Evaluation of bioelectronics sensor compared to other diagnostic test in diagnosis of Johne's disease in goats

A bioelectronics sensor has been developed and it is evaluated for the diagnosis of paratuberculosis in goats. Initially hematite nanoparticles were prepared and using this nanoparticles as core, electrically active polyaniline coated magnetic (EAPM) nanoparticles are synthesized from aniline monomer (made electrically active by acid doping). These EAPM nanoparticles were fabricated with rabbit anti-goat IgG for the detection of goat antibodies on the capture pad. The protoplasmic antigen of Mycobacterium avium subspecies paratuberculosis (MAP) immobilized onto the capture pad will detect the antibody against MAP in the goat sera samples. This bound goat antibody will be detected by the anti-goat IgG previously bound to EAPM. Upon detection the EAPM nanoparticles bridges an electric circuit between the silver electrodes, flanking the capture membrane. The electrical conductance, caused by EAPM, was measured as direct charge transfer between the electrodes. Testing of the biosensor with known Johne's disease (JD) positive and negative serum samples gave significant difference in the electrical conductance value. Further the efficacy of this biosensor was compared with other serological tests like agar gel immunodiffusion (AGID) and absorbed ELISA using field sera. Out of 265 goat sera tested, positive results recorded were; AGID 36 (13.59%), bioelectronics sensor 49 (19.14%), and absorbed ELISA 51 (19.25%). This biosensor was also compared in live animals using intradermal Johnin test and nested PCR (detecting mycobacterial DNA in feces) in 65 animals. Of which, positive results recorded in animals were; Johnin test 21 (32%), biosensor 26 (40%) and fecal PCR detected mycobacterial DNA in 28 (43%) animals. Though the nanobioelectronics sensor was slightly less sensitive (not statistically significant) compared to absorbed ELISA and fecal nested PCR for mycobacterial DNA but it was simple to perform in field conditions and requires less time. The speed of detection and the equipment involved would support its application toward the various point-of-care opportunities aimed at control and management of Johne's disease in goats.     

Rapid detection of Bacillus anthracis using monoclonal antibody functionalized QCM sensor

Since the anthrax spore bioterrorism attacks in America in 2001, the early detection of Bacillus anthracis spores and vegetative cells has gained significant interest. At present, many polyclonal antibody-based quartz crystal microbalance (QCM) sensors have been developed to detect B. anthracis simulates. To achieve a simultaneous rapid detection of B. anthracis spores and vegetative cells, this paper presents a biosensor that utilizes an anti-B. anthracis monoclonal antibody designated to 8G3 (mAb 8G3, IgG) functionalized QCM sensor. Having compared four kinds of antibody immobilizations on Au surface, an optimized mAb 8G3 was immobilized onto the Au electrode with protein A on a mixed self-assembled monolayer (SAM) of 11-mercaptoundecanoic acid (11-MUA) and 6-mercaptohexan-1-ol (6-MHO) as adhesive layer. The detection of B. anthracis was investigated under three conditions: dip-and-dry, static addition and flow through procedure. The results indicated that the sensor yielded a distinct response to B. anthracis spores or vegetative cells but had no significant response to Bacillus thuringiensis species. The functionalized sensor recognized B. anthracis spores and vegetative cells specifically from its homophylic ones, and the limit of detection (LOD) reached 10(3)CFU or spores/ml of B. anthracis in less than 30 min. Cyclic voltammogram (CV) and scanning electronic microscopy (SEM) were performed to characterize the surface of the sensor in variable steps during the modification and after the detection. The mAb functionalized QCM biosensor will be helpful in the fabrication of a similar biosensor that may be available in anti-bioterrorism in the future.     

