A cell-free biosensor for multiplexed and sensitive detection of biological warfare agents.

The rapid and precise detection of pathogenic agents is critical for public health and societal stability. The detection of biological warfare agents (BWAs) is especially vital within military and counter-terrorism contexts, essential in defending against biological threats. Traditional methods, such as polymerase chain reaction (PCR), are limited by their need for specific settings, impacting their adaptability and versatility. This study introduces a cell-free biosensor for BWA detection by converting the 16S rRNA of targeted pathogens into detectable functional protein molecules. The modular nature of this approach allows for the flexible configuration of pathogen detection, enabling the simultaneous identification of multiple pathogenic 16S rRNAs through customized reporter proteins for each targeted sequence. Furthermore, we demonstrate how this method integrates with techniques utilizing retroreflective Janus particles (RJPs) for facile and highly sensitive pathogen detection. The cell-free biosensor, employing RJPs to measure the reflection of non-chromatic white light, can detect 16S rRNA from BWAs at femtomolar levels, corresponding to tens of colony-forming units per milliliter of pathogenic bacteria. These findings represent a significant advancement in pathogen detection, offering a more efficient and accessible alternative to conventional methodologies.

Evaluation of sponge wipe surface sampling for collection of potential surrogates for non-spore-forming bioterrorism agents.

AIM: Evaluate the efficacy of sponge wipe sampling at recovering potential bacterial surrogates for Category A and B non-spore-forming bacterial bioterrorism agents from hard, nonporous surfaces. METHODS: A literature survey identified seven nonpathogenic bacteria as potential surrogates for selected Category A and B non-spore-forming bacterial agents. Small (2 × 4 cm) and large (35.6 × 35.6 cm) coupons made from either stainless steel, plastic, or glass, were inoculated and utilized to assess persistence and surface sampling efficiency, respectively. Three commercially available premoistened sponge wipes (3M™, Sani-Stick®, and Solar-Cult®) were evaluated. RESULTS: Mean recoveries from persistence testing indicated that three microorganisms (Yersinia ruckeri, Escherichia coli, and Serratia marcescens) demonstrated sufficient persistence across all tested material types. Sampling of large inoculated (≥107 CFU per sample) coupons resulted in mean recoveries ranging from 6.6 to 3.4 Log10 CFU per sample. Mean recoveries for the Solar-Cult®, 3M™ sponge wipes, and Sani-Sticks® across all test organisms and all material types were ≥5.7, ≥3.7, and ≥3.4 Log10 CFU per sample, respectively. Mean recoveries for glass, stainless steel, and ABS plastic across all test organisms and all sponge types were ≥3.8, ≥3.7, and ≥3.4 Log10 CFU per sample, respectively. CONCLUSIONS: Recovery results suggest that sponge wipe sampling can effectively be used to recover non-spore-forming bacterial cells from hard, nonporous surfaces such as stainless steel, ABS plastic, and glass.

Investigating the Potential Strategic Implications of COVID-19 for Biological Weapons Pursuit: A New Expert Simulation.

To investigate the impact of the COVID-19 pandemic on the strategic decisionmaking of leaders with respect to biological weapons, this study employed a prospective simulation technique called Asynchronous Strategic Dynamics Red Teaming. Using an immersive, multimedia simulation conducted remotely and asynchronously, the effort engaged 240 carefully selected and curated expert participants in either biological weapons or the countries of interest (as well as 60 naïve participants). Across our sample of 30 countries, simulated interest in pursuing some type of biological weapons program (defensive or offensive) remained low to moderate. While such interest increased after the simulated onset of the COVID-19 pandemic, it was limited overall, with only a handful of states showing salient increases in offensive biological weapon interest. When directly referencing why their countries might have changed their post-COVID-19 interest in biological weapons, the most commonly cited reasons were: (1) COVID-19 demonstrated the power of biological weapons to disrupt societies and cause large-scale economic harm, and (2) the pandemic revealed either the state's own or its rivals' vulnerability to diseases like COVID-19, as well as an inability to efficiently respond and contain such diseases. In sum, despite a global pandemic with massive consequences, the simulation revealed that most states are not likely to dramatically change their strategic posture regarding pursuit of offensive biological weapons.

