Case-case analyses of cryptosporidiosis and giardiasis using routine national surveillance data in the United States - 2005-2015.

Understanding endemic infectious disease risk factors through traditional epidemiological tools is challenging. Population-based case-control studies are costly and time-consuming. A case-case analyses using surveillance data addresses these limitations by using resources more efficiently. We conducted a case-case analyses using routine surveillance data reported by 16 U.S. states (2005-2015), wherein reported cases of salmonellosis were used as a comparison group to identify exposure associations with reported cases of cryptosporidiosis and giardiasis. Odds ratios adjusted for age and reporting state (aOR) and 95% confidence intervals (95% CI) were calculated. A total of 10 704 cryptosporidiosis cases, 17 544 giardiasis cases, and 106 351 salmonellosis cases were included in this analyses. When compared with cases of salmonellosis, exposure to treated recreational water (aOR 4.7, 95% CI 4.3-5.0) and livestock (aOR: 3.2; 95% CI: 2.9-3.5) were significantly associated with cryptosporidiosis and exposure to untreated drinking (aOR 4.1, 95% CI 3.6-4.7) and recreational water (aOR 4.1, 95% CI 3.7-4.5) were associated with giardiasis. Our analyses shows that routine surveillance data with standardised exposure information can be used to identify associations of interest for cryptosporidiosis and giardiasis.

Risk factors for sporadic Giardia infection in the USA: a case-control study in Colorado and Minnesota.

Giardia duodenalis is the most common intestinal parasite of humans in the USA, but the risk factors for sporadic (non-outbreak) giardiasis are not well described. The Centers for Disease Control and Prevention and the Colorado and Minnesota public health departments conducted a case-control study to assess risk factors for sporadic giardiasis in the USA. Cases (N = 199) were patients with non-outbreak-associated laboratory-confirmed Giardia infection in Colorado and Minnesota, and controls (N = 381) were matched by age and site. Identified risk factors included international travel (aOR = 13.9; 95% CI 4.9-39.8), drinking water from a river, lake, stream, or spring (aOR = 6.5; 95% CI 2.0-20.6), swimming in a natural body of water (aOR = 3.3; 95% CI 1.5-7.0), male-male sexual behaviour (aOR = 45.7; 95% CI 5.8-362.0), having contact with children in diapers (aOR = 1.6; 95% CI 1.01-2.6), taking antibiotics (aOR = 2.5; 95% CI 1.2-5.0) and having a chronic gastrointestinal condition (aOR = 1.8; 95% CI 1.1-3.0). Eating raw produce was inversely associated with infection (aOR = 0.2; 95% CI 0.1-0.7). Our results highlight the diversity of risk factors for sporadic giardiasis and the importance of non-international-travel-associated risk factors, particularly those involving person-to-person transmission. Prevention measures should focus on reducing risks associated with diaper handling, sexual contact, swimming in untreated water, and drinking untreated water.

Giardiasis outbreaks in the United States, 1971-2011.

Giardia intestinalis is the leading parasitic aetiology of human enteric infections in the United States, with an estimated 1·2 million cases occurring annually. To better understand transmission, we analysed data on all giardiasis outbreaks reported to the Centers for Disease Control and Prevention for 1971-2011. The 242 outbreaks, affecting ~41 000 persons, resulted from waterborne (74·8%), foodborne (15·7%), person-to-person (2·5%), and animal contact (1·2%) transmission. Most (74·6%) waterborne outbreaks were associated with drinking water, followed by recreational water (18·2%). Problems with water treatment, untreated groundwater, and distribution systems were identified most often during drinking water-associated outbreak investigations; problems with water treatment declined after the 1980s. Most recreational water-associated outbreaks were linked to treated swimming venues, with pools and wading pools implicated most often. Produce was implicated most often in foodborne outbreaks. Additionally, foods were most commonly prepared in a restaurant and contaminated by a food handler. Lessons learned from examining patterns in outbreaks over time can help prevent future disease. Groundwater and distribution system vulnerabilities, inadequate pool disinfection, fruit and vegetable contamination, and poor food handler hygiene are promising targets for giardiasis prevention measures.

Evolving epidemiology of reported cryptosporidiosis cases in the United States, 1995-2012.

