BACKGROUND: Clinical trials initiated during emerging infectious disease outbreaks must quickly enroll participants to identify treatments to reduce morbidity and mortality. This may be at odds with enrolling a representative study population, especially when the population affected is undefined. METHODS: We evaluated the utility of the Centers for Disease Control and Prevention's COVID-19-Associated Hospitalization Surveillance Network (COVID-NET), the COVID-19 Case Surveillance System (CCSS), and 2020 United States (US) Census data to determine demographic representation in the 4 stages of the Adaptive COVID-19 Treatment Trial (ACTT). We compared the cumulative proportion of participants by sex, race, ethnicity, and age enrolled at US ACTT sites, with respective 95% confidence intervals, to the reference data in forest plots. RESULTS: US ACTT sites enrolled 3509 adults hospitalized with COVID-19. When compared with COVID-NET, ACTT enrolled a similar or higher proportion of Hispanic/Latino and White participants depending on the stage, and a similar proportion of African American participants in all stages. In contrast, ACTT enrolled a higher proportion of these groups when compared with US Census and CCSS. The proportion of participants aged ≥65 years was either similar or lower than COVID-NET and higher than CCSS and the US Census. The proportion of females enrolled in ACTT was lower than the proportion of females in the reference datasets. CONCLUSIONS: Although surveillance data of hospitalized cases may not be available early in an outbreak, they are a better comparator than US Census data and surveillance of all cases, which may not reflect the population affected and at higher risk of severe disease.

BACKGROUND: Although several therapeutic agents have been evaluated for the treatment of coronavirus disease 2019 (Covid-19), no antiviral agents have yet been shown to be efficacious. METHODS: We conducted a double-blind, randomized, placebo-controlled trial of intravenous remdesivir in adults who were hospitalized with Covid-19 and had evidence of lower respiratory tract infection. Patients were randomly assigned to receive either remdesivir (200 mg loading dose on day 1, followed by 100 mg daily for up to 9 additional days) or placebo for up to 10 days. The primary outcome was the time to recovery, defined by either discharge from the hospital or hospitalization for infection-control purposes only. RESULTS: A total of 1062 patients underwent randomization (with 541 assigned to remdesivir and 521 to placebo). Those who received remdesivir had a median recovery time of 10 days (95% confidence interval [CI], 9 to 11), as compared with 15 days (95% CI, 13 to 18) among those who received placebo (rate ratio for recovery, 1.29; 95% CI, 1.12 to 1.49; P<0.001, by a log-rank test). In an analysis that used a proportional-odds model with an eight-category ordinal scale, the patients who received remdesivir were found to be more likely than those who received placebo to have clinical improvement at day 15 (odds ratio, 1.5; 95% CI, 1.2 to 1.9, after adjustment for actual disease severity). The Kaplan-Meier estimates of mortality were 6.7% with remdesivir and 11.9% with placebo by day 15 and 11.4% with remdesivir and 15.2% with placebo by day 29 (hazard ratio, 0.73; 95% CI, 0.52 to 1.03). Serious adverse events were reported in 131 of the 532 patients who received remdesivir (24.6%) and in 163 of the 516 patients who received placebo (31.6%). CONCLUSIONS: Our data show that remdesivir was superior to placebo in shortening the time to recovery in adults who were hospitalized with Covid-19 and had evidence of lower respiratory tract infection. (Funded by the National Institute of Allergy and Infectious Diseases and others; ACTT-1 ClinicalTrials.gov number, NCT04280705.).

Plasmodium cynomolgi causes zoonotic malarial infections in Southeast Asia and this parasite species is important as a model for Plasmodium vivax and Plasmodium ovale. Each of these species produces hypnozoites in the liver, which can cause relapsing infections in the blood. Here we present methods and data generated from iterative longitudinal systems biology infection experiments designed and performed by the Malaria Host-Pathogen Interaction Center (MaHPIC) to delve deeper into the biology, pathogenesis, and immune responses of P. cynomolgi in the Macaca mulatta host. Infections were initiated by sporozoite inoculation. Blood and bone marrow samples were collected at defined timepoints for biological and computational experiments and integrative analyses revolving around primary illness, relapse illness, and subsequent disease and immune response patterns. Parasitological, clinical, haematological, immune response, and -omic datasets (transcriptomics, proteomics, metabolomics, and lipidomics) including metadata and computational results have been deposited in public repositories. The scope and depth of these datasets are unprecedented in studies of malaria, and they are projected to be a F.A.I.R., reliable data resource for decades.

BACKGROUND: Two suspected shellfish poisoning events were reported in Cuddalore District in Tamil Nadu, India, between January and April 2015. OBJECTIVES: The study was conducted to confirm the outbreaks and to identify the source and risk factors. METHODS: For both outbreaks, a case was defined as a person with nausea, vomiting, or dizziness. Sociodemographic details and symptoms were noted down. Data were also collected in a standard 3-day food frequency questionnaire, along with a collection of clam samples. A case-control study was initiated in the April outbreak. Stool samples were collected from cases, and clam vendors were interviewed. RESULTS: In an outbreak that happened in January, all the twenty people reported to be consumed clams were diagnosed as cases (100% attack rate, 100% exposure rate). In the April outbreak, we identified 199 cases (95% attack rate). In both outbreaks, the clams were identified as genus Meretrix meretrix. The most common reported symptoms were dizziness and vomiting. The clams heated and consumed within 30-60 min. No heavy metals or chemicals were detected in the clams, but assays for testing shellfish toxins were unavailable. All 64 selected cases reported clam consumption (100% exposure rate) as did 11 controls (17% exposure rate). Illness was associated with a history of eating of clams (odds ratio = 314, 95% confidence interval = 39-512). Of the six stool samples tested, all were culture negative for Salmonella, Shigella, and Vibrio cholerae. The water at both sites was contaminated with garbage and sewage. CONCLUSION: Coordinated and timely efforts by a multidisciplinary team of epidemiologists, marine biologists, and food safety officers led to the outbreaks' containment.

