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PubMed2003

Development of a 5' nuclease assay to detect ciprofloxacin resistant isolates of the biowarfare agent Yersinia pestis.

The detection of antibiotic resistance in agents of biowarfare is extremely critical in order to start appropriate therapy in a timely manner. We have developed a 5' nuclease assay to detect ciprofloxacin resistance (Cip(r)) in Yersinia pestis. Two groups of fluorogenic probes were developed. The first group included a probe homologous to the wild type Y. pestis gyrA sequence with two corresponding probes that were homologous with two different mis-sense mutations in codon 81 of GyrA. The second group of probes included a wild type probe and two corresponding probes that recognized mis-sense mutations in codon 83 or gyrA. These probes specifically reacted only with the homologous DNA sequences. The 5' nuclease assay was sensitive to 1 pg (approximately 1 colony forming unit) of starting template and could be used on semi-purified DNA. We tested our optimized assay against a group of 38 Cip(r) Y. pestis isolates and demonstrated that it was accurate at determining the gyrA allele encoded by these strains. The 5' nuclease assay performed on the LightCycler proved to be sensitive, rapid and accurate at identification of Cip(r) Y. pestis and thus should be useful in characterization of this organism in the event of a future act of bioterrorism.

Animals↗

PubMed2004

The history of epidemic typhus.

Few infectious diseases have influenced human civilization to the same degree as louse-transmitted typhus. Rickettsia prowazekii continues to strikes tens to hundreds of thousands of persons who live with war, famine, crowding, and in squalid conditions associated with social unrest, with mortality rates in excess of 10% to 15%. Historical documents confirm that such devastation has been a continuous feature of human existence to the extent that typhus has been a major determinant in the outcome of many wars, altering human history in its wake-despite incomplete knowledge of its precise origin. In the twenty-first century, circumstances are still conductive for outbreak; the emerging threat of bioterrorism raises justifiable concerns that typhus could affect civilization just as greatly in the future as it has in the past.

Disease Outbreaks↗

PubMed2003

A handheld real time thermal cycler for bacterial pathogen detection.

The handheld advanced nucleic acid analyzer (HANAA) is a portable real time thermal cycler unit that weighs under 1 kg and uses silicon and platinum-based thermalcycler units to conduct rapid heating and cooling of plastic reaction tubes. Two light emitting diodes (LED) provide greater than 1 mW of electrical power at wavelengths of 490 nm (blue) and 525 nm (green), allowing detection of the dyes FAM and JOE/TAMRA. Results are displayed in real time as bar graphs, and up to three, 4-sample assays can be run on the charge of the 12 V portable battery pack. The HANAA was evaluated for detection of defined Escherichia coli strains, and wild-type colonies isolated from stream water, using PCR for the lac Z and Tir genes. PCR reactions using SYBR Green dye allowed detection of E. coli ATCC 11775 and E. coli O157:H7 cells in under 30 min of assay time; however, background fluorescence associated with dye binding to nonspecific PCR products was present. DNA extracted from three isolates of Bacillus anthracis Ames, linked to a bioterrorism incident in Washington DC in October 2001, were also successfully tested on the HANAA using primers for the vrrA and capA genes. Positive results were observed at 32 and 22 min of assay time, respectively. A TaqMan probe specific to the aroQ gene of Erwinia herbicola was tested on the HANAA and when 500 cells were used as template, positive results were observed after only 7 min of assay time. Background fluorescence associated with the use of the probe was negligible. The HANAA is unique in offering real time PCR in a handheld format suitable for field use; a commercial version of the instrument, offering six reaction chambers, is available as of Fall 2002.

Bacillus anthracis↗

PubMed2002

Whole genome amplification--applications and advances.

The concept of whole genome amplification is something that has arisen in the past few years as the polymerase chain reaction (PCR) has been adapted to replicate regions of genomes that are of biological interest. The applications are many--forensic science, embryonic disease diagnosis, bioterrorism genome detection, "immortalization" of clinical samples, microbial diversity, and genotyping. Several recent papers suggest that whole genomes can be replicated without bias or non-random distribution of the target, these findings open up a new avenue to molecular biology.

Animals↗

PubMed2002

Defining and using microbial spectral databases.

This work shows how fingerprints of mass spectral patterns from microbial isolates are affected by variations in instrumental condition, by sample environment, and by sample handling factors. It describes a novel method by which pattern distortions can be mathematically corrected for variations in factors not amenable to experimental control. One uncontrollable variable is "between-batch" differences in culture media. Another, relevant for determination of noncultured extracts, is differences between the cells' environmental experience (e.g., starved environmental extracts versus cultured standards). The method suggests that, after a single growth cycle on a solid medium (perhaps, a selective one), pyrolysis MS spectra of microbial isolates can be algorithmically compensated and an unknown isolate identified using a spectral database defined by culture on a different (perhaps, nonselective) medium. This reduces identification time to as few as 24 h from sample collection. The concept also proposes a possible way to compensate certain noncultured, nonisolated samples (e.g., cells concentrated from urine or impacted from aerosol or semi-selectively extracted by immunoaffinity methods from heavily contaminated matrices) for identification within half an hour. Using the method, microbial mass spectra from different labs can be assembled into coherent databases similar to those routinely used to identify pure compounds. This type of data treatment is applicable for rapid detection in biowarfare and bioterror events as well as in forensic, research, and clinical laboratory contexts.

