Protein biomarker elucidation for the verification of biological agents in the taxonomic group of Gammaproteobacteria using tandem mass spectrometry.

Some pathogenic microbes can be used for nefarious applications and instigate population-based fear. In a bio-threat scenario, rapid and accurate methods to detect biological agents in a wide range of complex environmental and clinical matrices, is of paramount importance for the implementation of mitigation protocols and medical countermeasures. This study describes targeted and shot-gun tandem MS based approaches for the verification of biological agents from the environmental samples. The marker proteins and peptides were elucidated by an exhaustive literature mining, in silico analysis of prioritized proteins, and MS/MS analysis of abundant proteins from selected bacterial species. For the shot-gun methodology, tandem MS analysis of abundant peptides was carried from spiked samples. The validation experiments employing a combination of shot-gun tandem MS analysis and a targeted search reported here is a proof of concept to show the applicability of the methodology for the unambiguous verification of biological agents at sub-species level, even with limited fractionation of crude protein extracts from environmental samples.

NOTIFy (non-toxic lyophilized field)-FISH for the identification of biological agents by Fluorescence in situ Hybridization.

The rapid and reliable diagnostics of highly pathogenic bacteria under restricted field conditions poses one of the major challenges to medical biodefense, especially since false positive or false negative reports might have far-reaching consequences. Fluorescence in situ hybridization (FISH) has the potential to represent a powerful microscopy-based addition to the existing molecular-based diagnostic toolbox. In this study, we developed a set of FISH-probes for the fast, matrix independent and simultaneous detection of thirteen highly pathogenic bacteria in different environmental and clinical sample matrices. Furthermore, we substituted formamide, a routinely used chemical that is toxic and volatile, by non-toxic urea. This will facilitate the application of FISH under resource limited field laboratory conditions. We demonstrate that hybridizations performed with urea show the same specificity and comparable signal intensities for the FISH-probes used in this study. To further simplify the use of FISH in the field, we lyophilized the reagents needed for FISH. The signal intensities obtained with these lyophilized reagents are comparable to freshly prepared reagents even after storage for a month at room temperature. Finally, we show that by the use of non-toxic lyophilized field (NOTIFy)-FISH, specific detection of microorganisms with simple and easily transportable equipment is possible in the field.

Elucidation of protein biomarkers for verification of selected biological warfare agents using tandem mass spectrometry.

Some pathogens and toxins have the potential to be used as weapons of mass destruction and instigate population-based fear. Efforts to mitigate biothreat require development of efficient countermeasures which in turn relies on fast and accurate methods to detect the biological agents in a range of complex matrices including environmental and clinical samples. We report here an mass spectrometry (MS) based methodology, employing both targeted and shot-gun approaches for the verification of biological agents from the environmental samples. Our shot-gun methodology relied on tandem MS analysis of abundant peptides from the spiked samples, whereas, the targeted method was based on an extensive elucidation of marker proteins and unique peptides resulting in the generation of an inclusion list of masses reflecting relevant peptides for the unambiguous identification of nine bacterial species [listed as priority agents of bioterrorism by Centre for Disease Control and Prevention (CDC)] belonging to phylogenetically diverse genera. The marker peptides were elucidated by extensive literature mining, in silico analysis, and tandem MS (MS/MS) analysis of abundant proteins of the cultivated bacterial species in our laboratory. A combination of shot-gun MS/MS analysis and the targeted search using a panel of unique peptides is likely to provide unambiguous verification of biological agents at sub-species level, even with limited fractionation of crude protein extracts from environmental samples. The comprehensive list of peptides reflected in the inclusion list, makes a valuable resource for the multiplex analysis of select biothreat agents and further development of targeted MS/MS assays.

Detecting Biothreat Agents: From Current Diagnostics to Developing Sensor Technologies.

