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Microbiological & Chemical Exposure Assessment Research Division Publications: 2004

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This page lists publication titles, citations and abstracts produced by NERL's Microbiological & Chemical Exposure Assessment Research Division for the year 2004, organized by Publication Type. Your search has returned 76 Matching Entries.

See also Microbiological & Chemical Exposure Assessment Research Division citations with abstracts: 1999,  2000,  2001,  2002,  2003,  2004,  2005,  2006,  2007,  2008,  2009

Technical Information Manager: Angela Moore - (513) 569-7341 or moore.angela@epa.gov

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Presented/Published
BOOK CHAPTER Possible Roles of Fungal Hemolysins in Sick Building Syndrome 09/27/2004
Vesper, S J. AND M. J. Vesper. Possible Roles of Fungal Hemolysins in Sick Building Syndrome. Advances in Applied Microbiology. Elsevier Science Ltd, London, Uk, 55:191-213, (2004).
Abstract: The World Health Organization (WHO) definition of SBS includes such symptoms in the occupants as headache, distraction, dizziness, fatigue, watery eyes, runny or blocked or bleeding nose, dry or sore throat and skin irritation. The WHO has set a criterion for a healthy building as "less than 20% of occupants complain" (WHO, 1995). Various chemicals, toxins and microorganisms have been suggested as causes of SBS. In this review, we will focus on exposures to fungi (molds) as causes of SBS, with emphasis on the possible role of proteinaceous fungal hemolysins in SBS.
Identifying and quantifying the great diversity of fungi found indoors had been one of the main limitations to addressing the cause-effect relationship between fungal exposures and SBS. The recent development of quantitative PCR (QPCR) analysis of molds (US. Patent 6,387,652) will dramatically improve fungal speciation and quantification, resulting in a highly standardized process for describing the indoor fungal population. Now that the populations of indoor fungi can be easily described, we need to identify possible causes for SBS associated with fungi. We suggest that fungal hemolysins may play a role in some symptoms observed in SBS.

Hemolysins are molecules that are designated as such because they have the ability to lyse red blood cells (RBCs). In this review, only proteinaceous hemolysins will be discussed, although the authors recognize that non-proteinaceous fungal molecules can have hemolytic effects, e.g. T-2 toxin causes hemolysis of red blood cells (Segal et al., 1983). For this review of fungal hemolysins, the artificial separation of macro-fungi (e. g. mushrooms) and micro-fungi (e.g. filamentous species and yeasts) will be used. Throughout this review, we will compare fungal hemolysins with bacterial hemolysins because bacterial hemolysins have been much more extensively studied and may provide useful insights into the nature and activities of fungal hemolysins.

There are primarily two types of hemolysins designated alpha and beta. Alpha hemolysins cause a partial lysis of the RBCs resulting in a darkening of the media around a colony on sheep's blood agar (SBA). The beta hemolysins produce a complete lysis of the RBCs, resulting in a clearing around the colony growing on SBA.

BOOK CHAPTER Inactivation and Removal of Enteric Protozoa in Water 03/01/2004
Schaefer III, F W., M. M. Marshall, AND J. L. Clancy. Inactivation and Removal of Enteric Protozoa in Water. , Chapter 9, C.R. Sterling, R. Adams (ed.), The Pathogenic Enteric Protozoa: Giardia, Entamoeba, Cryptosporidium, and Cyclospora. Kluwer Academic Publishers, Hingham, MA, 117-127, (2004).
Abstract: Protozoan parasites including Giardia, Cryptosporidium, and Entamoeba can be transmitted through water and cause disease in humans and animals. Control of waterborne infection can be accomplished through a variety of physical and chemical means, resulting in the production of safe drinking water and protection of public health. Coagulation, sedimentation, and filtration are the most commonly employed methods for physical removal of parasites, while chlorine-based compounds, ozone, and ultraviolet light are used for inactivation. Combinations of treatment technologies can result in parasite removal/inactivation greater than 6-log (10) resulting in reliable public health protection

BOOK CHAPTER Protozoa 01/01/2004
Schaefer III, F W. Protozoa. , 3rd., Chapter 11, Problem Organisms in Water: Identification and Treatment. American Water Works Association, Denver, CO, M7:69-78, (2004).
Abstract: There is no abstract available for this product. If further information is requested, please refer to the bibliographic citation and contact the person listed under Contact field.

EXTRAMURAL DOCUMENT Environmental Factors and Chemical and Microbiological Water Quality Constitutents Related to the Presence of Enteric Viruses in Ground Water from Small Public Water Supplies in Southeastern Michigan 06/01/2004
FRANCY, D. S., R. N. BUSHON, J. STOPAR, E. J. LUZANO, AND G. FOUT. Environmental Factors and Chemical and Microbiological Water Quality Constitutents Related to the Presence of Enteric Viruses in Ground Water from Small Public Water Supplies in Southeastern Michigan. U.S. Environmental Protection Agency, Washington, DC, 2004.
Abstract: A study of small public ground-water-supply wells that produce water from discontinuous sand and gravel aquifers was done from July 1999 through July 2001 in southeastern Michigan. Samples were collected to determine the occurrence of viral pathogens and microbiological indicators of fecal contamination (indicators), determine whether indicators are adequate predictors of the presence of enteric viruses, and determine the factors that affect the presence of enteric viruses. Small systems are those that serve less than 3,300 people. Samples were analyzed for specific enteric viruses by reverse transcriptase-polymerase chain reaction (RT-PCR), for culturable viruses by cell culture, and for the indicators total coliforms, Escherichia coli (E. coli), enterococci, and F-specific and somatic coliphage. Ancillary environmental and water-quality data were collected or compiled.
A total of 169 regular samples and 32 replicate pairs were collected from 38 wells. Replicate pairs were samples collected at the same well on the same date. One well was sampled 6 times, 30 wells were sampled five times, 6 wells were sampled twice, and 1 well was sampled once. By use of RT-PCR, enterovirus was found in four wells (10.5 percent) and hepatitis A virus (HAV) in five wells (13.2 percent). In two of these wells, investigators found both enterovirus and HAV, but on different sampling dates. Culturable viruses were found one time in two wells (5.9 percent), and neither of these wells was positive for viruses by use of RT-PCR on any sampling date. If results for all viruses are combined, 9 of the 38 small public-supply wells were positive for enteric viruses (23.7 percent) by either cell culture or RT-PCR.

One or more indicators were found in 18 of 38 wells. Total coliforms, E. coli, enterococci, and F-specific and somatic coliphage were found in 34.2, 10.5, 15.8, 5.9, and 5.9 percent, respectively, of the wells tested. In only 3 out of 18 wells were samples positive for an indicator on more than one date at the same well. The co-occurrence of enteric viruses and any indicator was 55.6 percent; five out of the nine virus-positive wells were also found to be positive for an indicator. Two wells with detections of viruses had a detection of total coliforms, one well had a detection of E. coli, one of enterococci, and one of F-specific coliphage. On a per sample basis, of 11 samples that were positive for enteric viruses, indicator bacteria co-occurred in only 2 samples, and coliphage were not present in any.

More virus-positive samples were found at sites served by septic systems than those served by sewerlines. Sampling condition (ground water or a mixture of tank and ground water), distance to septic system, type of and distance to nearest surface-water body, well characteristics, or land use were not related to the presence of viruses or indicators. Among continuous water-quality variables, statistically significant relations were found between total coliforms and dissolved organic carbon and between total coliforms and iron. There was a statistically significant relation between chloride concentrations >20 mg/L and detections of total coliforms. Presence of nitrate and nitrite was related to the presence of other indicators (E. coli, enterococci, and F-specific and somatic coliphage) or enteric viruses, but not to total coliforms. The data indicated that chloride-to-bromide (Cl:Br) ratios may be useful as a screening tool for total coliforms and enteric viruses but not for E. coli, enterococci, and F-specific and somatic coliphage.

This study provides evidence for fecal contamination of ground water from small public-supply wells, at least on an intermittent basis. Collecting data on multiple lines of evidence would be needed to reliably predict the presence of enteric viruses and protect public health. Future data collection toward this end could include repeat sampling several times a year for different indicators, measuring dissolved-organic carbon, nitrate plus nitrite, and (or) chloride concentrations, or determining Cl:Br ratios. The presence of a site served by a septic system is an indication that the well may be more vulnerable to contamination than a site served by a sewerline.

JOURNAL Use of Pharmacokinetic Modeling to Design Studies for Pathway-Specific Exposure Model Evaluation 12/01/2004
Hu, Y., G G. Akland, E. D. Pellizzari, M Berry, AND L J. Melnyk. Use of Pharmacokinetic Modeling to Design Studies for Pathway-Specific Exposure Model Evaluation. ENVIRONMENTAL HEALTH PERSPECTIVES. National Institute of Environmental Health Sciences (NIEHS), Research Triangle Park, NC, 112(17):1697-1703, (2004).
Abstract: Validating an exposure pathway model is difficult because the biomarker, which is often used to evaluate the model prediction, is an integrated measure for exposures from all the exposure routes/pathways. The purpose of this paper is to demonstrate a method to use pharmacokenetic modeling and computer simulation to guide the design of field studies to validate pathway models. The children's dietary intake model was discussed in detail as an example. Three important aspects were identified for a successful design to evaluate the children's dietary intake model: (1) longitudinal design of the study with alternating exposure status for the route/pathway of interest; (2) short biological half-life of the selected chemical; and (3) surface loading of selected chemical at sufficient levels. Exposure from other pathways, such as inhalation and non-dietary ingestion exposure, would not mask the dietary exposure under normal circumstances. This study proposed and demonstrated the use of a pharmacokinetic model in the design of a study to evaluate a path-specific exposure model, which has not been explored previously.

JOURNAL Genotypes of Cryptosporidium Infecting Fur-Bearing Mammals Differ from Those Infecting Humans 12/01/2004
Zhou, L., R. Fayer, J. Trout, F W. Schaefer III, AND L. Xiao. Genotypes of Cryptosporidium Infecting Fur-Bearing Mammals Differ from Those Infecting Humans. APPLIED AND ENVIRONMENTAL MICROBIOLOGY 70(12):7574-7577, (2004).
Abstract: To evaluate the public health significance of wildlife Cryptosporidium spp., five species of small wild mammals (beavers, muskrats, otters, raccoons, and foxes) living in Chesapeake Bay watersheds were examined in this study for the presence of Cryptosporidium spp., using a small subunit (SSU) rRNA gene-based PCR-restriction fragment length polymorphism technique (PCR-RFLP). A total of 471 fecal samples were collected, and 36 samples from four animal species (fox, raccoon, muskrat and beaver) were positive for five distinct Cryptosporidium spp.: C. canis dog genotype (1), C. canis fox genotype (4), Cryptosporidium muskrat genotype I (24), Cryptosporidium muskrat genotype II (6), and Cryptosporidium skunk genotype (2). Most animals had only one type of Cryptosporidium sp., with one muskrat having two types of Cryptosporidium (muskrat genotype I and II). Results of the study indicated that wildlife in watersheds carried host-adapted Cryptosporidium spp., which have no significant public health importance.

JOURNAL Glutathione Modulates Recominant Rat Arsenic (+3 Oxidation State) Methyltransferase-Catalyzed Formation of Trimethylarsine Oxide and Trimethylarsine 12/01/2004
Waters, S. B., V. Devesa, M. FRICKE, J. T. CREED, M. Styblow, AND D. J. THOMAS. Glutathione Modulates Recominant Rat Arsenic (+3 Oxidation State) Methyltransferase-Catalyzed Formation of Trimethylarsine Oxide and Trimethylarsine. CHEMICAL RESEARCH IN TOXICOLOGY. American Chemical Society, Washington, DC, 17(12):1621-1629, (2008).
Abstract: Humans and other species enzymatically convert inorganic arsenic (iAs) into methylated metabolites. Although the major metabolites are mono- and dimethylated arsenicals, trimethylated arsenicals have been detected in urine following exposure to iAs. The AS3MT gene encodes an arsenic (+3 oxidation state) methyltransferase, which catalyzes both the oxidative methylation of trivalent arsenicals and the reduction of pentavalent arsenicals. In reaction mixtures containing recombinant rat AS3MT (rrAS3MT) and radiolabeled arsenite, mono- and dimethylated arsenicals and a third radiolabeled product can be resolved by thin-layer chromatography. Hydride generation atomic absorption spectrometry and electrospray ionization mass spectrometry identified the third reaction product as trimethylarsine oxide. The addition of glutathione to reaction mixtures containing radiolabeled arsenite and rrAS3MT increased the yield of methylated and dimethylated arsenicals but suppressed the formation of trimethylarsine oxide. Although a dimethylarsenic-glutathione complex was rapidly converted to trimethylarsine oxide, the addition of a molar excess of glutathione to dimethylarsenic suppressed the production of trimethylarsine oxide. The nonquantitative recovery of radioarsenic from reaction mixtures suggested that AS3MT catalyzed the formation of a volatile arsenical. This volatile species was identified as trimethylarsine. Thus, AS3MT catalyzes the formation of all products in a reaction sequence leading from an inorganic to a volatile methylated arsenical. The regulation of this pathway by intracellular glutathione may be an important determinant of the pattern and extent of formation of arsenicals.

JOURNAL Extraction and Detection of a New Arsine Sulfide Containing Arsenosugar in Molluscs By Ic-ICP-MS and Ic-Esi-MS/MS 11/01/2004
Fricke, M., P A. Creed, A N. Parks, J A. Shoemaker, C A. Schwegel, AND J T. Creed. Extraction and Detection of a New Arsine Sulfide Containing Arsenosugar in Molluscs By Ic-ICP-MS and Ic-Esi-MS/MS. JOURNAL OF ANALYTICAL ATOMIC SPECTROMETRY 19(11):1454-1459, (2004).
Abstract: Using IC-ICP-MS and IC-ESI-MS/MS, an unknown arsenical compound in mollusks has been identified as a new arsine sulfide containing analog of a known arsenosugar and is referred to as As(498). This species has been observed in four separate shellfish species following a mild methanol/water extraction. As(498) is unstable, especially in acid, and converts to the arsine oxide containing arsenosugar As(482) over time.
Chromatographic retention of As(498) was observed on an anion exchanger ION-120 column but the species did not elute as a well defined peak from a PRP-X100. Mass spectrometric analysis of As(498) at pH 9.0 produced an [M-H]- species at a mass to charge of 497 in the negative-ion mode. A synthetic standard of As(498) was made by bubbling hydrogen sulfide into a stock solution of arsenosugar As(482). The retention time and ESI-MS/MS data were identical for the synthetic standard of As(498) and the unknown arsenical in s

JOURNAL Distribution of Six Virulence Factors in Aeromonas Species Isolated from US Drinking Water Utilities: A Pcr Identification 11/01/2004
Sen, K. AND M R. Rodgers. Distribution of Six Virulence Factors in Aeromonas Species Isolated from US Drinking Water Utilities: A Pcr Identification. JOURNAL OF APPLIED MICROBIOLOGY. Blackwell Publishing, Malden, MA, 97(5):1077-1086, (2004).
Abstract: Surveys of finished drinking water conducted by the U.S. EPA during 2000-2001, revealed 7 out of 16 water utilities encompassing four states, were contaminated with Aeromonas species. A Polymerase Chain reaction (PCR) based genetic characterization determined the presence of six virulence factors genes, elastase (ahyB), lipase, (pla/ lip/ lipH3/ alp-1) flagella A and B (flaA and fla B) genes, the enterotoxins, act , alt and ast genes, in these isolates. Primer sets were designed for all the target genes, except for act, which had been published previously. PCR was performed in three duplex assays using the primers to ahy B and lip, fla and alt genes, and act and ast genes together. ahyB, lip, fla alt, act, ast were present in 40 %, 86 %, 55% , 45 %, 69% and 35% of the strains, respectively. Only one isolate had all the virulence genes. More than one kind of species was isolated from some utilities. Also different combinations of virulence factors were present in different strains of the same species. However, a dominant strain having the same set of virulence factors, was usually isolated from different rounds of sampling from a single tap. These results suggest the importance of examining as many Aeromonas isolates as possible from a water sample. The results also suggest that the Aeromonas strains isolated from water utilities have the potential to be pathogenic, although, additional virulence factors, that have not yet been identified or characterized, may be needed to cause actual disease.

