Wildland firefighters (WFFs) are exposed to many inhalation hazards working in the wildland fire environment. To assess occupational exposures and acute and subacute health effects among WFFs, the wildland firefighter exposure and health effects study collected data for a 2-year repeated measures study. This manuscript describes the exposure assessment from one Interagency Hotshot Crew (N = 19) conducted at a wildfire incident. Exposures to benzene, toluene, ethylbenzene, xylene isomers, formaldehyde, acetaldehyde, and naphthalene were measured through personal air sampling each work shift. Biological monitoring was done for creatinine-adjusted levoglucosan in urine pre- and post-shift. For 3 days sampling at the wildfire incident, benzene, toluene, ethylbenzene, xylene isomers (m and p, and o) exposure was highest on day 1 (geometric mean [GM] = 0.015, 0.042, 0.10, 0.42, and 0.15 ppm, respectively) when WFFs were not exposed to smoke but used chainsaws to remove vegetation and prepare fire suppression breaks. Exposure to formaldehyde and acetaldehyde was highest on day 2 (GM = 0.03 and 0.036 ppm, respectively) when the WFFs conducted a firing operation and were directly exposed to wildfire smoke. The greatest difference of pre- and post-shift levoglucosan concentrations were observed on day 3 (pre-shift: 9.7 and post-shift: 47 μg/mg creatinine) after WFFs conducted mop up (returned to partially burned area to extinguish any smoldering vegetation). Overall, 65% of paired samples (across all sample days) showed a post-shift increase in urinary levoglucosan and 5 firefighters were exposed to benzene at concentrations at or above the National Institute for Occupational Safety and Health (NIOSH) recommended exposure limit. Our findings further demonstrate that exposure to inhalation hazards is one of many risks that wildland firefighters experience while suppressing wildfires.

Firefighters are at an increased risk of cancer due to their occupational exposure to combustion byproducts, especially when those compounds penetrate the firefighter personal protective equipment (PPE) ensemble. This has led to questions about the impact of base layers (i.e., shorts vs. pants) under PPE ensembles. This study asked 23 firefighters to perform firefighting activities while wearing one of three different PPE ensembles with varying degrees of protection. Additionally, half of the firefighters unzipped their jackets after the scenario while the other half kept their jackets zipped for five additional minutes. Several volatile organic compound (VOC) and naphthalene air concentrations outside and inside of hoods, turnout jackets, and turnout pants were evaluated; biological (urinary and exhaled breath) samples were also collected. VOCs and naphthalene penetrated the three sampling areas (hoods, jackets, pants). Significant (p-value < 0.05) increases from pre- to post-fire for some metabolites of VOCs (e.g., benzene, toluene) and naphthalene were found. Firefighters wearing shorts and short sleeves absorbed higher amounts of certain compounds (p-value < 0.05), and the PPE designed with enhanced interface control features appeared to provide more protection from some compounds. These results suggest that firefighters can dermally absorb VOCs and naphthalene that penetrate the PPE ensemble.

Practitioners in health departments, university extension programs, and nonprofit organizations working in public health face varied challenges to publishing in the peer-reviewed literature. These practitioners may lack time, support, skills, and efficacy needed for manuscript submission, which keeps them from sharing their wisdom and experience-based evidence. This exclusion can contribute to literature gaps, a failure of evidence-based practice to inform future research, reduced ability to educate partners, and delays in advancing public health practice. Our article describes the writing workshops offered to Division of Nutrition, Physical Activity, and Obesity (DNPAO), Centers for Disease Control and Prevention (CDC) funded programs in 2021. This project consisted of three 60-minute introductory writing webinars open to all recipients, followed by a Writing for Publications workshop, an 8- to 9-week virtual learning/writing intensive for selected writing team applicants. The Society for Public Health Education staff, consultants, and CDC/DNPAO staff developed, refined, and presented the curriculum. The workshop for public health practitioner writing teams was offered to two cohorts and included extensive coaching and focused on potential submission to a Health Promotion Practice supplement, "Reducing Chronic Disease through Physical Activity and Nutrition: Public Health Practice in the Field" (see Supplemental Material), which was supported by CDC/DNPAO. We describe the webinars, the workshop design, modifications, evaluation methods and results.

