The relationship between childhood atopic dermatitis and asthma in an under resourced community.

Background: Atopic dermatitis (AD) is an inflammatory skin disease caused by allergen exposures and estimated to affect ∼20% of children. Children in urban areas have a higher prevalence of AD compared with those living outside of urban areas. AD is believed to lead to asthma development as part of the "atopic march." Objective: Our objective was to determine the sequential and chronological relationships between AD and asthma for children in an under-resourced community. Methods: The progression from AD to asthma in the under-resourced, urban community of Sun Valley, Colorado, was examined by assessing Medicaid data for the years 2016 to 2019 for a diagnosis of AD or asthma in children 6 and 7 years old. Results: Pearson correlations between AD and asthma diagnoses were significant only with respect to AD at age 6 years compared with asthma 1 year later, at age 7 years. Conclusion: By studying a susceptible community with a consistent but mixed genetic background, we found sequential and chronological links between AD and asthma.

Comparative stability of assay results of enterococci measured by culture and qPCR over time in bathing beach waters.

The diurnal presence of the culturable bacterial indicators of fecal contamination in the water environment has been shown to be highly variable over time due to natural die-off and injury from effects of sunlight and other environmental stressors. Molecular analytes of a quantitative polymerase chain reaction (qPCR) method for measuring fecal contamination degrade considerably slower than the alternative of culturable fecal indicator bacteria. The rapid qPCR method holds the promise of more timely notification decisions with respect to postings or closure being made on the basis of microbial water quality samples collected earlier on the same day. In the case of culture-based methods requiring a 24 h or longer incubation period, decisions must be based on samples collected no sooner than the previous day. To examine the effect of this lag in assay results, temporal stability of a molecular Enterococci target analyte with that of traditional culture-based cells is compared using data from USEPA studies conducted between 2003 and 2007 on seven freshwater and marine beaches that were impacted by publicly-owned treatment works. Generally, levels of the molecular indicator were more consistent throughout the day between 8:00 am and 3:00 pm. The difference in temporal consistency is even more pronounced when the 24-h lag in culture-based results is taken into account.

Integrated preservation and sample clean up procedures for studying water ingestion by recreational swimmers via urinary biomarker determination.

The use of cyanuric acid as a biomarker for ingestion of swimming pool water may lead to quantitative knowledge of the volume of water ingested during swimming, contributing to a better understanding of disease resulting from ingestion of environmental contaminants. When swimming pool water containing chlorinated cyanurates is inadvertently ingested, cyanuric acid is excreted quantitatively within 24 h as a urinary biomarker of ingestion. Because the volume of water ingested can be quantitatively estimated by calculation from the concentration of cyanuric acid in 24 h urine samples, a procedure for preservation, cleanup, and analysis of cyanuric acid was developed to meet the logistical demands of large scale studies. From a practical stand point, urine collected from swimmers cannot be analyzed immediately, given requirements of sample collection, shipping, handling, etc. Thus, to maintain quality control to allow confidence in the results, it is necessary to preserve the samples in a manner that ensures as quantitative analysis as possible. The preservation and clean-up of cyanuric acid in urine is complicated because typical approaches often are incompatible with the keto-enol tautomerization of cyanuric acid, interfering with cyanuric acid sample preparation, chromatography, and detection. Therefore, this paper presents a novel integration of sample preservation, clean-up, chromatography, and detection to determine cyanuric acid in 24 h urine samples. Fortification of urine with cyanuric acid (0.3-3.0 mg/L) demonstrated accuracy (86-93% recovery) and high reproducibility (RSD < 7%). Holding time studies in unpreserved urine suggested sufficient cyanuric acid stability for sample collection procedures, while longer holding times suggested instability of the unpreserved urine. Preserved urine exhibited a loss of around 0.5% after 22 days at refrigerated storage conditions of 4 °C.

Immunoprevalence to Six Waterborne Pathogens in Beachgoers at Boquerón Beach, Puerto Rico: Application of a Microsphere-Based Salivary Antibody Multiplex Immunoassay.