Cryptosporidium is the leading aetiology of waterborne disease outbreaks in the United States. This report briefly describes the temporal and geographical distribution of US cryptosporidiosis cases and presents analyses of cryptosporidiosis case data reported in the United States for 1995-2012. The Cochran-Armitage test was used to assess changes in the proportions of cases by case status (confirmed vs. non-confirmed), sex, race, and ethnicity over the study period. Negative binomial regression models were used to estimate rate ratios (RR) and 95% confidence intervals (CI) for comparing rates across three time periods (1995-2004, 2005-2008, 2009-2012). The proportion of confirmed cases significantly decreased (P < 0·0001), and a crossover from male to female predominance in case-patients occurred (P < 0·0001). Overall, compared to 1995-2004, rates were higher in 2005-2008 (RR 2·92, 95% CI 2·08-4·09) and 2009-2012 (RR 2·66, 95% CI 1·90-3·73). However, rate changes from 2005-2008 to 2009-2012 varied by age group (P interaction < 0·0001): 0-14 years (RR 0·55, 95% CI 0·42-0·71), 15-44 years (RR 0·99, 95% CI 0·82-1·19), 45-64 years (RR 1·47, 95% CI 1·21-1·79) and ⩾65 years (RR 2·18, 95% CI 1·46-3·25). The evolving epidemiology of cryptosporidiosis necessitates further identification of risk factors in population subgroups. Adding systematic molecular typing of Cryptosporidium specimens to US national cryptosporidiosis surveillance would help further identify risk factors and markedly expand understanding of cryptosporidiosis epidemiology in the United States.

Communitywide cryptosporidiosis outbreak associated with a surface water-supplied municipal water system--Baker City, Oregon, 2013.

Cryptosporidium, a parasite known to cause large drinking and recreational water outbreaks, is tolerant of chlorine concentrations used for drinking water treatment. Human laboratory-based surveillance for enteric pathogens detected a cryptosporidiosis outbreak in Baker City, Oregon during July 2013 associated with municipal drinking water. Objectives of the investigation were to confirm the outbreak source and assess outbreak extent. The watershed was inspected and city water was tested for contamination. To determine the community attack rate, a standardized questionnaire was administered to randomly sampled households. Weighted attack rates and confidence intervals (CIs) were calculated. Water samples tested positive for Cryptosporidium species; a Cryptosporidium parvum subtype common in cattle was detected in human stool specimens. Cattle were observed grazing along watershed borders; cattle faeces were observed within watershed barriers. The city water treatment facility chlorinated, but did not filter, water. The community attack rate was 28·3% (95% CI 22·1-33·6), sickening an estimated 2780 persons. Watershed contamination by cattle probably caused this outbreak; water treatments effective against Cryptosporidium were not in place. This outbreak highlights vulnerability of drinking water systems to pathogen contamination and underscores the need for communities to invest in system improvements to maintain multiple barriers to drinking water contamination.

Neutralization of mitochondrial superoxide by superoxide dismutase 2 promotes bacterial clearance and regulates phagocyte numbers in zebrafish.

Mitochondria are known primarily as the location of the electron transport chain and energy production in cells. More recently, mitochondria have been shown to be signaling centers for apoptosis and inflammation. Reactive oxygen species (ROS) generated as by-products of the electron transport chain within mitochondria significantly impact cellular signaling pathways. Because of the toxic nature of ROS, mitochondria possess an antioxidant enzyme, superoxide dismutase 2 (SOD2), to neutralize ROS. If mitochondrial antioxidant enzymes are overwhelmed during severe infections, mitochondrial dysfunction can occur and lead to multiorgan failure or death. Pseudomonas aeruginosa is an opportunistic pathogen that can infect immunocompromised patients. Infochemicals and exotoxins associated with P. aeruginosa are capable of causing mitochondrial dysfunction. In this work, we describe the roles of SOD2 and mitochondrial ROS regulation in the zebrafish innate immune response to P. aeruginosa infection. sod2 is upregulated in mammalian macrophages and neutrophils in response to lipopolysaccharide in vitro, and sod2 knockdown in zebrafish results in an increased bacterial burden. Further investigation revealed that phagocyte numbers are compromised in Sod2-deficient zebrafish. Addition of the mitochondrion-targeted ROS-scavenging chemical MitoTEMPO rescues neutrophil numbers and reduces the bacterial burden in Sod2-deficient zebrafish. Our work highlights the importance of mitochondrial ROS regulation by SOD2 in the context of innate immunity and supports the use of mitochondrion-targeted ROS scavengers as potential adjuvant therapies during severe infections.

