Development of a methodology for the quantification of reaerosolization of a biological contaminate surrogate particle from a military uniform fabric.

In a mass casualty medical evacuation after a bioaerosol (BA) dispersal event, a decontamination (DC) method is needed that can both decontaminate and prevent biological particle (BP) re-aerosolization (RA) of contaminated clothes. However, neither the efficacy of current DC methods nor the risk of BP RA is greatly explored in the existing literature. The goals of this study were to develop a repeatable method to quantify the RA of a biological contaminant off military uniform fabric swatches and to test the efficacy of one DC protocol (high-volume, low-pressure water) using 1 µm polystyrene latex (PSL) spheres as a surrogate. A four-step methodology was developed: contamination using a Collison Nebulizer; RA using a laboratory mixer and aerosol collection using an inhalable air sampler with a polyvinyl chloride filter; DC using a gravity-fed water shower; and quantification using ultraviolet microscopy via both visual and computer techniques. All results for uncontaminated control samples showed little to no presence of PSL sphere-like particles, while the contaminated experimental trials showed that RA was much lower after DC with water at the 99% confidence level (p-value = 0.0081). The water DC showed an average ∼73% reduction in particle RA, along with a change in air sampler filter deposition patterns from aerosol-like (before DC) to droplet-like (after DC). The fluorescent sphere contamination method for testing the DC residual risk of RA was repeatable and successful.

Method development for the evaluation of emergency decontamination protocol effectiveness using an ultraviolet fluorescent aerosol.

After hazardous material incidents, it is important to perform emergency decontamination procedures to remove contamination from the body. As these emergency decontamination procedures are developed, it is important to understand the efficacy of a given protocol. This study discusses a method that was developed to evaluate the efficacy of decontamination procedures by using an ultraviolet fluorescent aerosol and an image analysis protocol. This method involves imaging a mannequin while both unclothed and clothed prior to exposure to the fluorescent aerosol. After exposure, it was imaged again, disrobed, and decontaminated following an unconscious patient wet decontamination method. This work describes in detail the materials and methods used to develop the final methodology. Two clothing types (black cotton and Tyvek) were used to simulate civilian and first responder casualties. Image analysis was used to measure the extent of contamination on the mannequin at each stage of the procedure. These measurements were then compared to determine decontamination efficacy for each step (disrobing, wet decontamination, and total removal). The exposure protocol was shown to provide repeatable deposition of aerosol onto the mannequin. Decontamination was also shown to be repeatable, with no trends toward efficacy changing over time.

Bioaerosol emissions from activated sludge basins: Characterization, release, and attenuation.

This article presents a critical review of the peer-reviewed literature related to bioaerosol generation from activated sludge basins. Characterization techniques include a variety of culture- and nonculture-based techniques, each with unique features. Bioaerosols contain a variety of clinical pathogens including Staphylococcus saprophyticus, Clostridium perfringens, and Salmonella enteritidis; exposure to these microorganisms increases human health risks. Release mechanisms involve splashing and bubble burst dynamics. Larger bubbles emit more aerosol particles than smaller ones. Attenuation strategies include covering sources with lids, adjusting the method and intensity of aeration, and using free-floating carrier media. Future studies should combine culture and non-culture based methods, and expand chemical databases and spectral libraries in order to realize the full power of real-time online monitoring.

Comparison and evaluation of a high volume air sampling system for the collection of Clostridioides difficile endospore aerosol in health care environments.