Waterborne infectious diseases are a major public health concern worldwide. Few methods have been established that are capable of measuring human exposure to multiple waterborne pathogens simultaneously using non-invasive samples such as saliva. Most current methods measure exposure to only one pathogen at a time, require large volumes of individual samples collected using invasive procedures, and are very labor intensive. In this article, we applied a multiplex bead-based immunoassay capable of measuring IgG antibody responses to six waterborne pathogens simultaneously in human saliva to estimate immunoprevalence in beachgoers at Boquerón Beach, Puerto Rico. Further, we present approaches for determining cutoff points to assess immunoprevalence to the pathogens in the assay. For the six pathogens studied, our results show that IgG antibodies against antigens from noroviruses GI.I and GII.4 were more prevalent (60 and 51.6%, respectively) than Helicobacter pylori (21.4%), hepatitis A virus (20.2%), Campylobacter jejuni (8.7%), and Toxoplasma gondii (8%) in the saliva of the study participants. The salivary antibody multiplex immunoassay can be used to examine immunoprevalence of specific pathogens in human populations.

Statistical approaches to developing a multiplex immunoassay for determining human exposure to environmental pathogens.

There are numerous pathogens that can be transmitted through water. Identifying and understanding the routes and magnitude of exposure or infection to these microbial contaminants are critical to assessing and mitigating risk. Conventional approaches of studying immunological responses to exposure or infection such as Enzyme-Linked Immunosorbent Assays (ELISAs) and other monoplex antibody-based immunoassays can be very costly, laborious, and consume large quantities of patient sample. A major limitation of these approaches is that they can only be used to measure one analyte at a time. Multiplex immunoassays provide the ability to study multiple pathogens simultaneously in microliter volumes of samples. However, there are several challenges that must be addressed when developing these multiplex immunoassays such as selection of specific antigens and antibodies, cross-reactivity, calibration, protein-reagent interferences, and the need for rigorous optimization of protein concentrations. In this study, a Design of Experiments (DOE) approach was used to optimize reagent concentrations for coupling selected antigens to Luminex™ xMAP microspheres for use in an indirect capture, multiplex immunoassay to detect human exposure or infection from pathogens that are potentially transmitted through water. Results from Helicobacter pylori, Campylobacter jejuni, Escherichia coli O157:H7, and Salmonella typhimurium singleplexes were used to determine the mean concentrations that would be applied to the multiplex assay. Cut-offs to differentiate between exposed and non-exposed individuals were determined using finite mixed modeling (FMM). The statistical approaches developed facilitated the detection of Immunoglobulin G (IgG) antibodies to H. pylori, C. jejuni, Toxoplasma gondii, hepatitis A virus, rotavirus and noroviruses (VA387 and Norwalk strains) in fifty-four diagnostically characterized plasma samples. Of the characterized samples, the detection rate was 87.5% for H. pylori, and 100% for T. gondii assays and 89% for HAV. Further, the optimized multiplex assay revealed exposure/infection to several other environmental pathogens previously uncharacterized in the samples.

A new in vitro model using small intestinal epithelial cells to enhance infection of Cryptosporidium parvum.

To better understand and study the infection of the protozoan parasite Cryptosporidium parvum, a more sensitive in vitro assay is required. In vivo, this parasite infects the epithelial cells of the microvilli layer in the small intestine. While cell infection models using colon, kidney, and stomach cells have been studied to understand the infectivity potential of the oocysts, an ideal in vitro model would be readily-available, human-derived, and originating from the small intestine. In this study, we developed a reproducible, quantitative infection model using a non-carcinoma, human small intestinal epithelial cell type, named FHs 74 Int. Our results show that FHs 74 Int cells are productively infected by viable oocysts, and exhibit higher levels of infection susceptibility compared to other cell types. Moreover, infection rate of the sporozoites on the monolayer was found to be comparable or better than other cell types. We furthermore demonstrate that infection can be improved by 65% when pre-treated oocysts are directly inoculated on cells, compared to inoculation of excysted sporozoites on cells. Identification of a better infection model, which captures the preferred site of infection in humans, will facilitate studies on the host pathogenesis mechanisms of this important parasitic human pathogen.

Equivalency of risk for a modified health endpoint: a case from recreational water epidemiology studies.

BACKGROUND: The United States Environmental Protection Agency (USEPA) and its predecessors have conducted three distinct series of epidemiological studies beginning in 1948 on the relationship between bathing water quality and swimmers' illnesses. Keeping pace with advances in microbial technologies, these studies differed in their respective microbial indicators of water quality. Another difference, however, has been their specific health endpoints. The latest round of studies, the National Epidemiological Assessment of Recreational (NEEAR) Water studies initiated in 2002, used a case definition, termed "NEEAR GI illness" (NGI), for gastrointestinal illness corresponding closely to classifications employed by contemporary researchers, and to that proposed by the World Health Organization. NGI differed from the previous definition of "highly credible gastrointestinal illness" (HCGI) upon which the USEPA's 1986 bathing water criteria had been based, primarily by excluding fever as a prerequisite. METHODS: Incidence of NGI from the NEEAR studies was compared to that of HCGI from earlier studies. Markov chain Monte Carlo method was used to estimate the respective beta binomial probability densities for NGI and HCGI establish credible intervals for the risk ratio of NGI to HCGI. RESULTS: The ratio of NGI risk to that of HCGI is estimated to be 4.5 with a credible interval 3.2 to 7.7. CONCLUSIONS: A risk level of 8 HCGI illnesses per 1000 swimmers, as in the 1986 freshwater criteria, would correspond to 36 NGI illnesses per 1000 swimmers. Given a microbial DNA-based (qPCR) water quality vs. risk relationship developed from the NEEAR studies, 36 NGI per 1000 corresponds to a geometric mean of 475 qPCR cell-equivalents per 100 ml.