BACKGROUND: Simultaneous infection with multiple malaria parasite strains is common in high transmission areas. Quantifying the number of strains per host, or the multiplicity of infection (MOI), provides additional parasite indices for assessing transmission levels but it is challenging to measure accurately with current tools. This paper presents new laboratory and analytical methods for estimating the MOI of Plasmodium falciparum. METHODS: Based on 24 single nucleotide polymorphisms (SNPs) previously identified as stable, unlinked targets across 12 of the 14 chromosomes within P. falciparum genome, three multiplex PCRs of short target regions and subsequent next generation sequencing (NGS) of the amplicons were developed. A bioinformatics pipeline including B4Screening pathway removed spurious amplicons to ensure consistent frequency calls at each SNP location, compiled amplicons by SNP site diversity, and performed algorithmic haplotype and strain reconstruction. The pipeline was validated by 108 samples generated from cultured-laboratory strain mixtures in different proportions and concentrations, with and without pre-amplification, and using whole blood and dried blood spots (DBS). The pipeline was applied to 273 smear-positive samples from surveys conducted in western Kenya, then providing results into StrainRecon Thresholding for Infection Multiplicity (STIM), a novel MOI estimator. RESULTS: The 24 barcode SNPs were successfully identified uniformly across the 12 chromosomes of P. falciparum in a sample using the pipeline. Pre-amplification and parasite concentration, while non-linearly associated with SNP read depth, did not influence the SNP frequency calls. Based on consistent SNP frequency calls at targeted locations, the algorithmic strain reconstruction for each laboratory-mixed sample had 98.5% accuracy in dominant strains. STIM detected up to 5 strains in field samples from western Kenya and showed declining MOI over time (q < 0.02), from 4.32 strains per infected person in 1996 to 4.01, 3.56 and 3.35 in 2001, 2007 and 2012, and a reduction in the proportion of samples with 5 strains from 57% in 1996 to 18% in 2012. CONCLUSION: The combined approach of new multiplex PCRs and NGS, the unique bioinformatics pipeline and STIM could identify 24 barcode SNPs of P. falciparum correctly and consistently. The methodology could be applied to field samples to reliably measure temporal changes in MOI.

Reactivation of Chagas disease has been described in immunosuppressed patients, but there is a paucity of literature describing reactivation in patients on immunosuppressive therapies for the treatment of autoimmune rheumatic diseases. We describe a case of Chagas disease reactivation in a woman taking azathioprine and prednisone for limited cutaneous systemic sclerosis (lcSSc). Reactivation manifested as indurated and erythematous cutaneous nodules. Sequencing of a skin biopsy specimen confirmed the diagnosis of Chagas disease. She was treated with benznidazole with clinical improvement in the cutaneous lesions. However, her clinical course was complicated and included disseminated CMV disease and subsequent septic shock due to bacteremia. Our case and review of the literature highlight that screening for Chagas disease should be strongly considered for patients who will undergo immunosuppression for treatment of autoimmune disease if epidemiologically indicated.

Within the last decade there has been a significant expansion in access to cannabis for medicinal and adult nonmedical use in the United States and abroad. This has resulted in a rapidly growing and diverse workforce that is involved with the growth, cultivation, handling, and dispensing of the cannabis plant and its products. The objective of this review was to educate physicians on the complexities associated with the health effects of cannabis exposure, the nature of these exposures, and the future practical challenges of managing these in the context of allergic disease. We will detail the biological hazards related to typical modern cannabis industry operations that may potentially drive allergic sensitization in workers. We will highlight the limitations that have hindered the development of objective diagnostic measures that are essential in separating “true” cannabis allergies from nonspecific reactions/irritations that “mimic” allergy-like symptoms. Finally, we will discuss recent advances in the basic and translational scientific research that will aid the development of diagnostic tools and therapeutic standards to serve optimal management of cannabis allergies across the occupational spectrum.

Cannabis is the most commonly used psychoactive drug. In recent years, Cannabis access has expanded for both medicinal and non-medicinal has grown. This is also marked with an increasing number of individuals gaining employment in this emerging industry. In this article, we briefly discuss the health hazards associated with Cannabis exposure with an emphasis on the potential for allergic reactions in workers who handle and process Cannabis plant.