JOURNAL Development of Homologous Viral Internal Controls for Use in Rt-Pcr Assays of Waterborne Enteric Viruses 10/01/2004
Parshionikar, S., J L. Cashdollar, AND G S. Fout. Development of Homologous Viral Internal Controls for Use in Rt-Pcr Assays of Waterborne Enteric Viruses. JOURNAL OF VIROLOGICAL METHODS 121(1):39-48, (2004).
Abstract: Enteric viruses often contaminate water sources causing frequent outbreaks of gastroenteritis. Reverse transcription-polymerase chain reaction (RT-PCR) assays are commonly used for detection of human enteric viruses in environmental and drinking water samples. RT-PCR provides a means to rapidly detect low levels of these viruses, but it is sensitive to inhibitors that are present in water samples. Inhibitors of RT-PCR are concentrated along with viruses during sample processing. While procedures have been developed to remove inhibitors, none of them completely remove all inhibitors from all types of water matrices. This problem requires that adequate controls be used to distinguish true from potentially false-negative results. To address this problem, we have developed homologous viral internal controls for hepatitis A virus (HAV), poliovirus, Norwalk virus and rotavirus. These internal controls can be used in RT-PCR assays for the detection of the above viruses by competitive amplification, thereby allowing the detection of false negatives in processed water samples. The internal controls developed in this study were successfully tested with virus-seeded environmental water sample concentrates.

JOURNAL Electrophoretic Mobility of Mycobacterium Avium Complex Organisms 09/01/2004
Lytle, D A., C Frietch, AND T C. Covert. Electrophoretic Mobility of Mycobacterium Avium Complex Organisms. APPLIED AND ENVIRONMENTAL MICROBIOLOGY 70(9):5667-5671, (2004).
Abstract: The electrophoretic mobilities (EPMs) of thirty Mycobacterium avium Complex (MAC) organisms isolated from clinical and environmental sources were measured in 9.15 mM KH2PO4 buffered water. The EPMs of fifteen clinical isolates ranged from -1.9 to -5.0 µm cm V-1 s-1 with a mean of -3.1 µm cm V-1 s-1, and the EPMs of fifteen environmental isolates ranged from -1.9 to -4.6 µm cm V-1 s-1 with a mean of -3.3 µm cm V-1 s-1 at pH 7. The EPM of MAC organisms were significantly more negative than other waterborne pathogens (E. coli 0157H:57, Vibrio cholerae, Cryptosporidium parvum) measured under similar experimental conditions.

JOURNAL Quantitative Pcr Analysis of House Dust Can Reveal Abnormal Mold Conditions 07/01/2004
Meklin, T, R A. Haugland, T. Reponen, M Varma, Z. Lummus, D. Bernstein, L J. Wymer, D. Dearborn, AND S J. Vesper. Quantitative Pcr Analysis of House Dust Can Reveal Abnormal Mold Conditions. JOURNAL OF ENVIRONMENTAL MONITORING. Royal Society of Chemistry, London, Uk, 6(7):615-620, (2004).
Abstract: Indoor mold populations were measured in the dust of homes in Cleveland and Cincinnati, OH, by quantitative PCR (QPCR) and, in Cincinnati, also by culturing. QPCR assays for 82 species (or groups of species) were used to identify and quantify indoor mold populations in moldy homes (MH) and reference homes (RH). About 65% of the species were never or only rarely detected. The ratios (MH geometric mean/ RH geometric mean) for 11 species (Aspergillus fumigatus, A. ochraceus, A. penicilliodes, A. restrictus, A. sclerotiorum, A. unguis, A. versicolor, Eurotium group, Penicillium purpurogenum, Cladosporium sphaerospermum and Stachybotrys chartarum) were >1 in both cities. However, the same ratios for 10 species (Aspergillus ustus, Penicillium chrysogenum type 2, Acremonium strictum, Alternaria alternata, Cladosporium cladosporiodes types 1 and 2, C. herbarum, Epicoccum nigrum, Mucor and Rhizopus group, and Rhizopus stolonifer were consistently <1. SAS Proc_Probit analysis of the sum of the logs of the populations of these two groups resulted in a 95% probability range for separating MH from RH. These results suggest that it may be possible to evaluate whether a home has an abnormal mold condition by quantifying a limited set of mold species in a dust sample.
Mold growth in homes, schools and buildings has become a controversial health issue with some studies suggesting negative health effects (1, 3, 6, 20, 25, 28, 29). Some of the reasons for the controversy may include the large number of molds that occur indoors and the difficulties involved with measuring their populations.
Traditionally indoor mold populations have been measured with culturing methods that are based on colony counting and microscopic identification (16, 18, 19, 21, 22, 27). However, culture methods are time consuming and normally only limited information on fungal species is obtained. In addition, viable microbes represent only a small fraction of the total molds present (26). More accurate, specific and faster methods are needed to analyze environmental samples for molds.
A DNA-based method for quantitative measurement of different species of indoor molds has been developed at the United States Environmental Protection Agency (11). The system is based on real time monitoring of the polymerase chain reaction (PCR) using fluorigenic 5' exonuclease chemistry (TaqManTM) (14) that allows for highly quantitative results (10, 12, 13, 23). This technology, called quantitative PCR (QPCR) is now being applied to environmental analysis of samples, including dust.
House dust samples may better represent cumulative exposures to molds compared to short air samples (5, 7). Findings showing the gradual increase of mold concentration in floor dust, despite of regular vacuuming (4), strengthen the idea of house dust samples as reservoir of fungal contamination. In this study, QPCR technology was used to quantify 82 mold species in dust samples in MH and RH in Cleveland and Cincinnati, OH.

JOURNAL Effects of Seeding Procedures and Water Quality on Recovery of Cryptosporidium Oocysts from Stream Water By Using U.S. Environmental Protection Agency Method 1623 07/01/2004
Francy, D. S., O. D. Simmons, M W. Ware, E. J. Granger, M. D. Sobsey, AND F W. Schaefer III. Effects of Seeding Procedures and Water Quality on Recovery of Cryptosporidium Oocysts from Stream Water By Using U.S. Environmental Protection Agency Method 1623. APPLIED AND ENVIRONMENTAL MICROBIOLOGY 70(7):4118-4128, (2004).
Abstract: U.S.EPA Methods 1622 and 1623 are used to detect and quantify Cryptosporidium oocysts in water. The protocol consists of filtration, immunomagnetic separation (IMS), staining with a fluorescent antibody, and microscopic analysis. Microscopic analysis includes detection by fluorescent antibody and confirmation by the demonstration of 1-4 sporozoites or nuclei after staining with 4',6-diamidino-2-phenyl indole dihydrochloride (DAPI). The purpose of this study was to evaluate a new IMS dissociation, a 10 minute incubation at 80 ° C. Heat dissociation improved the average oocyst recovery from 41% to 71% in seeded reagent water, and from 10% to 51% in seeded river samples. The average DAPI confirmation rate improved from 49 % to 93% in reagent water, and from 48% to 73% in river samples. This modification improved both oocyst recovery and confirmation.

JOURNAL Natural Atypical Listeria Innocua Strains With Listeria Monocytogenes Pathogenicity Island 1 Genes 07/01/2004
Johnson, J., J. Jinneman, G N. Stelma Jr., B G. Smith, D Lye, J W. Messer, J. Ulaszek, L. Evsen, S. Gendel, R. W. Bennett, B. Swaminathan, J. Pruckle, A. Steigerwalt, S. Kathariou, S. Yildirim, D. Volokhov, A. Rasooly, V. Chizhikov, M. Wiedmann, E. Fortes, R. E. Duvall, AND A. D. Hitchins. Natural Atypical Listeria Innocua Strains With Listeria Monocytogenes Pathogenicity Island 1 Genes. APPLIED AND ENVIRONMENTAL MICROBIOLOGY 70(7):4256-4266, (2004).
Abstract: The detection of the human foodborne pathogen, Listeria monocytogenes, in food, environmental samples and clinical specimens associated with cases of listeriosis, a rare but high mortality-rate disease, requires distinguishing the pathogen from other Listeria species. Speciation of bona fide Listeria isolates into the six species of the genus is normally a relatively simple few-test process. Occasionally aberrant isolates are found but even then their speciation is usually possible with only one or two extra confirmatory tests. We have discovered a hemolytic, rhamnose- and xylose-fermentation negative Listeria strain with surprising recalicitrance to speciation. The L. monocytogenes-negative results with two L. monocytogenes confirmatory tests, for which no false negatives have been reported to date, were contradicted by the L. monocytogenes -positive result of another confirmatory test for L. monocytogenes genes for which no false posititves results have been reported to date. The issue had to be resolved by using total DNA/DNA hybridization testing and then confirmed by further specific PCR tests made possible by the recently published gene sequences of L. monocytogenes and L. innocua. The results show that this isolate is indeed a novel one. It provides the first fully documented instance of the discovery of a hemolytic Listeria innocua strain. This species is typically non-hemolytic. The L. innocua isolate contains at least an overall representation, if not a facisimile, of the PrfA regulated virulance gnee cluster of L. monocytogenes. It apparently does not contain all the extra-cluster virulence genes because it is avirulent in the mouse pathogenicity test. At least one of these extra cluster virulence genes, the L. monocytogenes-specific allele of iap, which is not PrfA regulated, is not fully present, if at all. The virulence gene cluster contains hyl, the gene, which codes for the hemolysin (listeriolysin O) of the L. monocytogenes. In addition to the hemolysin at least one of the other genes of the cluster, plcA, is expressed in this strain of L. innocua. The detection of specific L. innocua genes in the strain is consistent with its L. innocua DNA/DNA hybridization identity. Further study of this strain may contribute to our understanding of evolution, particularly of virulence, in the genus Listeria.

JOURNAL Monitoring Aspergillus Species By Quantitative Pcr During Construction of a Multi-Story Hospital Building 06/14/2004
Morrison, J., C. S. Yang, K. Lin, R A. Haugland, A. N. Neely, AND S J. Vesper. Monitoring Aspergillus Species By Quantitative Pcr During Construction of a Multi-Story Hospital Building. JOURNAL OF HOSPITAL INFECTION 57(1):85-87, (2004).
Abstract: Noscomial fungal infections represent a persistent threat in hospitals. One of the major issues in fungal control has been monitoring these fungi in a timely manner. Quantitative polymerase chain reaction (QPCR) allows for the rapid (2 to 4 h), sensitive (often down to a single spore) detection and quantitifcation of fungi.
In this report, we describe the application of this technology to the analysis of Aspergillus contamination in a five-story addition to a community hospital in the upper mid-west of the US. While there have been many reports of fungal contamination associated with the remodeling of existing structures or with building demolition, we report here Aspergillus contamination associated with one of the risks of new construction: rain prior to building closure.

JOURNAL Measurement of Perchlorate in Water By Use of An 18o-Enriched Isotopic Standard and Ion Chromatography With Mass Spectrometric Detection 06/11/2004
Hedrick, E J. AND D. J. Munch. Measurement of Perchlorate in Water By Use of An 18o-Enriched Isotopic Standard and Ion Chromatography With Mass Spectrometric Detection. JOURNAL OF CHROMATOGRAPHY A 1039(1-2):83-88, (2004).
Abstract: Perchlorate (ClO4-) is a drinking water contaminant originating from the dissolution of the salts of ammonium, potassium, magnesium, or sodium in water. It is used primarily as an oxidant in solid propellant for rockets, missiles, pyrotechnics, as a component in air bag inflators, and in highway safety flares. Based on EPA Information Request Responses and occurrence monitoring, there have been 95 confirmed ClO4- releases in 25 states and 230 users or manufacturers in 40 states. From accidental releases and improper disposal practices of the past, ClO4- has become a contaminant in surface and ground waters where it is highly mobile and, due to its chemical stability, persists for decades. The primary human health effect is inhibition of iodide uptake by the thyroid gland. By disrupting thyroid hormone production, ClO4- interferes with metabolism and can affect brain development in fetuses and children, leading to mental impairment. There is now a need for a method that can confirm and quantify ClO4- at concentrations lower than what is achievable using ion chromatography with suppressed conductivity detection. Coupled with ion chromatographic separation, mass spectrometry is by far the most promising analytical tool available today for low-level identification and quantitation of ClO4- in drinking water. In this work, sub-ppb quantitation of ClO4- in drinking waters using ion chromatography electrospray ionization mass spectrometry (IC-ESI-MS) is demonstrated.
The primary mass of interest for ClO4- is 99 based on the 75.77% relative abundance of the chlorine-35 isotope. Mass 101 is a secondary mass of interest based on the 24.23% abundance of chlorine-37. In this work we demonstrate the feasibility of using oxygen-18 enriched perchlorate as an internal standard. The greatest benefit of such an internal standard is for the improvement of accuracy in the determination of perchlorate in waters with high total dissolved solids such as sulfate, carbonate and chloride. In the event that ClO4- is regulated in drinking water or that a second national occurrence survey is conducted, this research will lead to an inherently more specific and sensitive U.S. EPA method than is currently available.

JOURNAL Quantitative Pcr Analysis of Fungi in Dust from Homes of Infants Who Developed Idiopathic Pulmonary Hemorrhaging 06/06/2004
Vesper, S J., M Varma, L J. Wymer, D. Dearborn, J. Sobolewski, AND R A. Haugland. Quantitative Pcr Analysis of Fungi in Dust from Homes of Infants Who Developed Idiopathic Pulmonary Hemorrhaging. OCCUPATIONAL AND ENVIRONMENTAL MEDICINE 46(6):596-601, (2004).
Abstract: Fungal concentrations were measured in the dust of six homes in Cleveland, OH, where a child developed pulmonary hemorrhage (pulmonary hemorrhage homes, i.e. PHH), and 26 reference homes (RH) with no known fungal contamination. QPCR assays for 82 species (or assay groups) were used to identify and quantify fungal concentrations. The ratios of the geometric means of PHH to RH were >1 for 26 species (Group I) (Aspergillus flavus, A. fumigatus, A. niger, A. ochraceus, A. penicilliodes, A. restrictus, A.sydowii, A sclerotiorum, A. unguis, A. versicolor, Eurotium group, Penicillium brevicompactum, P. corylophilum, P. spinulosum, P. viaiable, P. purpurogenum, Penicillium group 2, Paecilomyces variotii, Aureobasidium pullans, Chaetomium globosum, Cladosporium sphaerospermum, Scopulariopsis brevicaulis, S. chartarum, Stachybotrys chartarum, Trichderma viride, and Wallemia sebi). However, the same ratios were <1 for 10 species (Group II) (Aspergillus ustus, Penicillium chrysogenum svar. 2, Acremonium strictum, Alternaria alternata, Cladosporium cladosporiodes svar 1 and 2, C. herbarum, Epicoccum nigrum, Mucor and Rhizopus group, and Rhizopus stolonifer). Probit analysis of the sum of the logs of the concentrations of these two groups resulted in a 95% probability range for separating PHH from RH homes. The same 82 fungal species were also tested for hemolysin production on sheep's blood agar (incubated at 37oC). Hemolysins were more commonly produced by group I species (42%) compared to Group II species (10%).

