180
COMPARISON OF REAL-TIME PCR AND DIRECT EXAMINATION ANALYSES USING THE VIACELL
AIR SAMPLING CASSETTE.
J. Dobranic, C. Cutler, EMSL Analytical Inc., Westmont, NJ.
A study was conducted comparing real-time PCR detection and quantification of mold spores and the traditional direct microscopic examination. ViaCell (Zefon International) spore trap cassettes were utilized in both laboratory and field samples. Initially, three replicate cassettes were sampled in the laboratory with individual pure cultures of Aspergillus versicolor, Cladosporium cladosporioides, and Penicillium chrysogenum followed by three cassettes with mixtures of these three fungi. The cassettes were first analyzed by direct microscopy then by PCR analysis to determine how comparable were the two techniques. Data will also be presented on a second study comparing three field samples taken in two mold contaminated homes that were again first analyzed by direct microscopy and then by real-time PCR.
The inherent limitations on identifications associated with the direct microscopic examination confines its use. PCR was able to provide much more valuable speciation identification especially of the Aspergillus/Penicillium group typically found on spore trap reports. The ViaCell can be effectively used for both direct examinations first, determining spore loads, and to pinpoint circumstances when PCR analysis is required on the same samples.
181
SAMPLING MEDIA EFFECTS ON THE CALCULATION OF MOLD SPORE EQUIVALENTS IN
QUANTITATIVE PCR.
D. Kahane, D. Cox, H. DeLucchi, M. Piercey, Forensic Analytical, Hayward, CA.
Airborne mold samples for quantitative real-time PCR (QPCR) analysis are typically collected on polycarbonate filters. In QPCR analysis, the sample filter is suspended in a buffer solution and a known aliquot of a Geotrichum candidum reference spore suspension is added to the sample. The cycle threshold (CT) values for the target species and the Geotrichum reference are detected, and the difference in CT values (ΔCT) of the reference and target species is used to calculate the quantity of target species spore equivalents in the air sample. An underlying assumption in the use of the ΔCT method to quantify species is that any condition in the analytical sample that would cause a “shift” in the CT value of Geotrichum would also cause the same direction and magnitude of shift in the CT value of any other species. However, we observed that the mere presence of a polycarbonate filter in the buffer solution sample suspension caused a shift in the CT value of the Geotrichum but no shift in the CT value of some other mold species. The result of media effects that differ between species has been the systematic underreporting of the number of spore equivalents of some species in samples collected on polycarbonate filters. For example, the result of air samples collected on polycarbonate filters and analyzed for Cladosporium cladosporioides were found to have been underreported by a factor of four because of the differing media effects. This paper presents the results of our studies of the media effects of polycarbonate filters and other typical sample collection media on the mold species most commonly analyzed by QPCR. Recommendations are made regarding correcting the results of QPCR analyses by taking into account known media effects.
182
WHEN YOU REALLY NEED TO KNOW: FUNGAL SPECIES IDENTIFICATION USING DNA SEQUENCING
AND ANALYSIS.
F. Wu, S. Huang, Aemtek Inc., Fremont, CA.
Sampling of fungi is often an integral part of indoor environmental quality assessment. Currently, spore trap, Andersen sampler, and dust check methods are widely used for fungal sampling. Subsequently, direct microscopy, culture morphology, and QPCR are used for fungal identification and analysis, respectively. Each of the methods has advantages and limitations and offers various degrees of resolution. In many cases, one or a combination of the methods mentioned above is sufficient. However, there are situations where none of the methods is adequate. The purpose of this presentation is to introduce DNA sequencing and analysis as an alternative for species identification. Comparative DNA sequence technology obtains and utilizes nucleic acid sequence information to achieve the most accurate and reproducible fungal identifications available. The procedure includes DNA extraction, PCR amplification, sequencing of the PCR products, and separation using automated DNA sequencer. Currently, the most widely used fungal sequences are ITS regions, 5.8S, and the subunits of ribosomal DNA. Mycological research in recent years has demonstrated that the ITS regions offer high level of interspecific specificity and low intraspecific variation in many groups of fungi. The sequence data is compared to databases to find the closest match. In addition to commercial databases, nucleic acid sequence data are regularly compiled in several publicly available databases. GenBank is the best known and most widely used. It currently has over 750,000 entries for fungal nucleic acids and represents the largest comparative data sets ever collected. The DNA sequencing technology works well with bulk samples of fungal colony and most of culturable samples. Viability or sporulation of the fungi is not required. Although this method is not for every investigation, it can be applied to situations where confirmative identification of unknown fungi is necessary to address health related issues.