Stachybotrys chartarum is a fungal contaminant within the built environment and a respiratory health concern in the United States. The objective of this study was to characterize the mechanisms influencing pulmonary immune responses to repeatedly inhaled S. chartarum. Groups of B6C3F1/N mice repeatedly inhaled viable trichothecene-producing S. chartarum conidia (Strain A or Strain B), heat-inactivated conidia, or HEPA-filtered air twice a week for 4 and 13 weeks. Strain A was found to produce higher amounts of respirable fragments compared to Strain B. Lung tissue, serum and bronchoalveolar lavage fluid (BALF) were collected at 24 and 48 hours following final exposure and processed for histology, flow cytometry, RNA, and proteomic analyses. At 4 weeks post-exposure, a Th2-mediated response was observed. After 13 weeks, a mixed T-cell response was observed following exposure to Strain A, compared to a Th2-mediated response following Strain B exposure. Following exposure, both strains induced pulmonary arterial remodeling at 13 weeks; however, Strain A-exposed mice progressed more quickly compared to Strain B-exposed mice. BALF was composed primarily of eosinophils, neutrophils, and macrophages. Both the immune response and the observed pulmonary arterial remodeling were supported by specific cellular, molecular, and proteomic profiles. The immunopathological responses occurred earlier in mice exposed to high fragment-producing Strain A. The rather striking induction of pulmonary remodeling by S. chartarum appears to be related to the presence of fungal fragments during exposure.

BACKGROUND: Ultra-Deep Sequencing (UDS) enabled identification of specific changes in human genome occurring in malignant tumors, with current approaches calling for the detection of specific mutations associated with certain cancers. However, such associations are frequently idiosyncratic and cannot be generalized for diagnostics. Mitochondrial DNA (mtDNA) has been shown to be functionally associated with several cancer types. Here, we study the association of intra-host mtDNA diversity with Hepatocellular Carcinoma (HCC). RESULTS: UDS mtDNA exome data from blood of patients with HCC (n = 293) and non-cancer controls (NC, n = 391) were used to: (i) measure the genetic heterogeneity of nucleotide sites from the entire population of intra-host mtDNA variants rather than to detect specific mutations, and (ii) apply machine learning algorithms to develop a classifier for HCC detection. Average total entropy of HCC mtDNA is 1.24-times lower than of NC mtDNA (p = 2.84E-47). Among all polymorphic sites, 2.09% had a significantly different mean entropy between HCC and NC, with 0.32% of the HCC mtDNA sites having greater (p < 0.05) and 1.77% of the sites having lower mean entropy (p < 0.05) as compared to NC. The entropy profile of each sample was used to further explore the association between mtDNA heterogeneity and HCC by means of a Random Forest (RF) classifier The RF-classifier separated 232 HCC and 232 NC patients with accuracy of up to 99.78% and average accuracy of 92.23% in the 10-fold cross-validation. The classifier accurately separated 93.08% of HCC (n = 61) and NC (n = 159) patients in a validation dataset that was not used for the RF parameter optimization. CONCLUSIONS: Polymorphic sites contributing most to the mtDNA association with HCC are scattered along the mitochondrial genome, affecting all mitochondrial genes. The findings suggest that application of heterogeneity profiles of intra-host mtDNA variants from blood may help overcome barriers associated with the complex association of specific mutations with cancer, enabling the development of accurate, rapid, inexpensive and minimally invasive diagnostic detection of cancer.

Background: Cutaneous anthrax in humans is associated with exposure to infected animals or animal products and has a case fatality rate of up to 20% if untreated. During May to June 2015, an outbreak of cutaneous anthrax was reported in Koraput district of Odisha, India, an area endemic for anthrax. We investigated the outbreak to identify risk factors and recommend control measures. Method: We defined a cutaneous anthrax case as skin lesions (e.g., papule, vesicle or eschar) in a person residing in Koraput district with illness onset between February 1 and July 15, 2015. We established active surveillance through a house to house survey to ascertain additional cases and conducted a 1:2 unmatched case control study to identify modifiable risk factors. In case control study, we included cases with illness onset between May 1 and July 15, 2015. We defined controls as neighbours of case without skin lesions since last 3 months. Ulcer exudates and rolled over swabs from wounds were processed in Gram stain in the Koraput district headquarter hospital laboratory. Result: We identified 81 cases (89% male; median age 38 years [range 5-75 years]) including 3 deaths (case fatality rate = 4%). Among 37 cases and 74 controls, illness was significantly associated with eating meat of ill cattle (OR: 14.5, 95% CI: 1.4-85.7) and with close handling of carcasses of ill animals such as burying, skinning, or chopping (OR: 342, 95% CI: 40.5-1901.8). Among 20 wound specimens collected, seven showed spore-forming, gram positive bacilli, with bamboo stick appearance suggestive of Bacillus anthracis. Conclusion: Our investigation revealed significant associations between eating and handling of ill animals and presence of anthrax-like organisms in lesions. We immediately initiated livestock vaccination in the area, educated the community on safe handling practices and recommended continued regular anthrax animal vaccinations to prevent future outbreaks.

Legalization of medicinal and recreational cannabis use in numerous states within the USA has resulted in the increased commercial cultivation of cannabis. Outdoor cannabis farming operations present a variety of potential physical, chemical, and biological hazards that currently remain uncharacterized. Worker exposures to endotoxins were evaluated at an outdoor US cannabis farm during harvesting and processing activities. Endotoxin area air sample concentrations ranged from below the limit of detection to 15 endotoxin units per cubic meter (EU/m3). Endotoxin breathing zone measurements (2.8–37 EU/m3) were below the Dutch Expert Committee on Occupational Safety occupational exposure limit of 90 /m3. During confidential medical interviews, no adverse health effects were reported by workers while harvesting or processing cannabis. Further endotoxin exposure assessments should be performed especially in larger, indoor cannabis operations where a confined environment may result in higher endotoxin exposures than observed in this outdoor environment.