JOURNAL Preservation of As(iii) and As(v) in Drinking Water Supply Samples from Across the United States Using Edta and Acetic Acid as a Means of Minimizing Iron-Arsenic CO-Precipitation 05/15/2004
Gallagher, P A., C A. Schwegel, A N. Parks, B. M. Gamble, L J. Wymer, AND J T. Creed. Preservation of As(iii) and As(v) in Drinking Water Supply Samples from Across the United States Using Edta and Acetic Acid as a Means of Minimizing Iron-Arsenic CO-Precipitation. ENVIRONMENTAL SCIENCE AND TECHNOLOGY 38(10):2919-2927, (2004).
Abstract: This paper evaluates seven different treatment/storage conditions for the preservation of the native As(III)/As(V) found in ten drinking water supplies from across the US. These ten waters were chosen because they have different As(III)/As(V) distributions; six of these waters contained enough iron to produce an iron precipitate during shipment. The waters were treated and stored under specific conditions and analyzed periodically over a span of approximately 75 days. Linear least squares (LLS) was used to estimate the change in As(III) and As(V) over the study period. Point estimates for the first and last analysis days and 95% confidence bounds were calculated from the LLS. The difference in the point estimates for the first and last day were then evaluated with respect to drinking water treatment decision making.
Three primary treatments were evaluated: EDTA/AcOH-treatment, AcOH treatment, as well as no treatment. The effect of temperature was explored for all treatments while the effect of aeration was evaluated for only the EDTA/AcOH treated samples. The non-treated samples experienced a 0-40% reduction in the native arsenic concentration due to the formation of Fe/As precipitates. The Fe/As precipitates were resolubilized and shown to contain elevated concentrations of As(V) relative to the native distribution. Once this Fe/As precipitate was removed from solution using a 0.45 and 0.2 m filter, the resulting arsenic concentration (As(III) + As(V)) was relatively constant (the largest LLS slope was -1.4 ng x 10-2 As/g water/day). The AcOH treatment eliminated the formation of the Fe/As precipitate observed in the non-treated samples. However, two of the AcOH water samples produced analytically significant changes in the As(III) concentration. The LLS slopes for these two waters were -5.7 x 10-2 ng As(III)/g water/day and -1.0 x 10-1 ng As(III)/g water/day. This corresponds to a -4.3 ng/g and a -7.8 ng/g change in the As(III) concentration over the study period, which is a 10% shift in the native distribution. The third and final treatment was EDTA/AcOH. This treatment eliminated the Fe/As precipitate that formed in the non-treated sample. The LLS slopes were less than -7.5x10-3 ng As(III)/g water/day for the above mentioned waters, corresponding to a 0.6 ng/g change over the study period. One of the EDTA/AcOH treated waters did indicate that using the 5 C storage temperature minimized the rate of conversion relative to 20 C storage.

JOURNAL Rare Occurrence of Heterotrophic Bacteria With Pathogenic Potential in Potable Water 05/01/2004
Stelma Jr., G N., D J. Lye, B G. Smith, AND J W. Messer. Rare Occurrence of Heterotrophic Bacteria With Pathogenic Potential in Potable Water. INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY 92(3):249-254, (2004).
Abstract: Since the discovery of Legionella pneumophila, an opportunistic pathogen that is indigenous to water, microbiologists have speculated that there may be other opportunistic pathogens among the numerous heterotrophic bacteria found in potable water. The USEPA developed a series of rapid in vitro assays to assess the virulence potential of large numbers of bacteria from potable water to possibly identify currently unknown pathogens. Results of surveys of potable water from several distribution systems using these tests showed that only 50 of the approximately 10,000 bacterial colonies expressed one or more virulence characteristics. In another study, 45 potable water isolates that were isolated from granular activated carbon filters and expressed multiple virulence factors were tested for pathogenicity in immunocompromised mice. None of the isolates infected mice that were compromised either by treatement with carrageenan, to induce susceptibility to facultative intracellular pathogens, or by cyclophosphamide, to induce susceptibility to extracellular pathogens. These results indicate that there are very few potential pathogens in potable water and that the currently developed in vitro virulence screening tests give an overestimation of the numbers of hetertrophic bacteria that may be pathogens. Current efforts are focused on using the animal models to screen concentrated samples of waters known to contain large numbers of heterotrophic bacteria and newly discovered Legionella-like organisms that parasitize amoebae.

JOURNAL Rapid Monitoring By Qpcr for Pathogenic Aspergillus During Carpet Removal from a Hospital 04/01/2004
Neely, A. N., V J. Gallardo, E F. Barth, R A. Haugland, G. D. Warden, AND S J. Vesper. Rapid Monitoring By Qpcr for Pathogenic Aspergillus During Carpet Removal from a Hospital. INFECTION CONTROL AND HOSPITAL EPIDEMIOLOGY 25(4):350-352, (2004).
Abstract: Monitoring for pathogenic Aspergillus species using a rapid, highly sensitive, quantitative polymerase chain reaction technique during carpet removal in a burn unit provided data which allowed the patients to be safely returned to the re-floored area sooner than if only conventional culture monitoring had been used.

JOURNAL Development of An Astrovirus Rt-Pcr Detection Assay for Use With Conventional, Real-Time, and Integrated Cell Culture/Rt-Pcr 04/01/2004
Grimm, A C., J L. Cashdollar, F P. Williams, AND G S. Fout. Development of An Astrovirus Rt-Pcr Detection Assay for Use With Conventional, Real-Time, and Integrated Cell Culture/Rt-Pcr. CANADIAN JOURNAL OF MICROBIOLOGY 50(4):269-278, (2004).
Abstract: Astrovirus is a common cause of gastroenteritis in humans that has been determined to be responsible for outbreaks of illness in several countries. Since astrovirus can be waterborne, it is important to be able to identify this virus in environmental water. We have developed and optimized an RT-PCR method that was able to amplify all eight astrovirus serotypes. In addition, an internal control was designed so that any inhibitors of this astrovirus assay could be detected. The assay was adapted for use in a real-time PCR assay and the sensitivity of these two methods was compared. The real-time assay was then combined with CaCo2 cell culture to produce an integrated cell culture/RT-PCR (ICC/RT-PCR) assay and the sensitivity of the combined assay was compared to RT-PCR alone. The methods were used to detect astrovirus in acute phase illness stool samples as well as to detect the virus in water samples spiked with astrovirus.

JOURNAL Overall Kinetics of Heterogeneous Elemental Mercury Reactions on Tio2 Sorbent Particles With UV Radiation 03/17/2004
Lee, T. G., P. Biswas, AND E J. Hedrick. Overall Kinetics of Heterogeneous Elemental Mercury Reactions on Tio2 Sorbent Particles With UV Radiation. INDUSTRIAL & ENGINEERING CHEMISTRY RESEARCH. American Chemical Society, Washington, DC, 43(6):1411-1417, (2001).
Abstract: A system consisting of a photochemical reaction was used to evaluate the kinetic parameters, such as reaction order and rate constant for the elemental mercury uptake by TiO2 in the presence of uv irradiation. TiO2 particles generated by an aerosol route were used in a fixed bed system along with commercial Degusa P25 TiO2 powders. In the presence of uv irradiation, the rate of Hg photooxidation was evaluated as a function of Hg concentration, uv intensity, and available surface area (actrivated sites). Reaction orders with respect to the gas phase mercury concentration and the uv intensity were found to be 1.40+/-0.1 and 0.35+/-0.05, respectively. For the case of in situ generated TiO2 particles, mercury concenration and uv intensity were found to be 1.1+/-0.1 and 0.390, respectively. From results of the experiments at various reaction temperatures, the apparent activation energy, and the experimentally observed rate constant were calculated.

JOURNAL A LC/MS Method for the Determination of Cyanobacteria Toxins in Water 03/01/2004
Maizels, M. AND W L. Budde. A LC/MS Method for the Determination of Cyanobacteria Toxins in Water. Analytical Chemistry 76(5):1342-1351, (2004).
Abstract: The cyanobacteria toxins anatoxin-a, microcystin-LR, microcystin-RR, microcystin-YR, and nodularin were separated in less than 30 minutes on several 1 mm x 15 cm reverse phase liquid chromatography (LC) columns, and their electrospray mass spectra were measured with 50 ng or less injected amounts with a bench-top time-of-flight (TOF) mass spectrometer. New data from this work includes: (a) the impact of acetic acid concentrations in the methanol-water mobile phase on measured ion abundances; (b) the performance of the electrospray-TOF mass spectrometer as an LC detector; (c) the accuracy and precision of exact m/z measurements after LC separation with a routinely-used mass spectrometer resolving power of 5000; and, (d) recoveries of the five toxins from reagent water, river waters, and sewage treatment plant effluent samples extractged with C-18 silica particles enmeshed in thin Teflon (trademark) membrane filter disks. This technique has the otential of providing a relatively simple and reasonable-cost sample preparation and LC/MS method that provides the sensitivity, selectivity, reliability, and information content needed for drinking water occurrence and human exposure studies.

JOURNAL Detection of Human Enteric Viruses in Stream Water With Rt-Pcr and Cell Culture 03/01/2004
DenisMize, K., G S. Fout, D R. Dahling, AND D. S. Francy. Detection of Human Enteric Viruses in Stream Water With Rt-Pcr and Cell Culture. JOURNAL OF WATER AND HEALTH 2(1):37-47, (2004).
Abstract: A multiplex RT-PCR method was used to measure virus occurrence at five stream water sites that span a range of hydroclimatic, water-quality, and land-use characteristics. The performance of the molecular method was evaluated in comparison to traditional cell culture and Escherichia coli membrane filtration assays. The study incorporated multiple quality controls and included a contol for virus recovery during the sampling procedure as well as controls to detect potentially false-negative and false-positive data. Poliovirus recovery ranged from 16 to 65% and was variable, even in samples collected within the same stream. All five sites were positive for viruses by both molecular and cell culture-based virus assays. Enteroviruses, reoviruses, rotaviruses, and hepatitis A viruses were detected, but the use of the quality controls proved critical for interpretation of the molecular data. All sites showed evidence of fecal contamination, and culturable viruses were detected in four samples that would have met the U. S. Environmental Protection Agency's recommended Escherichia coli standard for safe recreational water.

JOURNAL Quantitative Pcr of Selected Aspergillus, Penicillium and Paecilomyces Species 03/01/2004
Haugland, R A., M Varma, L J. Wymer, AND S J. Vesper. Quantitative Pcr of Selected Aspergillus, Penicillium and Paecilomyces Species. SYSTEMATIC AND APPLIED MICROBIOLOGY 27(2):198-210, (2004).
Abstract: A total of 65 quantitative PCR (QPCR) assays, incorporating fluorigenic 5' nuclease (TaqMan®) chemistry and directed at the nuclear ribosomal RNA operon, internal transcribed spacer regions (ITS1 or ITS2) was developed and tested for the detection of Aspergillus, Penicillium and Paecilomyces species that have been reported in indoor environments. The assays varied in specificity from species or subspecies to closely related species groups, subject to the amount of nucleotide sequence variation in the different organisms. A generic assay for all target species of Aspergillus, Penicillium and Paecilomyces was also developed and tested. Using a previously reported DNA extraction method, estimated conidia detection limits for target species ranged from less than one to several hundred per sample for the different assays. Conidia detection limits for non-target species were at least 1,000 fold higher in nearly all instances. The assays were used to analyze ten HVAC dust samples from different sources around the US. Total quantities of Aspergillus, Penicillium and Paecilomyces conidia in the samples, determined by the generic assay and the summed totals from the specific assays, were in general agreement, suggesting that all of the numerically dominant species in the samples were accounted for by the specific assays. The QPCR analyses detected from approximately 20 to 7500 fold higher levels of total target organisms than culture based enumerations on a commonly used growth medium in the different samples. QPCR analyses of these samples after spiking them with selected target organisms indicated that the enumeration results were within approximately a one-half log range of the expected values 95% of the time. Evidence is provided that the commonly used practices of enumerating Aspergillus and Penicillium as a single group or only by genus can be misleading in understanding the indoor populations of these organisms and their potential health risks.

JOURNAL A Mechanistic Model for Mercury Capture With in-Situ Generated Titania Particles: Role of Water Vapor 02/01/2004
Rodriguez, S., C. B. Almquist, T. G. Lee, M. Furuuchi, E J. Hedrick, AND P. Biswas. A Mechanistic Model for Mercury Capture With in-Situ Generated Titania Particles: Role of Water Vapor. JOURNAL OF AIR AND WASTE MANAGEMENT ASSOCIATION 54(2):149-156, (2004).
Abstract: A mechanistic model to predict the capture of gas phase mercury species using in-situ generated titania nanosize particles activated by UV irradiation is developed. The model is an extension of a recently reported model1 for photochemical reactions that accounts for the rates of electron-hole pair generation, adsorption of the compound to be oxidized and the adsorption of water vapor. The role of water vapor on the removal efficiency of Hg was investigated to evaluate the rates of mercury oxidation at different water vapor concentrations. As the water vapor concentration is increased, more OH-radical species are generated on the surface of the titania particle, increasing the number of active sites for the photo-oxidation and capture of mercury. At very high water vapor concentrations, competitive adsorption is expected to be important and reduce the number of sites available for photo-oxidation of mercury. The predictions of the developed phenomenological model agreed well with the measured mercury oxidation rates in this study, and with the data on oxidation of organic compounds reported in the literature.

JOURNAL Total Arsenic Determination and Speciation in Infant Food Products By Ion Chromatography Inductively Coupled Plasma Mass Spectrometry 01/01/2004
Vela, N. P. AND D. T. Heitkemper. Total Arsenic Determination and Speciation in Infant Food Products By Ion Chromatography Inductively Coupled Plasma Mass Spectrometry. JOURNAL OF AOAC INTERNATIONAL. AOAC International, Gaithersburg, MD, 87(1):244-252, (2004).
Abstract: Health risk associated with dietary arsenic intake may be different for infants and adults. Seafood is the main contributor to arsenic intake for adults while terrestrial-based food is the primary source for infants. Processed infant food products such as rice-based cereals, mixed rice/formula cereals, milk-based infant formula, applesauce and puree of peaches, pears, carrots, sweet potatoes, green beans, and squash were evaluated for total and speciated arsenic content. Arsenic concentrations found in rice-based cereals (63-320 ng/g dry weight) were similar to those reported for raw rice. Results for the analysis of powdered infant formula by inductively coupled plasma-mass spectrometry (ICP-MS) indicated a narrow and low arsenic concentration range (12 to 17 ng/g). Arsenic content in puree infant food products, including rice cereals, fruits and vegetables, varies from <1 to 24 ng/g wet weight. Sample treatment with trifluoroacetic acid at 100°C were an efficient and mild method for extraction of arsenic species present in different food matrixes as compared to alternative methods that included sonication and accelerated solvent extraction. Extraction recoveries from 94 to 128% were obtained when trhe summation of species was compared to total arsenic. The ion chromatography (IC)-ICP-MS method selected for arsenic speciation allowed for the quantitative determination of inorganic arsenic [As(III) + As(V)], dimethylarsnic acid (DMA), and methylarsonic acid (MMA). Inorganic arsenic and DMA are the main species found in rice-based and mixed rice/formula cereals, although traces of MMA were also detected. Inorganic arsenic was present in freeze-dried sweet potatoes, carrots, green beans, and peaches. MMA and DMA were not detected in these samples. Arsenic species in squash, pears, and applesauce were not detected above the mentioned detection limit.