INTRODUCTION: Firefighters are exposed to volatile organic compounds (VOCs) during structural fire responses and training fires, several of which (e.g., benzene, acrolein, styrene) are known or probable carcinogens. Exposure studies have found that firefighters can absorb chemicals like benzene even when self-contained breathing apparatus (SCBA) are worn, suggesting that dermal absorption contributes to potentially harmful exposures. However, few studies have characterized VOC metabolites in urine from firefighters. OBJECTIVES: We quantified VOC metabolites in firefighters' urine following live firefighting activity across two field studies. METHODS: In two separate controlled field studies, spot urine was collected before and 3 h after firefighters and firefighter students responded to simulated residential and training fires. Urine was also collected from instructors from the training fire study before the first and 3 h after the last training scenario for each day (instructors led three training scenarios per day). Samples were analyzed for metabolites of VOCs to which firefighters may be exposed. RESULTS: In the residential fire study, urinary metabolites of xylenes (2MHA), toluene (BzMA), and styrene (MADA) increased significantly (at 0.05 level) from pre- to post-fire. In the training fire study, MADA concentrations increased significantly from pre- to post-fire for both firefighter students and instructors. Urinary concentrations of benzene metabolites (MUCA and PhMA) increased significantly from pre- to post-fire for instructors, while metabolites of xylenes (3MHA+4MHA) and acrolein (3HPMA) increased significantly for firefighter students. The two highest MUCA concentrations measured post-shift from instructors exceeded the BEI of 500 μg/g creatinine. CONCLUSIONS: Some of the metabolites that were significantly elevated post-fire are known or probable human carcinogens (benzene, styrene, acrolein); thus, exposure to these compounds should be eliminated or reduced as much as possible through the hierarchy of controls. Given stringent use of SCBA, it appears that dermal exposure contributes in part to the levels measured here.

The wildland firefighter exposure and health effect (WFFEHE) study was a 2-year repeated-measures study to investigate occupational exposures and acute and subacute health effects among wildland firefighters. This manuscript describes the study rationale, design, methods, limitations, challenges, and lessons learned. The WFFEHE cohort included fire personnel ages 18-57 from six federal wildland firefighting crews in Colorado and Idaho during the 2018 and 2019 fire seasons. All wildland firefighters employed by the recruited crews were invited to participate in the study at preseason and postseason study intervals. In 2019, one of the crews also participated in a 3-day midseason study interval where workplace exposures and pre/postshift measurements were collected while at a wildland fire incident. Study components assessed cardiovascular health, pulmonary function and inflammation, kidney function, workplace exposures, and noise-induced hearing loss. Measurements included self-reported risk factors and symptoms collected through questionnaires; serum and urine biomarkers of exposure, effect, and inflammation; pulmonary function; platelet function and arterial stiffness; and audiometric testing. Throughout the study, 154 wildland firefighters participated in at least one study interval, while 144 participated in two or more study interval. This study was completed by the Centers for Disease Control and Prevention's National Institute for Occupational Safety and Health through a collaborative effort with the U.S. Department of Agriculture Forest Service, Department of the Interior National Park Service, and Skidmore College. Conducting research in the wildfire environment came with many challenges including collecting study data with study participants with changing work schedules and conducting study protocols safely and operating laboratory equipment in remote field locations. Forthcoming WFFEHE study results will contribute to the scientific evidence regarding occupational risk factors and exposures that can impact wildland firefighter health over a season and across two wildland fire seasons. This research is anticipated to lead to the development of preventive measures and policies aimed at reducing risk for wildland firefighters and aid in identifying future research needs for the wildland fire community.

Firefighters may encounter items containing flame retardants (FRs), including organophosphate flame retardants (OPFRs) and polybrominated diphenyl ethers (PBDEs), during structure fires. This study utilized biological monitoring to characterize FR exposures in 36 firefighters assigned to interior, exterior, and overhaul job assignments, before and after responding to controlled residential fire scenarios. Firefighters provided four urine samples (pre-fire and 3-h, 6-h, and 12-h post-fire) and two serum samples (pre-fire and approximately 23-h post-fire). Urine samples were analyzed for OPFR metabolites, while serum samples were analyzed for PBDEs, brominated and chlorinated furans, and chlorinated dioxins. Urinary concentrations of diphenyl phosphate (DPhP), a metabolite of triphenyl phosphate (TPhP), bis(1,3-dichloro-2-propyl) phosphate (BDCPP), a metabolite of tris(1,3-dichloro-2-propyl) phosphate (TDCPP), and bis(2-chloroethyl) phosphate (BCEtP), a metabolite of tris(2-chloroethyl) phosphate (TCEP), increased from pre-fire to 3-hr and 6-hr post-fire collection, but only the DPhP increase was statistically significant at a 0.05 level. The 3-hr and 6-hr post-fire concentrations of DPhP and BDCPP, as well as the pre-fire concentration of BDCPP, were statistically significantly higher than general population levels. BDCPP pre-fire concentrations were statistically significantly higher in firefighters who previously participated in a scenario (within the past 12 days) than those who were responding to their first scenario as part of the study. Similarly, firefighters previously assigned to interior job assignments had higher pre-fire concentrations of BDCPP than those previously assigned to exterior job assignments. Pre-fire serum concentrations of 2,3,4,7,8-pentachlorodibenzofuran (23478-PeCDF), a known human carcinogen, were also statistically significantly above the general population levels. Of the PBDEs quantified, only decabromodiphenyl ether (BDE-209) pre- and post-fire serum concentrations were statistically significantly higher than the general population. These results suggest firefighters absorbed certain FRs while responding to fire scenarios.