Algorithms↗

PubMed2003

Immunization issues for the 21st century.

OBJECTIVE: Review and discuss major issues of vaccination and immunization. The development and application of vaccination and immunization is one of the most remarkable successes of the 20th century. This is true both in the United States and worldwide. In the United States, a number of vaccine-preventable diseases have been all but eliminated through the development of a recommended childhood immunization schedule by governmental and nongovernmental organizations, education of providers about these recommendations, and enforcement of these recommendations by school and day care entry mandates. Despite these successes, vaccine-preventable diseases continue to occur, in part because of missed opportunities by health care providers, antivaccine forces empowered by misguided mass media, and parental ignorance. Important aspects of the 2002 recommended childhood immunization schedule are reviewed, including: birth dose hepatitis B, diphtheria underimmunization and tetanus overimmunization, increasing pertussis disease, the success of conjugate vaccines, the change in poliovirus vaccines, measles vaccine and autism, the safety of varicella vaccine, and adult vaccination recommendations. Finally, future prospects for vaccination and immunization are discussed, including: combination vaccines, vaccines against new diseases such as rotavirus, new routes of delivery of immunizing agents, the use of computerized vaccine registries to prevent missed opportunities, and vaccines against bioterrorism agents. CONCLUSIONS: A careful analysis of risk and benefit suggests that the benefit of vaccination far outweighs the risks from the utilization of immunizing agents. Vaccination delayed may be protection denied. The bottom line is that vaccines are good and disease is bad.

Child Welfare↗

PubMed2003

Innate and adaptive immune responses to an intracellular bacterium, Francisella tularensis live vaccine strain.

The immune response to intracellular bacterium, Francisella tularensis, which causes tularemia and is proposed to be a potential bioterrorism pathogen, has been studied in mice using the attenuated live vaccine strain (LVS). Here we review this infection model, which provides a convenient means of studying protective immune mechanisms not only for Francisella, but also for the large and important class of intracellular pathogens.

Animals↗

PubMed2002

Infectious disease 2001: drug resistance, new drugs.

The 41st Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC) was held in Chicago on 16-19 December 2001, rescheduled following the tragic events of 11th September. Nonetheless, it attracted thousands of delegates from industry and academia and covered, in over 2200 oral and poster presentations, topics ranging from bioterrorism, microbial pathogenesis and emerging pathogens, to infection control, vaccines, antibiotic resistance and new antimicrobial agents and treatment strategies. Summarized here are highlights on bacterial and fungal drug resistance and on new agents in preclinical and clinical development.

Animals↗

PubMed2004

Laboratory-acquired vaccinia infection.

BACKGROUND: Complications following vaccination with vaccinia virus have been well described but are not commonly observed. The use of vaccinia as a tool in molecular biology, in the development of therapeutics, and the anticipated increase of vaccinations in the general population due to the threat of bioterrorism have created a renewed awareness of the post-vaccination complications and the consequent need for clinical and laboratory diagnosis. OBJECTIVES: To report the clinical presentation and subsequent diagnosis of generalized vaccinia that resulted from a laboratory accident in an unvaccinated subject. STUDY DESIGN: The patient was seen by a local infectious disease's specialist and evaluated clinically and with laboratory support relative to a differential diagnosis. RESULTS: Careful assessment of the patient's history, an evaluation of the workplace, and the elimination of likely microbial etiologies led to the diagnosis of generalized vaccinia. Laboratory confirmation was obtained by use of electron microscopy (EM) to observe poxvirus particles in infected cell cultures. CONCLUSIONS: Exposure to vaccinia virus should raise the index of suspicion for patients with skin lesions. Rapid diagnosis may be accomplished by direct examination of lesion material by EM. The virus also readily replicates in commonly available cell cultures and in the absence of immune reagents, typical poxvirus particles may be observed in the infected cells by EM.

Adult↗

PubMed2003

Marine natural products and their potential applications as anti-infective agents.

