History of biological warfare and bioterrorism

Bioterrorism literally means using microorganisms or infected samples to cause terror and panic in populations. Bioterrorism had already started 14 centuries before Christ, when the Hittites sent infected rams to their enemies. However, apart from some rare well-documented events, it is often very difficult for historians and microbiologists to differentiate natural epidemics from alleged biological attacks, because: (i) little information is available for times before the advent of modern microbiology; (ii) truth may be manipulated for political reasons, especially for a hot topic such as a biological attack; and (iii) the passage of time may also have distorted the reality of the past. Nevertheless, we have tried to provide to clinical microbiologists an overview of some likely biological warfare that occurred before the 18th century and that included the intentional spread of epidemic diseases such as tularaemia, plague, malaria, smallpox, yellow fever, and leprosy. We also summarize the main events that occurred during the modern microbiology era, from World War I to the recent ‘anthrax letters’ that followed the World Trade Center attack of September 2001. Again, the political polemic surrounding the use of infectious agents as a weapon may distort the truth. This is nicely exemplified by the Sverdlovsk accident, which was initially attributed by the authorities to a natural foodborne outbreak, and was officially recognized as having a military cause only 13 years later.     

Laboratory diagnosis and biosafety issues of biological warfare agents

Bioterrorism events have been rare until recently. Many clinical laboratories may not be familiar with handling specimens from a possible bioterrorism attack. Therefore, they should be aware of their own responsibilities and limitations in the handling and treatment of such specimens, and what to do if they are requested to process clinical samples. The Centers for Disease Control and Prevention has developed the Laboratory Response Network to provide an organized response system for the detection and diagnosis of biological warfare agents based on laboratory testing abilities and facilities. There are potentially many biological warfare agents, but probably a limited number of agents would be encountered in case of an attack, and their identification and laboratory safety will be discussed.     

Biological warfare,bioterrorism, and biocrime

Biological weapons achieve their intended target effects through the infectivity of disease-causing infectious agents. The ability to use biological agents in warfare is prohibited by the Biological and Toxin Weapon Convention. Bioterrorism is defined as the deliberate release of viruses, bacteria or other agents used to cause illness or death in people, but also in animals or plants. It is aimed at creating casualties, terror, societal disruption, or economic loss, inspired by ideological, religious or political beliefs. The success of bioterroristic attempts is defined by the measure of societal disruption and panic, and not necessarily by the sheer number of casualties. Thus, making only a few individuals ill by the use of crude methods may be sufficient, as long as it creates the impact that is aimed for. The assessment of bioterrorism threats and motives have been described before. Biocrime implies the use of a biological agent to kill or make ill a single individual or small group of individuals, motivated by revenge or the desire for monetary gain by extortion, rather than by political, ideological, religious or other beliefs. The likelihood of a successful bioterrorist attack is not very large, given the technical difficulties and constraints. However, even if the number of casualties is likely to be limited, the impact of a bioterrorist attack can still be high. Measures aimed at enhancing diagnostic and therapeutic capabilities and capacities alongside training and education will improve the ability of society to combat ‘regular’ infectious diseases outbreaks, as well as mitigating the effects of bioterrorist attacks.     

Biological warfare in a historical perspective

There are some early examples of biological warfare (BW), but in modern times it was used first for sabotage by Germany during WWI. Development of biological weapons on a military significant scale was initiated in several countries in the period between the world wars. During WWII, several countries had active programs such as the USA, UK, Canada, Germany, Japan and the Soviet Union. It was only Japan that on a fairly large scale used BW. The US program continued until 1969, when President Nixon took a decision to end it in connection with signing the BTWC. The Soviet Union had also continued its program after the war, and this was enhanced after signing the BTWC: in the 1980s the program consisted of around fifty facilities and involved around 60 000 people. The Soviet Union produced and maintained a large stockpile of BW-agents. After the collapse of the Soviet Union, and due to pressure from USA and UK, President Yeltsin issued a decree in 1992 banning continued offensive BW activity. However, there are still concerns of residual activity in Russia. Another program of concern is the Iraqi BW-program. After 10 years of UN inspections that were stopped in 1998, there are still many unanswered questions concerning the BW program. There was also a covert BW-program in South Africa that was terminated aroud 1993. There have also been a number of allegations of alleged use or possession. In addition, there are indications that 10–12 states are now trying to acquire BW, and this assessment is based on intelligence information, mainly from the USA. For example Iraq, North Korea, Iran, Syria, Sudan and Libya. Another aspect is the strong driving force of technology developments to promote this type of program, opening new risks for future potential military misuse.     