The objective of this study was to evaluate the association between carbon nanotube and nanofiber (CNT/F) exposure and ex vivo responses of whole blood challenged with secondary stimulants, adjusting for potential confounders, in a cross-sectional study of 102 workers. Multi-day exposure was measured by CNT/F structure count (SC) and elemental carbon (EC) air concentrations. Demographic, lifestyle and other occupational covariate data were obtained via questionnaire. Whole blood collected from each participant was incubated for 18 hours with and without two microbial stimulants (lipopolysaccharide/LPS and staphylococcal enterotoxin type B/SEB) using TruCulture technology to evaluate immune cell activity. Following incubation, supernatants were preserved and analyzed for protein concentrations. The stimulant:null response ratio for each individual protein was analyzed using multiple linear regression, followed by principal component (PC) analysis to determine whether patterns of protein response were related to CNT/F exposure. Adjusting for confounders, CNT/F metrics (most strongly, the SC-based) were significantly (p < 0.05) inversely associated with stimulant:null ratios of several individual biomarkers: GM-CSF, IFN-gamma, interleukin (IL)-2, IL-4, IL-5, IL-10, IL-17, and IL-23. CNT/F metrics were significantly inversely associated with PC1 (a weighted mean of most biomarkers, explaining 25% of the variance in the protein ratios) and PC2 (a biomarker contrast, explaining 14%). Among other occupational exposures, only solvent exposure was significant (inversely related to PC2). CNT/F exposure metrics were uniquely related to stimulant responses in challenged whole blood, illustrating reduced responsiveness to a secondary stimulus. This approach, if replicated in other exposed populations, may present a relatively sensitive method to evaluate human response to CNT/F or other occupational exposures.

INTRODUCTION: Training fires may constitute a major portion of some firefighters' occupational exposures to smoke. However, the magnitude and composition of those exposures are not well understood and may vary by the type of training scenario and fuels. OBJECTIVES: To understand how structure fire training contributes to firefighters' and instructors' select chemical exposures, we conducted biological monitoring during exercises involving combustion of pallet and straw and oriented strand board (OSB) or the use of simulated smoke. METHODS: Urine was analyzed for metabolites of polycyclic aromatic hydrocarbons (PAHs) and breath was analyzed for volatile organic compounds (VOCs) including benzene. RESULTS: Median concentrations of nearly all PAH metabolites in urine increased from pre-to 3-hr post-training for each scenario and were highest for OSB, followed by pallet and straw, and then simulated smoke. For instructors who supervised three trainings per day, median concentrations increased at each collection. A single day of OSB exercises led to a 30-fold increase in 1-hydroxypyrene for instructors, culminating in a median end-of-shift concentration 3.5-fold greater than median levels measured from firefighters in a previous controlled-residential fire study. Breath concentrations of benzene increased 2 to 7-fold immediately after the training exercises (with the exception of simulated smoke training). Exposures were highest for the OSB scenario and instructors accumulated PAHs with repeated daily exercises. CONCLUSIONS: Dermal absorption likely contributed to the biological levels as the respiratory route was well protected. Training academies should consider exposure risks as well as instructional objectives when selecting training exercises.

To better understand the absorption of combustion byproducts during firefighting, we performed biological monitoring (breath and urine) on firefighters who responded to controlled residential fires and examined the results by job assignment and fire attack tactic. Urine was analyzed for metabolites of polycyclic aromatic hydrocarbons (PAHs) and breath was analyzed for volatile organic compounds (VOCs) including benzene. Median concentrations of PAH metabolites in urine increased from pre-firefighting to 3-h post firefighting for all job assignments. This change was greatest for firefighters assigned to attack and search with 2.3, 5.6, 3.9, and 1.4-fold median increases in pyrene, phenanthrene, naphthalene, and fluorene metabolites. Median exhaled breath concentrations of benzene increased 2-fold for attack and search firefighters (p < 0.01) and 1.4-fold for outside vent firefighters (p = 0.02). Compared to interior attack, transitional attack resulted in 50% less uptake of pyrene (p = 0.09), 36% less uptake phenanthrene (p = 0.052), and 20% less uptake of fluorene (p < 0.01). Dermal absorption likely contributed to firefighters' exposures in this study. Firefighters' exposures will vary by job assignment and can be reduced by employing a transitional fire attack when feasible.

Neisseria meningitidis serogroup B (MnB) was responsible for two independent meningococcal disease outbreaks at universities in the USA during 2013. The first at University A in New Jersey included nine confirmed cases reported between March 2013 and March 2014. The second outbreak occurred at University B in California, with four confirmed cases during November 2013. The public health response to these outbreaks included the approval and deployment of a serogroup B meningococcal vaccine that was not yet licensed in the USA. This study investigated the use of whole-genome sequencing(WGS) to examine the genetic profile of the disease-causing outbreak isolates at each university. Comparative WGS revealed differences in evolutionary patterns between the two disease outbreaks. The University A outbreak isolates were very closely related, with differences primarily attributed to single nucleotide polymorphisms/insertion-deletion (SNP/indel) events. In contrast, the University B outbreak isolates segregated into two phylogenetic clades, differing in large part due to recombination events covering extensive regions (>30 kb) of the genome including virulence factors. This high-resolution comparison of two meningococcal disease outbreaks further demonstrates the genetic complexity of meningococcal bacteria as related to evolution and disease virulence.

