H. Kenneth Dillon

Magnetic Bead Capture Eliminates PCR Inhibitors in Samples Collected from the Airborne Environment, Permitting Detection of Pneumocystis carinii DNA

ABSTRACT PCR detection methods are useful in studies of organisms not amenable to culture. Inhibitors in environmental samples can interfere with such assays. We describe a magnetic bead DNA capture protocol that removes inhibitors from outdoor air samples, maintaining the sensitivity of a 16S Pneumocystis carinii mitochondrial rRNA gene-based PCR.

Development of an HPLC Method for Simultaneous Analysis of Five Antineoplastic Agents

Simultaneous analysis of common antineoplastic agents potentially hazardous to healthcare workers is of much interest for the evaluation of the overall health risk to these workers. Such analysis could be applied to both air and surface monitoring samples to provide a broader indication of risk to combinations of these agents. It was determined that the ability to simultaneously evaluate five frequently used, potentially hazardous agents was sufficient for general evaluation of exposures to healthcare workers. The approach used to select the five agents was to obtain a list of the agents used most frequently in both a cancer hospital and an outpatient cancer treatment center, then review the list to determine which agents were potentially more hazardous to human health. From these reviews, it was decided to attempt to develop an analytical method able to detect and quantify the presence of 5-fluorouracil, ifosfamide, cyclophosphamide, doxorubicin HCl, and paclitaxel. A reverse-phase high performance liquid chromatograph (HPLC) with a Waters Symmetry C8 column and a UV wavelength of 195 nm was selected for method development. The mobile phase was 22.75 percent acetonitrile in water buffered to a pH of 6.0. The HPLC analytical method developed is able to detect all five agents of interest, and at minimum detectable concentrations of 0.5-microgram/mL for each of the five agents.

Development and Characterization of a Molecular Viability Assay for Pneumocystis carinii f sp hominis

Pneumocystis carinii pneumonia (PCP) remains the most common opportunistic infection among human immunodeficiency virus-infected persons. Despite this, little is known concerning the transmission dynamics of this infection. In the absence of a reliable method to isolate and culture P. carinii from environmental samples, it has not been possible to assess the importance of person-to-person transmission in the epidemiology of PCP. A molecular viability assay was developed for the human form of P. carinii (P. carinii f sp hominis) that is applicable to both clinical specimens and environmental samples. This assay will enable the evaluation of the spread and persistence of viable human P. carinii in the environment.

A New Monitoring Method Using Solid Sorbent Media for Evaluation of Airborne Cyclophosphamide and Other Antineoplastic Agents

Cyclophosphamide is a known human carcinogen. In July 1999, in a report at a conference on cytotoxic drugs in Sweden, it was indicated that cyclophosphamide (CP) was not effectively controlled by high efficiency particulate air (HEPA) filters.((1)) This then raised a concern that the existing air monitoring methods, which utilize polytetrafluoroethylene (a.k.a. PTFE, or Teflon) or glass fiber filters for evaluation of antineoplastics such as CP in air may also be ineffective for collection and quantification of such agents. It was decided that further evaluation of the existing filter method for monitoring antineoplastics in air be conducted. This evaluation determined that the filter method of monitoring was minimally effective for some antineoplastic agents, and that an alternate method of monitoring should be sought. The method subsequently developed utilizes a solid sorbent tube, Anasorb 708, a methacrylic acid polymer. Evaluation of this sorbent tube for adsorption and desorption properties found it had a greater than 90 percent recovery for both CP and ifosfamide. Other agents evaluated included 5-fluorouracil, doxorubicin, and paclitaxel. All three agents were able to be detected and measured by use of Anasorb 708 solid sorbent tube. Validation of the method was then conducted with air pulled through the tubes via attachment to an air manifold system at air flows ranging from 1.5 to approximately 4.0 liters per minute for up to 24 hours. This evaluation did validate the Anasorb 708 tube as an effective media for collection of airborne concentrations of CP from less than 1 microgram up to approximately 2 mg (2000 microgram) per tube. This corresponds to a concentration range of approximately 0.7 microgram/m(3) (0.0007 mg/m(3)) to 0.7 mg/m(3) in a 5.76 m(3) volume of air. This method can provide accurate information on airborne concentrations of CP for purposes of conducting risk assessments or evaluation of risk management methods.