Research considerations for more effective groundwater monitoring.

Since numerous pathogens occur in feces, water is monitored for fecal contamination using indicator organisms rather than individual pathogens. Although this approach is supported by health effects data in recreational waters, it is questionable when used for drinking water. Most outbreaks in groundwater occur in systems that have not violated the US EPA's maximum contaminant limit (MCL) for total coliforms within 12 months before the outbreak. Additionally, environmentally stable viruses and parasites are often detected in drinking water samples with no detectable indicators. Recent detections of Escherichia coli O157:H7 and Campylobacter jejuni in groundwaters in the apparent absence of indicators also cast some doubt on the worth of indicators for fecal bacterial pathogens. Individual pathogen monitoring is now technically achievable but currently unreasonable due to the number of possible pathogens and the costs involved. Several alternatives to pathogen monitoring could significantly reduce the frequency at which pathogens occur in waters testing negative for indicators: (i) increasing sample volumes for indicators, (ii) increasing monitoring frequency, (iii) using a suite of indicators, (iv) using a more conservative polymerase chain reaction (PCR) method, (v) sampling when fecal contamination is most likely present or (vi) any combination of these options.

Comparison of the multiple-sample means with composite sample results for fecal indicator bacteria by quantitative PCR and culture.

Few studies have addressed the efficacy of composite sampling for measuring indicator bacteria by quantitative PCR (qPCR). We compared results from composited samples with multiple-sample means for culture- and qPCR-based water quality monitoring. Results from composited samples for both methods were similarly correlated to multiple-sample means and predicted criteria exceedances equally.

Some chronic rhinosinusitis patients have elevated populations of fungi in their sinuses.

OBJECTIVES/HYPOTHESIS: To measure the populations of 36 fungi in the homes and sinuses of chronic rhinosinusitis (CRS) and non-CRS patients. STUDY DESIGN: Single-blind cross-sectional study. METHODS: Populations of 36 fungi were measured in sinus samples and in the home vacuum cleaner dust of CRS (n = 73) and non-CRS patients (n = 16) using quantitative polymerase chain reaction. Etest strips containing amphotericin B, anidulafungin, caspofungin, fluconazole, and voriconazole were used to test the susceptibility of seven potentially relevant fungi. RESULTS: Seven fungi (Alternaria alternata, Cladosporium cladosporioides types 1 and 2, Cladosporium herbarum, Penicillium brevicompactum, Penicillium crustosum, and Penicillium chrysogenum type 2) were discovered at very high concentrations in some CRS patients. In vitro antifungal susceptibility testing of these seven fungi demonstrated species specific sensitivities. Four CRS patients with marked elevations of fungal populations in their sinus samples underwent endoscopic sinus surgery. After surgical treatment, the fungal populations were reduced by several orders of magnitude. CONCLUSIONS: Seven fungi were found in very high concentrations in the sinuses of some CRS patients. Not one of the five common antifungal agents could control all seven of these fungi based on in vitro tests.

Sampling household surfaces for pesticide residues: comparison between a press sampler and solvent-moistened wipes.

A modified Press Sampler was evaluated to determine the efficiency of pesticide transfer from household surfaces to collection disks as compared to wiping with a solvent-moistened gauze pad. Organophosphate (OP), pyrazole, and pyrethroid pesticides were applied to three hard flooring materials and carpet at two loading rates. Surfaces were dried and press sampled using C(18), 100% cotton or polyurethane foam (PUF) for either 2 or 10 min or wiped with isopropanol-moistened gauze pads. Transfer efficiencies (TE, %) were calculated as a fraction of surface loadings captured simultaneously on foil deposition coupons. The highest mean TEs (17-55%) for the Press Sampler were observed for OPs from hard surfaces to C(18), considering both contact times. Cotton and PUF transferred 6-27% and 5-30% of OPs, respectively. Corresponding mean TEs for pyrazole and pyrethroid pesticides were only 3% (C(18)), 2-3% (cotton) and 1-2% (PUF). Wipes of hard surfaces removed 84-97% of all pesticides while wipes of carpet removed 31-39%, much higher than transferred to any Press Sampler materials. The mean TEs suggested that the extent of pesticide residue transfer was affected by surface type, pesticide class, and sampling procedure. Wiping was more efficient than press sampling for pesticide surface residue measurements, particularly for loading rates typical of residences.