Bioterrorismo y armas biológicas: caracterización y respuesta frente a las amenazas actuales / Bioterrorism and biological weapons: characterization and response to current threats

El uso de agentes biológicos con fines terroristas constituye una amenaza singular. Aunque poco probable, su materialización puede ser difícilmente evitable en el futuro. Este artículo revisa el fenómeno del bioterrorismo, examinando los posibles riesgos y vulnerabilidades, los mecanismos de respuesta y las nuevas amenazas para la bioseguridad. (AU)

The use of biological agents for terrorist purposes is a unique threat. Although unlikely, it may be difficult to prevent in the future. This article provides an overview of the phenomenon of bioterrorism, examining potential risks and vulnerabilities, response mechanisms and emerging threats to biosecurity. (AU)

Bioenergetic function is decreased in peripheral blood mononuclear cells of veterans with Gulf War Illness.

Gulf War Illness (GWI) is a major health problem for approximately 250,000 Gulf War (GW) veterans, but the etiology of GWI is unclear. We hypothesized that mitochondrial dysfunction is an important contributor to GWI, based on the similarity of some GWI symptoms to those occurring in some mitochondrial diseases; the plausibility that certain pollutants to which GW veterans were exposed affect mitochondria; mitochondrial effects observed in studies in laboratory models of GWI; and previous evidence of mitochondrial outcomes in studies in GW veterans. A primary role of mitochondria is generation of energy via oxidative phosphorylation. However, direct assessment of mitochondrial respiration, reflecting oxidative phosphorylation, has not been carried out in veterans with GWI. In this case-control observational study, we tested multiple measures of mitochondrial function and integrity in a cohort of 114 GW veterans, 80 with and 34 without GWI as assessed by the Kansas definition. In circulating white blood cells, we analyzed multiple measures of mitochondrial respiration and extracellular acidification, a proxy for non-aerobic energy generation; mitochondrial DNA (mtDNA) copy number; mtDNA damage; and nuclear DNA damage. We also collected detailed survey data on demographics; deployment; self-reported exposure to pesticides, pyridostigmine bromide, and chemical and biological warfare agents; and current biometrics, health and activity levels. We observed a 9% increase in mtDNA content in blood in veterans with GWI, but did not detect differences in DNA damage. Basal and ATP-linked oxygen consumption were respectively 42% and 47% higher in veterans without GWI, after adjustment for mtDNA amount. We did not find evidence for a compensatory increase in anaerobic energy generation: extracellular acidification was also lower in GWI (12% lower at baseline). A subset of 27 and 26 veterans returned for second and third visits, allowing us to measure stability of mitochondrial parameters over time. mtDNA CN, mtDNA damage, ATP-linked OCR, and spare respiratory capacity were moderately replicable over time, with intraclass correlation coefficients of 0.43, 0.44, 0.50, and 0.57, respectively. Other measures showed higher visit-to-visit variability. Many measurements showed lower replicability over time among veterans with GWI compared to veterans without GWI. Finally, we found a strong association between recalled exposure to pesticides, pyridostigmine bromide, and chemical and biological warfare agents and GWI (p < 0.01, p < 0.01, and p < 0.0001, respectively). Our results demonstrate decreased mitochondrial respiratory function as well as decreased glycolytic activity, both of which are consistent with decreased energy availability, in peripheral blood mononuclear cells in veterans with GWI.

ASSESSMENT OF THE CONSEQUENCES OF THE DETERIORATION OF THE EPIDEMIOLOGICAL SITUATION DURING HOSTILITIES.