Comparison of Detection Methods for Aspergillus fumigatus in Environmental Air Samples in an Occupational Environment

Methods to study occupational exposures to fungi and fungal materials in facilities where the dominant exposure is the pathogenic and allergenic fungus Aspergillus fumigatus are lacking. Air samples were collected near a conveyor in a wood chip recycling plant to compare methods that might be used to assess exposure to A. fumigatus or suitable proxies. These included total dust mass, total intact spores, culturable propagules growing > 35°C, ergosterol, A. fumigatus allergen Asp f1, and quantitative polymerase chain reaction for A. fumigatus. Of these measurements, Asp f1 showed the most promise based on its relative response to measurements where there is a long history of use in industrial hygiene practice (total mass, ergosterol, total intact spores, culturable propagules).

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A Compact Wind Tunnel for Evaluating Aerosol Samplers

Abstract A small wind tunnel (305 cm in length and with an inside diameter of 59 cm) was designed, fabricated, evaluated, and modified to meet flow field uniformity criteria consistent with those established by the U.S. Environmental Protection Agency for PM10 sampler testing. With an arrangement of three jet pumps located near the intake of the wind tunnel, velocity profiles in the plane of the test section near the exhaust of the wind tunnel demonstrated low variability. The coefficients of variation (CVs) of the mean velocities at traverse points within the test section were < 5 percent in the velocity range of 0.6 to 1.4 m/s. Stable particle size distributions of polydisperse aerosols of ceramic beads injected along the centerline of the wind tunnel through one of the jet pumps were generated with a vibrating feed generator [type 200 Zeeospheres®; count median aerodynamic diameter (CMAD), 1.6 to 1.8 μm; mass median aerodynamic diameter (MMAD), 5.1 μm; and geometric standard deviation (GSD), 1.7 to 1.8...

Laboratory Evaluation of a Novel Reactive Passive Sampler for the Quantitative Determination of Formaldehyde in Air

A newly developed color-indicating passive dosimeter for formaldehyde, the AirChem Technologies (ACT™) Monitoring Card System, was evaluated. Test atmospheres of formaldehyde were generated by the injection of formalin into a heated stream of nitrogen, with subsequent dilution with charcoal-filtered air to produce challenge concentrations in the range of 0.1 to 2 ppm. The temperature, relative humidity, and volume flow rate of the test mixture were regulated. The mixed gas stream was introduced into an insulated cylindrical test chamber equipped with a rotating disc sampler holder. The ACT card readings were highly correlated with results obtained with NIOSH Method 3500. A constant adjustment factor (0.04 ppm) yielded results at exposures ranging from 0.1 to 2 ppm and at exposure times of 4, 6, or 8 hours with accuracies that were well within ±25% at or above the Action Level and ±35% below the Action Level. For concentrations ≥0.3 ppm, the overall bias was 1% with a pooled CV of 5%. Elevated temperatures...

A comparison of two surface sample collection devices for use in polymerase chain reaction based detection of Pneumocystis carinii in house dust

A polymerase chain reaction assay was optimized to detect P. carinii cysts in composite dust samples. The optimal assay was capable of detecting as few as 10(3) P. carinii cysts in 50 mg of dust. Two dust collection devices were evaluated for efficiency and precision of collection of bulk dust and compatibility with the optimized PCR protocol for P. carinii DNA detection. A handheld vacuum cleaner equipped with a high-retention bag was found to be superior to a 37-mm filter cassette attached to an electrically powered vacuum pump in terms of dust collection efficiency (87% [n = 37] versus 81% [n = 35]), although the precision of the two devices as assessed by the standard deviation was similar (6.2% versus 6.3%). However, the vacuum cleaner method was not as compatible with the PCR-based detection assay as the filter cassette method. The filter cassette appears to be a better device for use in conjunction with PCR-based detection of P. carinii DNA in bulk dust samples from both smooth and carpeted surfaces.

An environmental chamber for investigating the evaporation of volatile chemicals

An inexpensive test chamber has been constructed that provides an environment appropriate for testing the effects of temperature and chemical interactions on gaseous emissions from test solutions. Temperature, relative humidity, and ventilation rate can be controlled and a well-mixed atmosphere can be maintained. The system is relatively simple and relies on heated tap water or ice to adjust the temperature. Temperatures ranging from 9 to 21 degrees C have been maintained. At an average temperature of 15.1 degrees C, temperatures at any location within the chamber vary by no more than 0.5 degree C, and the temperature of the test solution within the chamber varies by no more than 0.1 degree C. The temperatures within the chamber are stable enough to generate precise steady-state concentrations. The wind velocities within the chamber are reproducible from run to run. Consequently, the effect of velocity on the rate of evaporation of a test chemical is expected to be uniform from run to run. Steady-state concentrations can be attained in less than 1 hour at an air exchange rate of about 5 per hour.