Mumps, a vaccine-preventable disease, causes inflammation of salivary glands and may cause severe complications such as encephalitis, meningitis, deafness, and orchitis/oophoritis. In India, mumps vaccine is not included in the universal immunization program and during 2009-2014, 72 outbreaks with >1500 cases were reported. In August 2016, a suspected mumps outbreak was reported in Jaisalmer block, Rajasthan. We investigated to confirm the etiology, describe epidemiology, and recommend prevention and control measures. We defined a case as swelling in the parotid region in a Jaisalmer block resident between June 23 and September 10, 2016. We searched for cases in health facilities and house-to-house in affected villages and hamlets. We tested blood samples of cases for mumps IgM ELISA. We found 162 cases (60% males) with median age of 9.4 years (range: 7 month-38 years) and 65 (40%) were females. Symptoms included fever (70%) and bilateral swelling in neck (65%). None were vaccinated against mumps. Most (84%) cases were school-going children (3-16 years old). The overall attack rate was 2%. Village A, with two hamlets, had the highest attack rate (hamlet 1=13% and hamlet 2=12%). School A of village A, hamlet 1 which accommodated 200 children in two classrooms had an attack rate of 55%. Of 18 blood samples from cases, 11 tested positive for mumps IgM ELISA. This was a confirmed mumps outbreak in Jaisalmer bl0ock that disproportionately affected school-going children. We recommended continued surveillance, five-day absence from school, and vaccination. This article is protected by copyright. All rights reserved.

BACKGROUND/OBJECTIVE: Few studies have assessed the effect of ambient heat during the fetal development period on congenital heart defects (CHDs), especially in transitional seasons. We examined and compared the associations between extreme heat and CHD phenotypes in summer and spring, assessed their geographical differences, and compared different heat indicators. METHODS: We identified 5848 CHD cases and 5742 controls (without major structural defects) from the National Birth Defects Prevention Study, a US multicenter, population-based case-control study. Extreme heat events (EHEs) were defined by using the 95th (EHE95) or 90th (EHE90) percentile of daily maximum temperature and its frequency and duration during postconceptional weeks 3-8. We used a two-stage Bayesian hierarchical model to examine both regional and study-wide associations. Exposure odds ratios (ORs) were calculated using multivariate logistic regression analyses, while controlling for potential confounding factors. RESULTS: Overall, we observed no significant relationships between maternal EHE exposure and CHDs in most regions during summer. However, we found that 3-11days of EHE90 during summer and spring was significantly associated with ventricular septal defects (VSDs) study-wide (ORs ranged: 2.17-3.24). EHE95 in spring was significantly associated with conotruncal defects and VSDs in the South (ORs: 1.23-1.78). Most EHE indicators in spring were significantly associated with increased septal defects (both VSDs and atrial septal defects (ASDs)) in the Northeast. CONCLUSION: While generally null results were found, long duration of unseasonable heat was associated with the increased risks for VSDs and ASDs, mainly in South and Northeast of the US. Further research to confirm our findings is needed.

BACKGROUND: Aspergillus fumigatus induced allergic airway disease has been shown to involve conidial germination in vivo but the immunological mechanisms remain uncharacterized. OBJECTIVE: A subchronic murine exposure model was used to examine the immunological mediators that are regulated in response to either culturable or non-culturable A. fumigatus conidia. METHODS: Female B6C3F1/N mice were repeatedly dosed via inhalation with 1 x 105 viable or heat inactivated conidia (HIC), twice a week for 13 weeks (26 exposures). Control mice inhaled HEPA-filtered air. The influence of A. fumigatus conidial germination on the pulmonary immunopathological outcomes was evaluated by flow cytometry analysis of cellular infiltration in the airways, assessment of lung mRNA expression, and quantitative proteomics and histopathology of whole lung tissue. RESULTS: Repeated inhalation of viable conidia, but not HIC, resulted in allergic inflammation marked by vascular remodeling, extensive eosinophilia, and accumulation of alternatively activated macrophages (AAMs) in the murine airways. More specifically, mice that inhaled viable conidia resulted in a mixed TH1 and TH2 (IL-13) cytokine response. Recruitment of eosinophils corresponded with increased Ccl11 transcripts. Furthermore, genes associated with M2 or alternatively activated macrophage polarization (e.g. Arg1, Chil3 and Retnla) were significantly upregulated in viable A. fumigatus exposed mice. In mice inhaling HIC, CD4+ T cells expressing IFN-gamma (TH1) dominated the lymphocytic infiltration. Quantitative proteomics of the lung revealed metabolic reprogramming accompanied by mitochondrial dysfunction and endoplasmic reticulum stress stimulated by oxidative stress from repetitive microbial insult. CONCLUSION: Our studies demonstrate that A. fumigatus conidial viability in vivo is critical to the immunopathological presentation of chronic fungal allergic disease.