An integrated culture and real-time PCR method to assess viability of disinfectant treated Bacillus spores using robotics and the MPN quantification method.

Using robotics and the MPN technique, a 96-microwell method was developed to compare two procedures for enumeration of viable chlorine-treated B. atrophaeus spores: broth-culture enrichment followed by real-time polymerase chain reaction analysis; and filter plating on agar. Recoveries of chlorine-treated spores were improved by broth enrichment over filter plating, whereas recoveries of non-treated spores were not different in the two procedures.

Chemically and genetically immunocompromised mice are not more susceptible than immunocompetent mice to infection with Cryptosporidium muris.

The prevailing paradigm is that immunosuppressed individuals are more susceptible to infection and are at higher risk of infection from Cryptosporidium oocysts if present in drinking water. To test this hypothesis, three immune conditions were examined: genetically immunocompromised T cell deficient CD-1 nude mice, B and T cell deficient Fox Chase CB-17/IcrClB SCID mice, and chemically immunosuppressed C57Bl/6 mice. Chemical immunosuppression was induced with a single subcutaneous injection of methylprednisolone acetate (MPA) at 600 mg/kg. The MPA immunosuppressed C57Bl/6 mice were characterized by a sustained decrease in circulating CD3, CD4 and CD8 T-lymphocytes of greater than 80% and a similar decrease in B-lymphocytes. A sharp rise in circulating mature segmented neutrophils followed MPA injection, dropping sharply after 10-14 days, mirroring the decrease in lymphocytes. The cessation of oocyst production after MPA was not accompanied by a radical rise in circulating CD3 or CD4 T-lymphocytes, but rather a rise in CD8 T-lymphocytes. The ID50 for the MPA immunosuppressed C57Bl/6 mice was 122 oocysts, whereas the ID50 for the C57Bl/6 immunocompetent group was 44. The genetically immunocompromised mice showed similar differences. The ID50 for CD-1 nude mice was 166 oocysts compared to 64 in CD-1 immunocompetent mice. For Fox Chase CB-17/IcrClB SCID and the immunocompetent CB-17 mice, the ID50's were 83 and 60 oocysts, respectively. These results suggest that the lack of an immune response does not increase the ability of C. muris to establish a productive infection and produce oocysts.

Comment on "Derivation of numerical values for the World Health Organization guidelines for recreational waters".

The subject paper describes a procedure for adjusting a risk model based upon a measure of personal exposure (the "UK personal exposure model") in order to attribute an expected rate of gastroenteritis among a group of swimmers to a mean recreational water quality value (enterococci per 100mL). We term the resulting model for group risk the "UK ecologic exposure model." The distinction is essential to establishing recreational water quality guidelines because exposures of individual bathers are not known from a water monitoring program, the only assessment available being some form of ecologic exposure such as a mean log indicator density. While the authors of the subject paper solved the UK ecologic exposure model for only a single point (that value of mean log10 enterococcus density which is expected to result in five extra cases of gastroenteritis per 100 swimmers), we extend their model to show the entire curve over a relevant range of densities. The resulting exposure-response curve is seen to not differ substantially from the existing USEPA model for "highly credible gastrointestinal illness" in marine waters. However, particularly since such correspondence is not guaranteed for future studies or for other existing epidemiological studies, we recommend the direct approach to evaluating ecologic exposure, such as used in the USEPA studies, rather than the indirect approach of the UK ecologic exposure model, given the number of untested assumptions that are necessary for accomplishing the latter.

Indicator bacteria at five swimming beaches-analysis using random forests.

"Random forests," an extension of tree regression, provide a relatively new technique for exploring relationships of a response variable like the density of indicator bacteria in water to numerous potential explanatory variables. We used this tool to study relationships of indicator density at five beaches to numerous other variables and found that day of the week, indicator density 24h earlier, water depth at the sampling point, cloud cover, and others were related to density at one or more of the beaches. Using data from the first 52 days of measurement allowed predicting indicator densities in the following 10 days to order of magnitude at some of the beaches. Our analyses served to demonstrate the potential usefulness of this analytic tool for large data sets with many variables.