The oceans are a unique resource that provide a diverse array of natural products, primarily from invertebrates such as sponges, tunicates, bryozoans, and molluscs, and from marine bacteria and cyanobacteria. As infectious diseases evolve and develop resistance to existing pharmaceuticals, the marine environment provides novel leads against fungal, parasitic, bacterial, and viral diseases. Many marine natural products have successfully advanced to the late stages of clinical trials, including dolastatin 10, ecteinascidin-743, kahalalide F, and aplidine, and a growing number of candidates have been selected as promising leads for extended preclinical assessment. Although many marine-product clinical trials are for cancer chemotherapy, drug resistance, emerging infectious diseases, and the threat of bioterrorism have all contributed to the interest in assessing natural ocean products in the treatment of infectious organisms. In this review, we focus on the pharmacologically tested marine leads that have shown in-vivo efficacy or potent in-vitro activity against infectious and parasitic diseases.

Animals↗

PubMed2005

Smallpox vaccine and its stockpile in 2005.

Smallpox vaccine was the most important tool in the successful eradication of smallpox. In 1980, this achievement made it possible for all nations to cease smallpox vaccination. However, the threat of smallpox bioterrorism has made it necessary to reconsider the need for vaccination. Over the past 3 years, many nations have set up action plans for use in the event of such an attack. The setting up of these plans was not simple. Several factors needed to be considered, including the judgement of risk, vaccine complications, conventional vaccines versus new vaccines, optimal stockpile of smallpox vaccine, and its use for different target populations in different emergency situations. Here, I review measures taken by the USA, Japan, and other nations, and discuss likely national and global efforts in 2005 and subsequently, in view of the fact that half of the world's population is now apparently unvaccinated and that this proportion will increase with time.

Europe↗

PubMed2006

Yersinia pestis as a telluric, human ectoparasite-borne organism.

The classic epidemiological model of plague is an infection of rodents that is transmitted to human beings by rodent ectoparasites. This model fits with observations of sporadic and limited outbreaks, but hardly explains the persistence of plague foci for millennia or the epidemiological features drawn from the descriptions of historical pandemics. A comprehensive review of the published data, including scientific papers published in France between 1920 and 1940, allows the completion of the epidemiological chain by introducing soil as a reservoir, burrowing rodents as a first link, and human ectoparasites as the main driving force for pandemics. Modern studies are needed to confirm the validity of this controversial model and to assess the relative contribution of each link in the various epidemiological presentations of plague. If confirmed, these data should be taken into account to update public-health policies and bioterrorism risk management, particularly among ectoparasite-infested people.

Animals↗

PubMedDate not supplied

The Strategic National Stockpile: roles and responsibilities of health care professionals for receiving the stockpile assets.

In 1999, the US Congress created the National Pharmaceutical Stockpile Program (NPS) under the management of the Centers for Disease Control and Prevention (CDC) and the Department of Health and Human Services. On March 1, 2003, the National Pharmaceutical Stockpile Program became known as the Strategic National Stockpile (SNS) Program under the new Department of Homeland Security. The purpose of the SNS Program is to maintain a stockpile of pharmaceutical agents, vaccines, medical supplies, and equipment to augment state and local resources during a large-scale disaster or bioterrorism event. Upon request, the SNS Program will deliver materials anywhere in the United States within 12 or fewer hours. A brief overview of the SNS Program and the role of local and state health care providers for receiving the SNS assets are reviewed.

Anti-Bacterial Agents↗

PubMedDate not supplied

Terrorism in South Korea.

South Korea has experienced > 30 suspected terrorism-related events since 1958, including attacks against South Korean citizens in foreign countries. The most common types of terrorism used have included bombings, shootings, hijackings, and kidnappings. Prior to 1990, North Korea was responsible for almost all terrorism-related events inside of South Korea, including multiple assassination attempts on its presidents, regular kidnappings of South Korean fisherman, and several high-profile bombings. Since 1990, most of the terrorist attacks against South Korean citizens have occurred abroad and have been related to the emerging worldwide pattern of terrorism by international terrorist organizations or deranged individuals. The 1988 Seoul Olympic Games provided a major stimulus for South Korea to develop a national emergency response system for terrorism-related events based on the participation of multiple ministries. The 11 September 2001 World Trade Center and Pentagon attacks and the 2001 United States of America (US) anthrax letter attacks prompted South Korea to organize a new national system of emergency response for terrorism-related events. The system is based on five divisions for the response to specific types of terrorist events, involving conventional terrorism, bioterrorism, chemical terrorism, radiological terrorism, and cyber-terrorism. No terrorism-related events occurred during the 2002 World Cup and Asian Games held in South Korea. The emergency management of terrorism-related events in South Korea is adapting to the changing risk of terrorism in the new century.