Cannabis cultivation is an emerging industry within the United States. Organic dust derived in part from naturally occurring microorganisms is known to cause byssinosis in the hemp industry. In this pilot study, bacteria and fungi encountered by workers at an outdoor cannabis farm that utilized organic practices were elucidated by 1625% shaded blockS ribosomal RNA (rRNA) and Internal Transcribed Spacer (ITS) region sequencing, respectively. Area (n = 14) and personal air samples (n = 12) were collected during harvesting and processing activities. 1625% shaded blockS rRNA and ITS regions of extracted bacterial and fungal genomic DNA were amplified and sequenced using Sanger sequencing. Bacterial sequencing resolved 1077 sequences that were clustered into 639 operational taxonomic units (OTUs) and predominantly placed in the phylum, Actinobacteria (46%). Personal air samples revealed higher bacterial and Actinobacteria diversity compared to outdoor area samples collected within the facility (p<0.05). A high degree of dissimilarity between bacteria was identified within and between samples. Fungal sequences (n = 985) were identified and predominantly clustered in the phylum Ascomycota (53%). Of the 216 fungal OTUs elucidated, the cannabis plant pathogenic species, Botrytis cinerea, was the most prevalent and accounted for 34% of all fungal sequences. The relative abundance of B. cinerea was highest in personal air samples (59%) compared to area samples collected in the drying room (19%), greenhouse (18%) and outdoor environment (6%). There was 49% sample similarity between fungi identified within personal air samples, but higher dissimilarity coefficients were observed within and between greenhouse, drying room, and outdoor area air samples. The results of this pilot study suggest that the cannabis farm workers are potentially exposed to Actinobacteria as well as the cannabis plant pathogen, B. cinerea during harvesting, bud stripping, and hand trimming processes.

Cooling towers (CTs) are a leading source of outbreaks of Legionnaires' disease (LD), a severe form of pneumonia caused by inhalation of aerosols containing Legionella bacteria. Accordingly, proper maintenance of CTs is vital for the prevention of LD. The aim of this study was to determine the distribution of Legionella in a subset of regionally diverse US CTs and characterize the associated microbial communities. Between July and September of 2016, we obtained aliquots from water samples collected for routine Legionella testing from 196 CTs located in eight of the nine continental US climate regions. After screening for Legionella by PCR, positive samples were cultured and the resulting Legionella isolates were further characterized. Overall, 84% (164) were PCR-positive, including samples from every region studied. Of the PCR-positive samples, Legionella spp were isolated from 47% (78), L. pneumophila was isolated from 32% (53), and L. pneumophila serogroup 1 (Lp1) was isolated from 24% (40). Overall, 144 unique Legionella isolates were identified; 53% (76) of these were Legionella pneumophila. Of the 76 L. pneumophila isolates, 51% (39) were Lp1. Legionella were isolated from CTs in seven of the eight US regions examined. 16S rRNA amplicon sequencing was used to compare the bacterial communities of CT waters with and without detectable Legionella as well as the microbiomes of waters from different climate regions. Interestingly, the microbial communities were homogenous across climate regions. When a subset of seven CTs sampled in April and July were compared, there was no association with changes in corresponding CT microbiomes over time in the samples that became culture-positive for Legionella. Legionella species and Lp1 were detected frequently among the samples examined in this first large-scale study of Legionella in US CTs. Our findings highlight that, under the right conditions, there is the potential for CT-related LD outbreaks to occur throughout the US.

Plasmodium malariae is a protozoan parasite that can cause human malaria. The simian parasite Plasmodium brasilianum infects New World monkeys from Latin America and is morphologically indistinguishable from P. malariae Here, we report the first full draft genome sequence for P. brasilianum.

The anti-microbial compound triclosan is incorporated into numerous consumer products and is detectable in the urine of 75% of the general United States population. Recent epidemiological studies report positive associations with urinary triclosan levels and allergic disease. Although not sensitizing, earlier studies previously found that repeated topical application of triclosan augments the allergic response to ovalbumin (OVA) though a thymic stromal lymphopoietin (TSLP) pathway in mice. In the present study, early immunological effects following triclosan exposure were further evaluated following topical application in a murine model. These investigations revealed abundant expression of S100A8/A9, which reportedly acts as an endogenous ligand for Toll-like Receptor 4 (TLR4), in skin tissues and in infiltrating leukocytes during topical application of 0.75-3.0% triclosan. Expression of Tlr4 along with Tlr1, Tlr2 and Tlr6 increased in skin tissues over time with triclosan exposure; high levels of TLR4 were expressed on skin-infiltrating leukocytes. In vivo antibody blockade of the TLR4/MD-2 receptor complex impaired local inflammatory responses after four days, as evidenced by decreased Il6, Tnfalpha, S100a8, S100a9, Tlr1, Tlr2, Tlr4 and Tlr6 expression in the skin and decreased lymph node cellularity and production of IL-4 and IL-13 by lymph node T-cells. After nine days of triclosan exposure with TLR4/MD-2 blockade, impaired T-helper cell type 2 (TH2) cytokine responses were sustained, but other early effects on skin and lymph node cellularity were lost; this suggested alternative ligands/receptors compensated for the loss of TLR4 signaling. Taken together, these data suggest the S100A8/A9-TLR4 pathway plays an early role in augmenting immunomodulatory responses with triclosan exposure and support a role for the innate immune system in chemical adjuvancy.

