Evaluation of the rapid analyte measurement platform (RAMP) for the detection of Bacillus anthracis at a crime scene

The aim of this study was to evaluate the accuracy and reliability of the rapid analyte measurement platform (RAMP) for presumptive identification of Bacillus anthracis spores. Test samples consisted of serial dilutions of spore preparations of several Bacillus species, including B. anthracis, which were tested, using the RAMP Anthrax test cartridge, according to the manufacturer's instructions. The fluorescence labelled antibody-antigen complexes were detected in the portable reader after 15 min following sample addition. Dilutions of common environmental and household powders were also tested to identify possible false positive results. B. anthracis spores were identified reliably in test samples containing more than 6000 spores. The test kits were highly specific, showing no cross reactivity with other Bacillus species or any environmental powders tested. The RAMP system for detection of B. anthracis spores, from environmental samples, showed consistent results under a variety of analytical conditions, enabling the trained user to provide a rapid, accurate preliminary risk assessment of a suspected bioterrorism incident.     

Composition of bacillus species in aerosols from 11 U.S. cities

A PCR-based heteroduplex assay was used to determine the presence and composition of Bacillus species in 11,059 Environmental Protection Agency PM2.5 aerosol samples from 11 U.S. cities. The assay differentiated three groups: Type A containing Bacillus anthracis and very closely related, often pathogenic, Bacillus cereus and Bacillus thuringiensis strains; Type B containing other B. cereus and B. thuringiensis strains; and a third group of more-distantly related Bacillus species. Eight of the 11 cities were positive for Bacillus species in 50% or more of the samples, and the percent of aerosol samples that contained the HD Type A group ranged from 3% to 32%. Cities from the eastern half of the United States generally contained a higher frequency and broader diversity of Bacillus species than the western half of the United States. Positive samples were detected throughout the year. These results have implications for pathogen detection in environmental samples, understanding the natural evolution of new pathogenic strains, and incidence of infection caused by strains of the B. cereus subgroup.     

Environmental survey for four pathogenic bacteria and closely related species using phylogenetic and functional genes

Bacterial species with high DNA sequence similarity to pathogens could affect the specificity of assays designed to detect biological threat agents in environmental samples. The natural presence of four pathogenic bacteria, Bacillus anthracis, Clostridium perfringens, Francisella tularensis, and Yersinia pestis and their closely related species, was determined for a large collection of soil and aerosol samples. Polymerase chain reaction (PCR) and gene sequencing were used using group-specific 16S rRNA primers to identify pathogens and related species, and pathogen-specific virulence genes. Close relatives of B. anthracis (B. cereus group species) were detected in 37% of the soils and 25% of the aerosol samples. The B. anthracis protective antigen (pag) gene or a close homolog was detected in 16 of these samples. For the other three pathogen groups, the frequency of detection was much lower, and none of the samples were positive with both the phylogenetic and virulence gene primer sets.     

Stable isotope ratios as a tool in microbial forensics--Part 1. Microbial isotopic composition as a function of growth medium

The stable isotope ratios of a seized pathogen culture could potentially reveal information about the environment in which the agent was produced. In this paper we describe general relationships between stable isotopes of carbon, nitrogen, and hydrogen in bacteriological culture media and spores of Bacillus subtilis, an endospore-forming soil bacterium. In numerous media that varied both in nutrient composition and water stable isotope ratios, medium to spore enrichment in carbon isotopes was 0.3 +/- 2.0% per thousand (parts per thousand), and in nitrogen, 4.5 +/- 0.7% per thousand. We achieved mass balance for the contribution of hydrogen isotopes from nutrients (70%) and water (30%) to spores in independent experiments by varying the isotope ratios of nutrients or water. A model was derived for predicting the isotope ratio values of spores from those in nutrients and water.     