Background: Minority drug resistance mutations (DRMs) that are often missed by Sanger sequencing are clinically significant, as they can cause virologic failure in individuals treated with antiretroviral therapy (ART) drugs. Objective: This study aimed to estimate the prevalence of minor DRMs among patients enrolled in a Malawi HIV drug resistance monitoring survey at baseline and at one year after initiation of ART. Methods: Forty-one plasma specimens collected from HIV-1 subtype C-positive patients and seven clonal control samples were analysed using ultra-deep sequencing technology. Results: Deep sequencing identified all 72 DRMs detected by Sanger sequencing at the level of >/=20% and 79 additional minority DRMs at the level of < 20% from the 41 Malawian clinical specimens. Overall, DRMs were detected in 85% of pre-ART and 90.5% of virologic failure patients by deep sequencing. Among pre-ART patients, deep sequencing identified a statistically significant higher prevalence of DRMs to nucleoside reverse transcriptase inhibitors (NRTIs) compared with Sanger sequencing. The difference was mainly due to the high prevalence of minority K65R and M184I mutations. Most virologic failure patients harboured DRMs against both NRTIs and non-nucleoside reverse transcriptase inhibitors (NNRTIs). These minority DRMs contributed to the increased or enhanced virologic failures in these patients. Conclusion: The results revealed the presence of minority DRMs to NRTIs and NNRTIs in specimens collected at baseline and virologic failure time points. These minority DRMs not only increased resistance levels to NRTIs and NNRTIs for the prescribed ART, but also expanded resistance to additional major first-line ART drugs. This study suggested that drug resistance testing that uses more sensitive technologies, is needed in this setting.

Objectives In order to produce near real-time onsite results to detect surface contamination by antineoplastic drugs, the National Institute for Occupational Safety and Health developed monitors for 5-fluorouracil, which use surface wiping and lateral flow immunoassay for measurement. The monitors were tested in the laboratory to assess the sensitivity of detection on laboratory-produced contaminated surfaces. A field evaluation to assess the capability of the monitors to make measurements in healthcare workplaces was carried out in collaboration with a medical device company and the results are presented in this report. Methods The 5-fluorouracil monitor was evaluated in areas where oncology drugs were prepared and administered to patients at five different hospitals. The levels of contamination measured with the monitors were compared to levels measured with liquid chromatography-tandem mass spectrometry. Results The 5-fluorouracil values measured with the liquid chromatography-tandem mass spectrometry ranged from 0 to over 200,000 ng/100 cm(2). Measurements by the 5-fluorouracil monitors in the range 10-100 ng/100 cm(2) correlated with the liquid chromatography-tandem mass spectrometry. Receiver operating characteristic curves developed for the data indicated that a positive limit of 22 ng/100 cm(2) would give an acceptable level of false-positives while retaining most true-positive samples. If the liquid chromatography-tandem mass spectrometry measured greater than 100 ng/100 cm(2), then the monitors also measured levels greater than 100 ng/100 cm(2) for the majority of samples. Conclusion The data indicate that there are many areas in hospitals that are contaminated with 5-fluorouracil and the monitors will be useful in identifying this contamination.

A panel of experts was convened by the Infectious Diseases Society of America (IDSA) and Society for Healthcare Epidemiology of America (SHEA) to update the 2010 clinical practice guideline on Clostridium difficile infection (CDI) in adults. The update, which has incorporated recommendations for children (following the adult recommendations for epidemiology, diagnosis, and treatment), includes significant changes in the management of this infection and reflects the evolving controversy over best methods for diagnosis. Clostridium difficile remains the most important cause of healthcare-associated diarrhea and has become the most commonly identified cause of healthcare-associated infection in adults in the United States. Moreover, C. difficile has established itself as an important community pathogen. Although the prevalence of the epidemic and virulent ribotype 027 strain has declined markedly along with overall CDI rates in parts of Europe, it remains one of the most commonly identified strains in the United States where it causes a sizable minority of CDIs, especially healthcare-associated CDIs. This guideline updates recommendations regarding epidemiology, diagnosis, treatment, infection prevention, and environmental management.