183
PCR-BASED DETECTION OF MOLD WHEN CULTURE AND DIRECT MICROSCOPIC ANALYSIS REVEAL
NOTHING.
G. Saenz, EMSL Analytical Inc., South Pasadena, CA.
Culture and direct microscopic analysis are very good traditional methods of detecting and identifying molds in indoor environments and in human clinical specimens. However, positive identification using these traditional methods has its fallbacks. Culturing requires that the organisms collected remain viable from collection to analysis. Limitations of direct microscopic analysis include failing to detect fungal structures in low quantity and classifying spores into coarse taxonomic groups (e.g., Aspergillus/Penicillium) which provides little useful information from a clinical perspective. In two separate case studies, two patients presented antibodies to particular mold species in their blood. Because investigators were unable to find actual evidence of the molds themselves using traditional techniques, they turned to PCR as a more sensitive method to find these molds. In the case of the first patient, nine spore traps, nine swabs, and nine culturable air samples were taken from selected rooms in the home. All molds were identified to the species level when possible. Of the five mold species that were identified from antibodies in the blood, only two genera were found and none of the species were found. When PCR was used to analyze one air sample from the home, all five species were found. In the case of the second patient, the three mold genera detected from antibodies in the blood were also positively detected in environmental samples taken from the home. These same molds however, were not detectable in the patient’s tissues by direct microscopy. PCR analysis of the tissues was then requested to see if any of the molds could be detected using a DNA-based method. Using PCR, one species was positively detected in the patient’s tissue samples. These two case studies, although not common cases, demonstrate that PCR is an excellent method to detect molds that are undetectable by culture and/or direct exam.
184
COMPARISON OF N6 VIABLE SAMPLER WITH TWO TOTAL SPORE SAMPLERS, BURKARD AND
ALLERGENCO, IN ATMOSPHERES CONTAINING VIABLE AND NONVIABLE FUNGAL SPORES.
B. Samimi, D. Kernatzitskas, San Diego State University, San Diego, CA.
In this study, the Burkard and Allergenco MK-3 total spore samplers were compared to the Andersen N6 viable sampler. The objectives of the study were to compare the collection efficiency of the three samplers in atmospheres containing (1) freshly dispersed viable Aspergillus niger spores, (2) all nonviable A. niger spores, and (3) approximately half and half viable and nonviable spores. The samplers were operated under each of the above conditions in Phases I, II and III, respectively. Each trial run consisted of 20 simultaneous samples. Sampling runs were conducted within a controlled walk-in environmental chamber where fungal spores were introduced. Air samples from each sampler were analyzed in accordance with the manufacturers’ instructions.
In Phase I, introducing all viable spores, a significant correlation (R = 0.769; p = 0.009) was found between the N6 and the Burkard, whereas the correlation between the N6 and Allergenco was weak and insignificant. Among the three samplers, even in an all-viable spore atmosphere, the Burkard showed significantly highest spore collection efficiency. Allergenco was the second highest and N6 was the lowest. In Phase III, where one-half of the chamber atmosphere contained viable fungi, the difference between the N6 and the other two further widened. In Phase II, collecting nonviable spores, only the N6 samples, as expected, showed minimal collection (CFUs). The most significant findings of the study were (1) lower collection efficiency for N6 in a nearly 100% viable spores atmosphere, and (2) significantly higher collection efficiency of the Burkard sampler compared to the Allergenco sampler. It appears that the N6 sampler may underestimate the true concentration of viable spores, particularly for small fungal spores such as A. niger which may require higher impact velocity to develop adequate inertial force required to stop the particles of this nature.
185
COMPARISON OF THE THERMO-ANDERSEN N6, THE AEROTECH A6, THE SKC BIOSTAGE, AND THE
SKC MICROMEDIA VIABLE SAMPLERS IN COLLECTING AIRBORNE FUNGAL SPORES.