JOURNAL Analytical Mass Spectrometry of Herbicides 01/01/2004
Budde, W L. Analytical Mass Spectrometry of Herbicides. MASS SPECTROMETRY REVIEWS 23(1):1-24, (2004).
Abstract: Herbicides are chemical substances that are applied to agricultural soils, gardens, lawns, or plants to destroy or to prevent the growth of undesirable vegetation. The herbicides included in this review are generally syntehtic organic compuonds that are ingredients in commercial herbicide products that were designated active during late 2002 in the U.S. Environmental Protection Agency's database of registered and canceled pesticide products. The compounds are organized into 21 categories according to their general chemical structures or a common structural group. The herbicides in each category are discussed in terms of their structures, their database electron ionization mass spectra, their amenability to separation and measurement with gas chromatography, reverse phase liquid chromatography, and capillary electrophoresis combined with mass spectrometry. Ionization techniques ocnsidered are mainly electron ionization, electropray, and atmospheric pressure chemical ionization. Sixty-six references are provided to herbicide reviews, and to the recent herbicide analytical chemistry and mass spectrometry research literature.

PRESENTATION Current Vfars Ongoing Research and What Needs to Be Done to Evaluate the Vfars Concept and Determine Its Feasibility 11/14/2004
Stelma Jr., G N., S J. Vesper, S L. Hayes, D J. Lye, M J. Donohue, AND M R. Rodgers. Current Vfars Ongoing Research and What Needs to Be Done to Evaluate the Vfars Concept and Determine Its Feasibility. Presented at Water Quality Technology Conference, San Antonio, TX, November 14-18, 2004.
Abstract: The VFARs concept predicts that pathogens can be identified using structural similarities among virulence genes from diverse species. This concept is of interest to the EPA for several reasons: the Agency's need to discriminate between virulent and avirulent isolates of pathogen species listed on the Agency's Contaminant Candidate List (CCL), the Agency's desire to identify organisms for future CCLs and the Agency's desire to identify currently unrecognized waterborne pathogens. Several issues have created skepticism among some microbiologists concerning the immediate utility of the VFARs concept. (i) The degree to which the structure-function relationships seen in chemistry carry over into biology is unknown. Some identical or similar virulence genes may have arisen in diverse species due to parallel evolution; however, other virulence genes may express similar biological activities but may be structurally unique to one genus or species. (ii) The expression of an entire array of virulence genes is necessary for an organism to infect and cause illness. This means that gene probes must be available to test for all of the virulence genes, not just one or two critical genes. Many virulence genes are currently unknown; and it is unlikely that the entire arrays of virulence genes of most pathogens will be identified in the immediate future. (iii) nonpathogenic microorganisms occasionally carry key virulence genes but do not express them. This could cause gene probes to identify harmless strains as pathogens. (iv) The concept may not carry over to viruses and protozoan parasites because viruses and protozoa are all obligate intracellular pathogens and are all, therefore, virulent.
EPA is currently using several approaches to ascertain the long-term and short-term utility of the VFARs concept for evaluating CCL organisms and for identifying currently unknown pathogens from the environment. Our STAR Grants program has funded a data mining effort to find common DNA sequences among diverse species of pathogenic bacteria. One approach of our in-house research uses proteomics to examine and compare gene products from virulent and avirulent isolates of Aeromonas that are growing in cell culture. Another approach, which will be accomplished through an Interagency Agreement, will compare gene products and genes in Cryptosporidium strains that cause illness in humans with those from Cryptosporidium isolates that are avirulent in humans. Other protozoan species that infect humans will then be examined for genes that are analogous to those associated with those Cryptosporidium strains that are virulent to humans.

The most creative approach that EPA is currently comparing the responses of mammalian cells to pathogenic strains of Aeromonas to the responses observed with avirulent strains from the same genus. If the responses are significantly different, this approach should eliminate the need to identify the entire array of genes necessary for virulence in order to distinguish between virulent and avirulent isolates. This approach should also allow us to accurately characterize strains that carry one or more virulence genes but do not express them as avirulent. In addition, this approach should allow us to screen newly discovered microorganisms for virulence potential. The preliminary results obtained from these in-house studies are promising. Neonatal mice were challenged with a virulent isolate of A. hydrophila and an avirulent isolate of A. caviae. Five hours later microarrays were used to measure host mRNA responses. The differences were dramatic; over 400 genes were up-regulated after exposure of the host to the virulent strain; whereas, only 28 host genes were up-regulated after exposure to the avirulent strain. Furthermore, many of the genes that were induced by the virulent isolate were genes that are associated with immune responses. These experiments will be repeated using tissue culture and eventually using other genera of pathogens.

PRESENTATION What Goes Up Must Come Down: A Lagrangian Study of Potential Chemical Idicators of Fecal Contamination Downstream from Wastewater Plants 11/14/2004
Glassmeyer, S, E. T. Furlong, D. W. Kolpin, J. D. Cahill, S. D. Zaugg, S. L. Werner, L. B. Barber, AND D D. Kryak. What Goes Up Must Come Down: A Lagrangian Study of Potential Chemical Idicators of Fecal Contamination Downstream from Wastewater Plants. Presented at Society of Environmental Toxicology and Chemistry, Portland, OR, November 14-18, 2004.
Abstract: In earlier studies, we have determined that pharmaceuticals and other compounds found in household wastewater (such as surfactants, disinfectants, and scenting agents) are present in the effluent from wastewater treatment plants, and persist downstream from the facilities. To obtain a better understanding of the fate of these compounds, two wastewater dominated watersheds, Four Mile Creek in Ankeney, IA and Boulder Creek near Boulder, CO, were investigated using a Lagrangian sampling scheme. Prior to sampling, a dye tracer was released in each stream to determine the time it takes for the water to reach different points downstream. With this time of travel information, water was collected at the predetermined downstream points so that the same parcel of water was analyzed at all locations. This sample collection design allows for a better understanding of the processes that can remove the compounds from the stream, such as photodegradation, sorption onto particulates, and volatilization. Additionally, the kinetics of the combined removal processes can be approximated, so the average lifetime of each of these compounds in the watershed can be calculated. At each of the sample sites, a water sample was collected and field filtered. Two different samples were extracted using solid phase extraction, and analyzed using liquid chromatography/ mass spectrometry (pharmaceuticals) and gas chromatography/ mass spectrometry (wastewater compounds); between the two methods, there are over 85 analytes. The samples were also analyzed for two indicator bacteria, E. coli and enterococci. For most compounds, the concentration profiles were as expected: absent or very low concentrations in the upstream samples, with a noticeable increase in the effluent, and a decline in the downstream samples. The information gathered in this project will help determine which of these compounds might be useful as tracers of human fecal material. Disclaimer: Although this work was reviewed by EPA and approved for publication, it may not necessarily reflect official Agency policy.

PRESENTATION Virulence Factors of Aeromonas: A Genetic Characterization of Drinking Water Isolates 11/01/2004
Sen, K. AND M R. Rodgers. Virulence Factors of Aeromonas: A Genetic Characterization of Drinking Water Isolates. Presented at Water Quality Techology Conference, Philadelphia, PA, November 1-5, 2004.
Abstract: A survey of finished drinking water conducted by the US EPA during 2000-2001, revealed that 8 out of 18 water utilities encompassing several states (NY, KY, IA, OH) were contaminated with aeromonas species. Altogether 205 organisms were isolated by EPA method 1601. All of the species were Ampicillin and dextrin resistant and the major species isolated were A. bestiarum/ A salmonicida and A hydrophila (Birkenhauer et al, 2003). A. veronii , A. caviae, A. media and A. allosacharophila were the other species that were isolated in small numbers . As an initial attempt to determine the virulence of the different species present, a PCR based genetic characterization was done for the virulent factors elastase, lipase, flagella A and B genes, the cytolytic enterotoxin act ( achytoen) and the cytolytic enterotoxins, Alt and Ast. Disruption of any of these genes causes a loss or diminishes the virulence of the species. The achytoen gene primers were earlier described by Kingcombe et al ( ). For the other genes all the available sequences available in the genebank databases were aligned and primers were designed to conserved regions among respective similar genes, from different species. PCR was done by three duplex assays using the primers to elastase and lipase together, fla and alt together, and the act, ast genes together. Lipase was present in 86% of the strains; Act in 69% of the strains; Alt and Ast in 45% and 30% of the strains respectively, fla in 55% and Elastase in 40 % of the strains. Only one strain had all the virulent genes. From a given utility more than one kind of species were found. Also within the same contaminated sample of water from a given utility, different combination of virulent factors were present in different strains of the same species. These results indicated that the entire flora need to be tested from a water sample, for the different virulent factors, as within the same species the occurrence of certain virulence factors may vary . The results also suggest that the strains isolated have the potential to be pathogenic, although, additional virulence factors, that have not been characterized, may be needed to cause disease.

PRESENTATION Laboratory Capacity Needs Assessment of Drinking Water Utilities: A Global Perspective 11/01/2004
Rodgers, M R., S. Adrian, AND T. MacDonald. Laboratory Capacity Needs Assessment of Drinking Water Utilities: A Global Perspective. Presented at American Water Resources Association Annual Meeting, Orlando, FL, November 1-4, 2004.
Abstract: Fully-functioning analytical laboratories capable of producing quality data are essential components of well-run drinking water utilities. In Europe and the US, drinking water laboratory performance is closely monitored and regulated; this is not always the case in the less industrialized nations. Beginning with the US efforts to help Central America rebuild after the 2001 Hurricane Mitch, our team evaluated drinking water laboratories in El Salvador, Nicaragua and Honduras on their capacity and performance. On-site visits showed a lack of a functioning Quality Assurance Program, inadequate sampling/sample handling procedures, use of questionable methodologies, lack of proper equipment and the need for training of both managers and technicians. To address these problems, a series of training courses, designed and implemented in collaboration with the Pan American Health Organization, were conducted for health ministry officials and drinking water utility laboratory personnel from these three countries. Needed equipment was purchased and follow-up audits and performance evaluation samples were used to monitor progress. In India, our team has recently completed an evaluation of drinking water laboratories in two cities, with similar findings. The overall goal of these efforts is to prepare laboratories for accreditation and encourage national programs to institute such accreditation broadly. EPA is also working with the World Health Organization to further replicate such drinking water laboratory capacity through a global water quality partnership initiative launched by the WHO at the 2002 World Summit on Sustainable Development. EPA's laboratory program in India will serve as one component of this initiative. This work has been funded by the United States Environmental Protection Agency. It has been subjected to Agency review and approved for publication.

PRESENTATION Possible Role of Fungal Hemolysins in Sick Building Syndrome 10/30/2004
Vesper, S J. Possible Role of Fungal Hemolysins in Sick Building Syndrome. Presented at National Institute of Building Sciences Symposium: Mold, Moisture, Misery and Money plus Myth, San Diego, CA, October 30-31, 2004.
Abstract: Many fungi produce proteinaceous hemolytic agents. Like bacterial hemolysins, fungal hemolysins create pores or holes in membranes. Depending on which membranes are damaged, fungal hemolysins can produce a variety of effects. Fungal hemolysins can cause histamine release from mast cells and/or induce cytokine release from human or animal cells. These responses may result in some of the flu- or cold-like syptoms associated with sick building syndrome (SBS). Fungal hemolysins, like bacterial hemolysins, can also damage vascular tissues and thus cause some SBS symptoms like headache, dizziness and bleeding. The source of fungal hemolysins may be the colonization of the airway, especially nasopharynx, by indoor fungi and not simply from the inhaled spores. Measurements of fungal hemolysins in serum or other bodily fluid should prove useful in diagnosing SBS.

PRESENTATION Introduction to the Vfars Workshop 10/28/2004
Stelma Jr., G N. Introduction to the Vfars Workshop. Presented at VFARs Workshop, Baltimore, MD, October 28-29, 2004.
Abstract: A 1996 amendment to the Safe Drinking Water Act requires the EPA to periodically develop a list of currently unregulated contaminants and select five contaminants for regulatory decisions every five years. The National Research Council (NRC) was enlisted to assist the Agency in developing processes to develop new Contaminant Candidate Lists and to prioritize contaminants. The NRC suggested the premise that the architectural and biochemical components of microorganisms that cause disease are structurally related and that structure-activity relationships of virulence genes (VFARs) can be used to predict virulence in microorganisms the way that quantitative structure-activity relationships of chemicals (QSARs) can be used to predict toxicity. If this premise holds up, EPA should be able to use VFARs to develop new Contaminant Candidate Lists (CCLs), prioritize the pathogens on the list and possibly even to identify currently unrecognized pathogens. There are, however, some challenges that must be overcome before VFARs can be used in decision making. For example, we do not know the extent to which the biological universe parallels the chemical universe; nor do we know the entire array of virulence factors, except for a few pathogens. Host susceptibility factors and the presence of unexpressed virulence genes in some microorganisms must also be considered. This workshop is being held to allow EPA to get the advice from experts in the field of microbial pathogenesis and virulence regarding the types of research that is needed to evaluate the usefulness of VFARs in the CCL process.

PRESENTATION Investigation of Cyanobacteria Toxins By LC/MS 10/18/2004
Budde, W L., C. Diehnelt, AND N R. Dugan. Investigation of Cyanobacteria Toxins By LC/MS. Presented at U.S. EPA Region 6, 14th Annual Quality Assurance Conference, Dallas, TX, October 18, 2004.
Abstract: There is no abstract available for this product. If further information is requested, please refer to the bibliographic citation and contact the person listed under Contact field.

PRESENTATION Pesticide Transfer Efficiencies from Household Surfaces to Foods 10/17/2004
Bernard, C E., M R. Berry Jr., AND L J. Melnyk. Pesticide Transfer Efficiencies from Household Surfaces to Foods. Presented at International Society of Exposure Analysis National Meeting, Philadelphia, PA, October 17-21, 2004.
Abstract: Traditional dietary pesticide exposure assessments have focused on contamination during production (e.g., pesticides in agriculture). However, recent residential monitoring studies have demonstrated that a significant portion of infant and children's total exposure can result from food contamination in homes following residential pesticide usage. These exposures, termed excess dietary exposure, includes direct contact between foods handled by children and contaminated surfaces. Limited data exists concerning these transfers, but they have been shown to be a critical element in estimating children's excess dietary exposure. This study was conducted to determine the transfer efficiencies (%) of pesticides (organophosphate, pyrethroid, and pyrazole insecticides) from a household surface to bologna, apple, and Fruit Roll-Ups?, which are representative foods (i.e., meat, fruit, and snack foods) handled and eaten by children. The moisture and fat content of each food was also determined as tools for predicting transfer.
The highest transfer efficiencies were observed for the organophosphate pesticides to bologna (56-79%) with lower transfers to apples (20-37%) and Fruit Roll-Ups (6-17%). Transfer efficiencies of pyrethroid pesticides to bologna, apples, and Fruit Roll-Ups were 33-52%, 12-34% and 3%, respectively. Fipronil (pyrazole) had a similar transfer to pyrethroids (bologna 39-45%, apple 13-41%, and Fruit Roll-Up 4%). The average percent fat and moisture content for bologna, apples, and Fruit Roll-Ups were 30%, 1%, 7% and 54%, 85%, 11%, respectively. Although neither percent fat nor moisture content projected the ranking of transfer between the foods, combined they may provide a better prediction. These results suggest food type and chemical properties of the pesticide affect transfer. Study findings coupled with models under development will be used to generate more accurate estimates of excess dietary exposure to infants and children in homes where pesticides are used.