BACKGROUND: Environmental contamination of patient rooms and adjacent areas with C. difficile spores is a recognized transmission risk. Previous studies have shown that spores are aerosolized during patient care. These spores can remain airborne for extended periods and may contaminate distant surfaces. High-volume air sampling equipment allows for the collection of a large volume of air and was evaluated in the collection of C. difficile aerosol. METHOD: Air samplers evaluated in this research included the DFU-1000, XMX/2L-MIL, Biocapture-650, and a MB2. Aerosols of C. difficile were generated in a 5-m3 chamber and each air sampler sampled in the aerosol test chamber simultaneously with referee air samplers. RESULTS: The DFU-1000 achieved the highest efficiency of the 4 air samplers (P = .0145) with a mean efficiency of 38.60%. The relative efficiencies of the Biocapture-650, XMX/2L-MIL, and MB2 were 28.16%, 10.51%, and 3.05%, respectively. DISCUSSION/CONCLUSIONS: This study demonstrated high variation based on the sampling method employed. Based on the results of these studies, high-volume air samplers may be effectively applied to sample for airborne C. difficile in health care environments. The high sampling flow rate of the DFU-1000 would allow for the complete sampling of a patient room-sized volume in less than 1 hour.

Investigations of Amino Acids in the 5-Formyltetrahydrofolate Binding Site of 5,10-Methenyltetrahydrofolate Synthetase from Mycoplasma pneumonia.

5,10-Methenyltetrahydrofolate synthetase plays a significant role in folate metabolism by catalyzing the conversion of 5-formyltetrahydrofolate into 5,10-methenyltetrahydrofolate. The enzyme is important in some forms of chemotherapy, and it has been implicated in resistance to antifolate antibiotics. A co-crystal structure of the enzyme (1U3G) and primary sequence analysis were used to select highly conserved amino acids in close proximity to bound 5-formyltetrahydrofolate. The amino acids were then investigated using site directed mutagenesis and kinetics. Y123, E55, and F118 were concluded to be important for binding 5-formyltetrahydrofolate in the active site and/or for substrate turnover of the enzyme. Replacement of E55 or Y123 with alanine resulted in no detectable activity. The more subtle replacement of E55 with glutamine was also inactive suggesting an ionic interaction with 5-formyltetrahydrofolate. Mutations to F118 resulted in substantial increases in apparent Km for both 5-formyltetrahydrofolate and ATP, but did not substantially affect catalytic turnover. Outside the active site, the replacement of Q144 with alanine yielded an enzyme that bound the substrates of ATP and 5-formyltetrahydrofolate with higher apparent Km values than the wild-type enzyme, but demonstrated a 3.1 fold increase in kcat.

Toilet plume aerosol generation rate and environmental contamination following bowl water inoculation with Clostridium difficile spores.

INTRODUCTION: Clostridium difficile is the leading cause of health care-associated gastric illness. Environmental contamination with C difficile spores is a risk factor for contact transmission, and toilet flushing causes such contamination. This work explores toilet contamination persistence and environmental contamination produced over a series of flushes after contamination. METHODS: A flushometer toilet was seeded with C difficile spores in a sealed chamber. The toilet was flushed 24times, with postflush bowl water samples and settle plates periodically collected for culturing and counting. Air samples were collected after each of 12 flushes using rotating plate impactors. RESULTS: Spores were present in bowl water even after 24 flushes. Large droplet spore deposition accumulated over the 24-flush period. Droplet nuclei spore bioaerosol was produced over at least 12 flushes. CONCLUSIONS: Toilets contaminated with C difficile spores are a persistent source of environmental contamination over an extended number of flushes.

Comparison of high-volume air sampling equipment for viral aerosol sampling during emergency response.