OBJECTIVE: The aim: Scientific substantiation of the methodology for predicting the consequences of the worsening of the epidemic situation on the territory of Ukraine during military operations for the timely adoption of measures for the medical protection of military personnel in conditions of biological contamination. PATIENTS AND METHODS: Materials and methods: Determination and generalization of the impact of biological contamination due to the use of biological weapons were carried out considering the main determinants of the epidemic process using the index and coefficient of medical protection. Applied methods of scientific research: epidemiological, system, and information approach. RESULTS: Results: The authors proposed indicators that consider the pathogenicity of the infectious agent, contagiousness, the degree of non-specific protection of servicemen, specific protection of servicemen, and the sanitary-epidemiological state of the area of operations of troops (forces). Relevant epidemic situations were simulated, and the index and coefficient of medical protection were calculated to predict the consequences of the worsening of the epidemic situation to make timely decisions regarding the implementation of medical protection measures for military personnel in conditions of biological contamination during the repulsion of armed aggression. CONCLUSION: Conclusions: In the conditions of biological contamination, when biological weapons and biological terrorism are used, the epidemic process in the army is intensified, which requires timely decisions regarding the implementation of medical protection measures for military personnel in conditions of biological contamination.

Epidemiology of Pathogens Listed as Potential Bioterrorism Agents, the Netherlands, 2009‒2019.

We provide incidences (cases/10 million persons) in the Netherlands during 2009-2019 for pathogens listed as potential bioterrorism agents. We included pathogens from the highest categories of the European Medicines Agency or the US Centers for Disease Control and Prevention. Notifiable diseases and recently published data were used to calculate the average annual incidence. Coxiella burnetii had the highest incidence because of a Q fever epidemic during 2007-2010. Incidence then decreased to 10.8 cases/. Pathogens with an incidence >1 were Brucella spp. (2.5 cases), Francisella tularensis (1.3 cases), and Burkholderia pseudomallei (1.1 cases). Pathogens with an incidence <1 were hemorrhagic fever viruses (0.3 cases), Clostridium botulinum (0.2 cases), and Bacillus anthracis (0.1 cases). Variola major and Yersinia pestis were absent. The generally low incidences make it unlikely that ill-meaning persons can isolate these pathogens from natural sources in the Netherlands. However, the pathogens are stored in laboratories, underscoring the need for biosecurity measures.

Tularemia seroprevalence in humans in the region of the Hittite-Arzawa War (Inner Aegean Region), where the first biological weapon was used 3300 years ago.

BACKGROUND: : According to Egyptian records, tularemia emerged in the Canaan region, where it was first identified and spread to Anatolia over the Euphrates. It was used as an active biological weapon for the first time in the Hittite-Arzawa War in 1320-1318 BC. This study aimed to investigate the seroprevalence of tularemia in the Inner Aegean Region, which is thought to be the region where this war was fought 3300 years ago. METHODS: Tularemia seropositivity in humans was investigated in 27 villages/neighborhoods in 3 districts in each of Manisa, Kütahya, and Usak provinces. Before the study, the participants were informed about the disease via posters, and their blood samples were taken following filling out the questionnaire. Microagglutination tests were performed using in-house tularemia antigen and V plate for serological experiments. Rose-Bengal test was also performed on seropositive sera. RESULTS: Of the total of 410 people, 226 (55.12%) were male. The mean age of the volunteers was 43.72 years. The highest participation was from Kütahya Province. According to the results of the tularemia microagglutination test, seropositivity was detected in 6 cases. It was determined that all of the seropositive volunteers were in Kütahya. When the tularemia antibody titers were examined, seropositivity was determined at 1/20-1/160 titers. No positivity was detected in the Rose-Bengal test for cross-reaction. DISCUSSION: Kütahya has been identified as a risky region in terms of tularemia in the Inner Aegean Region. In order to use the resources in the country economically, first of all, the risk areas in terms of tularemia should be determined by serological studies in all regions. In order to increase awareness about the disease, physicians and filiation teams should be trained in risky areas. Surveillance studies should be conducted to identify and monitor possible sources in areas identified as risky.