Although a fundamental understanding of the pathogenicity of most biothreat agents has been elucidated and available treatments have increased substantially over the past decades, they still represent a significant public health threat in this age of (bio)terrorism, indiscriminate warfare, pollution, climate change, unchecked population growth, and globalization. The key step to almost all prevention, protection, prophylaxis, post-exposure treatment, and mitigation of any bioagent is early detection. Here, we review available methods for detecting bioagents including pathogenic bacteria and viruses along with their toxins. An introduction placing this subject in the historical context of previous naturally occurring outbreaks and efforts to weaponize selected agents is first provided along with definitions and relevant considerations. An overview of the detection technologies that find use in this endeavor along with how they provide data or transduce signal within a sensing configuration follows. Current "gold" standards for biothreat detection/diagnostics along with a listing of relevant FDA approved in vitro diagnostic devices is then discussed to provide an overview of the current state of the art. Given the 2014 outbreak of Ebola virus in Western Africa and the recent 2016 spread of Zika virus in the Americas, discussion of what constitutes a public health emergency and how new in vitro diagnostic devices are authorized for emergency use in the U.S. are also included. The majority of the Review is then subdivided around the sensing of bacterial, viral, and toxin biothreats with each including an overview of the major agents in that class, a detailed cross-section of different sensing methods in development based on assay format or analytical technique, and some discussion of related microfluidic lab-on-a-chip/point-of-care devices. Finally, an outlook is given on how this field will develop from the perspective of the biosensing technology itself and the new emerging threats they may face.

Beyond the Dirty Dozen: A Proposed Methodology for Assessing Future Bioweapon Threats.

Background: Defense policy planners and countermeasure developers are often faced with vexing problems involving the prioritization of resources and efforts. This is especially true in the area of Biodefense, where each new emerging infectious disease outbreak brings with it questions regarding the causative agent's potential for weaponization. Recent experience with West Nile Virus, Severe Acute Respiratory Syndrome, Monkeypox, and H1N1 Influenza highlights this problem. Appropriately, in each of these cases, the possibility of bioterrorism was raised, although each outbreak ultimately proved to have a natural origin. In fact, determining whether an outbreak has an unnatural origin can be quite difficult. Thus, the questions remain: could the causative agents of these and other emerging infectious disease outbreaks pose a future weaponization threat? And how great is that threat? Should precious resources be diverted from other defense efforts in order to prepare for possible hostile employment of novel diseases by belligerents? Answering such critical questions requires some form of systematic threat assessment. Methods: Through extensive collaborative work conducted within NATO's Biomedical Advisory Council, we developed a scoring matrix for evaluating the weaponization potential of the causative agents of such diseases and attempted to validate our matrix by examining the reproducibility of data using known threat agents. Our matrix included 12 attributes of a potential weapon and was provided, along with detailed scoring instructions, to 12 groups of biodefense experts in 6 NATO nations. Study participants were asked to score each of these 12 attributes on a scale of 0-3: Infectivity, Infection-to-Disease Ratio (Reliability), Predictability (& Incubation Period), Morbidity & Mortality (Virulence), Ease of Large-Scale Production & Storage, Aerosol Stability, Atmospheric Stability, Ease of Dispersal, Communicability, Prophylactic Countermeasure Availability, Therapeutic Countermeasure Availability, and Ease of Detection. Reproducibility of scoring data was assessed by examining the standard deviations (SD) of mean scores. Results: Our results were unexpected. Several familiar biothreat diseases such as anthrax and tularemia were judged, by our experts, to be less threatening than many others owing to a number of factors including ease of detection, lack of communicability, and the ready availability of countermeasures. Conversely, several toxins were judged by experts to have very high potential as threat agents owing, in part, to their reliability, virulence, and a lack of available countermeasures. Agreement among experts, as determined by lower SD about a mean score, was greater for more familiar threats. Discussion: Our study was designed to provide a concise and east-to-apply set of criteria that could be used by NATO nations to evaluate emerging infectious disease threats with respect to their weaponization potential. Our results were unexpected. We believe that a lack of appropriate weighting factors may explain these results and suggest that future studies weigh each of the 12 proposed criteria based on the intended use of the assessment data and other situational factors. We believe that the greatest value of our study lies in a codification of the attributes of a biological weapon.

Preparing for biological threats: Addressing the needs of pregnant women.