Detection of anthrax toxin genetic sequences by the solid phase oligo-probes

Purpose: There is an urgent need to detect a rapid field-based test to detect anthrax. We have developed a rapid, highly sensitive DNA-based method to detect the anthrax toxin lethal factor gene located in pXO1, which is necessary for the pathogenicity of Bacillus anthracis. Materials and Methods: We have adopted the enzyme-linked immunosorbent assay (ELISA) so that instead of capturing antibodies we capture the DNA of the target sequence by a rapid oligo-based hybridization and then detect the captured DNA with another oligoprobe that binds to a different motif of the captured DNA sequences at a dissimilar location. We chose anthrax lethal factor endopeptidase sequences located in pXO1 and used complementary oligoprobe, conjugated with biotin, to detect the captured anthrax specific sequence by the streptavidin-peroxidase-based colorimetric assay. Result: Our system can detect picomoles (pMoles) of anthrax (approximately 33 spores of anthrax) and is >1000 times more sensitive than the current ELISA, which has a detection range of 0.1 to 1.0 ng/mL. False positive results can be minimized when various parameters and the colour development steps are optimized. Conclusion: Our results suggest that this assay can be adapted for the rapid detection of minuscule amounts of the anthrax spores that are aerosolized in the case of a bioterrorism attack. This detection system does not require polymerase chain reaction (PCR) step and can be more specific than the antibody method. This method can also detect genetically engineered anthrax. Since, the antibody method is so specific to the protein epitope that bioengineered versions of anthrax may not be detected.     

Comparison of methods for measuring antibiotic consumption in an intensive care unit

Hospitals worldwide are working on minimizing unnecessary use of antimicrobials. To assess actual changes of antimicrobial usage, correct and precise measurements are necessary. This study aimed to compare data on the purchase of antibiotics from the pharmacy and the administration of antibiotics to patients, respectively, in an intensive care unit (ICU). Data were obtained from the Neurointensive Care Unit (NICU) at Rigshospitalet, Denmark. During a 23‐month period, comprising 10 770 bed‐days (BD), the ward purchased 16 908 defined daily doses (DDD) of antibiotics from the pharmacy, and 15 130 DDD and 41 304 individual doses were administered. Intraclass correlation coefficients (ICCs) were calculated; control and runcharts and a Bland–Altman plot were constructed. Pharmacy sales and drug administration data showed no systematic variation over time with a monthly overestimation of pharmacy sales data of 10% (95% confidence interval (CI), 6.20–14.3%) for all antibiotics, and 7% (95% CI: 1.81–11.1%) for broad‐spectrum antibiotics. The antibiotic consumption, without bed‐days, has a clinically acceptable ICC of >0.70 and no systematic difference is suggested by the Bland–Altman plot. In this study of a large NICU, whose antibiotic consumption varied at random, pharmacy sales data were an acceptable approximation of the actual summarized drug consumption.     