Immunosensors for Assay of Toxic Biological Warfare Agents.

An immunosensor for the assay of toxic biological warfare agents is a biosensor suitable for detecting hazardous substances such as aflatoxin, botulinum toxin, ricin, Shiga toxin, and others. The application of immunosensors is used in outdoor assays, point-of-care tests, as a spare method for more expensive devices, and even in the laboratory as a standard analytical method. Some immunosensors, such as automated flow-through analyzers or lateral flow tests, have been successfully commercialized as tools for toxins assay, but the research is ongoing. New devices are being developed, and the use of advanced materials and assay techniques make immunosensors highly competitive analytical devices in the field of toxic biological warfare agents assay. This review summarizes facts about current applications and new trends of immunosensors regarding recent papers in this area.

Analysis of the Virus SARS-CoV-2 as a Potential Bioweapon in Light of International Literature.

INTRODUCTION: As of early 2022, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic still represents a worldwide medical emergency situation. The ongoing vaccination programs can slow down the spread of the virus; however, from time to time, the newly emerging variants of concern and antivaccination movements carry the possibility for the disease to remain in our daily lives. After the appearance of SARS-CoV-2, there was scholarly debate whether the virus was of natural origin, or it emerged from a laboratory, some even thinking the agent's potential biological weapon properties suggest the latter scenario. Later, the bioweapon theory was dismissed by the majority of experts, but the question remains that despite its natural origin, how potent a biological weapon the SARS-CoV-2 virus can become over time. MATERIALS AND METHODS: Based on 12 bioweapon threat assessment criteria already published in 2018, we performed a literature search and review, focusing on relevant potential bioweapon properties of the virus SARS-CoV-2. Instead of utilizing a survey among experts, we tried to qualify and quantify characteristics according to the available data found in peer-reviewed papers. We also identified other key elements not mentioned in the original 12 bioweapon criteria, which can play an important role in assessing future biological weapons. RESULTS: According to the international literature we analyzed, SARS-CoV-2 is a moderately infectious agent (ID50 estimated between 100 and 1,000), with high infection-to-disease ratio (35%-45% rate of asymptomatic infected) and medium incubation period (1-34 days, mean 6-7 days). Its morbidity and mortality rate can be categorized as medium (high morbidity rate with significant mortality rate). It can be easily produced in large quantities, has high aerosol stability, and has moderate environmental stability. Based on laboratory experiments and statistical model analysis, it can form and is contagious with droplet nuclei, and with spray technique utilization, it could be weaponized effectively. Several prophylactic countermeasures are available in the form of vaccines; however, specific therapeutic options are much more limited. In connection with the original assessment criteria, the SARS-CoV-2 only achieved a "0" score on the ease of detection because of readily available, relatively sensitive, and specific rapid antigen tests. Based on the pandemic experience, we also propose three new assessment categories: one that establishes a mean to measure the necessary quarantine restrictions related to a biological agent, another one that can represent the personal protective equipment required to work safely with a particular agent, and a third one that quantifies the overall disruptive capability, based on previous real-life experiences. These factors could further specify the threat level related to potential biological weapons. CONCLUSIONS: Our results show that the virus can become a potent bioweapon candidate in the future, achieving a total score of 24 out of 36 on the original 12 criteria. The SARS-CoV-2 has already proven its pandemic generating potential and, despite worldwide efforts, still remains an imminent threat. In order to be prepared for the future possibility of the virus arising as a bioweapon, we must remain cautious and take the necessary countermeasures.

Gain of Function and Loss of Control: Genetic Modification of Microbial Agents - Viruses "On Steroids".