Nanowire labeled direct-charge transfer biosensor for detecting Bacillus species

A direct-charge transfer (DCT) biosensor was developed for the detection of the foodborne pathogen, Bacillus cereus. The biosensor was fabricated using antibodies as the sensing element and polyaniline nanowire as the molecular electrical transducer. The sensor design consisted of four membrane pads, namely, sample application, conjugate, capture and absorption pads. Two sets of polyclonal antibodies, secondary antibodies conjugated with polyaniline nanowires and capture antibodies were applied to the conjugate and the capture pads of the biosensor, respectively. The detection technique was based on capillary flow action which allowed the liquid sample to move from one membrane to another. The working principle involved antigen-antibody interaction and direct electron charge flow to generate a resistance signal that was being recorded. Detection from sample application to final results was completed in 6 min in a reagentless process. Experiments were conducted to find the best performance of the biosensors by varying polyaniline types and concentrations. Polyaniline protonated with hydrochloric acid, emeraldine salt and polyaniline protonated with perchloric acid were the three kinds of polyaniline used in this study. The biosensor sensitivity in pure cultures of B. cereus was found to be 10(1) to 10(2)CFU/ml. Results indicated that using emeraldine salt at a concentration of 0.25 g/ml gave the best biosensor performance in terms of sensitivity. The biosensor was also found to be specific in detecting the presence of B. cereus in a mixed culture of different Bacillus species and other foodborne pathogens. The speed, sensitivity and ease-of-use of this biosensor make it a promising device for rapid field-based diagnosis towards the protection of our food supply chain. The phenotypic and genotypic similarities between B. cereus and Bacillus anthracis will also allow this biosensor to serve as an excellent model for the detection of B. anthracis.     

DNA probe functionalized QCM biosensor based on gold nanoparticle amplification for Bacillus anthracis detection

The rapid detection of Bacillus anthracis, the causative agent of anthrax disease, has gained much attention since the anthrax spore bioterrorism attacks in the United States in 2001. In this work, a DNA probe functionalized quartz crystal microbalance (QCM) biosensor was developed to detect B. anthracis based on the recognition of its specific DNA sequences, i.e., the 168 bp fragment of the Ba813 gene in chromosomes and the 340 bp fragment of the pag gene in plasmid pXO1. A thiol DNA probe was immobilized onto the QCM gold surface through self-assembly via Au-S bond formation to hybridize with the target ss-DNA sequence obtained by asymmetric PCR. Hybridization between the target DNA and the DNA probe resulted in an increase in mass and a decrease in the resonance frequency of the QCM biosensor. Moreover, to amplify the signal, a thiol-DNA fragment complementary to the other end of the target DNA was functionalized with gold nanoparticles. The results indicate that the DNA probe functionalized QCM biosensor could specifically recognize the target DNA fragment of B. anthracis from that of its closest species, such as Bacillus thuringiensis, and that the limit of detection (LOD) reached 3.5 × 10(2)CFU/ml of B. anthracis vegetative cells just after asymmetric PCR amplification, but without culture enrichment. The DNA probe functionalized QCM biosensor demonstrated stable, pollution-free, real-time sensing, and could find application in the rapid detection of B. anthracis.     

The effect of salt and phage concentrations on the binding sensitivity of magnetoelastic biosensors for Bacillus anthracis detection

This article presents an investigation of the effect of salt and phage concentrations on the binding affinity of magnetoelastic (ME) biosensors. The sensors were fabricated by immobilizing filamentous phage on the ME platform surface for the detection of Bacillus anthracis spores. In response to the binding of spores to the phage on the ME biosensor, a corresponding decrease occurs in resonance frequency. Transmission electron microscopy (TEM) was used to verify the structure of phage under different combinations of salt/phage concentration. The chemistry of the phage solution alters phage bundling characteristics and, hence, influences both the sensitivity and detection limit of the ME biosensors. The frequency responses of the sensors were measured to determine the effects of salt concentration on the sensors' performance. Scanning electron microscopy (SEM) was used to confirm and quantify the binding of spores to the sensor surface. This showed that 420 mM salt at a phage concentration of 1 x 10(11) vir/mL results in an optimal distribution of immobilized phages on the sensor surface, consequently promoting better binding of spores to the biosensor's surface. Additionally, the sensors immobilized with phage under this condition were exposed to B. anthracis spores in different concentrations ranging from 5 x 10(1) to 5 x 10(8) cfu/mL in a flowing system. The results showed that the sensitivity of this ME biosensor was 202 Hz/decade.     