Clinical anatomy as the basis for clinical examination: development and evaluation of an Introduction to Clinical Examination in a problem-oriented medical curriculum

Clinical anatomy is usually defined as anatomy applied to patient care. The question is asked whether students of a new horizontally and vertically integrated medical curriculum recognize the subject as the basis for clinical examination. A clinical anatomy practicum was developed in the special activity, "Introduction to Clinical Medicine," held in the second year of the Pretoria medical curriculum. The practicum was conducted on a station basis to anatomically prepare the student for the inspection, palpation, percussion, and auscultation of the cardiovascular, respiratory, abdominal, and urogenital systems. A total of 23 stations consisting of eight cardiovascular, seven respiratory, and eight abdominal/urogenital stations were designed. Standardized patients, cadavers, skeletons, prosected specimens, x-rays, computed tomography (CT) scans, magnetic resonance imaging (MRI), multimedia programs, and clinical case studies were used as resources. A Likert-type questionnaire was used for student evaluation of the practicum. Most students realized the importance of surface anatomy for a family physician. More than two-thirds thought the practicum improved their understanding of the anatomical basis for clinical examination. The minority of students were stimulated to do further reading on clinical examination. The students' response to their ability to integrate the clinical examination with the radiological anatomy was average. Most students were continuously aware of the appropriateness of the practicum for their future career. We conclude that medical students recognize the importance of anatomy as the basis for clinical examination when exposed to an appropriate integrated presentation format.     

Clinical utility of an enhanced human immunodeficiency virus type 1 p24 antigen capture assay

The presence of p24 core antigen in the serum of individuals with human acquired immunodeficiency syndrome has been used as one of the important prognostic markers of HIV-1 infection and also as an end point in evaluating antiviral drugs and vaccines. Unfortunately the majority of p24 antigen present in serum exists as an antigenantibody complex and is not detected with the commercial kits currently available to measure p24 antigen. In this study, we report a simple procedure utilizing treatment of serum samples with glycine buffer (pH 1.85) to dissociate antigen-antibody complexes prior to assaying for p24 antigen. A 300% increase in the number of p24-reactive samples and a 3- to 12-fold increase in the quantity of antigen detected were observed when samples were pretreated with 1.5M glycine buffer (pH 1.85) for 1 hr. Glycine treatment of samples did not result in nonspecific positive tests and samples previously shown to be reactive remained positive. In reconstruction experiments the release of antigen was found to be inversely proportional to the amount of p24 antibody present in the serum. The percentage of HIV-1-infected patients positive for p24 antigen was clearly a function of CD4 count. Forty-nine percent of patients with more than 500 CD4 cells and 100% of patients with less than 200 CD4 were p24 positive. The improved sensitivity for detection of p24 provided by this procedure enhances our understanding of the pathogenesis of AIDS by showing that the majority of patients with HIV-1 infection is p24 positive and facilitates the analysis of data obtained in clinical trials involving anti-HIV compounds.     

Clinical symptoms,immune factors,and molecular characteristics of an adult male in Shenzhen,China infected with influenza virus H5N1

On December 29, 2011, a man infected with a subclade of the H5N1 virus was confirmed in Shenzhen, China. The clinical symptoms and immune factors of the patient were investigated and the phylogenetic and molecular characteristics of the virus were analyzed. High fever, rapid development of serious pneumonia and multi‐organ failure were the main clinical symptoms. Arterial blood gas analysis showed that PaCO₂ rose sharply and PO₂ decreased. Leukocyte and platelet counts decreased rapidly. Peripheral blood lymphocyte counts indicated lymphopenia and inverted ratios of CD4⁺ to CD8⁺ cells. Cytokine analysis showed that the levels of serum IL‐6, IL‐10, and IFN‐r continued to increase, whereas the levels of IL‐12 and TNFs decreased during the clinical course. MCP‐1 and IP‐10 remained at a high level after infection. Phylogenetic analysis confirmed that the virus A/Shenzhen/1/2011 belongs to the new subclade 2.3.2.1. An Arg (R) insertion at P6 and an RP8I substitution in the HA cleavage site motif were detected in the virus. Compared to the vaccine strain, 16 specific substitutions occurred in the HA1 protein. Some of them were located on the receptor‐binding site, glycosylation site and the region of the antigenic determinant. In summary, serious complications and immune system disorders were the main features of the infection with H5N1. Gene variation did not weaken the highly pathogenic features of viruses and the pathogenicity and antigenicity of the new subclade virus were changed. J. Med. Virol. 85:760–768, 2013. © 2013 Wiley Periodicals, Inc.