Gain of Function refers to genetic modification to enhance certain properties of a biological agent. "Dual use research" refers to experiments which have a primary goal of benefitting humanity, but which could produce harm if misapplied. So, for example, a virus which was being genetically modified (GM) for altruistic reasons might become more transmissible or resistant to vaccines or antimicrobial medications. Such a GM virus has bioterrorism potential. The UN Biological Weapons Convention has not been universally approved and 10 States are not signatories to the Convention. The control of such experiments is variously controlled in certain jurisdictions but in Australia these experiments are well regulated through the Gene Technology Act 2000 (Cth), the National Health Security Act 2007 (Cth) and the Crimes (Biological Weapons) Act 1976 (Cth). The controls on such experiments in Europe and the United States are less precise. There are examples in the United States and Europe where the security provisions to contain microorganisms undergoing research including genetic modification have been breached. This threatens the health and safety of laboratory workers and the wider community.

The Biological Weapons Convention should endorse the Tianjin Biosecurity Guidelines for Codes of Conduct.

The Tianjin Biosecurity Guidelines for Codes of Conduct for Scientists are a set of ten principles designed to promote responsible science and strengthen biosecurity governance. They should be broadly adopted, including being endorsed by the Biological Weapons Convention at its 9th Review Conference in November 2022.

Systematic Review of Hospital Treatment Outcomes for Naturally Acquired and Bioterrorism-Related Anthrax, 1880-2018.

BACKGROUND: Bacillus anthracis can cause anthrax and is a potential bioterrorism agent. The 2014 Centers for Disease Control and Prevention recommendations for medical countermeasures against anthrax were based on in vitro data and expert opinion. However, a century of previously uncompiled observational human data that often includes treatment and outcomes is available in the literature for analysis. METHODS: We reviewed treatment outcomes for patients hospitalized with anthrax. We stratified patients by meningitis status, route of infection, and systemic criteria, then analyzed survival by treatment type, including antimicrobials, antitoxin/antiserum, and steroids. Using logistic regression, we calculated odds ratios and 95% confidence intervals to compare survival between treatments. We also calculated hospital length of stay. Finally, we evaluated antimicrobial postexposure prophylaxis (PEPAbx) using data from a 1970 Russian-language article. RESULTS: We identified 965 anthrax patients reported from 1880 through 2018. After exclusions, 605 remained: 430 adults, 145 children, and 30 missing age. Survival was low for untreated patients and meningitis patients, regardless of treatment. Most patients with localized cutaneous or nonmeningitis systemic anthrax survived with 1 or more antimicrobials; patients with inhalation anthrax without meningitis fared better with at least 2. Bactericidal antimicrobials were effective for systemic anthrax; addition of a protein synthesis inhibitor(s) (PSI) to a bactericidal antimicrobial(s) did not improve survival. Likewise, addition of antitoxin/antiserum to antimicrobials did not improve survival. Mannitol improved survival for meningitis patients, but steroids did not. PEPAbx reduced risk of anthrax following exposure to B. anthracis. CONCLUSIONS: Combination therapy appeared to be superior to monotherapy for inhalation anthrax without meningitis. For anthrax meningitis, neither monotherapy nor combination therapy were particularly effective; however, numbers were small. For localized cutaneous anthrax, monotherapy was sufficient. For B. anthracis exposures, PEPAbx was effective.

Clinical Features of Patients Hospitalized for All Routes of Anthrax, 1880-2018: A Systematic Review.

