Jim Ho

Design of an instrument for real-time detection of bioaerosols using simultaneous measurement of particle aerodynamic size and intrinsic fluorescence.

A prototype instrument has been constructed to measure individual airborne particles based on their aerodynamic size and their intrinsic fluorescence at selected excitation and emission wavelength bands. The instrument combines features of an aerodynamic particle sizing device with capabilities similar to those of a liquid flow cytometer. The goal of the instrument is to provide real-time data indicative of particle characteristics, and it is especially targeted to respond to bioaerosols from 0.5 to 10 micrometers (aerodynamic diameter) with intrinsic fluorescence exited at a wavelength of 325 nm and emitting from 420 to 580 nm. This size range covers individual airborne bacteria and bacteria clusters, and the fluorescence sensitivity is selected for biological molecules commonly found in cellular systems, for example, reduced nicotinamide adenine dinucleotide phosphate [NAD(P)H] and riboflavin. Initial tests with nebulised Bacillus subtilis var. niger (BG, ATCC 9372) spores have shown that, for both individual spores and spore clumps, a low level of fluorescence is detected from 17% of the particles. This detection percentage is on the same order as previous experiments that have measured viability of about 12% for mechanically dispersed BG spores (Ho and Fisher (1993) Defense Research Establishment Suffield Memorandum 1421) and suggests a need for further investigation into the possible relationship between the detected fluorescence and viability of bacterial spores.

Detection of Airborne Severe Acute Respiratory Syndrome (SARS) Coronavirus and Environmental Contamination in SARS Outbreak Units

Abstract Severe acute respiratory syndrome (SARS) is characterized by a risk of nosocomial transmission; however, the risk of airborne transmission of SARS is unknown. During the Toronto outbreaks of SARS, we investigated environmental contamination in SARS units, by employing novel air sampling and conventional surface swabbing. Two polymerase chain reaction (PCR)–positive air samples were obtained from a room occupied by a patient with SARS, indicating the presence of the virus in the air of the room. In addition, several PCR-positive swab samples were recovered from frequently touched surfaces in rooms occupied by patients with SARS (a bed table and a television remote control) and in a nurses’ station used by staff (a medication refrigerator door). These data provide the first experimental confirmation of viral aerosol generation by a patient with SARS, indicating the possibility of airborne droplet transmission, which emphasizes the need for adequate respiratory protection, as well as for strict surface hygiene practices

Bacillus anthracis aerosolization associated with a contaminated mail sorting machine.

On October 12, 2001, two envelopes containing Bacillus anthracis spores passed through a sorting machine in a postal facility in Washington, D.C. When anthrax infection was identified in postal workers 9 days later, the facility was closed. To determine if exposure to airborne B. anthracis spores continued to occur, we performed air sampling around the contaminated sorter. One CFU of B. anthracis was isolated from 990 L of air sampled before the machine was activated. Six CFUs were isolated during machine activation and processing of clean dummy mail. These data indicate that an employee working near this machine might inhale approximately 30 B. anthracis-containing particles during an 8-h work shift. What risk this may have represented to postal workers is not known, but the risk is approximately 20-fold less than estimates of sub-5 micron B. anthracis-containing particles routinely inhaled by asymptomatic, unvaccinated workers in a goat-hair mill.

Standoff sensing of bioaerosols using intensified range-gated spectral analysis of laser-induced fluorescence

In atmospheric sensing, one application that has demonstrated several impressive successes over the last two decades is the light detection and ranging (LIDAR). Elastic LIDAR has shown an important capability in providing aerosol density and spatial distribution from a standoff position. However, it provides limited information on the material composition of the aerosol component. On the other hand, inelastic LIDARs (including laser-induced fluorescence and Raman LIDARs) measure the spectrally distributed returned signal that may contain important clues about the nature of the scatterers. In order to investigate the capability of these LIDARs in characterizing bioaerosols from a standoff position, Defence Research & Development Canada initiated a three-year program in spring 1999, named SINBAHD (Standoff Integrated Bioaerosol Active Hyperspectral Detection). The aim of the program was to investigate the sensitivity and discrimination capabilities of an inelastic LIDAR based on the intensified range-gated spectral detection of laser-induced fluorescence. An exploratory prototype based on this technique has shown sensitivity of a few living bioaerosol particles per liter of air for a range of 1.4 km at night. Furthermore, based on spectral signatures measured during open-air releases, good discrimination capabilities were obtained between Bacillius subtilis var globiggi (BG) and Erwinia herbicola (EH). These results agree well with a performance model using Raman returns from atmospheric nitrogen as a calibration tool.

Measurement of Biological Aerosol with a Fluorescent Aerodynamic Particle Sizer (Flaps): Correlation of Optical Data with Biological Data

Biological aerosol measurement in real time is an urgent military requirement that also has many potential non-military applications. Such detection capabilities will be useful in environmental monitoring, for example, in gathering information in perceived hazardous areas like housing developments downwind of sewage treatment plants.

Effect of growth media and washing on the spectral signatures of aerosolized biological simulants

We have evaluated the influence of growth media and washing on the laser-induced fluorescence spectra of bacteria. Three different bacterial simulants were cultured in three types of growth media. Three kinds of samples were generated from each culture: the culture itself, the growth medium alone, and a triple-washed sample. The materials were injected as aerosols in a lab-sized lidar aerosol chamber to obtain their spectra. Using two different analysis approaches, signature variations were observed between the three kinds of samples for most combinations of growth media/bacteria. This study concludes that the culture media used influences the spectral signatures.