Firefighters' skin may be exposed to chemicals via permeation/penetration of combustion byproducts through or around personal protective equipment (PPE) or from the cross-transfer of contaminants on PPE to the skin. Additionally, volatile contaminants can evaporate from PPE following a response and be inhaled by firefighters. Using polycyclic aromatic hydrocarbons (PAHs) and volatile organic compounds (VOCs) as respective markers for non-volatile and volatile substances, we investigated the contamination of firefighters' turnout gear and skin following controlled residential fire responses. Participants were grouped into three crews of twelve firefighters. Each crew was deployed to a fire scenario (one per day, four total) and then paired up to complete six fireground job assignments. Wipe sampling of the exterior of the turnout gear was conducted pre- and post-fire. Wipe samples were also collected from a subset of the gear after field decontamination. VOCs off-gassing from gear were also measured pre-fire, post-fire, and post-decon. Wipe sampling of the firefighters' hands and neck was conducted pre- and post-fire. Additional wipes were collected after cleaning neck skin. PAH levels on turnout gear increased after each response and were greatest for gear worn by firefighters assigned to fire attack and to search and rescue activities. Field decontamination using dish soap, water, and scrubbing was able to reduce PAH contamination on turnout jackets by a median of 85%. Off-gassing VOC levels increased post-fire and then decreased 17-36 minutes later regardless of whether field decontamination was performed. Median post-fire PAH levels on the neck were near or below the limit of detection (< 24 micrograms per square meter [microg/m2]) for all positions. For firefighters assigned to attack, search, and outside ventilation, the 75th percentile values on the neck were 152, 71.7, and 39.3 microg/m2, respectively. Firefighters assigned to attack and search had higher post-fire median hand contamination (135 and 226 microg/m2, respectively) than other positions (< 10.5 microg/m2). Cleansing wipes were able to reduce PAH contamination on neck skin by a median of 54%.

The gut microbiome community structure and development are associated with several health outcomes in young children. To determine the household influences of gut microbiome structure, we assessed microbial sharing within households in western Kenya by sequencing 16S rRNA libraries of fecal samples from children and cattle, cloacal swabs from chickens, and swabs of household surfaces. Among the 156 households studied, children within the same household significantly shared their gut microbiome with each other, although we did not find significant sharing of gut microbiome across host species or household surfaces. Higher gut microbiome diversity among children was associated with lower wealth status and involvement in livestock feeding chores. Although more research is necessary to identify further drivers of microbiota development, these results suggest that the household should be considered as a unit. Livestock activities, health and microbiome perturbations among an individual child may have implications for other children in the household.

Vaccinia virus (VACV) has been implicated in infections of dairy cattle and humans, and outbreaks have substantially impacted local economies and public health in Brazil. During a 2005 outbreak, a VACV strain designated Serro 2 virus (S2V) was collected from a 30-year old male milker. Our aim was to phenotypically and genetically characterize this VACV Brazilian isolate. S2V produced small round plaques without associated comets when grown in BSC40 cells. Furthermore, S2V was less virulent than the prototype strain VACV-Western Reserve (WR) in a murine model of intradermal infection, producing a tiny lesion with virtually no surrounding inflammation. The genome of S2V was sequenced by primer walking. The coding region spans 184,572 bp and contains 211 predicted genes. Mutations in envelope genes specifically associated with small plaque phenotypes were not found in S2V; however, other alterations in amino acid sequences within these genes were identified. In addition, some immunomodulatory genes were truncated in S2V. Phylogenetic analysis using immune regulatory-related genes, besides the hemagglutinin gene, segregated the Brazilian viruses into two clusters, grouping the S2V into Brazilian VACV group 1. S2V is the first naturally-circulating human-associated VACV, with a low passage history, to be extensively genetically and phenotypically characterized.

The complete genomes of a skunkpox, volepox, and raccoonpox virus were sequenced and annotated. Phylogenetic analysis of these genomes indicates that although these viruses are all orthopoxviruses, they form a distinct clade to the other known species. This supports the ancient divergence of the North American orthopoxviruses from other members of the orthopoxviruses. Only two open reading frames appear to be unique to this group of viruses, but a relatively small number of insertions/deletions contribute to the varied gene content of this clade. The availability of these genomes will help determine whether skunkpox and volepox viruses share the characteristics that make raccoonpox a useful vaccine vector.

Here, we report the 187.8-kb genome sequence of Vaccinia virus Lister-Butantan, which was used in Brazil during the WHO smallpox eradication campaign. Its genome showed an average similarity of 98.18% with the original Lister isolate, highlighting the low divergence among related Vaccinia virus vaccine strains, even after several passages in animals and cell culture.