BACKGROUND: Anthrax is a toxin-mediated zoonotic disease caused by Bacillus anthracis, with a worldwide distribution recognized for millennia. Bacillus anthracis is considered a potential biowarfare agent. METHODS: We completed a systematic review for clinical and demographic characteristics of adults and children hospitalized with anthrax (cutaneous, inhalation, ingestion, injection [from contaminated heroin], primary meningitis) abstracted from published case reports, case series, and line lists in English from 1880 through 2018, assessing treatment impact by type and severity of disease. We analyzed geographic distribution, route of infection, exposure to anthrax, and incubation period. RESULTS: Data on 764 adults and 167 children were reviewed. Most cases reported for 1880 through 1915 were from Europe; those for 1916 through 1950 were from North America; and from 1951 on, cases were from Asia. Cutaneous was the most common form of anthrax for all populations. Since 1960, adult anthrax mortality has ranged from 31% for cutaneous to 90% for primary meningitis. Median incubation periods ranged from 1 day (interquartile range [IQR], 0-4) for injection to 7 days (IQR, 4-9) for inhalation anthrax. Most patients with inhalation anthrax developed pleural effusions and more than half with ingestion anthrax developed ascites. Treatment and critical care advances have improved survival for those with systemic symptoms, from approximately 30% in those untreated to approximately 70% in those receiving antimicrobials or antiserum/antitoxin. CONCLUSIONS: This review provides an improved evidence base for both clinical care of individual anthrax patients and public health planning for wide-area aerosol releases of B. anthracis spores.

A Collection of Molecular Fingerprints of Single Aerosol Particles in Air for Potential Identification and Detection Using Optical Trapping-Raman Spectroscopy.

Characterization, identification, and detection of aerosol particles in their native atmospheric states remain a challenge. Recently, optical trapping-Raman spectroscopy (OT-RS) has been developed and demonstrated for characterization of single, airborne particles. Such particles in different chemical groups have been characterized by OT-RS in recent years and many more are being studied. In this work, we collected single-particle Raman spectra measured using the OT-RS technique and began construction of a library of OT-RS fingerprints that may be used as a reference for potential detection and identification of aerosol particles in the atmosphere. We collected OT-RS fingerprints of aerosol particles from eight different categories including carbons, bioaerosols (pollens, fungi, vitamins, spores), dusts, biological warfare agent surrogates, etc. Among the eight categories, spectral fingerprints of six groups of aerosol particles have been published previously and two other groups are new. We also discussed challenges, limitations, and advantages of using single-particle optical trapping-Raman spectroscopy for aerosol-particle characterization, identification, and detection.

Monkeypox, a Literature Review: What Is New and Where Does This concerning Virus Come From?

Among the Poxviridae family, orthopoxvirus is the most notorious genus. Several DNA viruses belonging to this group are known to produce human disease from the life-threatening variola virus (VARV) (the causative agent of smallpox), monkeypox virus (MPXV), cowpox virus (CPXV), and vaccinia virus (VACV). These orthopoxviruses still remain a public health concern as VACV or CPXV still cause emerging endemic threads, especially in developing countries. MPXV is able to cause sporadic human outbreaks of a smallpox-like zoonotic disease and, in May 2022, hundreds of cases related to MPXV have been reported from more than 30 countries around the globe. At the end of July, monkeypox (MPX) outbreak was even declared a global health emergency by the World Health Organization (WHO). Many aspects remain unclear regarding this outbreak and a deep understanding of orthopoxvirus might have crucial and evident implications. During the era in which people under 45 years old are not protected against VACV, the potential use of orthopoxviruses as a biological weapon raises global concern considering the rapid spreading of the current MPX outbreak in vulnerable populations. Hence, we review the most recent evidence about phylogenesis, pathogenesis, prevention, and treatment for this concerning disease.

Smallpox as a Bioagent: A Refresher and Update for the SOF Provider.

Smallpox plagued humans for millennia until its eradication in 1980 following a successful global campaign led by the World Health Organization (WHO). It is the first known biological weapon to be used in war and has been weaponized in the past by the former Soviet Union. To date, smallpox remains a Category A Bioagent and is assessed to be a relevant threat to US military personnel. Given that the last natural case of smallpox occurred more than 40 years ago, a high level of suspicion along with a substantial understanding of the disease process are required to recognize potential future cases. While available countermeasures are limited, several new agents have recently become available for the prevention and treatment of smallpox and have been added to the strategic national stockpile. This review serves as a refresher and update for the clinical disease, to include its epidemiology and management with updated FDA-approved countermeasures.