Detection of frequency resonance energy transfer pair on double-labeled microsphere and Bacillus anthracis spores by flow cytometry

Development of an ultrasensitive biosensor for biological hazards in the environment is a major need for pollutant control and for the detection of biological warfare. Fluorescence methods combined with immunodiagnostic methods are the most common. To minimize background noise, arising from the unspecific adsorption effect, we have adapted the FRET (frequency resonance energy transfer) effect to the immunofluorescence method. FRET will increase the selectivity of the diagnosis process by introducing a requirement for two different reporter molecules that have to label the antigen surface at a distance that will enable FRET. Utilizing the multiparameter capability of flow cytometry analysis to analyze the double-labeling/FRET immunostaining will lead to a highly selective and sensitive diagnostic method. This work examined the FRET interaction of fluorescence-labeled avidin molecules on biotin-coated microspheres as a model system. As target system, we have used labeled polyclonal antibodies on Bacillus anthracis spores. The antibodies used were purified immunoglobulin G (IgG) molecules raised in rabbits against B. anthracis exosoporium components. The antibodies were fluorescence labeled by a donor-acceptor chromophore pair, alexa488 as a donor and alexa594 as an acceptor. On labeling the spores with alexa488-IgG as a donor and alexa594-IgG as an acceptor, excitation at 488 nm results in quenching of the alexa-488 fluorescence (E(q) = 35%) and appearance of the alexa594 fluorescence (E(s) = 22%), as detected by flow cytometry analysis. The FRET effect leads to a further isolated gate (FL1/FL3) for the target spores compared to competitive spores such as B. thuringiensis subsp. israelensis and B. subtilis. This new approach, combining FRET labeling and flow cytometry analysis, improved the selectivity of the B. anthracis spores by a factor of 10 with respect to B. thuringiensis subsp. israelensis and a factor of 100 with respect to B. subtilis as control spores.     

Evaluation of five commercial nucleic acid extraction kits for their ability to inactivate Bacillus anthracis spores and comparison of DNA yields from spores and spiked environmental samples

This study evaluated five commercial extraction kits for their ability to recover DNA from Bacillus anthracis spores and spiked environmental samples. The kits evaluated represent the major types of methodologies which are commercially available for DNA or total nucleic acid extraction, and included the ChargeSwitch gDNA Mini Bacteria Kit, NucliSens Isolation Kit, Puregene Genomic DNA Purification Kit, QIAamp DNA Blood Mini Kit, and the UltraClean Microbial DNA Isolation Kit. Extraction methods were performed using the spores of eight virulent strains of B. anthracis. Viability testing of nucleic acid extracts showed that the UltraClean kit was the most efficient at depleting samples of live B. anthracis spores. TaqMan real-time PCR analysis revealed that the NucliSens, QIAamp and UltraClean kits yielded the best level of detection from spore suspensions. Comparisons of processed samples from spiked swabs and three powder types indicated that DNA extraction using the UltraClean kit resulted in the most consistently positive results and the lowest limit of detection. This study demonstrated that different nucleic extraction methodologies, represented here by various commercial extraction kits, differ in their ability to inactivate live B. anthracis spores as well as DNA yield and purity. In addition, the extraction method used can influence the sensitivity of real-time PCR assays for B. anthracis.     

A combined immunomagnetic separation and lateral flow method for a sensitive on-site detection of Bacillus anthracis spores – assessment in water and dairy products