Comparison of quantitative PCR and culture-based methods for evaluating dispersal of Bacillus thuringiensis endospores at a bioterrorism hoax crime scene

Since the anthrax mail attacks of 2001, law enforcement agencies have processed thousands of suspicious mail incidents globally, many of which are hoax bioterrorism threats. Bio-insecticide preparations containing Bacillus thuringiensis (Bt) spores have been involved in several such threats in Australia, leading to the requirement for rapid and sensitive detection techniques for this organism, a close relative of Bacillus anthracis. Here we describe the development of a quantitative PCR (qPCR) method for the detection of Bt crystal toxin gene cry1, and evaluation of the method's effectiveness during a hoax bioterrorism event in 2009. When combined with moist wipe sampling, the cry1 qPCR was a rapid, reliable, and sensitive diagnostic tool for detecting and quantifying Bt contamination, and mapping endospore dispersal within a mail sorting facility. Results from the cry1 qPCR were validated by viable counts of the same samples on Bacillus-selective agar (PEMBA), which revealed a similar pattern of contamination. Extensive and persistent contamination of the facility was detected, both within the affected mailroom, and extending into office areas up to 30m distant from the source event, emphasising the need for improved containment procedures for suspicious mail items, both during and post-event. The cry1 qPCR enables detection of both viable and non-viable Bt spores and cells, which is important for historical crime scenes or scenes subjected to decontamination. This work provides a new rapid method to add to the forensics toolbox for crime scenes suspected to be contaminated with biological agents.     

Fatty Acid Profiles for Differentiating Growth Medium Formulations Used to Culture Bacillus cereus T‐strain Spores

Microbial biomarkers that indicate aspects of an organism's growth conditions are important targets of forensic research. In this study, we examined fatty acid composition as a signature for the types of complex nutrients in the culturing medium. Bacillus cereus T‐strain spores were grown in medium formulations supplemented with one of the following: peptone (meat protein), tryptone (casein protein), soy protein, and brain–heart infusion. Cellular biomass was profiled with fatty acid methyl ester (FAME) analysis. Results showed peptone cultures produced spores enriched in straight‐chained lipids. Tryptone cultures produced spores enriched in branched‐odd lipids when compared with peptone, soy, and brain–heart formulations. The observed FAME variation was used to construct a set of discriminant functions that could help identify the nutrients in a culturing recipe for an unknown spore sample. Blinded classification tests were most successful for spores grown on media containing peptone and tryptone, showing 88% and 100% correct identification, respectively.     

Pancuronium bromide (Pavulon) isolation and identification in aged autopsy tissues and fluids

The isolation and detection of pancuronium bromide was developed for aged autopsy samples to identify and confirm this compound in questioned tissue samples. A novel protocol was optimized for the isolation of the target drug in highly decomposed tissues. Solid-phase extraction (SPE) cartridges containing styrene-divinylbenzene were investigated. This polymer retained quaternary drugs and facilitated sequential elution upon washing with commonly available solvents. The semi-purified SPE samples were prescreened by pyrolysis GC-MS. A candidate specimen was then confirmed by microbore high-performance liquid chromatography/electrospray-ionization/mass spectrometry (microHPLC-ESI-MS/MS) with a triple-quadrupole mass spectrometer. The developed procedures provided a qualitative or semiquantitative (at best) basis for the investigation of difficult cases involving overdoses of polar drugs.     

Effect of Sterilants on Amplification and Detection of Target DNA from Bacillus cereus Spores

To conceal criminal activity of a bioterrorist or agroterrorist, the site of pathogen generation is often treated with sterilants to kill the organisms and remove evidence. As dead organisms cannot be analyzed by culture, this study examined whether DNA from sterilant‐treated Bacillus cereus spores was viable for amplification. The spores were exposed to five common sterilants: bleach, Sterilox®, oxidizer foam (L‐Gel), a peroxyacid (Actril®), and formaldehyde vapor. The spores were inoculated on typical surfaces found in offices and laboratories to test for environmental effects. It was found that the surface influenced the efficiency of recovery of the organisms. The DNA isolated from the recovered spores was successfully detected using RT‐qPCR for all treatments except for formaldehyde, by amplifying the phosphatidylinositol phospholipase C and sphingomyelinase genes. The results demonstrated that evidence from sites treated with sterilants can still provide information on the uncultured organism, using DNA amplification.     