B. Samimi, A. Shufutinsky, San Diego State University, San Diego, CA.
Recently several samplers that mimic the Andersen N6 viable sampler have been placed on the market. However, there have been no original studies to compare the performance of these samplers against the traditional Andersen N6. This study compares the performance of the Aerotech A6, the SKC BioStage, the SKC Micromedia BioStage, and the Thermo Electron/Andersen N6 with respect to collection efficiency for sampling viable airborne fungal spores. A total of 480 side-by-side samples were taken in high and low concentrations in one controlled and two uncontrolled contaminated environments. The null hypothesis was that there is no significant difference in collection efficiency between the four samplers. For the controlled experiment, Aspergillis niger spores were introduced into a walk-in environmental chamber. Uncontrolled environments included a contaminated residential kitchen cabinet and an outdoor residential environment. Using NIOSH Method 0800, 20 samples were taken per sampler in each testing condition. After the sampling periods, the MEA contained plates incubated at 25oC for five days. The colony forming units (CFUs) were counted, adjusted using the positive-hole correction factors, and calculated to determine the concentrations of CFU/M3 of air in the environment.
In comparing the samplers against each other, there was no particular trend among the samplers and no single sampler had higher counts in every environment. The resulting data was analyzed using the nonparametric Mann-Whitney U-test, Pearson Correlation, and Linear Regression. All analysis groups had Mann-Whitney significance levels that well exceeded the 0.05 alpha values. Acceptance of the null hypothesis indicated that there is no statistically significant difference in collection efficiency of airborne fungi between the above tested viable samplers for collecting viable airborne fungal spores. IAQ professionals using any of these samplers should be able to get fairly equal results in their attempt to evaluate air contamination to viable fungal spores.
186
EVALUATION OF TOTAL MOLD SPORE COUNTS USING LIGHT MICROSCOPY AND EPIFLUORESCENCE.
T. Godish, Ball State University, Muncie, IN.
This study was conducted to evaluate the relative effectiveness of several different microscopic techniques in determining mold spore levels in air samples collected by spore trap methods. Sample slides were stained with aniline blue solution which increases mold structure visibility under light microscopy and forms a fluorochrome with glucans in the cell walls of mold spores and hyphae and fluorescing under blue light. Such fluorescence is enhanced when mold structures are viable. All counts were conducted at 1000X magnification since its greater resolving power (compared to lower magnifications) allows for identifying and counting small mold spores and yeast cells. The use of epifluorescence to count mold spores at 1000x in most cases resulted in significantly higher count numbers (typically 20–100%) than using light microscopy. Differences were small with low particle density sample collections but increasingly became greater as particle density increased. Epifluorescence and associated light filter use provided increased clarity for the identification of small mold spore/particle structures and yeast cells as well as those in various stages of decomposition.
187
HOW VARIABLE IS THE ANALYSIS OF SPORE TRAP SAMPLES FOR TOTAL FUNGI?
P. Heinsohn, Micro Bios, Pacifica, CA; D. Sime, AeroBio Lab Inc., La Honda, CA; T. Carroll, University of Nevada, Las Vegas, Las Vegas, NV.
Like all industrial hygiene data, interpretation of spore trap data for total fungal spores requires knowledge of analytical variability. The purpose of this work was to assess the inter- and intralaboratory variability of counting and identifying fungi in spore trap samples. Understanding the variability of such data is critical in determining if indoor air quality is degraded, which may result in an atypical occupant inhalation exposure.
Zefon Air-O-Cell spore trap samples were collected in indoor and outdoor air. Each sample was stained with lacto-cotton blue, permanently mounted onto individual glass microscope slides, cover slipped, and labeled. Randomly selected identification numbers providing no qualifying information as to sample location were used. Samples were priority mailed to six AIHA accredited laboratories for analysis. Participating laboratories reported raw counts of each fungal spore identified to genus level and a total concentration using their own analytical method.
Analysis of variance and Spearman rank correlation were used to compare the 120 observations. There were significant differences in the means of the samples for 14 taxa/categories, including Cladosporium, Penicillium/Aspergillus, Stachybotrys, and total spore count. There were significant differences in the mean spore counts for analysts and between laboratories for the total spore count. Thus, there were significant differences in procedures between two laboratories. Also, for five taxa, including Cladosporium and Penicillium/Aspergillus, we failed to reject the null hypothesis that the rankings of counts were random.
The variability reported here is due to differences in counting methodologies between laboratories, and the spore identification skills of analysts. Importantly, both are independent of environmental conditions and sampling design strategy. These data argue strongly for collection of replicate samples, standardization of a counting procedure, consistent and competent classification, and interpretation of spore trap data taking the reported variability into account.
Posted May 30, 2005