Comparison of Enterococcus measurements in freshwater at two recreational beaches by quantitative polymerase chain reaction and membrane filter culture analysis.

Cell densities of the fecal pollution indicator genus, Enterococcus, were determined by a rapid (3 h or less) quantitative polymerase chain reaction (QPCR) analysis method in 100 ml water samples collected from recreational beaches on Lake Michigan and Lake Erie during the summer of 2003. Measurements by this method were compared with counts of Enterococcus colony-forming units (CFU) determined by Method 1600 membrane filter (MF) analysis using mEI agar. The QPCR method had an estimated 95% confidence, minimum detection limit of 27 Enterococcus cells per sample in analyses of undiluted DNA extracts and quantitative analyses of multiple lake water samples, spiked with known numbers of these organisms, gave geometric mean results that were highly consistent with the spike levels. At both beaches, the geometric means of ambient Enterococcus concentrations in water samples, determined from multiple collection points during each sampling visit, showed approximately lognormal distributions over the study period using both QPCR and MF analyses. These geometric means ranged from 10 to 8548 cells by QPCR analysis and 1-2499 CFU by MF culture analysis in Lake Michigan (N=56) and from 8 to 8695 cells by QPCR and 3-1941 CFU by MF culture in Lake Erie (N=47). Regression analysis of these results showed a significant positive correlation between the two methods with an overall correlation coefficient (r) of 0.68.

Quantitative PCR analysis of house dust can reveal abnormal mold conditions.

Indoor mold concentrations were measured in the dust of moldy homes (MH) and reference homes (RH) by quantitative PCR (QPCR) assays for 82 species or related groups of species (assay groups). About 70% of the species and groups were never or only rarely detected. The ratios (MH geometric mean : RH geometric mean) for 6 commonly detected species (Aspergillus ochraceus, A. penicillioides, A. unguis, A. versicolor, Eurotium group, and Cladosporium sphaerospermum) were >1 (Group I). Logistic regression analysis of the sum of the logs of the concentrations of Group I species resulted in a 95% probability 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 number of mold species in a dust sample. Also, four common species of Aspergillus were quantified by standard culturing procedures and their concentrations compared to QPCR results. Culturing underestimated the concentrations of these four species by 2 to 3 orders of magnitude compared to QPCR.

Quantitative polymerase chain reaction analysis of fungi in dust from homes of infants who developed idiopathic pulmonary hemorrhaging.

Fungal concentrations were measured in the dust of 6 homes in Cleveland, Ohio, where an infant developed pulmonary hemorrhage (pulmonary hemorrhage homes [PHH]) and 26 reference homes (RH) with no known fungal contamination. Quantitative polymerase chain reaction 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). However, the same ratios were <1 for 10 species (group II). Probit analysis of the sum of the logs of the concentrations of these 2 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 37 degree C). Hemolysins were more commonly produced by group I species (42%) compared with group II species (10%).

Quantitative PCR analysis of selected Aspergillus, Penicillium and Paecilomyces species.

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 selected Aspergillus, Penicillium and Paecilomyces species. 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. 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.

Evaluation of a rapid, quantitative real-time PCR method for enumeration of pathogenic Candida cells in water.

Quantitative PCR (QPCR) technology, incorporating fluorigenic 5' nuclease (TaqMan) chemistry, was utilized for the specific detection and quantification of six pathogenic species of Candida (C. albicans, C. tropicalis, C. krusei, C. parapsilosis, C. glabrata and C. lusitaniae) in water. Known numbers of target cells were added to distilled and tap water samples, filtered, and disrupted directly on the membranes for recovery of DNA for QPCR analysis. The assay's sensitivities were between one and three cells per filter. The accuracy of the cell estimates was between 50 and 200% of their true value (95% confidence level). In similar tests with surface water samples, the presence of PCR inhibitory compounds necessitated further purification and/or dilution of the DNA extracts, with resultant reductions in sensitivity but generally not in quantitative accuracy. Analyses of a series of freshwater samples collected from a recreational beach showed positive correlations between the QPCR results and colony counts of the corresponding target species. Positive correlations were also seen between the cell quantities of the target Candida species detected in these analyses and colony counts of Enterococcus organisms. With a combined sample processing and analysis time of less than 4 h, this method shows great promise as a tool for rapidly assessing potential exposures to waterborne pathogenic Candida species from drinking and recreational waters and may have applications in the detection of fecal pollution.