Toluene diisocyanate (TDI) is a potent low molecular weight chemical sensitizer and a leading cause of chemical-induced occupational asthma. The regulatory potential of microRNAs (miRNAs) has been recognized in a variety of disease states, including allergic disease; however, the roles of miRNAs in chemical sensitization are largely unknown. In a previous work, increased expression of multiple miRNAs during TDI sensitization was observed and several putative mRNA targets identified for these miRNAs were directly related to regulatory T-cell (Treg) differentiation and function including Foxp3 and Runx3. In this work, we show that miR-210 expression is increased in the mouse draining lymph node (dLN) and Treg subsets following dermal TDI sensitization. Alterations in dLN mRNA and protein expression of Treg related genes/putative miR-210 targets (foxp3, runx3, ctla4, and cd25) were observed at multiple time points following TDI exposure and in ex vivo systems. A Treg suppression assay, including a miR-210 mimic, was utilized to investigate the suppressive ability of Tregs. Cells derived from TDI sensitized mice treated with miR-210 mimic had less expression of miR-210 compared to the acetone control suggesting other factors, such as additional miRNAs, might be involved in the regulation of the functional capabilities of these cells. These novel findings indicate that miR-210 may have an inhibitory role in Treg function during TDI sensitization. Because the functional roles of miRNAs have not been previously elucidated in a model of chemical sensitization, these data contribute to the understanding of the potential immunologic mechanisms of chemical induced allergic disease.

BACKGROUND: Personal exposure to fungal bioaerosols derived from contaminated building materials or agricultural commodities may induce or exacerbate a variety of adverse health effects. The genomic mechanisms that underlie pulmonary immune responses to fungal bioaerosols have remained unclear. OBJECTIVE: The impact of fungal viability on the pulmonary microRNA and messenger RNA profiles that regulate murine immune responses was evaluated following subchronic inhalation exposure to Aspergillus fumigatus conidia. METHODS: Three groups of naive B6C3F1/N mice were exposed via nose-only inhalation to A. fumigatus viable conidia, heat-inactivated conidia, or HEPA-filtered air twice a week for 13 weeks. Total RNA was isolated from whole lung 24 and 48 hours post final exposure and was further processed for gene expression and microRNA array analysis. The molecular network pathways between viable and heat-inactivated conidia groups were evaluated. RESULTS: Comparison of datasets revealed increased Il4, Il13, and Il33 expression in mice exposed to viable versus heat-inactivated conidia. Of 415 microRNAs detected, approximately 50% were altered in mice exposed to viable versus heat-inactivated conidia 48 hours post exposure. Significantly downregulated (P < 0.05) miR-29a-3p was predicted to regulate TGF-beta3 and Clec7a, genes involved in innate responses to viable A. fumigatus. Also significantly downregulated (P < 0.05), miR-23b-3p regulates genes involved in pulmonary IL-13 and IL-33 responses and SMAD2, downstream of TGF-beta signaling. Using Ingenuity Pathway Analysis, a novel interaction was identified between viable conidia and SMAD2/3. CONCLUSION AND CLINICAL RELEVANCE: Examination of the pulmonary genetic profiles revealed differentially expressed genes and microRNAs following subchronic inhalation exposure to A. fumigatus. MicroRNAs regulating genes involved in the pulmonary immune responses were those with the greatest fold change. Specifically, germinating A. fumigatus conidia were associated with Clec7a and were predicted to interact with Il13 and Il33. Furthermore, altered microRNAs may serve as potential biomarkers to evaluate fungal exposure.

BACKGROUND: Epidemiological surveys indicate that occupants of mold contaminated environments are at increased risk of respiratory symptoms. The immunological mechanisms associated with these responses require further characterization. OBJECTIVE: The aim of this study was to characterize the immunotoxicological outcomes following repeated inhalation of dry Aspergillus fumigatus spores aerosolized at concentrations potentially encountered in contaminated indoor environments. METHODS: A. fumigatus spores were delivered to the lungs of naive BALB/cJ mice housed in a multi-animal nose-only chamber twice a week for a period of 13 weeks. Mice were evaluated at 24 and 48 hours post-exposure for histopathological changes in lung architecture, recruitment of specific immune cells to the airways, and serum antibody responses. RESULT: Germinating A. fumigatus spores were observed in lungs along with persistent fungal debris in the perivascular regions of the lungs. Repeated exposures promoted pleocellular infiltration with concomitant epithelial mucus hypersecretion, goblet cell metaplasia, subepithelial fibrosis and enhanced airway hyperreactivity. Cellular infiltration in airways was predominated by CD4+ T cells expressing the pro-allergic cytokine IL-13. Furthermore, our studies show that antifungal T cell responses (IFN-gamma+ or IL-17A+ ) co-expressed IL-13, revealing a novel mechanism for the dysregulated immune response to inhaled fungi. Total IgE production was augmented in animals repeatedly exposed to A. fumigatus. CONCLUSIONS & CLINICAL RELEVANCE: Repeated inhalation of fungal aerosols resulted in significant pulmonary pathology mediated by dynamic shifts in specific immune populations and their cytokines. These studies provide novel insights into the immunological mechanisms and targets that govern the health outcomes that result from repeated inhalation of fungal bioaerosols in contaminated environments. This article is protected by copyright. All rights reserved.