During 2010 and 2012, California and Vermont, respectively, experienced statewide epidemics of pertussis with differences seen in the demographic affected, case clinical presentation, and molecular epidemiology of the circulating strains. To overcome limitations of the current molecular typing methods for pertussis, we utilized whole-genome sequencing to gain a broader understanding of how current circulating strains are causing large epidemics. Through the use of combined next-generation sequencing technologies, this study compared de novo, single-contig genome assemblies from 31 out of 33 Bordetella pertussis isolates collected during two separate pertussis statewide epidemics and 2 resequenced vaccine strains. Final genome architecture assemblies were verified with whole-genome optical mapping. Sixteen distinct genome rearrangement profiles were observed in epidemic isolate genomes, all of which were distinct from the genome structures of the two resequenced vaccine strains. These rearrangements appear to be mediated by repetitive sequence elements, such as high-copy-number mobile genetic elements and rRNA operons. Additionally, novel and previously identified single nucleotide polymorphisms were detected in 10 virulence-related genes in the epidemic isolates. Whole-genome variation analysis identified state-specific variants, and coding regions bearing nonsynonymous mutations were classified into functional annotated orthologous groups. Comprehensive studies on whole genomes are needed to understand the resurgence of pertussis and develop novel tools to better characterize the molecular epidemiology of evolving B. pertussis populations. IMPORTANCE Pertussis, or whooping cough, is the most poorly controlled vaccine-preventable bacterial disease in the United States, which has experienced a resurgence for more than a decade. Once viewed as a monomorphic pathogen, B. pertussis strains circulating during epidemics exhibit diversity visible on a genome structural level, previously undetectable by traditional sequence analysis using short-read technologies. For the first time, we combine short- and long-read sequencing platforms with restriction optical mapping for single-contig, de novo assembly of 31 isolates to investigate two geographically and temporally independent U.S. pertussis epidemics. These complete genomes reshape our understanding of B. pertussis evolution and strengthen molecular epidemiology toward one day understanding the resurgence of pertussis.

Here, we report the complete genome sequences of three Legionella pneumophila subsp. pascullei strains (including both serogroup 1 and 5 strains) that were found in the same health care facility in 1982 and 2012.

BACKGROUND: Infections with Taenia solium are the most common cause of adult acquired seizures worldwide, and are the leading cause of epilepsy in developing countries. A better understanding of the genetic diversity of T. solium will improve parasite diagnostics and transmission pathways in endemic areas thereby facilitating the design of future control measures and interventions. Microsatellite markers are useful genome features, which enable strain typing and identification in complex pathogen genomes. Here we describe microsatellite identification and characterization in T. solium, providing information that will assist in global efforts to control this important pathogen. METHODS: For genome sequencing, T. solium cysts and proglottids were collected from Huancayo and Puno in Peru, respectively. Using next generation sequencing (NGS) and de novo assembly, we assembled two draft genomes and one hybrid genome. Microsatellite sequences were identified and 36 of them were selected for further analysis. Twenty T. solium isolates were collected from Tumbes in the northern region, and twenty from Puno in the southern region of Peru. The size-polymorphism of the selected microsatellites was determined with multi-capillary electrophoresis. We analyzed the association between microsatellite polymorphism and the geographic origin of the samples. RESULTS: The predicted size of the hybrid (proglottid genome combined with cyst genome) T. solium genome was 111 MB with a GC content of 42.54%. A total of 7,979 contigs (>1,000 nt) were obtained. We identified 9,129 microsatellites in the Puno-proglottid genome and 9,936 in the Huancayo-cyst genome, with 5 or more repeats, ranging from mono- to hexa-nucleotide. Seven microsatellites were polymorphic and 29 were monomorphic within the analyzed isolates. T. solium tapeworms were classified into two genetic groups that correlated with the North/South geographic origin of the parasites. CONCLUSIONS/SIGNIFICANCE: The availability of draft genomes for T. solium represents a significant step towards the understanding the biology of the parasite. We report here a set of T. solium polymorphic microsatellite markers that appear promising for genetic epidemiology studies.

Burkholderia pseudomallei strain Bp1651, a human isolate, is resistant to all clinically relevant antibiotics. We report here on the finished genome sequence assembly and annotation of the two chromosomes of this strain. This genome sequence may assist in understanding the mechanisms of antimicrobial resistance for this pathogenic species.

Surface contamination from methamphetamine in meth labs continues to be a problem. We had previously developed a lateral flow assay cassette for field detection of methamphetamine contamination that is commercially available and has been used by a number of groups to assess contamination. This cassette uses the complete disappearance of the test line as an end point for detection of 50 ng/100 cm2 of methamphetamine contamination for surface sampling with cotton swabs. In the present study, we further evaluate the response of the cassettes using an electronic lateral flow reader to measure the intensities of the test and control lines. The cassettes were capable of detecting 0.25 ng/ml for calibration solutions. For 100 cm2 ceramic tiles that were spiked with methamphetamine and wiped with cotton-tipped wooden swabs wetted in assay/sampling buffer, 1 ng/tile was detected using the reader. Semi-quantitative results can be produced over the range 0-10 ng/ml for calibration solutions and 0-25 ng/tile for spiked tiles using either a 4-parameter logistic fit of test line intensity versus concentration or spiked mass or the ratio of the control line to the test line intensity fit to concentration or spiked mass. Recovery from the tiles was determined to be about 30% using the fitted curves. Comparison of the control line to the test line was also examined as a possible visual detection end point and it was found that the control line became more intense than the test line at 0.5 to 1 ng/ml for calibration solutions or 1 to 2 ng/tile for spiked tiles. Thus the lateral flow cassettes for methamphetamine have the potential to produce more sensitive semi-quantitative results if an electronic lateral flow reader is used and can be more sensitive for detection if the comparison of the control line to the test line is used as the visual end point.