Thermal and Residual Excited-State Population in a 3D Transmon Qubit.

Remarkable advancements in coherence and control fidelity have been achieved in recent years with cryogenic solid-state qubits. Nonetheless, thermalizing such devices to their milliKelvin environments has remained a long-standing fundamental and technical challenge. In this context, we present a systematic study of the first-excited-state population in a 3D transmon superconducting qubit mounted in a dilution refrigerator with a variable temperature. Using a modified version of the protocol developed by Geerlings et al., we observe the excited-state population to be consistent with a Maxwell-Boltzmann distribution, i.e., a qubit in thermal equilibrium with the refrigerator, over the temperature range 35-150 mK. Below 35 mK, the excited-state population saturates at approximately 0.1%. We verified this result using a flux qubit with ten times stronger coupling to its readout resonator. We conclude that these qubits have effective temperature T(eff)=35 mK. Assuming T(eff) is due solely to hot quasiparticles, the inferred qubit lifetime is 108 µs and in plausible agreement with the measured 80 µs.

Phase-dependent resetting of the adrenal clock by ACTH in vitro.

The adrenal cortex has a molecular clock that generates circadian rhythms in glucocorticoids, yet how the clock is synchronized to the external environment is unknown. Using mPER2::Luciferase (mPER2Luc) knockin mice, in which luciferase is rhythmically expressed under the control of the mouse Per2 clock gene, we hypothesized that ACTH transmits entrainment signals to the adrenal. Adrenal explants were administered ACTH at different phases of the mPER2Luc rhythm. Treatment with ACTH 1-39 produced a phase delay that was phase-dependent, with a maximum at circadian time (CT)18; ACTH did not alter the period or amplitude of the rhythm. Forskolin produced a parallel response, suggesting that the phase delay was cAMP-mediated. The response to ACTH was concentration-dependent and peptide-specific. Pulse administration (60 min) of ACTH 1-39 also produced phase delays restricted to late CTs. In contrast to ACTH 1-39, other ACTH fragments, including α-melanocyte-stimulating hormone, which do not activate the melanocortin 2 (MC2/ACTH) receptor, had no effect. The finding that ACTH in vitro phase delays the adrenal mPER2luc rhythm in a monophasic fashion argues for ACTH as a key resetter, but not the sole entrainer, of the adrenal clock.

Chronic subordination stress phase advances adrenal and anterior pituitary clock gene rhythms.

Circadian rhythms in glucocorticoids are the product of interactions between the hypothalamic-pituitary-adrenal (HPA) axis and the mammalian clock gene system. The adrenal clock can generate the glucocorticoid rhythm that in turn synchronizes other peripheral clocks to maintain homeostasis. Stress acutely activates and chronically upregulates the HPA axis, suggesting that the adrenal clock could be modulated by stress. However, there is no direct evidence that stress affects the adrenal clock rhythm. We tested the hypothesis that a model of chronic subordination stress (CSS) that has a major impact on HPA axis regulation, metabolism, and emotional behavior alters adrenal and pituitary clock gene rhythms. Clock gene rhythms were assessed using mPER2::Luciferase (PER2Luc) knockin mice in which in vitro bioluminescence rhythms reflect the Per2 clock gene expression. PER2Luc mice that experienced CSS for 2 wk showed positive energy balance reflected by increased body weight and food intake. Additionally, CSS phase advanced the adrenal (∼2 h) and the pituitary (∼1 h) PER2Luc rhythm compared with control mice. The activity rhythm was not affected. The adrenal clock phase shift was associated with increased feed conversion efficiency, suggesting that the metabolic phenotype in CSS mice may be related to altered adrenal clock rhythmicity. Interestingly, a single subordination experience followed by 8 h sensory housing also phase advanced the adrenal, but not the pituitary, PER2Luc rhythm. Overall, these data demonstrate a stress-induced phase shift in a peripheral clock gene rhythm and differential stress sensitivity of two peripheral clocks within the HPA axis, suggesting a link between clock desynchrony and individual vulnerability to stress.

