and (iii) common to all samples used to train the SVM algorithm for HID (termed selected). The SNPs chosen based on criteria for overall, per marker, and selected methods resulted in an accuracy of 92.00%, 94.77%, and 88.00%, respectively. The results support that estimates of FST, combined with SVM, can notably improve forensic HID via skin microbiome profiling. IMPORTANCE There is a need for additional genetic information to help identify the source of biological evidence found at a crime scene. The human skin microbiome is a potentially abundant source of DNA that can enable the identification of a donor of biological evidence. With microbial profiling for human identification, there will be an additional source of DNA to identify individuals as well as to exclude individuals wrongly associated with biological evidence, thereby improving the utility of forensic DNA profiling to support criminal investigations.

Asunto(s)

RESUMEN

Information on the time when a stain was deposited at a crime scene can be valuable in forensic investigations. It can link a DNA-identified stain donor with a crime or provide a post-mortem interval estimation in cases with cadavers. The available methods for estimating stain deposition time have limitations of different types and magnitudes. In this proof-of-principle study we investigated for the first time the use of microbial DNA for this purpose in human saliva stains. First, we identified the most abundant and frequent bacterial species in saliva using publicly available 16S rRNA gene next generation sequencing (NGS) data from 1,848 samples. Next, we assessed time-dependent changes in 15 identified species using de-novo 16S rRNA gene NGS in the saliva stains of two individuals exposed to indoor conditions for up to 1 year. We selected four bacterial species, i.e., Fusobacterium periodonticum, Haemophilus parainfluenzae, Veillonella dispar, and Veillonella parvula showing significant time-dependent changes and developed a 4-plex qPCR assay for their targeted analysis. Then, we analyzed the saliva stains of 15 individuals exposed to indoor conditions for up to 1 month. Bacterial counts generally increased with time and explained 54.9% of the variation (p = <2.2E-16). Time since deposition explained ≥86.5% and ≥88.9% of the variation in each individual and species, respectively (p = <2.2E-16). Finally, based on sample duplicates we built and tested multiple linear regression models for predicting the stain deposition time at an individual level, resulting in an average mean absolute error (MAE) of 5 days (ranging 3.3-7.8 days). Overall, the deposition time of 181 (81.5%) stains was correctly predicted within 1 week. Prediction models were also assessed in stains exposed to similar conditions up to 1 month 7 months later, resulting in an average MAE of 8.8 days (ranging 3.9-16.9 days). Our proof-of-principle study suggests the potential of the DNA profiling of human commensal bacteria as a method of estimating saliva stains time since deposition in the forensic scenario, which may be expanded to other forensically relevant tissues. The study considers practical applications of this novel approach, but various forensic developmental validation and implementation criteria will need to be met in more dedicated studies in the future.

RESUMEN

The spectrum of emerging new diseases as well as re-emerging old diseases is broadening as infectious agents evolve, adapt, and spread at enormous speeds in response to changing ecosystems. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recent phenomenon and may take a while to understand its transmission routes from less traveled territories, ranging from fomite exposure routes to wastewater transmission. The critical challenge is how to negotiate with such catastrophic pandemics in high-income countries (HICs ~20% of the global population) and low-and middle-income countries (LMICs ~ 80% of the global population) with a total global population size of approximately eight billion, where practical mass testing and tracing is only a remote possibility, particularly in low-and middle-income countries (LMICs). Keeping in mind the population distribution disparities of high-income countries (HICs) and LMICs and urbanisation trends over recent years, traditional wastewater-based surveillance such as that used to combat polio may help in addressing this challenge. The COVID-19 era differs from any previous pandemics or global health challenges in the sense that there is a great deal of curiosity within the global community to find out everything about this virus, ranging from diagnostics, potential vaccines/therapeutics, and possible routes of transmission. In this regard, the fact that the gut is the common niche for both poliovirus and SARS-CoV-2, and due to the shedding of the virus through faecal material into sewerage systems, the need for long-term wastewater surveillance and developing early warning systems for better preparedness at local and global levels is increasingly apparent. This paper aims to provide an insight into the ongoing COVID-19 crisis, how it can be managed, and what measures are required to deal with a current global international public health concern. Additionally, it shed light on the importance of using wastewater surveillance strategy as an early warning practical tool suitable for massive passive screening, as well as the urgent need for microfluidic technology as a rapid and cost-effective approach tracking SARS-CoV-2 in wastewater.

RESUMEN

BACKGROUND: Tracking dispersal of microbial populations in the environment requires specific detection methods that discriminate between the target strain and all potential natural and artificial interferents, including previously utilized tester strains. Recent work has shown that genomic insertion of short identification tags, called "barcodes" here, allows detection of chromosomally tagged strains by real-time PCR. Manual design of these barcodes is feasible for small sets, but expansion of the technique to larger pools of distinct and well-functioning assays would be significantly aided by software-guided design. RESULTS: Here we introduce barCoder, a bioinformatics tool that facilitates the process of creating sets of uniquely identifiable barcoded strains. barCoder utilizes the genomic sequence of the target strain and a set of user-specified PCR parameters to generate a list of suggested barcode "modules" that consist of binding sites for primers and probes, and appropriate spacer sequences. Each module is designed to yield optimal PCR amplification and unique identification. Optimal amplification includes metrics such as ideal melting temperature and G+C content, appropriate spacing, and minimal stem-loop formation; unique identification includes low BLAST hits against the target organism, previously generated barcode modules, and databases (such as NCBI). We tested the ability of our algorithm to suggest appropriate barcodes by generating 12 modules for Bacillus thuringiensis serovar kurstaki-a simulant for the potential biowarfare agent Bacillus anthracis-and three each for other potential target organisms with variable G+C content. Real-time PCR detection assays directed at barcodes were specific and yielded minimal cross-reactivity with a panel of near-neighbor and potential contaminant materials. CONCLUSIONS: The barCoder algorithm facilitates the generation of synthetically barcoded biological simulants by (a) eliminating the task of creating modules by hand, (b) minimizing optimization of PCR assays, and (c) reducing effort wasted on non-unique barcode modules.

Asunto(s)

RESUMEN

Microbial Forensics is a field that continues to grow in interest and application among the forensic community. This review, divided into two sections, covers several topics associated with this new field. The first section presents a historic overview concerning the use of microorganisms (or its product, i.e. toxins) as harmful biological agents in the context of biological warfare (biowarfare), bioterrorism, and biocrime. Each case is illustrated with the examination of case reports that span from prehistory to the present day. The second part of the manuscript is devoted to the role of MF and highlights the necessity to prepare for the pressing threat of the harmful use of biological agents as weapons. Preventative actions, developments within the field to ensure a timely and effective response and are discussed herein.

Asunto(s)