OBJECTIVES: To identify a pediatric ventilator-associated condition definition for use in neonates and children by exploring whether potential ventilator-associated condition definitions identify patients with worse outcomes. DESIGN: Retrospective cohort study and a matched cohort analysis. SETTING: Pediatric, cardiac, and neonatal ICUs in five U.S. hospitals. PATIENTS: Children 18 years old or younger ventilated for at least 1 day. INTERVENTIONS, MEASUREMENTS AND MAIN RESULTS: We evaluated the evidence of worsening oxygenation via a range of thresholds for increases in daily minimum fraction of inspired oxygen (by 0.20, 0.25, and 0.30) and daily minimum mean airway pressure (by 4, 5, 6, and 7 cm H2O). We required worsening oxygenation be sustained for at least 2 days after at least 2 days of stability. We matched patients with a ventilator-associated condition to those without and used Cox proportional hazard models with frailties to examine associations with hospital mortality, hospital and ICU length of stay, and duration of ventilation. The cohort included 8,862 children with 10,209 hospitalizations and 77,751 ventilator days. For the fraction of inspired oxygen 0.25/mean airway pressure 4 definition (i.e., increase in minimum daily fraction of inspired oxygen by 0.25 or mean airway pressure by 4), rates ranged from 2.9 to 3.2 per 1,000 ventilator days depending on ICU type; the fraction of inspired oxygen 0.30/mean airway pressure 7 definition yielded ventilator-associated condition rates of 1.1-1.3 per 1,000 ventilator days. All definitions were significantly associated with greater risk of hospital death, with hazard ratios ranging from 1.6 (95% CI, 0.7-3.4) to 6.8 (2.9-16.0), depending on thresholds and ICU type. Each definition was associated with prolonged hospitalization, time in ICU, and duration of ventilation, among survivors. The advisory board of the study proposed using the fraction of inspired oxygen 0.25/mean airway pressure 4 thresholds to identify pediatric ventilator-associated conditions in ICUs. CONCLUSIONS: Pediatric patients with ventilator-associated conditions are at substantially higher risk for mortality and morbidity across ICUs, regardless of thresholds used. Next steps include identification of risk factors, etiologies, and preventative measures for pediatric ventilator-associated conditions.

OBJECTIVES: Contamination of workplace surfaces by antineoplastic drugs presents an exposure risk for healthcare workers. Traditional instrumental methods to detect contamination such as gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS) are sensitive and accurate but expensive and incapable of producing results in real time. This limits their utility in preventing worker exposure. We are currently developing monitors based on lateral flow immunoassay that can detect drug contamination in near real time. In this report, we describe the laboratory performance of a 5-fluorouracil (5-FU) monitor. METHODS: The monitor was evaluated by spiking ceramic, vinyl, composite, stainless steel, and glass surfaces of 100 cm2 area with 5-FU masses of 0, 5, 10, 25, 50, and 100 ng. The surface was sampled with a wetted cotton swab, the swab was extracted with buffer, and the resulting solution was applied to a lateral flow monitor. Two ways of evaluating the response of these monitors were used: an electronic method where a lateral flow reader was used for measuring line intensities, and a visual method where the intensity of the test line was visually compared to the control line. RESULTS: The 5-FU monitor is capable of detecting 10 ng/100 cm2 (0.1 ng/cm2) using the electronic reader and 25 ng/100 cm2 (0.25 ng/cm2) using the visual comparison method for the surfaces studied. The response of the monitors was compared to LC-MS/MS results for the same samples for validation and there was good correlation of the two methods but some differences in absolute response, especially at higher spiking levels for the surface samples.

Legionella pneumophila is the leading etiology of legionellosis infections in North America and Europe. Here we report the draft genome sequence of L. pneumophila D-5864, a serogroup 6 strain, which was isolated from a bronchial alveolar lavage specimen of a male patient from Arizona in 2009. Genes within the lipopolysaccharide (LPS)-biosynthesis region could potentially be determinants of serogroup specificity.