PRESENTATION Pesticide Residue Recoveries from Surface Wipes 10/17/2004
Bernard, C E. AND L J. Melnyk. Pesticide Residue Recoveries from Surface Wipes. Presented at International Society of Exposure Analysis National Meeting, Philadelphia, PA, October 17-21, 2004.
Abstract: Human exposure is a consequence of pesticide use indoors with a primary source resulting from residue deposition on household surfaces. Accurate measurements of surface residues is essential for estimating exposure from different routes. Various procedures have been developed for measuring pesticides including surface wipes with solvent moistened gauze pads, which is commonly used by the USEPA. However, surface wiping procedures differ in sampling material (e.g., gauze brand), solvent type, solvent volume, and number of wipes collected per sample. A standardized surface wipe sampling procedure is needed in order to provide surface measurements that may be compared between exposure studies.
USEPA's National Exposure Research Laboratory (Cincinnati) has developed a procedure that uses a cotton gauze pad moistened with 10 mL isopropanol (IPA) wiped across a designated sampling area followed by a second dry wipe in the same direction. The sampling is repeated using a moistened pad (10 mL IPA) followed by a dry wipe in the vertical direction. This method was evaluated using both hard (i.e., ceramic tile) and soft (i.e., carpet) household surfaces contaminated with organophosphate, pyrethroid, and pyrazole pesticides.

Residues were recovered from hard surfaces at mean levels of 94 ? 14% (organophosphates), 98 ? 18% (pyrethroids), and 97 ?21% (pyrazole). Lower recoveries were captured from carpet at 35 ? 7% (organophosphates), 33 ? 5% (pyrethroids), and 35 ? 8% (pyrazole). In comparison, lower recoveries of similar pesticides from carpet have been reported for procedures using smaller IPA volumes and without the 2 dry wipes. Analytical interferences from extracts have also been observed for different gauze pad brands. The method described provides a consistent, reliable surface wipe sampling procedure, without interferences, that can be used in a laboratory or field study.

PRESENTATION Estimating Excess Dietary Exposures of Young Children 10/17/2004
Melnyk, L J., C E. Bernard, AND J. Raymer. Estimating Excess Dietary Exposures of Young Children. Presented at International Society of Exposure Analysis, Philadelphia, PA, October 17-21, 2004.
Abstract: Nine children in a daycare that routinely applied the pesticide, esfenvalerate, were studied to assess excess dietary exposures. Surface wipes, a standard food item of processed American cheese slice pressed on the surface and handled by the child, an accelerometer reading, and questionnaires were collected for each participant. These measurements were used to assess potential indirect dietary exposures to esfenvalerate from activities the child displayed during a routine day. The cheese slice was used to obtain esfenvalerate transferred from the hands and surfaces due to food contact. Accelerometers were used to indicate each child's activity level. Both transfer and activity are essential parameters when determining the total dietary exposure of a child.
The children in the daycare were visited six times. Each visit allowed certain monitoring to take place. Questionnaires were given to the teachers and parents of the participants. Surface wipes indicated between 0.3 to 62.7 ng/cm2 of the pesticide with the highest levels found on the interior floor. Surface transfer to cheese measured 0.2 - 1.9 ng/cm2 with the highest level at the interior floor. Handled cheese contained 1 - 24 ng/g of transferable pesticide. The video taping and questionnaire responses were used to interpret the accelerometer counts. Results indicated that the cheese slice was successfully used to collect transfer information from hands and surfaces, accelerometer placement on non-dominant hand produced the highest readings but required more time on participants to obtain useful data, and questionnaire responses by the teacher agreed more closely with the video and standard food results.

PRESENTATION The EPA Microbial Source Tracking Document 10/14/2004
Stelma Jr., G N. The EPA Microbial Source Tracking Document. Presented at EPA's National Beaches Conference, San Diego, CA, October 14, 2004.
Abstract: Beach closures or violations of total maximum daily loads of fecal organisms in watersheds frequently generate a need to identify the major sources of contamination or, at least, determine whether the source is human or animal. A few years ago E. coli ribotyping was the only method available for microbial source tracking (MST). Recently, however, a number of diverse methods are reported to be effective for MST; and it has become difficult for beach managers and other local officials to choose the method that is best for their specific needs. The USEPA is writing a guidance document to assist the users of MST methods in choosing the most appropriate method for their individual beaches or watersheds. The MST guide document contains descriptions of each published method, including references; the assumptions on which the methods are based; the limitations of each method; data collection and analyses and method performance. The final chapter provides decision criteria and includes a "decision tree" which guides the reader through the various scenarios in which MST may be useful. Each decision point in the tree contains a menu of the most appropriate methods for the user's needs. The document is comprehensive, including both library-dependent and library-independent molecular methods, as well as library-dependent phenotypic methods.

PRESENTATION Persistence of Pharmaceuticals and Other Wastewater Related Compounds 10/13/2004
Glassmeyer, S, E. T. Furlong, D. W. Kolpin, I. FerrerFelis, J. D. Cahill, S. D. Zaugg, S. L. Werner, AND D D. Kryak. Persistence of Pharmaceuticals and Other Wastewater Related Compounds. Presented at 4th International Conference on Pharmaceuticals and Encocrine Disrupting Chemicals in Water, Minneapolis, MN, October 13-15, 2004.
Abstract: The quality of drinking and recreational water is currently ascertained using indicator bacteria. However, the tests to analyze for these bacteria require a considerable length of time to complete, and do not discriminate between human and animal fecal material sources. One solution is to use chemicals found in human wastewater which would have the advantage of requiring shorter analysis times, and can be selected to be human specific. At ten locations, water samples were collected upstream, and at two successive points downstream from wastewater treatment facility. A treated effluent sample was also collected at each location. This longitudinal sampling scheme was used to determine the persistence of the target compounds in streams. The water samples were extracted using either solid phase or liquid-liquid extraction and were analyzed using either liquid chromatography/mass spectrometry or gas chromatography/mass spectrometry. Of the 95 chemical analytes investigated in this project, 78 were found in at least one sample. Seventeen of the 95 compounds were classified as either a prescription or non-prescription pharmaceutical. Of these, nine were found in at least 50 % of the samples and thirteen were found in at least 10 % of the samples. While most concentrations of all of the compounds were in the range of 0.1 to 1.0 g/ L, in some of the more highly contaminated samples, concentrations were in the range of 5-20 g/ L. The concentrations of the majority of the chemical compounds present in the samples generally followed a reasonable trend: they were either non-existent or at only trace levels in the upstream samples, had their maximum values in the wastewater effluent samples, and then declined in the two downstream samples. This work indicates that these chemical analytes do have utility as tracers of human wastewater discharge.

PRESENTATION Preliminary Health Burden Analysis for Epidemiologic Recreational Water Studies Mceard 10/13/2004
SAMS, E. A., A. H. WILLIAMS, T. J. WADE, M. BEACH, K. P. BRENNER, AND A. P. DUFOUR. Preliminary Health Burden Analysis for Epidemiologic Recreational Water Studies Mceard. Presented at National Beaches Conference, San Diego, CA, October 13 - 15, 2004.
Abstract: Presented at the National Beaches Conference, San Diego, California, October 13-15, 2004

PRESENTATION Survey of Environmental Exposure and Health Outcomes at Beaches for the National Epidemiological and Environmental Assessment of Recreational (Neear) Water Study Mceard 10/13/2004
TORRE, K. D., S. K. DIETZ, K. PATRIZI, E. A. SAMS, K. P. BRENNER, T. J. WADE, A. H. WILLIAMS, AND M. BEACH. Survey of Environmental Exposure and Health Outcomes at Beaches for the National Epidemiological and Environmental Assessment of Recreational (Neear) Water Study Mceard. Presented at National Beaches Conference, San Diego, CA, October 13 - 15, 2004.
Abstract: Presented at the National Beaches Conference, San Diego, CA, October 13-15, 2004

PRESENTATION Rapid Measurement of Bacterial Fecal Pollution Indicators at Recreational Beaches By Quantitative Polymerase Chain Reaction 10/13/2004
Haugland, R A., S D. Siefring, K Brenner, A P. Dufour, J. A. Griffith, K. Schiff, S. B. Weisberg, J. Paar, AND K. G. Field. Rapid Measurement of Bacterial Fecal Pollution Indicators at Recreational Beaches By Quantitative Polymerase Chain Reaction. Presented at National Beaches Conference, San Diego, CA, October 13-15, 2004.
Abstract: Previous studies have demonstrated that measurements by the membrane filtration (MF) method of the bacterial indicators Enterococcus and E. coli in recreational beach water samples are correlated with swimming-associated gastroenteritis. These relationships currently serve as the basis for recommended guidance by the USEPA on unacceptable health risks associated with swimming in natural waters. The MF method, however, requires at least 24 hours for results and during this delay, swimmers may be exposed to unsafe waters. The quantitative polymerase chain reaction (QPCR) method is presently being evaluated as a possible alternative to MF. Water analyses using this technology can provide results in approximately 2 hours. In the summer of 2003, studies were conducted by several organizations including USEPA, Office of Research and Development, USEPA Region I, and the Southern California Coastal Water Research Project at both freshwater and marine beaches to determine the correlation between results of the QPCR and MF methods. Two of these studies also tested a newly developed assay for fecal indicator bacteria in the class Bacteriodetes and collected data on swimmer illness rates that have been compared with the QPCR and MF results. Positive correlations were observed in each of these studies and continuing evaluations of the QPCR method are being performed in 2004. Based on the performance of this method to date, the USEPA Office of Water is currently considering its potential use as a reference method in performance evaluations of other alternative nucleic acid tests for fecal contamination in ambient waters.

PRESENTATION Using Today's Data to Close the Beach Today. Quantitative Polymerase Chain Reaction (Qpcr) Rapid Beach Closing Tool 10/06/2004
Haugland, R A. Using Today's Data to Close the Beach Today. Quantitative Polymerase Chain Reaction (Qpcr) Rapid Beach Closing Tool. Presented at ORD Product Expo at Region 5, Chicago, IL, October 6, 2004.
Abstract: Recreational beaches are an important economic and aesthetic asset to communities, states and the nation as a whole. Considerable resources are expended each year in the measurement of fecal indicator bacteria concentrations in the water at these beaches to determine whether these waters are safe for public use. These measurements are presently performed by culture-based methods such as membrane filtration that require at least 24 hr before results are obtained. As a result, these methods often may not allow notification of potential health risks to swimmers until after exposures have occurred. Several molecular microbial analysis technologies have the capability of circumventing this deficiency by providing results in shorter time periods. One particularly rapid technology is the quantitative polymerase chain reaction (QPCR). Water analyses using this technology can provide results in approximately 2 hours. This technology has now been adapted for the measurement of the EPA-recommended fecal indicator organisms, Eschericia coli and Enterococcus, in surface water samples. In the summer of 2003, studies were initiated by the USEPA, Office of Research and Development, at two Great Lakes beaches, to determine the correlation between QPCR and EPA Method 1600 (membrane filtration) measurements of enterococci in water samples and swimmer illness rates. Positive correlations were observed between illness rates and the results of both methods and continuing evaluations are being performed in 2004. This presentation will provide an overview of the principles of the QPCR method, describe its application for beach water quality analysis and provide current data on the relationship between results obtained by this method and membrane filtration.

PRESENTATION The Challenge of Molds for the U.S. Army 08/08/2004
Vesper, S J. The Challenge of Molds for the U.S. Army. Presented at 7th Annual Air Force Health Protection Conference, Albuquerque, NM, August 6-12, 2004.
Abstract: The US Army and all armies have been interested in molds since there were armies. The most obvious interest was human infections by molds like trench foot. Then there were losses of military animals and contamination of their fodder, most notably the Soviet loss of thousands of horses in the late 1930's, as Russia prepared for war. Molds destroy human food supplies also. As Napoleon said "armies march on their stomachs." Of course, military equipment was destroyed by molds. In the tropics, tents and clothing would disappear under relentless mold attack. Armies have conquered many of these challenges from mold. Today, the US Army faces a new challenge from molds found in its buildings and family housing.

PRESENTATION Mold Specific Quantitative Pcr for Rapid Identification and Enumeration 08/06/2004
Vesper, S J. Mold Specific Quantitative Pcr for Rapid Identification and Enumeration. Presented at 7th Annual Army Force Health Protection (FHP) Confernce, Albuquerque, NM, August 6-12, 2004.
Abstract: There is growing awareness that indoor molds/fungi may be connected to such conditions as asthma, allergies, hemorrhaging, chronic rhinosinusitis, memory loss, and a symptom complex called sick-building-syndrome. In addition, molds cause frequently fatal nosocomical infections. The US EPA has developed a mold specific quantitative PCR (MSQPCR) process that relies of the unique DNA signature of each mold species to do the identification and quantification. Assays are now available for more than 100 of the most common and relevant molds. These assays are standardized, specific, and sensitive (usually down to a single spore). The analysis can be completed in 2 to 3 hours. An automated platform can be used to perform these analyses. Examples of the use of the technology will be described in the presentation.

PRESENTATION The National Epidemiological and Environmental Assessment of Recreational Waters: Results from the First Summer Full-Scale Studies 08/01/2004
WADE, T. J., R. L. CALDERON, E. A. SAMS, K. P. BRENNER, M. BEACH, A. H. WILLIAMS, AND A. P. DUFOUR. The National Epidemiological and Environmental Assessment of Recreational Waters: Results from the First Summer Full-Scale Studies. Presented at International Society for Environmental Epidemiologists, New York, NY, August 01 - 04, 2004.
Abstract: Introduction
The National Epidemiological and Environmental Assessment of Recreational Waters (NEEAR) is a multi-year study of recreational water conducted by the United States Environmental Protection Agency and the Centers for Disease Control and Prevention (CDC), designed to evaluate new rapid and specific indicators of recreational water quality and to determine their relationship to health effects. These studies are the first to evaluate the association between health effects and novel rapid indicators of recreational water quality.

Methods

We conducted epidemiologic studies at a Lake Michigan beach and a Lake Erie beach during the summer of 2004. Interviewers approached beach goers and solicited their enrollment in the study. Interviewers asked beach-goers to return to the interview station to complete a series questions relating to whether or not the beach-goers went in the water and what other types of activities they did on the beach as the left the beach for the day. Ten to 12 days after the beach interview, interviewers telephoned each household to ascertain health symptoms (gastrointestinal, respiratory, eye, ear, skin) experienced in the days following the beach interview.

At each beach water samples were collected at several transects (three at Lake Michigan and five at Lake Erie) at two depths, 0.3 m (shin depth) and 1 m (knee depth), three times a day. Samples were tested for enterococci using the standard method (Method 1600) and for enterococci and Bacteroides sp. using novel methods including rapid PCR and an antibody based, fiber-optics system.

Results

At the Lake Michigan Beach, we conducted surveys on 20 days between June 1 and August 3. 2834 household interviews were attempted, 1683 household interviews were completed. Data were available on 2877 individuals. At the Lake Erie beach, we conducted surveys on 13 days between August 2 and September 14. 2962 households interviews were attempted, and 1634 household interviews were completed. Data were available for 2840 individuals.

Both beaches experienced significant variability in water quality over the summer. At Lake Michigan, daily geometric means of enterococci (Method 1600) ranged from 1 to over 1000 with and 3 days exceeded the current US EPA guideline value. At Lake Erie daily geometric means of enterococci ranged from 1 to over 800 and 6 days exceeded. Gastrointestinal, ear, eye, and skin rash symptoms were significantly associated with swimming. The relationships between health symptoms and the traditional and rapid indicators will be fully evaluated and presented in detail.