Method validation of a survey of thevetia cardiac glycosides in serum samples

A sensitive and specific liquid chromatography tandem mass spectrometry (HPLC-ESI(+)-MS/MS) procedure was developed and validated for the identification and quantification of thevetin B and further cardiac glycosides in human serum. The seeds of Yellow Oleander (Thevetia peruviana) contain cardiac glycosides that can cause serious intoxication. A mixture of six thevetia glycosides was extracted from these seeds and characterized. Thevetin B, isolated and efficiently purified from that mixture, is the main component and can be used as evidence. Solid phase extraction (SPE) proved to be an effective sample preparation method. Digoxin-d3 was used as the internal standard. Although ion suppression occurs, the limit of detection (LOD) is 0.27 ng/ml serum for thevetin B. Recovery is higher than 94%, and accuracy and precision were proficient. Method refinement was carried out with regard to developing a general screening method for cardiac glycosides. The assay is linear over the range of 0.5-8 ng/ml serum. Finally, the method was applied to a case of thevetia seed ingestion.     

Validity testing of commercial urine cocaine metabolite assays: II. Sensitivity, specificity, accuracy, and confirmation by gas chromatography/mass spectrometry

A comprehensive validity assessment study was performed on eight commercial urine assays for detection of cocaine use. Sensitivity, specificity, and accuracy of each assay were evaluated by analyzing, in random order and under blind conditions, specimens spiked with known drug concentrations and clinical specimens obtained from human subjects after intravenous cocaine use. Commercial assay results were compared with gas chromatography/mass spectrometry (GC/MS) assay of the same specimens for benzoylecgonine. All of the assays examined were determined to have utility in screening for cocaine use, with the exception of the KDI Quik Test, which was not a reliable test for detection of cocaine use. Major differences in sensitivity, specificity, and confirmation rate by GC/MS were noted among the assays, differences which should be taken into consideration when implementing a urine screening test for cocaine use or interpreting test results involving use of these assays.     

Bioterrorism: processing contaminated evidence, the effects of formaldehyde gas on the recovery of latent fingermarks

In the present age of heightened emphasis on counter terrorism, law enforcement and forensic science are constantly evolving and adapting to the motivations and capabilities of terrorist groups and individuals. The use of biological agents on a population, such as anthrax spores, presents unique challenges to the forensic investigator, and the processing of contaminated evidence. In this research, a number of porous and nonporous items were contaminated with viable anthrax spores and marked with latent fingermarks. The test samples were then subjected to a standard formulation of formaldehyde gas. Latent fingermarks were then recovered post decontamination using a range of methods. Standard fumigation, while effective at destroying viable spores, contributed to the degradation of amino acids leading to loss of ridge detail. A new protocol for formaldehyde gas decontamination was developed which allows for the destruction of viable spores and the successful recovery of latent marks, all within a rapid response time of less than 1 h.     

Pathogen detection using a liquid array technology

Abstract: Low concentrations of microbial pathogens in pure and mixed samples were detected using a bead‐based, liquid array technology. A 20‐bp sequence in the 23S rRNA gene, rrl, was amplified in four microorganisms: Bacillus cereus, Escherichia coli, Salmonella enterica and Staphylococcus aureus. PCR products were positively identified with the Luminex^® 100? system. The system could detect very low amounts of DNA and the instrument response was proportional to the input concentration. The lower limit of detection (LLD) was determined to be 0.5 ng for B. cereus and E. coli and 2 ng for S. enterica. The LLD for S. aureus was not determined as the instrument response was still above the threshold when quantities of DNA as low as 0.25 ng were used. The platform positively identified organisms present in mixed samples even when the minor component was overshadowed by a 10‐fold excess of the major component.     