Whole genome sequencing of Cryptosporidium spp. is hampered by difficulties in obtaining sufficient, highly pure genomic DNA from clinical specimens. In this study, we developed procedures for the isolation and enrichment of Cryptosporidium genomic DNA from fecal specimens and verification of DNA purity for whole genome sequencing. The isolation and enrichment of genomic DNA were achieved by a combination of three oocyst purification steps and whole genome amplification (WGA) of DNA from purified oocysts. qPCR analysis of WGA products was used as an initial quality assessment of amplified genomic DNA. The purity of WGA products was assessed by Sanger sequencing of cloned products. Next generation sequencing tools were used in final evaluations of genome coverage and extent of contamination. Altogether, 24 fecal specimens of Cryptosporidium parvum, C. hominis, C. andersoni, C. ubiquitum, C. tyzzeri, and Cryptosporidium chipmunk genotype I were processed with the procedures. As expected, WGA products with low Ct values (<16.0) yielded mostly Cryptosporidium sequences in Sanger sequencing. The cloning-sequencing analysis, however, showed significant contamination in 5 WGA products (percentage of positive colonies derived from Cryptosporidium genomic DNA ≤ 25%). Following this strategy, 20 WGA products from six Cryptosporidium species/genotypes with low Ct values (mostly <14.0) were submitted to whole genome sequencing, generating sequence data covering 94.5-99.7% Cryptosporidium genomes, with mostly minor contamination from bacterial, fungal, and host DNA. These results suggest that the described strategy can be used effectively for the isolation and enrichment of Cryptosporidium DNA from fecal specimens for whole genome sequencing.

OBJECTIVES: Contamination of workplace surfaces by antineoplastic drugs presents an exposure risk for healthcare workers. Traditional instrumental methods to detect contamination such as liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) are sensitive and accurate but expensive. Since immunochemical methods may be cheaper and faster than instrumental methods, we wanted to explore their use for routine drug residue detection for preventing worker exposure. METHODS: In this study we examined the feasibility of using fluorescence covalent microbead immunosorbent assay (FCMIA) for simultaneous detection and semi-quantitative measurement of three antineoplastic drugs (5-fluorouracil, paclitaxel, and doxorubicin). The concentration ranges for the assay were 0-1000 ng/ml for 5-fluorouracil, 0-100 ng/ml for paclitaxel, and 0-2 ng/ml for doxorubicin. The surface sampling technique involved wiping a loaded surface with a swab wetted with wash buffer, extracting the swab in storage/blocking buffer, and measuring drugs in the extract using FCMIA. RESULTS: There was no significant cross-reactivity between these drugs at the ranges studied indicated by a lack of response in the assay to cross analytes. The limit of detection (LOD) for 5-fluorouracil on the surface studied was 0.93 ng/cm2 with a limit of quantitation (LOQ) of 2.8 ng/cm2, the LOD for paclitaxel was 0.57 ng/cm2 with an LOQ of 2.06 ng/cm2, and the LOD for doxorubicin was 0.0036 ng/cm2 with an LOQ of 0.013 ng/cm2. CONCLUSION: The use of FCMIA with a simple sampling technique has potential for low cost simultaneous detection and semi-quantitative measurement of surface contamination from multiple antineoplastic drugs.

Three vancomycin-resistant streptococcal strains carrying vanG elements (two invasive Streptococcus agalactiae isolates [GBS-NY and GBS-NM, both serotype II and multilocus sequence type 22] and one Streptococcus anginosus [Sa]) were examined. The 45,585-bp elements found within Sa and GBS-NY were nearly identical (together designated vanG-1) and shared near-identity over an ~15-kb overlap with a previously described vanG element from Enterococcus faecalis. Unexpectedly, vanG-1 shared much less homology with the 49,321-bp vanG-2 element from GBS-NM, with widely different levels (50% to 99%) of sequence identity shared among 44 related open reading frames. Immediately adjacent to both vanG-1 and vanG-2 were 44,670-bp and 44,680-bp integrative conjugative element (ICE)-like sequences, designated ICE-r, that were nearly identical in the two group B streptococcal (GBS) strains. The dual vanG and ICE-r elements from both GBS strains were inserted at the same position, between bases 1328 and 1329, within the identical RNA methyltransferase (rumA) genes. A GenBank search revealed that although most GBS strains contained insertions within this specific site, only sequence type 22 (ST22) GBS strains contained highly related ICE-r derivatives. The vanG-1 element in Sa was also inserted within this position corresponding to its rumA homolog adjacent to an ICE-r derivative. vanG-1 insertions were previously reported within the same relative position in the E. faecalis rumA homolog. An ICE-r sequence perfectly conserved with respect to its counterpart in GBS-NY was apparent within the same site of the rumA homolog of a Streptococcus dysgalactiae subsp. equisimilis strain. Additionally, homologous vanG-like elements within the conserved rumA target site were evident in Roseburia intestinalis. IMPORTANCE: These three streptococcal strains represent the first known vancomycin-resistant strains of their species. The collective observations made from these strains reveal a specific hot spot for insertional elements that is conserved between streptococci and different Gram-positive species. The two GBS strains potentially represent a GBS lineage that is predisposed to insertion of vanG elements.

Shiga toxin-producing Escherichia coli (STEC) is an important food-borne pathogen. Here, we report the draft whole-genome sequences of nine STEC strains isolated from clinical cases in the United States. This is the first report of such information for STEC of serotypes O69, H11, O145:H25, O118:H16, O91:H21, O146:H21, O45:H2, O128:H2, and O121:H19.