Aim:  Combination of immunomagnetic separation (IMS) and lateral flow device (LFD) assays for the development of a sensitive, rapid, on-site methodology that enables concentration and detection of Bacillus anthracis spores in complex samples.
Methods and Results:  The data presents the development of an optimized, 30 min, IMS assay, with about 95% capture of B. anthracis spores from different dairy products ( n  = 38). No cross reactivity was detected with typical milk flora and some closely related Bacilli. To enable direct application of the IMS captured spores on the LFD, spores were eluted from the bead–spore complex utilizing 95% (v/v) formamide-10 mmol l⁻¹ EDTA for 30 s in a microwave oven. Detached spores were analysed on LFD enabling detection within 10 min. The combined IMS–LFD methodology (40 min) demonstrates a 60-fold improvement in sensitivity, relative to samples that were applied directly on the LFD without the IMS concentrating step.
Conclusions:  The IMS–LFD method is a powerful platform, combining rapidity, specificity and efficiency for concentrating and detecting B. anthracis from water and milk contaminated samples.
Significant and Impact of the Study:  The combination of IMS and LFD enhances the sensitivity and flexibility of B. anthracis spore detection from complex samples. This method can potentially be extended to other toxins and micro-organisms in a variety of matrices.     

Sequential detection of Salmonella typhimurium and Bacillus anthracis spores using magnetoelastic biosensors

Multiple phage-based magnetoelastic (ME) biosensors were simultaneously monitored for the detection of different biological pathogens that were sequentially introduced to the measurement system. The biosensors were formed by immobilizing phage and 1mg/ml BSA (blocking agent) onto the magnetoelastic resonator's surface. The detection system included a reference sensor as a control, an E2 phage-coated sensor specific to S. typhimurium, and a JRB7 phage-coated sensor specific to B. anthracis spores. The sensors were free standing during the test, being held in place by a magnetic field. Upon sequential exposure to single pathogenic solutions, only the biosensor coated with the corresponding specific phage responded. As the cells/spores were captured by the specific phage-coated sensor, the mass of the sensor increased, resulting in a decrease in the sensor's resonance frequency. Additionally, non-specific binding was effectively eliminated by BSA blocking and was verified by the reference sensor, which showed no frequency shift. Scanning electron microscopy was used to visually verify the interaction of each biosensor with its target analyte. The results demonstrate that multiple magnetoelastic sensors may be simultaneously monitored to detect specifically targeted pathogenic species with good selectivity. This research is the first stage of an ongoing effort to simultaneously detect the presence of multiple pathogens in a complex analyte.     

Fluorescent europium-modified polymer nanoparticles for rapid and sensitive anthrax sensors

Novel fluorescent polyacrylonitrile nanoparticles were synthesized by microemulsion polymerization and Schiff base modification. By further modification with europium, the polyacrylonitrile nanoparticles could be used as a highly sensitive and rapid sensor for Bacillus anthracis spore detection in aqueous solution. The europium-modified polyacrylonitrile nanoparticles were readily combined with dipicolinic acid as a unique biomarker of B. anthracis, leading to high fluorescence emission. These nanoparticles enabled ratiometric detection without instrument-specific calibration due to the internal fluorescence reference. Additionally, the europium-modified polyacrylonitrile nanoparticle sensors exhibited a remarkable limit of detection (10pM) for dipicolinic acid and outstanding selectivity (160×) over aromatic ligands in aqueous solution. The ultrafine nanoparticle sensor showed a high capability for detecting anthrax due to the increased surface area-to-volume ratio and enhanced dispersibility.     

Rapid detection of Bacillus anthracis spores directly from powders with an evanescent wave fiber-optic biosensor

There currently are no rapid, sensitive tests to directly and reliably detect Bacillus anthracis spores in common powders. Traditional culture is time consuming and molecular techniques cannot directly process powders. This study describes a biosensor assay that detects B. anthracis at concentrations of 3.2 x 10(5) spores/mg or higher in spiked powders in less than 1 h with minimal sample preparation.     

Bacillus anthracis multiplication, persistence, and genetic exchange in the rhizosphere of grass plants