PRESENTATION GIS Analysis for Epidemiologic Recreational Water Studies 08/01/2004
SAMS, E. A., R. L. CALDERON, T. J. WADE, M. BEACH, K. P. BRENNER, A. H. WILLIAMS, AND A. P. DUFOUR. GIS Analysis for Epidemiologic Recreational Water Studies. Presented at International Society for Environmental Epidemiologists, New York, NY, August 01 - 04, 2004.
Abstract: Presented at the International Society for Environmental Epidemiologists, New York, NY, August 1-4, 2004

PRESENTATION Standardized Mold Identification and Enumeration 07/28/2004
Vesper, S J. Standardized Mold Identification and Enumeration. Presented at ASTM International Conference, Boulder, CO, July 25-30, 2004.
Abstract: There are probably at least 100,000 species of molds or fungi. But there are actually about 100 typically found indoors. Some pose a threat to humans and animals and others don't. We need to know what molds are present indoors and their concentrations. The older methods of culturing molds on media or counting spores are slow, laborious and subjective. Quantitative or real time PCR is objective because it is based on the DNA sequences unique to the different mold species.

PRESENTATION Quantification of Enterovirus and Hepatitis a Viruses in Wells and Springs in East Tennessee Using Real-Time Reverse Transciption Pcr 07/19/2004
Baldwin, T., A. C. Layton, L. D. McKay, S. Jones, V. Garrett, G. Johnson, AND G S. Fout. Quantification of Enterovirus and Hepatitis a Viruses in Wells and Springs in East Tennessee Using Real-Time Reverse Transciption Pcr. Presented at 4th International Groundwater Quality Conference, Ontario, Canada, July 19-22, 2004.
Abstract: This project involves development, validation testing and application of a fast, efficient method of quantitatively measuring occurrence and concentration of common human viral pathogens, enterovirus and hepatitis A virus, in ground water samples using real-time reverse transcription-polymerase chain reaction. Development of the real time RT-PCR assays for enterovirus and hepatitis A virus are complete and they are being verified by comparison with cell culture assays. The validation and application phase of the project includes monitoring viral occurrence and concentration in samples from six community water supply wells or springs in karst settings in east Tennessee. The wells/springs were chosen on the basis of prior monitoring of fecal indicator bacteria and geochemical parameters (temperature, turbitity, DO, etc.), as well as the presence or absence of likely input sources of human sewage. The wells/springs used for sampling include two sites that have a relatively high likelihood of containing human pathogens, two that have a very low likelihood, and two intermediate sites. Sampling for viruses is scheduled to begin in January 2004 and these are expected to be the first measurements of viral occurrence in groundwater in the state of Tennessee.

PRESENTATION The Determination of Pyrethroid and Prethrin Insecticides in Foods 07/18/2004
Brisbin, J. AND J N. Morgan. The Determination of Pyrethroid and Prethrin Insecticides in Foods. Presented at 2004 Florida Pesticide Residue Workshop, Orlando, FL, July 18-21, 2004.
Abstract: Pyrethrins, and the more light stable synthetic pyrethroids, are insecticides that are effective against many pests. They have been used for many years and are relatively non-toxic to warm blooded animals. The residue analysis of pyrethrins and pyrethroids is of interest to the U.S. Environmental Protection Agency's (USEPA) National Exposure Research Laboratory (NERL) in relation to multimedia/multi-pathway (aggregate) exposure studies and assessments for the purpose of determining an individual's exposure to various environmental contaminants. In this work an accelerated solvent extraction (ASE) and residue clean-up procedure, previously shown effective in determining a broad range of pesticides in composite diets, were shown effective in extracting pyrethrins and pyrethroids from fortified composite diets. Determinations performed by gas chromatography/electron capture detection (GC/?ECD) showed recoveries ranging from 111% to 130% for the 11 insecticides tested. Determinations by gas chromatography/mass spectrometry (GC/MS) showed recoveries ranging from 94 to 119% for 8 of 13 insecticides tested. Recoveries were higher for allethrin, bifenthrin, cypermethrin and the pyrethrins, but lower for resmethrin. The % relative standard deviation (%RSD) for all of the insecticides was below 15% using GC/ECD and below 10% for all except 3 insecticides using GC/MS. Further determinations of samples fortified at various levels using matrix matched calibration standards are required.

PRESENTATION Analysis of Toxicant(s) on Building Surfaces Following Chemical Attack 07/18/2004
Bernard, C E., L J. Melnyk, AND J N. Morgan. Analysis of Toxicant(s) on Building Surfaces Following Chemical Attack. Presented at 2004 Florida Pesticide Residue Workshop, Orlando, FL, July 18-21, 2004.
Abstract: There is no abstract available for this product. If further information is requested, please refer to the bibliographic citation and contact the person listed under Contact field.

PRESENTATION Evaluating Excess Dietary Exposure of Young Children Eating in Contaminated Environments 06/13/2004
Melnyk, L J., C E. Bernard, AND M R. Berry Jr. Evaluating Excess Dietary Exposure of Young Children Eating in Contaminated Environments. Presented at 5th European Pesticide Residue Workshop, Stockholm, Sweden, June 13-16, 2004.
Abstract: The United States' Food Quality Protection Act of 1996 requires more accurate assessment of children's aggregate exposures to environmental contaminants. Since children have unstructured eating behaviors, their excess exposures, caused by eating activities, becomes an important factor in determining dietary exposures of the most highly exposed children. A deterministic model was developed to more accurately estimate dietary intakes of a chemical contaminant by young children. The sum of three terms determine total intake: the original contaminant residue on food, surface-to-food contamination, and surface-to-hand-to-food contamination. Transfer of the contaminant from surfaces (including hands) to food and activity level of the child are the dominant factors for food consumed in highly-contaminated environments. Pesticide transfer from surfaces to foods has been measured to establish relationships between different pesticide classes, surface types, and contact duration. Transfer efficiencies from hardwood flooring surfaces were 30 to 57% of applied pesticide concentrations. The corresponding transfer efficiencies from carpet were much less. Transfer to foods was greater with both increased contact force and duration. Limited data are available for assessing factors associated with children's dietary activities. Videotaping allows frequencies and durations of hand and food contacts to be recorded and translated by a computerized software program. However, video analysis is labor intensive and new approaches to define children's activity patterns are being evaluated including accelerometers, handling of a standard food, and questionnaires. Research directed at developing measurement approaches to evaluate children's dietary exposures, including factors associated with additional contamination caused by surface contacts and eating activities, will be presented.

PRESENTATION Pesticide Analytical Methods to Support Duplicate-Diet Human Exposure Measurements 06/13/2004
Morgan, J N., P Kauffman, T E. Hieber, AND J. Brisbin. Pesticide Analytical Methods to Support Duplicate-Diet Human Exposure Measurements. Presented at 5th European Pesticide Residue Workshop, Stockholm, Sweden, June 13-16, 2004.
Abstract: Historically, analytical methods for determination of pesticides in foods have been developed in support of regulatory programs and are specific to food items or food groups. Most of the available methods have been developed, tested and validated for relatively few analytes and food items. Method performance for composited duplicate diet samples, as collected in USEPA residential-based exposure monitoring programs, is largely unknown. Several considerations impact development of methods to support duplicate diet human exposure measurements. The methods must generate useful dietary exposure data. Consequently, they must be sufficiently sensitive to detect pesticides in composite diet samples collected from the general population and they must be both accurate and precise. Detection limits should be comparable to those for other media collected in a total exposure monitoring program. Additionally, the methods should be as simple and cost effective as possible to minimize the costs associated with analysis of large numbers of samples.
A multi-class, multi-residue method utilizing a temperature-programmable pre-separation column in the gas chromatographic injection port for determination of a wide range of semi-volatile pesticides has been developed. Detection is performed by gas chromatography/mass spectrometry in the selected ion monitoring mode. Method detection limits are at or below 2 ng/g for most pesticides studied, with recovery between 70 and 125%. Sample preparation time and solvent consumption are reduced using this technique. Various extraction techniques have also been evaluated in conjunction with this method.
An organophosphate pesticide method has been developed which utilizes pressurized fluid extraction (PFE), diatomaceous earth and C18 clean-up and detection by gas chromatography with flame photometric detection. Detection limits for most analytes are in the low part per billion range with recoveries typically between 70 and 130%. Pulsed flame photometric and atomic emission detection have been evaluated in conjunction with this method. Additionally, PFE with in-line clean-up has been studied.
An overview of recent method development efforts, as summarized above, will be presented. Current research which focuses on determination of synthetic pyrethroids at sub-ppb levels by gas chromatography tandem mass spectrometry will also be discussed.

PRESENTATION Overview of Proposed EPA Method 330.0: Determination of Perchlorate in Drinking Water By Suppressed Conductivity Ion Chromatography and Mass Spectrometric Detection 06/08/2004
Hedrick, E J. Overview of Proposed EPA Method 330.0: Determination of Perchlorate in Drinking Water By Suppressed Conductivity Ion Chromatography and Mass Spectrometric Detection. Presented at Louisville Analytical Conference, Louisville, KY, June 8-9, 2004.
Abstract: There is no abstract available for this product. If further information is requested, please refer to the bibliographic citation and contact the person listed under Contact field.

PRESENTATION The Determination of Non-Pesticidal and Pesticidal Organotin Compounds in Water By Gas Chromatography With [pulsed] Flame Photometric Detection (Gs/Pfpd): the Effects of "MASS" Discrimination 06/02/2004
Evans, O M., P Kauffman, AND J N. Morgan. The Determination of Non-Pesticidal and Pesticidal Organotin Compounds in Water By Gas Chromatography With [pulsed] Flame Photometric Detection (Gs/Pfpd): the Effects of "MASS" Discrimination. Presented at American Chemical Society 36th Central Regional Meeting, Indianapolis, IN, June 2-4, 2004.
Abstract: Capillary gas chromatography with GC/PFPD was used in the development of analytical methodology for determining both non-pesticidal and pesticidal organotin compounds in drinking water and other aqueous matrices. The method involves aqueous ethylation of organotin analytes with sodium tetraethylborate, extraction into hexane and analysis by GC/PFPD. A PFPD is an equimolar response detector, i.e., the same quantity of tin reaching the detector should produce the same response for all organotin compounds. In general, the lower boiling and lower molecular weight non-pesticidal organotin compounds give analytical signal responses that are linearly related to the concentration of the analytes over the range studied. The higher boiling and higher molecular weight pesticidal organotin analytes, on the other hand, can give erratic peak area and peak height responses that are too small, and may result in non-linear signals, and lower slopes, as compared to their more volatile counterparts over the same concentration range. The fully alkylated, non-derivatized, quantitative internal standards, tetrabutyltin and tetrapentyltin, added to the extract in the same relative amount, may yield divergent peak area and peak height responses. An investigation of the needle handling techniques, i.e., "cold needle" injection vs. "hot needle" injection and injection speed, reveal the problem may be, in part, attributable to "needle" or "mass" discrimination between the [more volatile] lower boiling and lower molecular weight non-pesticidal organotins and some of the higher boiling and higher molecular weight pesticidal organotin compounds. Additionally, data will be presented to show: a) a minimum reaction time of 5 minutes for sodium tetraethylborate alkylation, b) derivatization yields greater than 50%, subsequent to alkylation, for most of the organotins, c) multiple internal standard calibration and calibration checks over a 21 day period, d) results of the analysis of very hard water [hardness: 325 to 350 mg/L], fortified with organotin analytes from 2.5 to10 parts-per-trillion, yielding recoveries ranging from to 71 to121% for 9 out of 10 compounds, e) method detection limits for organotins, ranging from 0.01 to 0.18 parts-per trillion, using the EPA single concentration procedure vs. the alternative Hubaux-Vos calibration based graphical approach, and f) the analytical response and correlation coefficient data for each analyte over the desired concentration range of 0.2 to 10.0 parts-per-billion, subsequent to extraction and concentration.

PRESENTATION What Rapid-Response Technologies Are Available to Determine If Our Beaches Are Safe? 06/01/2004
Haugland, R A. What Rapid-Response Technologies Are Available to Determine If Our Beaches Are Safe? Presented at EPA Science Forum 2004, Washington, DC, June 1-2, 2004.
Abstract: There is no abstract available for this product. If further information is requested, please refer to the bibliographic citation and contact the person listed under Contact field.

PRESENTATION Development of a Better Method to Identify and Measure Perchlorate in Drinking Water 06/01/2004
Hedrick, E J., D. J. Munch, AND T D. Behymer. Development of a Better Method to Identify and Measure Perchlorate in Drinking Water. Presented at EPA Science Forum 2004, Washington, DC, June 1-3, 2004.
Abstract: Perchlorate (ClO4 -) is an oxidant used primarily in solid propellant for rockets, missiles, pyrotechnics, as a component in air bag inflators, and in highway safety flares. Perchlorate tainted water has been found throughout the southwestern United States where its source has often been traced to defense industry activity or to manufactures who supply the defense industry. While there are other sources of perchlorate in drinking water, the connection of perchlorate to the defense industry has garnered increased media attention to the issue of perchlorate contamination. Perchlorate is a known thyroid hormone inhibitor; however, it was not until the mid-1990s that analytical methods were available for the detection of perchlorate in drinking water at levels of human health concern. As analytical methods have improved in sensitivity, the discovery of perchlorate in drinking waters at lower and lower concentrations has increased. Collaboration between EPA's Office of Research and Development, the Office of Water and an instrument manufacturer has produced a method that is capable of identifying concentrations of perchlorate in drinking water ten times lower than previous methods. The most distinguishing feature of this method, which is not available in existing methods, is the capability to identify perchlorate by its chlorine isotope ratio. This isotopic ratio, in addition to separation of perchlorate from interferences, virtually eliminates the likelihood of mistaken identity. Should perchlorate become regulated in drinking water, this method will enable definitive identification and quantitation of perchlorate at concentrations as low as 0.1 part per billion.

PRESENTATION Exposure of Children to Indoor Molds 06/01/2004
Frederick, R, E J. Furtaw Jr., R A. Haugland, K. Hudnell, L. Kolb, M Y. Menetrez, B. Macler, M K. Selgrade, J A. Shoemaker, J M. Van Emon, S J. Vesper, M. W. Ward, AND L J. Wymer. Exposure of Children to Indoor Molds. Presented at EPA Science Forum 2004, Washington, DC, June 1-3, 2004.
Abstract: Children now spend more than 90% of their time indoors. Thus, any exposure to indoor pollutants may be critical to their health. Molds are one of the most important pollutants children are exposed to indoors. Molds produce hundreds of allergens and toxins. These products have been shown to cause many serious health problems, including allergy and asthma. Asthma causes thousands of deaths and cost billions of dollars to treat. Mold exposures can not be completely eliminated, so it is critical to determine which molds are problematic and at what concentrations and to seek methods to reduce their occurrence.
A team of EPA researchers from NERL, NHEERL, NRMRL and NCEA have made significant progress in understanding mold exposures and their impact on children's health. So far this team has:
Patented and applied molecular technology (QPCR) to identify and quantify indoor molds.
Licensed and trained more than 15 commercial firms in US and UK in using "mold technology" for analyzing environmental samples.
Identified mold proteins that induce allergic asthma-like responses in mouse model.
Discovered that many indoor molds produce hemolytic agents
Tested chemical and physical controls for indoor molds and determined which ones work.
Applied the non-invasive visual contrast sensitivity test to show neurotoxicity from mold exposure
Based on these results, a risk assessment/ risk model for mold exposures is being developed.

