Erik A. Henchal

The evolving field of biodefence: therapeutic developments and diagnostics

The threat of bioterrorism and the potential use of biological weapons against both military and civilian populations has become a major concern for governments around the world. For example, in 2001 anthrax-tainted letters resulted in several deaths, caused widespread public panic and exerted a heavy economic toll. If such a small-scale act of bioterrorism could have such a huge impact, then the effects of a large-scale attack would be catastrophic. This review covers recent progress in developing therapeutic countermeasures against, and diagnostics for, such agents.

Comparison of Dissociation-Enhanced Lanthanide Fluorescent Immunoassays to Enzyme-Linked Immunosorbent Assays for Detection of Staphylococcal Enterotoxin B, Yersinia pestis-Specific F1 Antigen, and Venezuelan Equine Encephalitis Virus

ABSTRACT The dissociation-enhanced lanthanide fluorescent immunoassays (DELFIA) were developed for the detection of staphylococcal enterotoxin B, Yersinia pestis-specific F1 antigen, and Venezuelan equine encephalitis virus. These assays were compared to previously developed enzyme-linked immunosorbent assays (ELISAs) by determining the sensitivity or limit of detection (LOD), the dynamic range, and the reproducibility of each assay in a number of different sample matrices. The sensitivity and specificity of each assay were then determined by using a small panel of blinded spiked and nonspiked samples. All three DELFIAs demonstrated at least 1 log greater sensitivity than corresponding ELISAs utilizing the same reagents and showed an increase in dynamic range of at least 2 log10 concentrations. This increased LOD resulted in higher sensitivity rates for the DELFIA. The specificity of all of the assays evaluated was 100%, and no sample matrix effects were observed in either format. However, the reproducibility of the DELFIA was poor due to randomly distributed wells exhibiting excessive background signal (hot wells), which occurred throughout the evaluation. As this technology matures, the reproducibility of these assays should improve, as will the ability to identify hot wells. Despite its sensitivity, the logistical burden associated with the DELFIA and the technical expertise required to complete assays and interpret the data limit the application of this technology to reference or large clinical laboratories.

Viral Replication and Host Gene Expression in Alveolar Macrophages Infected with Ebola Virus (Zaire Strain)

ABSTRACT In order to characterize the cellular response to and identify potential diagnostic markers for the early detection of Ebola virus, an in vitro culture system involving nonhuman primate alveolar macrophages was developed. Ebola virus replication in the alveolar macrophages was characterized by plaque assay, immunohistochemical analysis, and in situ hybridization. Fluorogenic 5′ -nuclease assays specific for nonhuman primate proinflammatory cytokines and chemokines were designed and used to evaluate mRNA transcription in macrophages infected with Ebola virus. Transient increases in cytokine and chemokine mRNA levels were observed immediately following exposure to Ebola virus. At 2 h postexposure, levels of cytokine and chemokine mRNAs were markedly reduced. Although Ebola virus infection of alveolar macrophages failed to induce a sustained increase in proinflammatory cytokine and chemokine mRNA transcription (potentially reducing the use of these markers as diagnostic tools), the fluorogenic 5′-nuclease assays developed may have prognostic value for individuals infected with Ebola virus. Recently published data have indicated that persons who remain asymptomatic after exposure to Ebola virus are capable of mounting an early proinflammatory cytokine response and that those who become clinically ill are not. If implemented immediately after exposure, these assays could be used to predict which individuals will be more likely to remain asymptomatic as opposed to those who will be more likely to develop clinical signs and eventually succumb to the virus.