Detection of Anthrax and Other Pathogens Using a Unique Liquid Array Technology

A bead‐based liquid hybridization assay, Luminex^® 100?, was used to identify four pathogenic bacteria, Bacillus anthracis, Clostridium botulinum, Francisella tularensis subsp. tularensis, and Yersinia pestis, and several close relatives. Hybridization between PCR‐amplified target sequences and probe sequences (located within the 23S ribosomal RNA gene rrl and the genes related to the toxicity of each bacterium) was detected in single‐probe or multiple‐probe assays, depending on the organism. The lower limits of detection (LLDs) for the probes ranged from 0.1 to 10 ng. Sensitivity was improved using lambda exonuclease to digest the noncomplementary target strand. All contributors in 33 binary, ternary, and quaternary mixtures in which all components were present in a 1:1 ratio were identified with an 80% success rate. Twenty‐eight binary mixtures in which the two components were combined in various ratios were further studied. All target sequences were detected, even when the minor component was overshadowed by a tenfold excess of the major component.     

Evaluation of desorption/ionization mass spectrometric methods in the forensic applications of the analysis of inks on paper.

Fast atom bombardment and laser desorption mass spectrometry (LDMS) provide molecular level information concerning an ink's composition. Two ink-jet printer inks, Ink A containing the cationic dye Methyl Violet 2B, and Ink B containing the anionic dye, Solvent Black, were studied. Both positive and negative ion detection modes of the mass spectrometer were used. LD may be used for the analysis of inks on paper. Once on paper, the ink's solvent system has evaporated, leaving mainly the dyes behind, which are detected using LDMS. An ink fades with time, indicating that the dyes are degrading. Preliminary results from an accelerated aging study of ballpoint pen ink using UV irradiation confirm that dye degradation products are formed. The degradation chemistry follows an oxidative demethylation process for which all products formed are detected using LDMS. Results suggest that LDMS may be developed to determine the relative age of inks.     

Toxicological analysis of thiamylal in biological materials by gas chromatography/mass spectrometry

A reliable and sensitive method to analyze thiamylal in biological materials was developed, using gas chromatography/mass spectrometry (GC/MS). A quantitative determination was made by use of mass fragmentography with the lower detection limit of 0.01 microgram/g. Thiopental was used as the internal standard. Distribution of the drug in the blood and body tissues of rats was examined. The method was then used to detect thiamylal in tissues from an autopsied patient and concentration of this drug in the body materials was evaluated, from medico-legal aspects.     

Comparison of glass fragments using particle-induced X-ray emission (PIXE) spectrometry

A procedure has been developed to analyze the trace element concentrations in glass fragments using particle-induced X-ray emission (PIXE) spectrometry. This method involves using accelerated protons to excite inner-shell electronic transitions of target atoms and recording the resultant X-rays to characterize the trace element concentrations. The protocol was able to identify those glass fragments that originated from different sources based on their elemental analyses. The protocol includes specific approaches to calculating uncertainties and handling measurements below the level of detection. The results indicate that this approach has increased sensitivity for several elements with higher atomic number compared with X-ray fluorescence methods. While not as sensitive as laser-ablation or inductively coupled plasma mass spectrometry methods of dissolved samples, it is entirely nondestructive and entails a much simpler sample preparation process that may be used to presort glass fragments for more comprehensive elemental analysis. As such, the technique described may have a niche role in forensic glass analysis.     

Validity testing of commercial urine cocaine metabolite assays: III. Evaluation of an enzyme-linked immunosorbent assay (ELISA) for detection of cocaine and cocaine metabolite

A validity assessment study was performed on the Genetic Diagnostic Enzyme Immunoassay test kit, a new enzyme-linked immunosorbent assay (GDC ELISA) for detection of cocaine and cocaine metabolite in urine. A set of 290 urine specimens, comprised of clinical cocaine urines collected from 5 male subjects who had received single doses of intravenous cocaine, drug-free urines spiked with cocaine, cocaine metabolites, cocaine isomers, and other drugs of abuse, were assayed by GDC ELISA. The results were compared with results by gas chromatography/mass spectrometry (GC/MS) assay for benzoylecgonine. Concordance was high between the GDC ELISA assay and GC/MS and with results reported earlier for other commercial assays. Detection times and specificity of the GDC ELISA antibody were most similar to those of the Abuscreen radioimmunoassay for cocaine metabolite. Overall, the assay produced no false negative or false positive results and appeared to be a reliable screening test for detection of cocaine and benzoylecgonine in human urine.     