Bacillus anthracis, the causative agent of anthrax, is known for its rapid proliferation and dissemination in mammalian hosts. In contrast, little information exists regarding the lifestyle of this important pathogen outside of the host. Considering that Bacillus species, including close relatives of B. anthracis, are saprophytic soil organisms, we investigated the capacity of B. anthracis spores to germinate in the rhizosphere and to establish populations of vegetative cells that could support horizontal gene transfer in the soil. Using a simple grass plant-soil model system, we show that B. anthracis strains germinate on and around roots, growing in characteristic long filaments. From 2 to 4 days postinoculation, approximately one-half of the B. anthracis CFU recovered from soil containing grass seedlings arose from heat-sensitive organisms, while B. anthracis CFU retrieved from soil without plants consisted of primarily heat-resistant spores. Co-inoculation of the plant-soil system with spores of a fertile B. anthracis strain carrying the tetracycline resistance plasmid pBC16 and a selectable B. anthracis recipient strain resulted in transfer of pBC16 from the donor to the recipient as early as 3 days postinoculation. Our findings demonstrate that B. anthracis can survive as a saprophyte outside of the host. The data suggest that horizontal gene transfer in the rhizosphere of grass plants may play a role in the evolution of the Bacillus cereus group species.     

Fast and Sensitive Detection of Bacillus anthracis Spores by Immunoassay

Bacillus anthracis is one of the most dangerous potential biological weapons, and it is essential to develop a rapid and simple method to detect B. anthracis spores in environmental samples. The immunoassay is a rapid and easy-to-use method for the detection of B. anthracis by means of antibodies directed against surface spore antigens. With this objective in view, we have produced a panel of monoclonal antibodies against B. anthracis and developed colorimetric and electrochemiluminescence (ECL) immunoassays. Using Meso Scale Discovery ECL technology, which is based on electrochemiluminescence (ECL) detection utilizing a sulfo-Tag label that emits light upon electrochemical stimulation (using a dedicated ECL plate reader, an electrical current is placed across the microplate with electrodes integrated into the bottom of the plate, resulting in a series of electrically induced reactions leading to a luminescent signal), a detection limit ranging between 0.3 × 10(3) and 10(3) CFU/ml (i.e., 30 to 100 spores per test), depending on the B. anthracis strain assayed, was achieved. In complex matrices (5 mg/ml of soil or simulated powder), the detection level (without any sample purification or concentration) was never altered more than 3-fold compared with the results obtained in phosphate-buffered saline.     

Sensitive and rapid quantitative detection of anthrax spores isolated from soil samples by real-time PCR

Quantitative analysis of anthrax spores from environmental samples is essential for accurate detection and risk assessment since Bacillus anthracis spores have been shown to be one of the most effective biological weapons. Using TaqMan real-time PCR, specific primers and probes were designed for the identification of pathogenic B. anthracis strains from pag gene and cap gene on two plasmids, pXO1 and pXO2, as well as a sap gene encoded on the S-layer. To select the appropriate lysis method of anthrax spore from environmental samples, several heat treatments and germination methods were evaluated with multiplex-PCR. Among them, heat treatment of samples suspended with sucrose plus non-ionic detergent was considered an effective spore disruption method because it detected up to 10(5) spores/g soil by multiplex-PCR. Serial dilutions of B. anthracis DNA and spore were detected up to a level of 0.1 ng/ microliters and 10 spores/ml, respectively, at the correlation coefficient of 0.99 by real-time PCR. Quantitative analysis of anthrax spore could be obtained from the comparison between C(T) value and serial dilutions of soil sample at the correlation coefficient of 0.99. Additionally, spores added to soil samples were detected up to 10(4) spores/g soil within 3 hr by real-time PCR. As a consequence, we established a rapid and accurate detection system for environmental anthrax spores using real-time PCR, avoiding time and labor-consuming preparation steps such as enrichment culturing and DNA preparation.     

Interdigitated array microelectrode based impedance biosensor coupled with magnetic nanoparticle-antibody conjugates for detection of Escherichia coli O157:H7 in food samples