Intentional release of infectious agents and biological weapons to cause illness and death has the potential to greatly impact pregnant women and their fetuses. We review what is known about the maternal and fetal effects of seven biological threats: Bacillus anthracis (anthrax); variola virus (smallpox); Clostridium botulinum toxin (botulism); Burkholderia mallei (glanders) and Burkholderia pseudomallei (melioidosis); Yersinia pestis (plague); Francisella tularensis (tularemia); and Rickettsia prowazekii (typhus). Evaluating the potential maternal, fetal, and infant consequences of an intentional release of an infectious agent requires an assessment of several key issues: (1) are pregnant women more susceptible to infection or illness compared to the general population?; (2) are pregnant women at increased risk for severe illness, morbidity, and mortality compared to the general population?; (3) does infection or illness during pregnancy place women, the fetus, or the infant at increased risk for adverse outcomes and how does this affect clinical management?; and (4) are the medical countermeasures recommended for the general population safe and effective during pregnancy? These issues help frame national guidance for the care of pregnant women during an intentional release of a biological threat. Birth Defects Research 109:391-398, 2017.© 2017 Wiley Periodicals, Inc.

[On-site detection of bioterrorism-relevant agents : Rapid detection methods for viruses, bacteria and toxins - capabilities and limitations]. / Vor-Ort-Nachweis bioterroristisch relevanter Agenzien : Schnelle Nachweismethoden für Viren, Bakterien und Toxine ­ Potenziale und Grenzen.

In Europe, besides the threat of terrorist attacks involving conventional methods such as explosive devices and automatic weapons, there is also a potential threat of terrorist groups using non-conventional material like biological agents in the scope of future attacks. Consequently, rapid and reliable detection systems for biological agents are being developed and tested continuously to inform crisis management. For environmental detection, a broad spectrum of different laboratory-based techniques has been developed for relevant biological agents. However for environmental samples, fast and reliable on-site detection methods are desired by first responders for rapid assessment.Based on different functional principles, generic, immunological and nucleic-acid-based on-site detection methods can be distinguished. Those should be facile, fast, sensitive, and specific. However, commercially available kits usually have limited sensitivity and often have not been validated independently. Furthermore in this context, the multitude of relevant biological agents that potentially have to be considered present in complex environmental matrices poses a serious challenge for reliable detection. Therefore, detailed knowledge of the specific scope of applications and the limitations of different analytical systems is necessary to evaluate the results obtained purposefully.The aim of this article is to provide an overview of the analytical principles, benefits and limitations of prevailing on-site environmental detection systems for bioterrorism-relevant viruses, bacteria and toxins. Despite promising developments the informative value of currently available on-site tests is still limited. Thus, expert laboratories have to conduct confirmatory testing.

Electrochemiluminescence DNA sensor array for multiplex detection of biowarfare agents.

Development of a fully automated electrochemiluminescence (ECL) DNA assay for multiplex detection of six biowarfare agents is described. Aminated-DNA capture probes were covalently immobilised on activated-carbon electrodes and subsequently hybridised to target strands. Detection was achieved via a sandwich-type assay after Ru(bpy)3(2+)-labelled reporter probes were hybridised to the formed probe-target complexes. The assay was performed in an automated microsystem in a custom designed ECL detection box with integrated fluidics, electronics,and movable photomultiplier detector. The obtained limits of detection were 0.6-1.2 nmol L(-1) for six targets ranging from 50 to 122 base pairs in size, with linear range 1-15 nmol L(-1). Non-specific adsorption and cross-reactivity were very low. Detection of six targets on a single chip was achieved with subnanomolar detection limits.

A proposal for the classification of biological weapons sensu lato.

Due to historical and legislation reasons, the category of bioweapons is rather poorly defined. Authors often disagree on involving or excluding agents like hormones, psychochemicals, certain plants and animals (such as weeds or pests) or synthetic organisms. Applying a wide definition apparently threatens by eroding the regime of international legislation, while narrow definitions abandon several important issues. Therefore, I propose a category of 'biological weapons sensu lato' (BWsl) that is defined here as any tool of human aggression whose acting principle is based on disciplines of biology including particularly microbiology, epidemiology, medical biology, physiology, psychology, pharmacology and ecology, but excluding those based on inorganic agents. Synthetically produced equivalents (not necessarily exact copies) and mock weapons are also included. This definition does not involve any claim to subject all these weapons to international legislation but serves a purely scholarly purpose. BWsl may be properly categorized on the base of the magnitude of the human population potentially targeted (4 levels: individuals, towns, countries, global) and the biological nature of the weapons' intended effects (4 levels: agricultural-ecological agents, and non-pathogenic, pathogenic, or lethal agents against humans).

Love-wave sensors combined with microfluidics for fast detection of biological warfare agents.