OBJECTIVE: This study compared the performance of two high-volume bioaerosol air samplers for viable virus to an accepted standard low-volume sampler. In typical bioaerosol emergency response scenarios, highvolume sampling is essential for the low infective concentrations and large air volumes involved. DESIGN: Two high-volume air samplers (XMX/2LMIL and DFU-1000) were evaluated alongside a lowvolume sample (BioSampler). Low and high concentrations (9.3-93.2 agent containing particles per liter of air [ACPLA]) of male-specific coliphage 2 (MS2) virus were released into a 12 m3 aerosol test chamber and collected using the air samplers. The collection media from the samplers were then processed and viable virus was assessed via plaque assay. SETTING: Aerosol test chamber. SUBJECTS, PARTICIPANTS: None. INTERVENTIONS: Collection media and flow rate were modified for the XMX/2L-MIL sampler for viable analysis. MAIN OUTCOME MEASURES: Concentration estimates in units of plaque forming units per liter of air (PFU/liter) assessed by the samplers as compared to the levels inside the chamber as evaluated with a slit to agar plate in units of ACPLA. Comparison was made via one-way analysis of variance. RESULTS: Both the XMX/2L-MIL and DFU-1000 achieved collection effectiveness equal to or greater than the low-volume air sampler for the evaluated MS2 concentrations. The XMX/2L-MIL reliably collected quantifiable low concentrations of MS2, but the DFU-1000 was unable to do so. CONCLUSIONS: For emergency response to suspected bioaerosols, the evaluated high-volume samplers are as effective as the standard low-flow sampler and should be considered in conducting a health risk assessment. If low concentrations are expected, then high-flow samplers using liquid collection are preferred.

Genome scans and gene expression microarrays converge to identify gene regulatory loci relevant in schizophrenia.

Multiple linkage regions have been reported in schizophrenia, and some appear to harbor susceptibility genes that are differentially expressed in postmortem brain tissue derived from unrelated individuals. We combined traditional genome-wide linkage analysis in a multiplex family with lymphocytic genome-wide expression analysis. A genome scan suggested linkage to a chromosome 4q marker (D4S1530, LOD 2.17, theta = 0) using a dominant model. Haplotype analysis using flanking microsatellite markers delineated a 14 Mb region that cosegregated with all those affected. Subsequent genome-wide scan with SNP genotypes supported the evidence of linkage to 4q33-35.1 (LOD = 2.39) using a dominant model. Genome-wide microarray analysis of five affected and five unaffected family members identified two differentially expressed genes within the haplotype AGA and GALNT7 (aspartylglucosaminidase and UDP-N-acetyl-alpha-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase 7) with nominal significance; however, these genes did not remain significant following analysis of covariance. We carried out genome-wide linkage analyses between the quantitative expression phenotype and genetic markers. AGA expression levels showed suggestive linkage to multiple markers in the haplotype (maximum LOD = 2.37) but to no other genomic region. GALNT7 expression levels showed linkage to regulatory loci at 4q28.1 (maximum LOD = 3.15) and in the haplotype region at 4q33-35.1 (maximum LOD = 2.37). ADH1B (alcohol dehydrogenase IB) was linked to loci at 4q21-q23 (maximum LOD = 3.08) and haplotype region at 4q33-35.1 (maximum LOD = 2.27). Seven differentially expressed genes were validated with RT-PCR. Three genes in the 4q33-35.1 haplotype region were also differentially expressed in schizophrenia in postmortem dorsolateral prefrontal cortex: AGA, HMGB2, and SCRG1. These results indicate that combining differential gene expression with linkage analysis may help in identifying candidate genes and potential regulatory sites. Moreover, they also replicate recent findings of complex trans- and cis- regulation of genes.

Suggestive linkage of schizophrenia to 5p13 in Costa Rica.

Schizophrenia afflicts roughly 1% of all people worldwide. Remarkably, despite differing cultures and environments, the expression of illness is essentially the same. Family, twin, and adoption studies identify schizophrenia as a genetically influenced disease. Linkage studies suggest many positive regions of interest, but as a complex genetic disorder most of the pathogenic loci have not yet been found. Isolated populations are commonly used to study rare Mendelian inherited diseases due to the more homogenous genetic background of the subjects and are thought to be useful for detecting linkage in complex genetic disorders such as schizophrenia. This study aims to define areas of the genome that exhibit co-inheritance with schizophrenia in one large, Mendelian-like family from the central valley of Costa Rica. The whole genome scan analysis of this pedigree, which included 11 cases of schizophrenia and schizoaffective disorder, identified a number of markers on chromosome 5p that appear to co-segregate with the disease with a maximum lod score of 2.70 at marker D5S426. Current studies include investigating additional Costa Rican pedigrees to replicate these findings and identify additional loci linked to the disease.