An impedance biosensor based on interdigitated array microelectrode (IDAM) coupled with magnetic nanoparticle-antibody conjugates (MNAC) was developed and evaluated for rapid and specific detection of E. coli O157:H7 in ground beef samples. MNAC were prepared by immobilizing biotin-labeled polyclonal goat anti-E. coli antibodies onto streptavidin-coated magnetic nanoparticles, which were used to separate and concentrate E. coli O157:H7 from ground beef samples. Magnitude of impedance and phase angle were measured in a frequency range of 10 Hz to 1 MHz in the presence of 0.1M mannitol solution. The lowest detection limits of this biosensor for detection of E. coli O157:H7 in pure culture and ground beef samples were 7.4 x 10(4) and 8.0 x 10(5)CFU ml(-1), respectively. The regression equation for the normalized impedance change (NIC) versus E. coli O157:H7 concentration (N) in ground beef samples was NIC=15.55 N-71.04 with R(2)=0.95. Sensitivity of the impedance biosensor was improved by 35% by concentrating bacterial cells attached to MNAC in the active layer of IDAM above the surface of electrodes with the help of a magnetic field. Based on equivalent circuit analysis, it was observed that bulk resistance and double layer capacitance were responsible for the impedance change caused by the presence of E. coli O157:H7 on the surface of IDAM. Surface immobilization techniques, redox probes, or sample incubation were not used in this impedance biosensor. The total detection time from sampling to measurement was 35 min.     

Gamma irradiation can be used to inactivate Bacillus anthracis spores without compromising the sensitivity of diagnostic assays

The use of Bacillus anthracis as a biological weapon in 2001 heightened awareness of the need for validated methods for the inactivation of B. anthracis spores. This study determined the gamma irradiation dose for inactivating virulent B. anthracis spores in suspension and its effects on real-time PCR and antigen detection assays. Strains representing eight genetic groups of B. anthracis were exposed to gamma radiation, and it was found that subjecting spores at a concentration of 10(7) CFU/ml to a dose of 2.5 x 10(6) rads resulted in a 6-log-unit reduction of spore viability. TaqMan real-time PCR analysis of untreated versus irradiated Ames strain (K1694) spores showed that treatment significantly enhanced the detection of B. anthracis chromosomal DNA targets but had no significant effect on the ability to detect targets on the pXO1 and pXO2 plasmids of B. anthracis. When analyzed by an enzyme-linked immunosorbent assay (ELISA), irradiation affected the detection of B. anthracis spores in a direct ELISA but had no effect on the limit of detection in a sandwich ELISA. The results of this study showed that gamma irradiation-inactivated spores can be tested by real-time PCR or sandwich ELISA without decreasing the sensitivity of either type of assay. Furthermore, the results suggest that clinical and public health laboratories which test specimens for B. anthracis could potentially incorporate gamma irradiation into sample processing protocols without compromising the sensitivity of the B. anthracis assays.     

Low-level detection of a bacillus anthracis simulant using Love-wave biosensors on 36 degrees YX LiTaO3

We present an acoustic Love-wave biosensor for detection of the Bacillus anthracis simulant, Bacillus thuringiensis at or below inhalational infectious levels. The present work is an experimental study of 36 degrees YX cut LiTaO3 based Love-wave devices for detection of pathogenic spores in aqueous conditions. Given that the detection limit (D1) of Love-wave-based sensors is a strong function of the overlying waveguide, two waveguide materials have been investigated, which are polyimide and polystyrene. To determine the mass sensitivity of Love-wave sensor, bovine serum albumin (BSA) protein was injected into the Love-wave test cell while recording the magnitude and phase shift across each sensor. Polyimide had the lowest mass detection limit with an estimated value of 1.0-2.0 ng/cm2, as compared to polystyrene where D1 = 2.0 ng/cm2. Suitable chemistries were used to orient antibodies on the Love-wave sensor using protein G. The thickness of each biofilm was measured using ellipsometry from which the surface concentrations were calculated. The monoclonal antibody BD8 with a high degree of selectivity for anthrax spores was used to capture the non-pathogenic simulant B. thuringiensis B8 spores. Bacillus subtilis spores were used as a negative control to determine whether significant non-specific binding would occur. Spore aliquots were prepared using an optical counting method, which permitted removal of background particles for consistent sample preparation. This work demonstrates that Love-wave biosensors are promising for low-level detection for whole-cell biological pathogens.     