Evaluation of Commercial‐off‐the‐Shelf Materials for the Preservation of Bacillus anthracis Vegetative Cells for Forensic Analysis,

Environmental surface sampling is crucial in determining the zones of contamination and overall threat assessment. Viability retention of sampled material is central to such assessments. A systematic study was completed to determine viability of vegetative cells under nonpermissive storage conditions. Despite major gains in nucleic acid sequencing technologies, initial positive identification of threats must be made through direct culture of the sampled material using classical microbiological methods. Solutions have been developed to preserve the viability of pathogens contained within clinical samples, but many have not been examined for their ability to preserve biological agents. The purpose of this study was to systematically examine existing preservation materials that can retain the viability of Bacillus anthracis vegetative cells stored under nonpermissive temperatures. The results show effectiveness of five of seventeen solutions, which are capable of retaining viability of a sporulation deficient strain of B. anthracis Sterne when stored under nonrefrigerated conditions.     

Forensic intoxication with clobazam: HPLC/DAD/MSD analysis

Clobazam (Castillium, Urbanil), a benzodiazepine often used as an anxiolytic and in the treatment of epilepsy, is considered a relatively safe drug. The authors present a fatal case with a 49-year-old female, found dead at home. She had been undergoing psychiatric treatment and was a chronic alcoholic. The autopsy findings were unremarkable, except for multivisceral congestion, steatosis and a small piece of a plastic blister pack in the stomach. Bronchopneumonia, bronchitis and bronchiolitis were also diagnosed. Anhigh-performance liquid chromatography (HPLC)/diode array detector (DAD)/mass spectrometry detection (MSD) with electrospray method was developed in order to detect, confirm and quantify clobazam in the post-mortem samples. In the chromatographic separation, a reversed-phase column C18 (2.1 x 150 mm, 3.5 microm) was used with a mobile phase of methanol and water, at a 0.25 ml/min flow rate. Carbonate buffer (pH 10.5) and 20 microl of prazepam (100 microg/ml) as internal standard were added to the samples. A simple and reliable liquid-liquid extraction method for the determination of clobazam in post-mortem samples was described. Calibration curves for clobazam were performed in blood, achieving linearity between 0.01 and 10 microg/ml and a detection limit of 1.0 ng/ml. The clobazam concentration found in post-mortem blood was 3.9 microg/ml, higher than the reported therapeutic concentration (0.1-0.4 microg/ml). The simultaneous acquisition by photodiode array detection and mass spectrometry detection results allowed benzodiazepines to be identified with sufficient certainty. An examination of all the available information suggested that death resulted from respiratory depression due to clobazam toxicity.     

Evaluation of Four Fingerprint Development Methods for Touch Chemistry Using Matrix‐Assisted Laser Desorption Ionization/Time‐of‐Flight Mass Spectrometry,

Four preparation techniques for MALDI/TOF mass spectrometry were compared to determine the ability to gather intelligence for investigations through the chemical analysis of latent fingerprints, defined as “touch chemistry.” Compatible fingerprint development processes used for identification along with new techniques are necessary to evaluate touch chemistry. Ten volunteers deposited fingerprints from solvent residues containing drugs and explosives onto microscope slides. The developers included (A) fingerprint powder, (B) MALDI matrix, (C) fingerprint powder and lifting, and (D) cyanoacrylate fuming with fingerprint powder. Qualitative identification was based on ion images and spectra. The highest average detection rates (88%) were found using methods A and B. Methods C (52%) or D (18%) had limited success. Results demonstrate the importance of imaging coupled to extracted mass spectral data in detecting analytes in deposited fingerprints. Overall, the results suggest continued development of touch chemistry applications could prove useful for gathering intelligence and forensically relevant information.