Diisocyanates (dNCOs) are low molecular weight chemical sensitizers that react with autologous proteins to produce neoantigens. dNCO-haptenated proteins have been used as immunogens for generation of dNCO-specific antibodies and as antigens to screen for dNCO-specific antibodies in exposed individuals. Detection of dNCO-specific antibodies in exposed individuals for diagnosis of dNCO asthma has been hampered by poor sensitivities of the assay methods in that specific IgE can only be detected in approximately 25% of the dNCO asthmatics. Apart from characterization of the conjugates used for these immunoassays, the choice of the carrier protein and the dNCO used are important parameters that can influence the detection of dNCO-specific antibodies. Human serum albumin (HSA) is the most common carrier protein used for detection of dNCO specific-IgE and -IgG but the immunogenicity and/or antigenicity of other proteins that may be modified by dNCO in vivo is not well documented. In the current study, 2,4-toluene diisocyanate (TDI) and 1,6-hexamethylene diisocyanate (HDI) were reacted with HSA and human hemoglobin (Hb) and the resultant adducts were characterized by (i) HPLC quantification of the diamine produced from acid hydrolysis of the adducts, (ii) 2,4,6-trinitrobenzene sulfonic acid (TNBS) assay to assess extent of cross-linking, (iii) electrophoretic migration in polyacrylamide gels to analyze intra- and inter-molecular cross-linking, and (iv) evaluation of antigenicity using a monoclonal antibody developed previously to TDI conjugated to Keyhole limpet hemocyanin (KLH). Concentration-dependent increases in the amount of dNCO bound to HDI and TDI, cross-linking, migration in gels, and antibody-binding were observed. TDI reactivity with both HSA and Hb was significantly higher than HDI. Hb-TDI antigenicity was approximately 30% that of HSA-TDI. In conclusion, this data suggests that both, the extent of haptenation as well as the degree of cross-linking differs between the two diisocyanate species studied, which may influence their relative immunogenicity and/or antigenicity.

BACKGROUND: The Polio Endgame strategy includes phased withdrawal of oral poliovirus vaccines (OPV) coordinated with introduction of inactivated poliovirus vaccine (IPV) to ensure population immunity. The impact of IPV introduction into a primary OPV series of immunizations in a developing country is uncertain. METHODS: Between May 2011 and November 2012, we enrolled 700 Bangladeshi infant-mother dyads from Dhaka slums into an open-label randomized controlled trial to test whether substituting an injected IPV dose for the standard Expanded Program on Immunization (EPI) fourth tOPV dose at infant age 39 weeks would reduce fecal shedding and enhance systemic immunity. The primary endpoint was mucosal immunity to poliovirus at age one year, measured by fecal excretion of any Sabin virus at five time points up to 25 days post-52 week tOPV challenge, analyzed by the intention to treat principle. FINDINGS: We randomized 350 families to the tOPV and IPV vaccination arms. Neither study arm resulted in superior intestinal protection at 52 weeks measured by the prevalence of infants shedding any of three poliovirus serotypes, but the IPV dose induced significantly higher seroprevalence and seroconversion rates. This result was identical for poliovirus detection by cell culture or RT-qPCR. The non-significant estimated culture-based shedding risk difference was -3% favoring IPV, and the two vaccination schedules were inferred to be equivalent within a 95% confidence margin of -10% to +4%. Results for shedding analyses stratified by poliovirus type were similar. CONCLUSIONS: Neither of the vaccination regimens is superior to the other in enhancing intestinal immunity as measured by poliovirus shedding at 52 weeks of age and the IPV regimen provides similar intestinal immunity to the four tOPV series, although the IPV regimen strongly enhances humoral immunity. The IPV-modified regimen may be considered for vaccination programs without loss of intestinal protection.

The parasite Cyclospora cayetanensis causes foodborne diarrheal illness. Here, we report draft genome sequences obtained from C. cayetanensis oocysts purified from a human stool sample. The genome assembly consists of 865 contigs with a total length of 44,563,857 bases. These sequences can facilitate the development of subtyping tools to aid outbreak investigations.

Background: Environmental enteropathy (EE) is a subclinical enteric condition found in low-income countries that is characterized by intestinal inflammation, reduced intestinal absorption, and gut barrier dysfunction. We aimed to assess if EE impairs the success of oral polio and rotavirus vaccines in infants in Bangladesh. Methods: We conducted a prospective observational study of 700 infants from an urban slum of Dhaka, Bangladesh from May 2011 to November 2014. Infants were enrolled in the first week of life and followed to age one year through biweekly home visits with EPI vaccines administered and growth monitored. EE was operationally defied as enteric inflammation measured by any one of the fecal biomarkers reg1B, alpha-1-antitrypsin, MPO, calprotectin, or neopterin. Oral polio vaccine success was evaluated by immunogenicity, and rotavirus vaccine response was evaluated by immunogenicity and protection from disease. This study is registered with ClinicalTrials.gov, number NCT01375647. Findings: EE was present in greater than 80% of infants by 12. weeks of age. Oral poliovirus and rotavirus vaccines failed in 20.2% and 68.5% of the infants respectively, and 28.6% were malnourished (HAZ. <. -2) at one year of age. In contrast, 0%, 9.0%, 7.9% and 3.8% of infants lacked protective levels of antibody from tetanus, Haemophilus influenzae type b, diphtheria and measles vaccines respectively. EE was negatively associated with oral polio and rotavirus response but not parenteral vaccine immunogenicity. Biomarkers of systemic inflammation and measures of maternal health were additionally predictive of both oral vaccine failure and malnutrition. The selected biomarkers from multivariable analysis accounted for 46.3% variation in delta HAZ. 24% of Rotarix® IgA positive individuals can be attributed to the selected biomarkers. Interpretation: EE as well as systemic inflammation and poor maternal health were associated with oral but not parenteral vaccine underperformance and risk for future growth faltering. These results offer a potential explanation for the burden of these problems in low-income problems, allow early identification of infants at risk, and suggest pathways for intervention. Funding: The Bill and Melinda Gates Foundation (OPP1017093). © 2015 The Authors.