Detection and Identification of Ciprofloxacin-Resistant Yersinia pestis by Denaturing High-Performance Liquid Chromatography

ABSTRACT Denaturing high-performance liquid chromatography (DHPLC) has been used extensively to detect genetic variation. We used this method to detect and identify Yersinia pestis KIM5 ciprofloxacin-resistant isolates by analyzing the quinolone resistance-determining region (QRDR) of the gyrase A gene. Sequencing of the Y. pestis KIM5 strain gyrA QRDR from 55 ciprofloxacin-resistant isolates revealed five mutation types. We analyzed the gyrA QRDR by DHPLC to assess its ability to detect point mutations and to determine whether DHPLC peak profile analysis could be used as a molecular fingerprint. In addition to the five mutation types found in our ciprofloxacin-resistant isolates, several mutations in the QRDR were generated by site-directed mutagenesis and analyzed to further evaluate this method for the ability to detect QRDR mutations. Furthermore, a blind panel of 42 samples was analyzed by screening for two mutant types to evaluate the potential diagnostic value of this method. Our results showed that DHPLC is an efficient method for detecting mutations in genes that confer antibiotic resistance.

Use of Denaturing High-Performance Liquid Chromatography To Identify Bacillus anthracis by Analysis of the 16S-23S rRNA Interspacer Region and gyrA Gene

ABSTRACT Denaturing high-performance liquid chromatography (DHPLC) was evaluated as a method for identifying Bacillus anthracis by analyzing two chromosomal targets, the 16S-23S intergenic spacer region (ISR) and the gyrA gene. The 16S-23S ISR was analyzed by this method with 42 strains of B. anthracis, 36 strains of Bacillus cereus, and 12 strains of Bacillus thuringiensis; the gyrA gene was analyzed by this method with 33 strains of B. anthracis, 27 strains of B. cereus, and 9 strains of B. thuringiensis. Two blind panels of 45 samples each were analyzed to evaluate the potential diagnostic capability of this method. Our results show that DHPLC is an efficient method for the identification of B. anthracis.

Early indicators of exposure to biological threat agents using host gene profiles in peripheral blood mononuclear cells

BackgroundEffective prophylaxis and treatment for infections caused by biological threat agents (BTA) rely upon early diagnosis and rapid initiation of therapy. Most methods for identifying pathogens in body fluids and tissues require that the pathogen proliferate to detectable and dangerous levels, thereby delaying diagnosis and treatment, especially during the prelatent stages when symptoms for most BTA are indistinguishable flu-like signs.MethodsTo detect exposures to the various pathogens more rapidly, especially during these early stages, we evaluated a suite of host responses to biological threat agents using global gene expression profiling on complementary DNA arrays.ResultsWe found that certain gene expression patterns were unique to each pathogen and that other gene changes occurred in response to multiple agents, perhaps relating to the eventual course of illness. Nonhuman primates were exposed to some pathogens and the in vitro and in vivo findings were compared. We found major gene expression changes at the earliest times tested post exposure to aerosolized B. anthracis spores and 30 min post exposure to a bacterial toxin.ConclusionHost gene expression patterns have the potential to serve as diagnostic markers or predict the course of impending illness and may lead to new stage-appropriate therapeutic strategies to ameliorate the devastating effects of exposure to biothreat agents.

Requirements for Biological Threat Identification Systems

Rapid identification of bioterrorism or biological warfare agents is most urgent within the first 24 h after an attack (1–3). After that period, the ability to affect the prognosis of patients that have been infected with a highly virulent organism, such as Bacillus anthracis, sharply declines (2). For less virulent organisms, identification during the pre-symptomatic stage is critical because the clinical picture is often confusing. Some patients may present with “flu-like” symptoms, rash or unusual skin lesions, respiratory or gastrointestinal illness (1,4–5). Definitive identification of biological threats requires an integrated approach (see Fig. 1). No single identification technology is sufficient to definitively identify biological threats because of the microbial diversity, an often confusing clinical presentation, the close antigenic and genetic relatedness of some biological agents (see Table 1) and the consequences of misidentification (1,4–8). A system of overlapping technologies and approaches are required to achieve the highest level of confidence and decrease the number of “false alarms” (9). The Food and Drug Administration (FDA) has not reviewed the performance of many technologies proposed for the rapid identification of biological threat agents. All identifications must be confirmed by using “gold standard” or FDA-approved methods, such as bacterial culture. In the future, first responders, medical care providers, and laboratories will need a combination of overlapping diagnostic approaches tied together by a robust information management system when responding to a bioterrorism attack (9).