Disaster Planning↗

PubMed2004

Identification of biomarkers of whole Coxiella burnetii phase I by MALDI-TOF mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) specific biomarkers have been shown to be an effective tool for identifying microorganisms. In this study, we demonstrate the feasibility of using this technique to detect the obligate intracellular bacterium Coxiella burnetii, a category B bioterrorism agent. Specific biomarkers were detected in C. burnetii Nine Mile phase I (NMI) strain purified from embryonated egg yolk sac preparations. Whole organisms were applied directly to the MALDI target. MALDI-TOF MS analysis of C. burnetii NMI grown and purified at different times and places revealed a group of unique, characteristic, and reproducible spectral markers in the mass range of 1000-25000 Da. Statistical analysis of the averaged centroided masses uncovered at least 24 peptides or biomarkers. Three biomarkers observed in the MALDI-TOF MS spectrum consistently matched proteins that had been previously described in C. burnetii, one of them being the small cell variant protein A. MALDI-TOF MS analysis of whole organisms represents a sensitive and specific option for characterizing C. burnetii isolates, especially when coupled with antigen capture techniques. The method also has potential for several applications in basic microbial research, including regulation of gene expression.

Animals↗

PubMed2005

Comparison of glycosphingolipids and antibodies as receptor molecules for ricin detection.

Glycosphingolipids (GSLs) have been shown to undergo strong interactions with a number of protein toxins, including potential bioterrorism agents such as ricin and botulinum neurotoxin. Characterization of this interaction in recent years has led to a number of studies where GSLs were used as the recognition molecules for biosensing applications. Here, we offer a comparison of quartz crystal microbalance (QCM) sensors for the detection of ricin using antibodies and the GSLs GM1 and asialoGM1, which have been shown to undergo strong interactions with ricin. The presence, orientation, and activity of the GSL and antibody films were confirmed using ellipsometry, Fourier transform infrared spectroscopy (FT-IR), and QCM. It was found that the GSLs offered more sensitive detection limits when directly compared with antibodies. Both GSLs had lower detection limits at 5 microg/mL, approximately 5 times lower than were found for antibodies (25 microg/mL), and their linear detection range extended to the highest concentrations tested (100 microg/mL), almost an order of magnitude beyond the saturation point for the antibody sensors. Potential sites for nonspecific adsorption were blocked using serum albumin without sacrificing toxin specificity.

Biosensing Techniques↗

PubMed2005

Low-level detection of viral pathogens by a surface-enhanced Raman scattering based immunoassay.

The need for rapid, highly sensitive, and versatile diagnostic tests for viral pathogens spans from human and veterinary medicine to bioterrorism prevention. As an approach to meet these demands, a diagnostic test employing monoclonal antibodies (mAbs) for the selective extraction of viral pathogens from a sample in a chip-scale, sandwich immunoassay format has been developed using surface-enhanced Raman scattering (SERS) as a readout method. The strengths of SERS-based detection include its inherent high sensitivity and facility for multiplexing. The capability of this approach is demonstrated by the capture of feline calicivirus (FCV) from cell culture media that is exposed to a gold substrate modified with a covalently immobilized layer of anti-FCV mAbs. The surface-bound FCVs are subsequently coupled with an extrinsic Raman label (ERL) for identification and quantification. The ERLs consist of 60-nm gold nanoparticles coated first with a layer of Raman reporter molecules and then a layer of mAbs. The Raman reporter molecule is strategically designed to chemisorb as a thiolate adlayer on the gold nanoparticle, to provide a strong and unique spectral signature, and to covalently link a layer of mAbs to the gold nanoparticle. The last feature provides a means to selectively tag substrate-bound FCV. This paper describes the development of the assay, which uses cell culture media as a sample matrix and has a linear dynamic range of 1 x 10(6)-2.5 x 10(8) viruses/mL and a limit of detection of 1 x 10(6) viruses/mL. These results reflect the findings from a detailed series of investigations on the effects of several experimental parameters (e.g., salt concentration, ERL binding buffer, and sample agitation), all of which were aimed at minimizing nonspecific binding and maximizing FCV binding efficiency. The performance of the assay is correlated with the number of captured FCV, determined by atomic force microscopy, as a means of method validation.

Buffers↗

PubMed2005

Aflatoxin screening by MALDI-TOF mass spectrometry.

Efficient detection of aflatoxins B1, B2, G1, and G2 has been performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using a UV-absorbing ionic liquid matrix to obtain "matrix-free" mass spectra and addition of NaCl to enhance sensitivity via Na+ cationization. Using ionic alpha-cyano-4-hydroxycinnamic acid (Et3N-alpha-CHCA) as the matrix, matrix-free mass spectra in the m/z range of interest are acquired, and the B1, B2, G1, and G2 aflatoxins are readily detected with an LOD as low as 50 fmol. The technique is fast, requires little sample preparation and no derivatization or chromatographic separation, and seems therefore to be suitable for high-throughput aflatoxin screening. It should be easily extended to other micotoxins and provide an attractive technique to control the quality of major crops subjected to huge world commercial trades such as peanuts, corn, and rice as well as to monitor bioterrorism threats by micotoxin poisoning.

Aflatoxins↗