Submicronic fungal fragments have been observed in in vitro aerosolization experiments. The occurrence of these particles has therefore been suggested to contribute to respiratory health problems observed in mold contaminated indoor environments. However, the role of submicronic fragments in exacerbating adverse health effects has remained unclear due to limitations associated with detection methods. In the present study, we report the development of an indirect immunodetection assay that utilizes chicken polyclonal antibodies developed against spores from Aspergillus versicolor and high resolution field emission scanning electron microscopy (FESEM). Immunolabeling was performed with A. versicolor fragments immobilized and fixed onto poly-L-lysine coated polycarbonate filters. Ninety percent of submicronic fragments and 1-2 mum fragments, compared to 100% of >2 mum fragments generated from pure freeze dried mycelial fragments of A. versicolor, were positively labeled. In proof of concept experiments, air samples collected from moldy indoor environments were evaluated using the immunolabelling technique. Our results indicated that 13% of total collected particles were derived from fungi. This fraction comprises 79% of fragments that were detected by immunolabelling and 21% spore particles that were morphologically identified. The methods reported in this study, enable the enumeration of fungal particles, including submicronic fragments, in a complex heterogeneous environmental sample.

Triclosan is an antimicrobial chemical incorporated into many personal, medical and household products. 75% of the U.S. population has detectable levels of triclosan in their urine and although it is not typically considered a contact sensitizer, recent studies have begun to link triclosan exposure with augmented allergic disease. We examined the effects of dermal triclosan exposure on the skin and lymph nodes of mice and in a human skin model to identify mechanisms for augmenting allergic responses. Triclosan (0-3%) was applied topically at 24 hour intervals to the ear pinnae of OVA-sensitized BALB/c mice. Skin and draining lymph nodes were evaluated for cellular responses and cytokine expression over time. The effects of triclosan (0-0.75%) on cytokine expression in a human skin tissue model was also examined. Exposure to triclosan increased the expression of TSLP, IL-1beta and TNF-alpha in the skin with concomitant decreases in IL-25, IL-33 and IL-1alpha. Similar changes in TSLP, IL1B and IL33 expression occurred in human skin. Topical application of triclosan also increased draining lymph node cellularity consisting of activated CD86+GL-7+ B cells, CD80+CD86+ dendritic cells, GATA-3+OX-40+IL-4+IL-13+ Th2 cells and IL-17A+ CD4 T cells. In-vivo antibody blockade of TSLP reduced skin irritation, IL-1beta expression, lymph node cellularity, and Th2 responses augmented by triclosan. Repeated dermal exposure to triclosan induces TSLP expression in skin tissue as a potential mechanism for augmenting allergic responses.

Natural rubber latex (NRL) from Hevea brasiliensis (Euphorbiaceae) constitutes an important cause of allergy with distinct phenotypes depending on the sensitization route and profile of the patient.1 In health care workers NRL allergy is dominated by sensitization to the proteins Hev b 5, 6.01, 6.02, and 6.03.2 Because of the ubiquity of these proteins in the plant kingdom, patients frequently display cross-reactivity to various fruits and vegetables. In patients who have undergone multiple operations, such as those with spina bifida, cross-reactivity is rare because NRL allergy mainly results from sensitization to components without homologues in edible plants, namely Hev b 1 and Hev b 3.1 | However, NRL allergy has recently been confirmed in patients without clear sensitization to the components Hev b 1, 3, 5, 6, 7, 8, 9, 10, and 11.1 The exact explanation for this observation probably relates to the fact that the panel of recombinant proteins currently available for diagnostics does not cover the entire NRL IgE response. Although Beezhold et al3 identified the nonspecific lipid transfer protein (nsLTP) Hev b 12 from NRL as a potential cross-reactive panallergen more than a decade ago, literature about the clinical significance of Hev b 12 sensitization is limited, and no Hev b 12 sensitization was demonstrable in 25 children with latex allergy.4 Rihs et al5 observed Hev b 12–specific IgE (sIgE) reactivity in only 6 (12.5%) of 48 patients with fruit allergy but without symptoms on latex exposure. In these patients Hev b 12 sensitization seemed attributable to cross-reactive nsLTPs from different fruits and apparently of limited role in NRL allergy.

This work demonstrates that a full laboratory-quality immunoassay can be run on a smartphone accessory. This low-cost dongle replicates all mechanical, optical, and electronic functions of a laboratory-based enzyme-linked immunosorbent assay (ELISA) without requiring any stored energy; all necessary power is drawn from a smartphone. Rwandan health care workers used the dongle to test whole blood obtained via fingerprick from 96 patients enrolling into care at prevention of mother-to-child transmission clinics or voluntary counseling and testing centers. The dongle performed a triplexed immunoassay not currently available in a single test format: HIV antibody, treponemal-specific antibody for syphilis, and nontreponemal antibody for active syphilis infection. In a blinded experiment, health care workers obtained diagnostic results in 15 min from our triplex test that rivaled the gold standard of laboratory-based HIV ELISA and rapid plasma reagin (a screening test for syphilis), with sensitivity of 92 to 100% and specificity of 79 to 100%, consistent with needs of current clinical algorithms. Patient preference for the dongle was 97% compared to laboratory-based tests, with most pointing to the convenience of obtaining quick results with a single fingerprick. This work suggests that coupling microfluidics with recent advances in consumer electronics can make certain laboratory-based diagnostics accessible to almost any population with access to smartphones.