The following paper examines a time-efficient method for detecting biological warfare agents (BWAs). The method is based on a system of a Love-wave immunosensor combined with a microfluidic chip which detects BWA samples in a dynamic mode. In this way a continuous flow-through of the sample is created, promoting the reaction between antigen and antibody and allowing a fast detection of the BWAs. In order to prove this method, static and dynamic modes have been simulated and different concentrations of BWA simulants have been tested with two immunoreactions: phage M13 has been detected using the mouse monoclonal antibody anti-M13 (AM13), and the rabbit immunoglobulin (Rabbit IgG) has been detected using the polyclonal antibody goat anti-rabbit (GAR). Finally, different concentrations of each BWA simulants have been detected with a fast response time and a desirable level of discrimination among them has been achieved.

Normativas internacionais de proteção contra bioterrorismo e biocrimes: lacunas e vulnerabilidades no Brasil / International standards for protection against bioterrorism and biocrimes: gaps and vulnerabilities in Brazil

Saúde Global pode ser entendida como questões de saúde que transcendem fronteiras nacionais e demandam intervenções nos assuntos que determinam a saúde das populações. Atualmente, os Estados deparam-se seguidamente com problemas e crises relacionadas à área da saúde. Em relação à segurança nacional, essa preocupação se manifesta sob a forma de ameaças de proliferação de Armas Biológicas e de Bioterrorismo. A comunidade internacional tem se esforçado para propor normas que previnam tais atos. Os melhores exemplos são a Convenção para a Proibição de Armas Biológicas e suas Toxinas (CPAB), a Resolução 1540 do Conselho de Segurança das Nações Unidas (Res 1540/CSNU) e o Regulamento Sanitário Internacional (RSI). Contudo, existem várias lacunas e vulnerabilidades que podem ser exploradas nesses documentos. O escopo desse estudo é propor questionamentos a partir da Análise Documental desses tratados, ponderando a articulação entre os órgãos nacionais com a responsabilidade de prevenção, controle e resiliência contra ataques biológicos. O Brasil é signatário das principais normativas internacionais direcionadas à prevenção e controle de uso intencional de agentes biológicos e vem respondendo a contento à comunidade internacional. Ainda assim, é necessário mais que uma resposta formal a entidades supranacionais para prover, efetivamente, a devida proteção à população. O País possui capacidades técnicas estruturadas de forma isolada e fragmentada em diversos órgãos inexistindo um sistema formalmente instituído para a prevenção,resposta e controle de ataques bioterroristas...

Global Health can be understood as health issues that transcend national borders andrequire intervention in matters that determine the health of populations. Currently, States face crisis and problems related to health realm. Regarding national security, thisconcern is manifested in the form of threats of Biological Weapons proliferation and Bioterrorism. The international community has endeavored to propose regulations that prevent such acts. The best examples of it are the Convention for the Prohibition ofBiological Weapons and their Toxins (BWC), the United Nations Security Council Resolution 1540 (Res 1540/UNSC) and the International Health Regulations (IHR). However, there are several gaps and vulnerabilities that can be exploited in thesedocuments. The scope of this study is questioning these treaties using DocumentAnalysis, pondering the link between national entities responsible for prevention,control and resilience against biological attacks. Brazil is signatory of the major international instruments aimed at the prevention and control of biological agentsintentional use and has responded satisfactorily to the international community. Never theless, it takes more than a formal supranational response to provide effectivelyappropriate protection to population. The country owns technical capabilities not connected, distributed in several organs and lacks a system for the prevention, responseand control of bioterrorist attacks. Likewise, there is no national protocol established inorder to coordinate the activities of the bodies responsible for controlling the threat. Factors that determine individuals or groups to employ biological agents and toxins intheir violent attacks are not fully understood. Thus, more reckless than exacerbate therisk of intentional actions with biological agents is not being properly prepared toprevent and control such acts...

Development of biosensors for the detection of biological warfare agents: its issues and challenges.