PRESENTATION Transfer Efficiences of Pesticides from Household Ceramic Tile to Foods 06/01/2004
Bernard, C E. Transfer Efficiences of Pesticides from Household Ceramic Tile to Foods. Presented at EPA Science Forum 2004, Washington, DC, June 1-3, 2004.
Abstract: Traditional assessments of pesticide exposure through diet have focused on contamination during production (e.g., pesticides in agriculture). However, recent residential monitoring studies have demonstrated that a significant portion of total exposure to infants and children can result from food contamination in homes following residential pesticide usage. The US EPA's Office of Research Development (ORD) is generating exposure data and models specifically concerning these exposures for accurate assessments. These exposures, termed excess dietary exposure, occur from foods handled by children in homes were pesticides have been applied. Foods become contaminated from either direct contact with contaminated surfaces (surface-to-food) and/or through an intermediate such as hands (surface-to-hands-to-food). Excess dietary exposure estimates require empirically determined measurements of transfer efficiencies of pesticides from contaminated household surfaces to foods handled by children. Limited data exists concerning these transfers, but they have been shown to be a critical element in estimating children's excess dietary exposure. This study was conducted to determine the transfer efficiencies (%) of pesticides (organophosphate, pyrethroid, and pyrazole insecticides) from ceramic tile, a common household surface, to three different types of foods (bologna, apple, and Fruit Roll-Ups?) that are representative of items (i.e., meat, fruit, and snack foods) typically handled and eaten by children. The study also determined the moisture and fat content of each food as tools for predicting transfer efficiencies.
The highest transfer efficiencies were observed for the organophosphate pesticides to bologna (56-79%). Lower transfer efficiencies were observed for apples (20-37%) and Fruit Roll-Ups (6-17%). Transfer efficiencies of pyrethroid pesticides to bologna were 33-52%. The corresponding transfer efficiencies to apples and Fruit Roll-Ups were 12-34% and 3%, respectively. Fipronil (pyrazole insecticide) had a similar transfer to that of the pyrethroids (bologna 39-45%, apple 13-41%, and Fruit Roll-Up 4%). The average percent fat content for bologna, apples, and Fruit Roll-Ups were 30%, 1%, and 7%, respectively. The corresponding average percent moisture content were 54%, 85%, and 11%, respectively. Although neither percent fat nor moisture content directly project the ranking of transfer between the foods, these two factors combined may provide a better prediction of transfer. Ultimately, these results suggest the extent of transfer is affected by the food type and chemical properties of the pesticide itself. Findings from this study in conjunction with models under development will be used to generate more accurate estimates of excess dietary exposure to infants and children in homes where pesticides are used.

PRESENTATION Re-Entering Building Following Chemical Attack: Measuring the Effectiveness of Surface Decontamination 06/01/2004
Morgan, J N. Re-Entering Building Following Chemical Attack: Measuring the Effectiveness of Surface Decontamination. Presented at EPA Science Forum 2004, Washington, DC, June 1-3, 2004.
Abstract: Prior to re-entering a building following a chemical attack, decontamination and testing must be conducted to determine whether toxic agents have been eliminated or reduced to safe levels. Building contents must also be decontaminated and tested or destroyed. Recent incidents in the news, including the ricin scare in a Senate office building and an incident in Belgium involving hydrazine and phenarsazine, serve to remind us that chemical attacks are a very real possibility. The Belgium incident also serves to emphasize the fact that readily available common industrial chemicals may be used in an attack, not just traditional chemical weapons. In order to ensure safe re-entry into a building following such an attack, methods are needed for measuring the levels of a variety of industrial chemicals on building surfaces following the decontamination process. The objective is to develop rapid and sensitive procedures for determination of widely used and commercially available pesticides on representative hard surfaces commonly found in building structures and contents.
Readily available pesticides, including carbaryl, bendiocarb, propoxur, imidacloprid and diphacinone, were applied to hard surfaces using a customized spray chamber. Hard surfaces wre chosen to be representative of building contents and structural materials and included formica, plastic, glass and galvanized steel. Surface sampling consisted of wiping with organic solvent moistened gauze pads. The gauze pads were extracted using pressurized fluid extraction. The extract was analyzed by liquid chromatography tandem mass spectrometry.

Products developed from this project will include protocols for conducting surface wipes to collect toxicant(s) from contaminated building surfaces, as well as the detection and quantitation of compounds captured in them. Since the selected toxic compounds in this study will encompass a wide range of physical and chemical properties, the techniques could be applied to a wide variety of industrial chemicals and other chemical agents. These protocols and analytical procedures will be available for use by EPA and its partners in an emergency response to chemical terrorism and will provide tools for ensuring that buildings and contents are safe for re-entry and re-use following chemical attack; thereby, eliminating the tremendous financial burden of acquiring new materials for use by building occupants.

PRESENTATION It's in the Chips: Development of a Microarray Genechip Approach to Dete T and Type Waterborne Viruses 06/01/2004
Brinkman, N. It's in the Chips: Development of a Microarray Genechip Approach to Dete T and Type Waterborne Viruses. Presented at EPA Science Forum 2004, Washington, DC, June 1-3, 2004.
Abstract: Human caliciviruses, specifically members of the genus Norovirus, have been documented as a culprit for drinking water-related outbreaks of acute gastroenteritis in the United States. In addition, these viruses are believed to be one of the major causes of waterborne disease. Due to the potential risk these viruses pose to the public, they have been placed on the EPA's Contaminant Candidate List (CCL). The CCL was initiated in response to the 1996 Amendment to the Safe Drinking Water Act to aid in setting priorities of unregulated microbiological and chemical contaminants in the Agency's drinking water program. The CCL is published in the Federal Register (63 FR 10274).
Despite the fact that Noroviruses are highly infectious to people of all ages, qualitative risk assessments have been hindered because these viruses cannot be cultured by traditional methods, nor can they be cultivated through an animal model. Methods for identifying Noroviruses currently rely on molecular approaches, such as Reverse-Transcriptase-Polymerase Chain Reaction (RT-PCR) followed by product confirmation via hybridization with genogroup probes. Although this approach works, it is not conducive to specific Norovirus subtype identification in a timely fashion. Subtyping is important since it will allow waterborne isolates to be compared to clinical specimens in outbreak situations for identification of the source of contamination. In addition, it has been proposed that Norovirus subtypes may differ in their virulence properties. If this is the case, subtype identification will aid in accurately assessing the risk posed by these viruses. Subtyping can rapidly be achieved using a microarray platform. Affymetrix's GenFlex GeneChip allows the flexibility of designing a microarray to type Norovirus isolates in a high throughput and time-effective manner. While this technique is in the evaluation phase, the data shows that it is likely to be a valuable tool for typing viruses.

PRESENTATION Development of Aptamers to Waterborne Parasites 06/01/2004
Nichols, T L. Development of Aptamers to Waterborne Parasites. Presented at EPA Science Forum 2004, Washington, DC, June 1-3, 2004.
Abstract: The Safe Drinking Water Act Amendment of 1996 mandates that the U. S. Environmental Protection Agency (EPA) evaluate public health risks associated with drinking water contaminants to include waterborne parasites, such as Cryptosporidium and Giardia. Additionally, the Agency establishes a list of unregulated microbiological contaminants that have potential pathogenic transmission via water. This list is referred to as the Candidate Contaminant List (CCL). Although free-living stages of protozoans are generally larger microorganisms that are susceptible to filtration and sedimentation, many protozoans have spore or cyst stages that are not susceptible to filtration and/or disinfection. Therefore, the detection of pathogenic parasites is essential for maintaining safe drinking water. Detection of waterborne parasites is primarily based on antibody-antigen reaction assays which vary in sensitivity and specificity. Also, antigenic cross-reactivity between pathogenic and non-pathogenic species represents a problem when exclusively using these assays to monitor safe drinking water. Our goal is to provide an improved detection system based on a new technology termed "Systematic Evolution of Ligands by Exponential enrichment (SELEX). The final products of SELEX are short single-stranded oligonucleotides termed "aptamers," which form secondary structures that exhibit high affinity to targeted proteins and/or organisms. In this study, the SELEX process is being employed in the development of an aptamer to the EPA-regulated pathogenic parasite, Giardia lamblia. Briefly, the development of a specific aptamer begins with a large combinatorial library of randomized sequences that is mixed with G. lamblia. Aptamers that have bound Giardia cysts are selected and amplified via the polymerase chain reaction (PCR) for further enrichment cycles. Candidate aptamers will be cloned and sequenced. These sequences will be evaluated for similar motifs and structures for potential assay development utilizing fluorochromes and/or immuno-magnetic detection systems. Aptamers offer advantages over traditional antibody (protein) -based systems in cost and flexibility of assay platforms as well as in sensitivity and specificity. Once developed, this technique will allow the EPA to develop more sensitive and specific methods to detect pathogenic parasites in environmental waters.

PRESENTATION Arsenic Mobility from Iron Oxide Solids Produced During Water Treatment 05/24/2004
Whiting, J., P A. Gallagher, C A. Schwegel, AND J T. Creed. Arsenic Mobility from Iron Oxide Solids Produced During Water Treatment. Presented at Remediation of Chlorinated and Recalcitrant Compounds: The Fourth International Conference, Monterey, CA, May 24-27, 2004.
Abstract: The Arsenic Rule under the Safe Drinking Water Act will require certain drinking water suppliers to add to or modify their existing treatment in order to comply with the new 10 ppb arsenic standard. One of the treatment options is co-precipitation of arsenic with iron. This treatment is attractive because arsenic and iron are geologically correlated so that well waters containing arsenic have a propensity to contain iron. Specifically, iron can be precipitated from water as iron oxide (AFeAsOOH@) via aeration and in the process co-precipitate some of the arsenic leading to arsenic removal. In addition, adsorptive media can be used to remove arsenic from the drinking water supplies. In either case arsenic containing solids are generated and these solids can be discharged to the local wastewater treatment plant, deposited in landfills, or discharged to the local waterway.
The distribution of As(III) and As(V) on these solids may aid in estimating the mobility of the arsenic when these solids are exposed to different environmental factors. The research presented will attempt to estimate the distribution of As(III) and As(V) on these treatment solids using an acetic acid / EDTA extraction solvent. Desorption experiments will be conducted to evaluate the species specific mobility of the leachable arsenic. The ultimate goal of the research is to produce an improved understanding of the environmental factors which may affect the mobility of the arsenic.

PRESENTATION Identification of Microcystin Toxins from a Strain of Mycrocystis Aeruoginosa 05/23/2004
Diehnelt, C., N R. Dugan, AND W L. Budde. Identification of Microcystin Toxins from a Strain of Mycrocystis Aeruoginosa. Presented at 52nd American Society for Mass Spectrometry Conference, Nashville, TN, May 23-27, 2004.
Abstract: There is no abstract available for this product. If further information is requested, please refer to the bibliographic citation and contact the person listed under Contact field.

PRESENTATION Development of Multiplex Rt-Pcr for the Detection of Reovirus, Hepatitis a Virus, Poliovirus, Norwalk Virus and Rotavirus 05/23/2004
Parshionikar, S., J L. Cashdollar, AND G S. Fout. Development of Multiplex Rt-Pcr for the Detection of Reovirus, Hepatitis a Virus, Poliovirus, Norwalk Virus and Rotavirus. Presented at Annual Meeting of the American Society for Microbiology, New Orleans, LA, May 23-27, 2004.
Abstract: Water sources are often found to be contaminated by enteric viruses. This is a public health concern as food and waterborne outbreaks caused by enteric viruses such as noroviruses, rotaviruses, hepatitis A virus (HAV) and enteroviruses are a common occurrence. All of these viruses are transmitted by the oral-fecal route. Cell culture detection of these viruses is time consuming and not possible for viruses that cannot be cultured (for example, noroviruses). Reverse transcription-polymerase chain reaction (RT-PCR) assays are commonly used for detection of human enteric viruses in environmental water samples. RT-PCR provides a means to detect low levels of these viruses rapidly. Multiplex RT-PCR is a modification of RT-PCR by which several viruses can be amplified simultaneously in a single reaction using multiple primers pairs. We report the development of multiplex RT-PCR assays to detect reovirus, rotavirus, HAV, poliovirus and Norwalk virus. Primer pairs for all the viruses were designed such that most of the existing strains of each virus could be detected. The method was optimized for Mg+2 concentration, relative primer concentration, template concentration and annealing time and temperature. Mg+2 and relative primer concentration seemed to be the most crucial factors in the success of the assay. The presence of the viruses was confirmed by dot blot hybridizations. The method was shown to be as sensitive as the monoplex RT-PCR for each virus. The method when applied to seeded Ohio River water samples yielded positive results for three viruses. Multiplex RT-PCR format which, when used for detection of viruses in environmental water sources, is a useful tool as it reduces the number of assays needed for each sample thereby facilitating virus detection in a rapid and economical manner.

PRESENTATION Evaluation of Spiking Procedures for Recovery of Cryptosporidium in Stream Waters Using USEPA Method 1623 05/23/2004
Simmons, O. D., D. S. Francy, E. J. Granger, M W. Ware, M. D. Sobsey, AND F W. Schaefer III. Evaluation of Spiking Procedures for Recovery of Cryptosporidium in Stream Waters Using USEPA Method 1623. Presented at 104th General Meeting of the American Society for Microbiology, New Orleans, LA, May 23-27, 2004.
Abstract: U.S. Environmental Protection Agency Method 1623 is widely used to monitor source waters and drinking water supplies for Cryptosporidium oocysts. Analyzing matrix spikes is an important component of Method 1623. Matrix spikes are used to determine the effect of the environmental matrix on the method's recovery efficiency for the target organism and require the collection and analysis of two environmental samples. Using a new product, ColorSeed, spiked organisms can be differentiated from unmodified organisms and thus recovery efficiencies can be determined by analyzing only one environmental sample. Twenty-nine stream-water samples and one effluent sample were collected to compare ColorSeed to traditional spiking procedures. Adjusted recoveries of oocysts ranged from 2.8 to 49 percent for viable oocysts (traditional spiking procedure) and 3.1 to 59 percent for ColorSeed. The recoveries between the two spiking procedures were highly correlated (r = 0.802) and were not found to be significantly different. Recoveries using ColorSeed, therefore, were comparable to recoveries using traditional spiking procedures. Collecting and processing these samples afforded the opportunity to address other issues regarding the use of Method 1623 for monitoring environmental waters. In examining the effects of water-qualiy variables on method recovery efficiency of oocysts, significant negative correlations were found between average oocyst recovery and turbidity or suspended sediment; this was especially apparent in samples with turbidities greater than 100 NTU and suspended-sediment concentrations greater than 100 mg/L. Because oocysts were found in a small percentage of samples (16.7 percent), the presence of Cryptosporidium was qualitatively compared to concentrations of microbiological indicators and to concentrations of chemical constituents known to be associated with fecal contamination. Concentrations of E. coli, F-specific coliphage, and total phosphorus were above the median non-detection concentrations in all samples with detections of Cryptosporidium, but were not above the median for Cl. perfringens, somatic coliphage, chloride, and nitrate.

PRESENTATION Analysis of Aeromonas By Mass Spectrometry: Speciation and Virulence Factors 05/22/2004
Donohue, M J., J A. Shoemaker, A W. Smallwood, AND M R. Rodgers. Analysis of Aeromonas By Mass Spectrometry: Speciation and Virulence Factors. Presented at 52nd ASMS Conference, Nashville, TN, May 22-27, 2004.
Abstract: Introduction:
A number of bacteria, including Aeromonas hydrophila, are listed on the Environmental Protection Agency's 1998 Contaminant Candidate List (CCL) as research needs. One research priority designated by the CCL is the identification of virulence activity factor relationships (VFARs). VFARs may provide a more rapid means of identifying waterborne pathogens than the current process which relies on exposure and health effects as the two primary categories for screening potential microbial drinking water contaminants. The first step in the research project is to obtain the MALDI-MS spectra of the available virulent and avirulent species/strains of Aeromonas, and identify the masses which are unique to virulent Aeromonas species and strains.