Evaluation of the plasmid copy number in B. cereus spores, during germination, bacterial growth and sporulation using real-time PCR.

Only a small number of studies have measured the plasmid copy number (PCN) variation during bacterial growth. Besides, information about the PCN in spores is still rare. In this work, we utilized a real-time PCR assay to evaluate the PCN of four different plasmids in Bacillus cereus. The PCN was measured in spores as well as during germination, active bacterial growth, and sporulation. Plasmid stability was also evaluated to ensure that plasmid loss does not affect the accuracy of the PCN measurement. We demonstrated that the PCN of low and high copy number plasmids varies with growth phase as well as culture media over B. cereus life cycle. The PCN was minimum during the germination and maximum during the stationary growth phase for all plasmids tested. We also demonstrated that the use of antibiotic in the culture media is not enough to ensure stable inheritance in spores of plasmids carrying antibiotic resistance genes. Moreover, we revealed that the PCN in spores is related to the PCN during endospores formation. Therefore, the plasmid partitioning during sporulation is not influenced by the unequal-size of the forespores and the mother cells, even for a plasmid distributed randomly.

Anthrax Letters: Personal Exposure, Building Contamination, and Effectiveness of Immediate Mitigation Measures

This report is the first detailed and quantitative study of potential mitigation procedures intended to deal with anthrax letters using a simulated anthrax letter release within an actual office building. Spore aerosols were created by opening letters containing 0.1 g of dry powdered Bacillus atrophaeus spores. Culturable aerosol samples were collected using slit-to-agar and filter-based samplers. Five test scenarios were designed to determine whether simple mitigation procedures or activities carried out by the person who opened the letter made a significant difference to aerosol concentrations in comparison to a control scenario where no activity took place. Surface contamination of the letter opener was measured at 10 body points for Scenarios 1 to 4. A sixth scenario, based on published Centers for Disease Control and Prevention anthrax letter response guidelines, used letters containing 1 g of spores. Results demonstrated that the spore aerosol spread throughout the building in less than 4.5 min. Potential mitigation techniques such as closing the office door or shutting off the ventilation system were not effective. Activities carried out by the letter opener including moving, walking to another location, and spraying water onto the contaminated desk with a hand sprayer all resulted in significantly higher aerosol concentrations in comparison to control. The potential total inhalational hazard for the letter opener during the five test scenarios ranged from 4.1 × 10 5 to 1.6 × 10 6 colony forming units (CFU) compared to 3.9 × 10 5 CFU for the control. Surface contamination of the letter opener (Scenarios 1 to 4) was highest on the right hip (4.8 × 10 4 to 1.0 × 10 5 CFU/cm − 2 ) and lowest on the right or left side of the head (2.2 × 10 2 to 3.7 × 10 3 CFU/cm −2 ). The statistically based methodology used in this study provided the means to objectively assess anthrax letter protocols to determine their effectiveness under realistic conditions. Potential mitigation procedures tested in this study did not reduce aerosol hazard or surface contamination.

Permeabilization and hybridization protocols for rapid detection of Bacillus spores using fluorescence in situ hybridization.

BACKGROUND Fluorescence in situ hybridization (FISH) is not adapted for the detection of bacterial spores because of their resistance to conventional permeabilization treatments. Since spore-forming bacteria have important ecological, economical, and medical roles, their in situ detection needs to be improved. The aim of this study was to develop rapid and effective protocols to permeabilize Bacillus spores in order to apply the FISH technique. METHODS Permeabilization protocols were developed for three species of Bacillus spores. Hybridization was performed using universal and specific probes. Surface structural analysis of the permeabilization treatments was performed using scanning electron microscopy. RESULTS With the proposed protocols, Bacillus spores can be labeled in less than 1 h. The scanning microscopy showed some visible structural differences between the permeabilized spores compared to intact ones. CONCLUSION For the first time, rapid and effective protocols to detect Bacillus spores by FISH were developed and can be applied to study Bacillus spores using in situ labeling within 1 h. Previously published in situ hybridization protocols have never reached or been close to the currently described rapidity. This work will contribute to the possibility of near real time detection of biological threats that may be present as spores.

Neural network recognition and classification of aerosol particle distributions measured with a two-spot laser velocimeter.

This paper describes the use of a neural computational network model for pattern recognition and classification of aerodynamic particle size distributions associated with a number of environmental, bacterial, and artificial aerosols. The aerodynamic particle size distributions are measured in real time with high resolution using a two-spot He-Ne laser velocimeter. The technique employed here for the recognition and classification of aerosols of unknown origin is based on a three-layered neural network that has been trained on a training set consisting of 75 particle size distributions obtained from three distinct types of aerosols. The training of the neural network was accomplished with the back-propagation learning algorithm. The effects of the number of processing units in the hidden layer and the level of noise corrupting the training set, the test set, and the connection weights on the learning rate and classification efficiency of the neural network are studied. The ability of the trained network to generalize from the finite number of size distributions in the training set to unknown size distributions obtained from uncertain and unfamiliar environments is investigated. The approach offers the opportunity of recognizing, classifying, and characterizing aerosol particles in real time according to their aerodynamic particle size spectrum and its high recognition accuracy shows considerable promise for applications to rapid real-time air monitoring in the areas of occupational health and air pollution standards.