This review discusses current development in biosensors for the detection of biological warfare agents (BWAs). BWAs include bacteria, virus and toxins that are added deliberately into air water and food to spread terrorism and cause disease or death. The rapid and unambiguous detection and identification of BWAs with early warning signals for detecting possible biological attack is a major challenge for government agencies particularly military and health. The detection devices--biosensors--can be classified (according to their physicochemical transducers) into four types: electrochemical, nucleic acid, optical and piezoelectric. Advantages and limitations of biosensors are discussed in this review followed by an assessment of the current state of development of different types of biosensors. The research and development in biosensors for biological warfare agent detection is of great interest for the public as well as for governments.

[Characteristic and classification of biological agents]. / Charakterystyka i klasyfikacja czynników biologicznych.

Respiratory tract infections are caused by biological pathogens, which are found in the environment around us. They cause inflammation of varying severity, threatening the health and lives. For this reason they can be used as a biological weapon. Dealing with victims requires special diagnostic and therapeutic procedures. In some cases, it is necessary to quarantine patients and apply an intensive treatment in specialized centers.

Antibodies for biodefense.

Potential bioweapons are biological agents (bacteria, viruses, and toxins) at risk of intentional dissemination. Biodefense, defined as development of therapeutics and vaccines against these agents, has seen an increase, particularly in the US following the 2001 anthrax attack. This review focuses on recombinant antibodies and polyclonal antibodies for biodefense that have been accepted for clinical use. These antibodies aim to protect against primary potential bioweapons, or category A agents as defined by the Centers for Disease Control and Prevention (Bacillus anthracis, Yersinia pestis, Francisella tularensis, botulinum neurotoxins, smallpox virus, and certain others causing viral hemorrhagic fevers) and certain category B agents. Potential for prophylactic use is presented, as well as frequent use of oligoclonal antibodies or synergistic effect with other molecules. Capacities and limitations of antibodies for use in biodefense are discussed, and are generally applicable to the field of infectious diseases.

Cutaneous manifestations of category A bioweapons.

The 2001 anthrax attacks on the United States brought bioterrorism to the forefront of the medical community. Because many bioterrorist agents produce cutaneous disease, dermatologists will likely be first responders during a future attack. Despite this, many dermatologists are not adequately prepared to deal with a bioterrorism attack. It is critical that all first responders be able to recognize symptoms of bioterrorism-related disease and prepared to respond to a bioterrorist attack to minimize threats to public health. This article reviews the diagnosis and treatment of diseases caused by potential biowarfare agents that produce cutaneous manifestations, and also provides information regarding reporting and containment of possible bioterrorism-related diseases.

[Biological weapons]. / Biologische Waffen.

Biological weapons are weapons of mass destruction that use pathogens (bacteria, viruses) or the toxins produced by them to target living organisms or to contaminate non-living substances. In the past, biological warfare has been repeatedly used. Anthrax, plague and smallpox are regarded as the most dangerous biological weapons by various institutions. Nowadays it seems quite unlikely that biological warfare will be employed in any military campaigns. However, the possibility remains that biological weapons may be used in acts of bioterrorism. In addition all diseases caused by biological weapons may also occur naturally or as a result of a laboratory accident. Risk assessment with regard to biological danger often proves to be difficult. In this context, an early identification of a potentially dangerous situation through experts is essential to limit the degree of damage.

[The dirty dozen]. / Krankenhaushygiene--Das dreckige Dutzend.

The term "the dirty dozen" refers to the 12 biological agents such as, for example, the anthrax bacillus or botulinum toxin that could be used as biological weapons. Even if their military use is considered unlikely physicians should be familiar with the problematic because these pathogens can also occur naturally or, respectively, cause disease as the consequence of a laboratory accident. It is of decisive importance to make the diagnosis as quickly as possible in order to limit any further spread of the disease.

Reclassifying bioterrorism risk: are we preparing for the proper pathogens?

Existing classifications of potential biological weapons, acknowledge only limited important parameters of biological weapon potential. Certain pathogen factors would further influence the outcome of a potential attack in context with social and political aspects of the time and space of the attack. The importance of these factors was investigated through various attack scenarios that have been developed by the authors, and an individual score for each of these factors was calculated, based on the overall effect their variation had in the scenario outcome. A new classification score for potential biological weapons was subsequently developed, one, which drastically alters the perception of risk for certain pathogens, such as filoviruses and anthrax. This frame further allows for more accurate evaluation of the bioweapon potential of agents such as avian flu. Recognition of intervening factors and proper assessment of the actual risk might augment in proper distribution of interest and funds on relevant medical research.