Methods and Instrumentation:

Chosen ATCC strains and field samples of the microorganism were plated onto 5% sheep blood agar plate and incubated for 16 hr at 35 C. The cells were subsequently harvested, and the optical density of the solutions were then adjusted to an O.D.600 of 0.5. The harvested cells were then washed three times with water.
A 1 L aliquot of the bacterial sample was applied to the target and allowed to air dry before an 1.5 L aliquot of sinapinic acid matrix was overlaid on top. All spectra were generated on a Bruker BiFlex III MALDI-MS, operated in positive linear mode. The spectra for all samples was obtained by summing four scans of a 100 shots.

Preliminary Data:

The long range goal of this project is to identify novel virulence factors for Aeromonas using ESI-MS and MALDI-MS. In the process, the mass spectral fingerprints obtained can be used to determine if rapid species/strain differentiation is possible using MALDI-MS. Nine species of Aeromonas have been analyzed by MALDI-MS. The procedure provides a unique mass spectral fingerprint for each of the chosen isolates. The spectra obtained allow identification of the species differentiation in the genus Aeromonas. Unique masses were observed in each of the Aeromonas isolates providing a mean to differentiate between Aeromonas species by MALDI-MS. To test the method, a blind study was done using field samples. The MALDI analysis of the field samples was able to correctly identify all the Aeromonas species/isolates previously identified by PCR analysis. Further research is being conducted on identifying the proteins unique to virulent Aeromonas using these MALDI-MS mass spectral fingerprints.

PRESENTATION Overview of a New EPA Method: Determination of Perchlorate in Drinking Water, Groundwater and High Salinity Water By Ion Chromatography, Suppressed Conductivity With Electrospray Ionization Mass Spectrometric Detection 05/03/2004
Hedrick, E J. Overview of a New EPA Method: Determination of Perchlorate in Drinking Water, Groundwater and High Salinity Water By Ion Chromatography, Suppressed Conductivity With Electrospray Ionization Mass Spectrometric Detection. Presented at Texas Commission on Environmental Quality, Environmental Trade Fair and Conference, Austin, TX, May 3-5, 2004.
Abstract: In this presentation the analytical instrumentation and procedures necessary to qualitatively and quantitatively determine low levels of perchlorate (ClO4-) in drinking waters using ion chromatography with electrolytic conductivity suppression, electrospray ionization mass spectrometric detection (IC-ESI-MS) will be described. Using an anion exchange separator column, ClO4- is separated from constituent cations and anions in natural water samples using a potassium hydroxide mobile phase. During the time frame that ClO4- elutes from the column, the eluate is passed through an electrolytic conductivity suppressor to remove the K+ ions of the mobile phase and the analyte counter ions prior to the eluate entering the mass spectrometer. The masses of interest for ClO4- are 99 and 101, based on the 75.53% relative abundance of the chlorine-35 isotope and 24.47% abundance of the chlorine-37 isotope, respectively. Accurate quantitation is made possible by using an oxygen-18 enriched ClO4- internal standard which has a mass-to-charge ratio of 107. Qualitative identification of ClO4- is made by retention time and mass ratio confirmation (99:101) within predetermined quality control limits.
U.S. EPA type method detection limits are between 0.02 - 0.06 ppb whether quantifying on the mass 99 or mass 101 ion. The lowest concentration minimum reporting limit (LCMRL) using the mass 99 ion is 0.15 ppb. The LCMRL is defined as the lowest concentration for which the 99% prediction interval for results is within a predefined acceptance limit of 50-150% recovery. The method is applicable to waters containing up to 3,000 ppm total dissolved solids without sample dilution or sample pretreatment to remove the common anions chloride, sulfate or carbonate (Cl-, SO42-, CO32-). Of the common anions, Cl-, SO42-, CO32-, sulfate is the most problematic due to an isotope of sulfur contributing to the background at mass 99 (HSO4-). For samples containing more than 1,000 ppm sulfate, pretreatment to remove the sulfate is recommended to maintain optimal method performance. Analysis of perchlorate concentrations less than1.0 ppb is possible in high salinity samples (>30,000 ppm TDS) by sample dilution and in some cases sample pretreatment to reduce sulfate levels.

PRESENTATION Identification of Allergens from Metarhizium Anisopliae Using Mass Spectrometry 04/19/2004
Shoemaker, J A. AND M J. Donohue. Identification of Allergens from Metarhizium Anisopliae Using Mass Spectrometry. Presented at 5th International Symposium on the Interface between Analytical Chemistry and Microbiology, Richland, WA, April 19-21, 2004.
Abstract: Background The U.S. EPA, under the "Children at Risk" Program, is currently addressing the problem of indoor fungal bioaerosol contamination. The fungus Metarhizium Anisopliae has been used as a bio-pesticide for insect control since the 1800's. Recent studies have shown that exposure to this micoroorganism can cause an immediate hyper-immunosensitivity or Type I allergenic response. Currently, the proteins which cause this allergenic response to M. Anisopliae are unknown. The goal of this project is the identification and characterization of allergens from M. Anisopliae using mass spectrometry (MS). Matrix assisted laser desorption ionization MS (MALDI-MS) and electrospray tandem MS (ESI-MS/MS) were used to analyze the fungal peptides and proteins isolated by 2D gel electrophoresis and immuno-blot analysis.

Methods
Total protein extracts of M. Anisopliae were separated using 2-D gel electrophoresis. Allergenic proteins were identified by immuno-blot analysis, using mouse hyperimmune serum against M. anisopliae (primary antibody) and anti-mouse immunoglobulin E (secondary antibody) as the detection system. Gel spots that reacted with the IgE were excised and in-gel digested. The peptides were extracted and analyzed by MALDI-MS and ESI-MS/MS. The MS data on molecular weight, peptide profiles and amino acid sequences were used to mine databases for potential sequence or domain homology to known allergens. Peptide sequences were analyzed using the MASCOT� data mining algorithm.

Results
Eight proteins were identified as allergenic proteins based on IgE reactivity in the immuno-blot assay. MALDI-MS peptide fingerprints have been obtained on all eight gel spots. Using these mass fingerprints to mine non-redundant databases yielded no significant matches. Five of the allergenic gel spots were partially sequenced by ESI-MS/MS and tentatively identified as catalases. The identification was confirmed on four of the five spots using a polyclonal catalase antibody. Catalase serves to protect the cell from the toxic effects of hydrogen peroxide by catalyzing its decomposition into molecular oxygen and water.

Conclusions
Eight proteins were identified in M. Anisopliae as allergenic by gel electrophoresis and immuno-blot analysis. Five of the allergic proteins in M. Anisopliae have been partially sequenced by MS and identified as catalases. The next step will be to study other fungi or molds to determine potential similarities in the allergenic proteins identified. The identification of fungal proteins that induce IgE responses can eventually aid in the prevention or treatment of allergies.

PRESENTATION The United States EPA Concept for Deriving Water Quality Guidelines for Recreational Waters 03/28/2004
Dufour, A P. The United States EPA Concept for Deriving Water Quality Guidelines for Recreational Waters. Presented at Workshop: "Monitoring the Quaity of Recreational Waters: Do we need a change of paragdigms?", Tuebingen, Germany, March 28-31, 2004.
Abstract: The guidelines developed by the US EPA for controlling the quality of recreational waters are based on protecting the health of swimmers and other recreationists who may be exposed to waters contaminated by human and animal excreta. Risks to swimmers were determined through a series of epidemiological studies at marine and freshwater beaches where the relationships between water quality and symptomatic swimming-associated illness were defined. The data, which showed that there was an association between gastrointestinal illness and swimming, and also that as the density of fecal indicator microbes in the water increased, so did the frequency of swimming-associated illness, were used to set a tolerable limit to the level of illness in swimmers. The limit was related to the geometric mean of indicator bacteria in at least five water samples per month and also a single sample value. These water quality limiting values are recommended to the states in the US, who have the authority to set guideline values into state standards.

PRESENTATION Comparison of Enteroccocus Densities Determined By Culture and Qpcr Analyses in Water Samples from Two Recreation Beaches 03/17/2004
Haugland, R A., S D. Siefring, L J. Wymer, K Brenner, AND A P. Dufour. Comparison of Enteroccocus Densities Determined By Culture and Qpcr Analyses in Water Samples from Two Recreation Beaches. Presented at New England Association of Environmental Biologists (NEAEB), Hancock, MA, March 17-19, 2004.
Abstract: Previous studies conducted by the U.S. Environmental Protection Agency (USEPA) have demonstrated that cell densities of the bacterial genus Enterococcus in water samples are directly correlated with gastroenteritis illness rates in swimmers at both marine and fresh water beaches. Based on these data, guidance has been proposed that unacceptable health risks may be associated with swimming in waters containing concentrations of these organisms in excess of per 104 colony forming units (CFU)/100 ml, as determined from one-time measurements. An improved, selective culture method (mEI agar membrane filter method) has been developed by the U.S. EPA for measuring Enterococcus concentrations in recreational water samples, however, this method still requires at least 24 hours to obtain results and, hence, may not allow timely notification of potential hazards to bathers. The U.S. EPA has initiated a new recreational water health study to evaluate the correlation between illness rates in swimmers and fecal indicator concentrations, determined by the mEI agar membrane filter method and several new technologies providing near real-time results. One of the new technologies being evaluated is quantitative polymerase chain reaction (QPCR) analysis.
Water samples from two Great Lakes freshwater beaches were collected three times daily on weekends during the summer of 2003. Samples were collected at beach 1 (West Beach, Indiana Dunes National Lakeshore) over a 10 week period and at beach 2 (Huntington Beach, Bay Village, Ohio) over an 8 week period. Totals of 336 and 420 samples were analyzed at beach 1 and beach 2, respectively. Preliminary analyses of the data have shown that Enterococcus concentrations at beach 1 ranged from <1 to 3.7 x 103 CFU/100 ml, as determined by the mEI agar filter method, and from non-detectable to 1.7 x 104 cells/100 ml, as determined by the QPCR method. Enterococcus concentrations at beach 2 ranged from <1 to 4.8 x 104 CFU/100 ml by the filter method and from non-detectable to 1.3 x 105 cells/100 ml by the QPCR method. The extrapolated detection limit of the QPCR method averaged 1 cell/100 ml water sample using 45 amplification cycles. Overall correlation factors, determined by regression analysis, for the log-transformed results of the two methods were 0.41 at beach 1 and 0.59 at beach 2. Of all the individual samples from both beaches, 17% contained Enterococcus concentrations > 104 CFU/100 ml, and of these, 95% also contained > 104 cells/100 ml determined by QPCR analysis. Determinations of correlations between illness rates in swimmers and Enterococcus measurements by the two methods are ongoing.

PRESENTATION Analytical Organic Mass Spectrometry 03/09/2004
Budde, W L. Analytical Organic Mass Spectrometry. Presented at Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Chicago, IL, March 9, 2004.
Abstract: There is no abstract available for this product. If further information is requested, please refer to the bibliographic citation and contact the person listed under Contact field.

PRESENTATION Ord/NERL Current Vrars Research 03/02/2004
Stelma Jr., G N. AND S J. Vesper. Ord/NERL Current Vrars Research. Presented at Drinking Water Progress Review, Washington, DC, March 2, 2004.
Abstract: Virulence is the degree of pathogenicity of a microorganism and virulence factors are the components of an organism that contribute to virulence. Identifying microorganisms using known virulence factors is one method used by microbiologists to distinguish pathogenic isolates from non-pathogens. This does not always work because an a sequential series of these factors is often needed for an organism to be pathogenic and to date most of these factors are still unknown. Virulence factor activity relationships (VFARs) refers to the hypothesized structure-activity relationships among virulence factors possessed by different pathogens. By examining VFARs, we could potentially identify pathogenic strains within a species and possibly currently unrecognized pathogens. There are some limitations to this approach in the immediate future because so many virulence factors are still unknown and we do not know the extent to which the biological world parallels the chemical world with respect to structure-activity relationships. We do know that some virulence factors are genetically similar in species that are not closely related; whereas, others appear to be unique to one genus or species. NERL researchers have developed an approach that could potentially circumvent the need to know all of the virulence factors possessed by a particular genus or species. This approach involves measuring the responses of host cells to known pathogenic strains and compare them to the response of non-pathogens. This is being accomplished by infecting cell cultures and quantitatively measuring the specific messenger RNAs that are produced in each case, using microarrays that include the entire genome of the host species. Although pathogens with different types of virulence mechanisms, may elicit somewhat different messenger RNA signals, it is hoped that these signals will at least allow us to identify broad classes of pathogens. The end result will be a database of RNA response profiles from known pathogenic strains that can be compared to similarly derived profiles from uncharacterized strains

PRESENTATION Nontuberculous Mycobacyeria Spp Isolated from Residents of King County, Washington, 1999-2002 02/29/2004
Hilborn, E D., T C. Covert, M. Yakrus, G N. Stelma Jr., AND M Schmitt. Nontuberculous Mycobacyeria Spp Isolated from Residents of King County, Washington, 1999-2002. Presented at International Conference on Emerging and Infectious Diseases, Atlanta, GA, February 29-March 3, 2004.
Abstract: Background: Pathogenic nontuberculous Mycobacteria spp. (NTM) are not known to be transmitted among persons, but may be acquired from exposure to contaminated media such as soil, food and water. We examined the spectrum of NTM isolated from human specimens in King County, WA.Methods: NTM were isolated from clinical specimens collected during 1999 - 2002 from patients residing in King Co., Washington. Participating laboratories collected isolates, the date and anatomical site of specimen collection, and the home zip code of the patient. NTM species were identified using multiple laboratory methods (molecular probe, biochemical characterization, high performance liquid chromatography). Data were analyzed with SAS 8.2. Isolates were described by species and anatomic site. Analyses of potential differences among specimen dates of collection were performed.
Results: A total of 507 NTM isolates were obtained from specimens collected during 1999 - 2002. NTM isolate numbers ranged from: 113 isolated from specimens collected during 2000, to 152 isolated from specimens collected during 1999. Complex or species level identification occurred in 94% (476/507) of isolates. Slow-growers comprised 83% (396/476) of isolates. Mycobacterium avium complex (MAC) comprised 65% (256/396) of the slow-growers. MAC were isolated from 203 respiratory sites (80%), 37 sterile sites (14%), 1 stool (0.4%) and 15 other sites (6%). The 80 rapid growers were isolated from 56 respiratory sites (70%), 9 sterile sites (11%), and 14 other sites (18%); 1 site was not recorded. Rapid growers were not all identified to the species level. No differences were seen in the numbers of isolates when compared among month or season of specimen collection.
Conclusions: Slow-growing species were identified in the majority of isolates. MAC was the most common NTM isolated from clinical specimens and from sterile sites. Respiratory specimens yielded the most NTM isolates, including those species not typically associated with infection. We found no evidence of seasonal differences among collected specimens.



PRESENTATION US EPA Drinking Water Certification Program 01/19/2004
Rodgers, M R. US EPA Drinking Water Certification Program. Presented at Seminar in Delhi, Bangalore and Hyderabad, India, Delhi, Bangalore and Hyderabad, India, January 19-February 4, 2004.
Abstract: There is no abstract available for this product. If further information is requested, please refer to the bibliographic citation and contact the person listed under Contact field.

 

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