炭疽杆菌检测方法的研究现状与展望

炭疽是严重威胁人类健康的烈性传染病,其病原体为炭疽芽孢杆菌。炭疽芽孢杆菌在我国公布的《人间传染的病原微生物名录》中被列为第二类病原微生物(高致病性病原微生物),其芽孢可作为生物战剂和生物恐怖的原材料,因此,发展灵敏、高效的炭疽杆菌检测方法十分重要和紧迫。按检测的靶标分类,针对炭疽杆菌的检测方法主要有四大类:针对炭疽杆菌芽孢的检测方法,针对细菌繁殖体的检测方法,针对炭疽杆菌基因的检测方法和针对炭疽毒素蛋白的检测方法。其中,针对炭疽杆菌芽孢和细菌繁殖体的检测已经有比较成熟的方法,但其在特异性以及临床的实用性方面难以令人满意;针对炭疽杆菌基因的检测技术在特异性和灵敏度上有较大的提高,但在临床诊断等方面还有欠缺;而针对炭疽毒素蛋白的检测技术的发展,使得直接对炭疽杆菌的主要致病因子的检测成为可能,这对于临床诊断以及流行病学研究具有重要意义。本文对当前炭疽杆菌检测方法的最新进展做了简要的归纳,关注了不同检测方法的适用范围和检测能力,并展望了相关领域的发展趋势,希望能为从事炭疽杆菌检测方法研究的同行提供参考和帮助。     

Evaluation of the Cepheid GeneXpert system for detecting Bacillus anthracis

AIMS: The Cepheid GeneXpert is a four-site, automated sample preparation and real-time PCR detection system. In this study, the capability of the GeneXpert to isolate and detect nucleic acid from Bacillus anthracis Ames spores was assessed. METHODS AND RESULTS: A four-plex, dried-down bead cartridge containing PCR reagents specific for the pXO1 and pXO2 plasmids as well as sample processing and inhibition controls was evaluated. For B. anthracis Ames spores harbouring pXO1 and pXO2, samples containing 68 CFU per ml (148 spores per ml) were positive in all four replicates. A limited cross-reactivity panel, which included closely related Bacillus species, was also tested to determine the specificity of the pXO1 and pXO2 assays. No cross-reactivity occurred. Further, B. anthracis Sterne spore samples were analysed to compare results when processed using the GeneXpert to those run directly on the Cepheid SmartCycler without sample processing. The GeneXpert detection capability was three logs lower than the SmartCycler indicating the benefit of incorporating a nucleic acid extraction procedure. CONCLUSIONS: This study demonstrates that the GeneXpert is a rapid and reliable system for simultaneously detecting the B. anthracis virulence plasmids pXO1 and pXO2. SIGNIFICANCE AND IMPACT OF THE STUDY: The GeneXpert is the only platform currently available that is capable of both nucleic acid purification and real-time PCR detection enclosed within a single system. Further, all sample manipulations are automated, thus reducing errors associated with manual processing.     

Bacillus anthracis contamination and inhalational anthrax in a mail processing and distribution center

AIMS: Four inhalational anthrax cases occurred in a large mail processing and distribution center in Washington, DC, after envelopes containing Bacillus anthracis spores were processed. This report describes the results of sampling for B. anthracis spores during investigations conducted in October and December 2001. METHODS AND RESULTS: Wet swabs, wet wipes, vacuum sock, and air-filter samples were collected throughout the facility to characterize the extent of building contamination. The results showed widespread contamination of B. anthracis spores, particularly associated with one delivery bar code sorter (DBCS) machine that had sorted the spore-containing envelopes and an area where the envelopes were handled by postal workers. Spore concentrations decreased as distance from the DBCS machine increased, but spores were widely dispersed into surrounding areas. CONCLUSION: The spatial distribution of culture positive samples was closely related to the work areas of the inhalational anthrax cases and supported epidemiological evidence that the workers became ill from exposure to B. anthracis spores in areas where the contaminated envelopes had travelled. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this investigation were used to guide decontamination efforts and provided baseline spore concentrations for follow-up measurements after the building had been cleaned. Implementing methods to reduce aerosolization and dispersion of dust within the facility would reduce postal workers' potential exposures to bioterrorism agents.