Polarization enhancement technique for nuclear quadrupole resonance detection.

We demonstrate a dramatic increase in the signal-to-noise ratio (SNR) of a nuclear quadrupole resonance (NQR) signal by using a polarization enhancement technique. By first applying a static magnetic field to pre-polarize one spin subsystem of a material, and then allowing that net polarization to be transferred to the quadrupole subsystem, we increased the SNR of a sample of ammonium nitrate by one-order of magnitude.

Case definitions, diagnostic algorithms, and priorities in encephalitis: consensus statement of the international encephalitis consortium.

BACKGROUND: Encephalitis continues to result in substantial morbidity and mortality worldwide. Advances in diagnosis and management have been limited, in part, by a lack of consensus on case definitions, standardized diagnostic approaches, and priorities for research. METHODS: In March 2012, the International Encephalitis Consortium, a committee begun in 2010 with members worldwide, held a meeting in Atlanta to discuss recent advances in encephalitis and to set priorities for future study. RESULTS: We present a consensus document that proposes a standardized case definition and diagnostic guidelines for evaluation of adults and children with suspected encephalitis. In addition, areas of research priority, including host genetics and selected emerging infections, are discussed. CONCLUSIONS: We anticipate that this document, representing a synthesis of our discussions and supported by literature, will serve as a practical aid to clinicians evaluating patients with suspected encephalitis and will identify key areas and approaches to advance our knowledge of encephalitis.

Effects of gene flow on phenotype matching between two varieties of Joshua tree (Yucca brevifolia; Agavaceae) and their pollinators.

In animal-pollinated plants, local adaptation to pollinator behaviour or morphology can restrict gene flow among plant populations; but gene flow may also prevent divergent adaptation. Here, we examine possible effects of gene flow on plant-pollinator trait matching in two varieties of Joshua tree (Agavaceae: Yucca brevifolia). The two varieties differ in strikingly in floral morphology, which matches differences in the morphology of their pollinators. However, this codivergence is not present at a smaller scale: within the two varieties of Joshua tree, variation in floral morphology between demes is not correlated with differences in moth morphology. We use population genetic data for Joshua tree and its pollinators to test the hypotheses that gene flow between Joshua tree populations is structured by pollinator specificity, and that gene flow within the divergent plant-pollinator associations 'swamps' fine-scale coadaptation. Our data show that Joshua tree populations are structured by pollinator association, but the two tree varieties are only weakly isolated - meaning that their phenotypic differences are maintained in the face of significant gene flow. Coalescent analysis of gene flow between the two Joshua tree types suggests that it may be shaped by asymmetric pollinator specificity, which has been observed in a narrow zone of sympatry. Finally, we find evidence suggesting that gene flow among Joshua tree sites may shape floral morphology within one plant-pollinator association, but not the other.

Common source outbreaks of Campylobacter infection in the USA, 1997-2008.

Campylobacter is a common but decreasing cause of foodborne infections in the USA. Outbreaks are uncommon and have historically differed from sporadic cases in seasonality and contamination source. We reviewed reported outbreaks of campylobacteriosis. From 1997 to 2008, 262 outbreaks were reported, with 9135 illnesses, 159 hospitalizations, and three deaths. The annual mean was 16 outbreaks for 1997-2002, and 28 outbreaks for 2003-2008. Almost half occurred in warmer months. Foodborne transmission was reported in 225 (86%) outbreaks, water in 24 (9%), and animal contact in seven (3%). Dairy products were implicated in 65 (29%) foodborne outbreaks, poultry in 25 (11%), and produce in 12 (5%). Reported outbreaks increased during a period of declining overall incidence, and seasonality of outbreaks resembled that of sporadic infections. Unlike sporadic illnesses, which are primarily attributed to poultry, dairy products are the most common vehicle identified for outbreaks.

Application of qRT-PCR and RNA-Seq analysis for the identification of housekeeping genes useful for normalization of gene expression values during Striga hermonthica development.

Striga is a root parasitic weed that attacks many of the staple crops in Africa, India and Southeast Asia, inflicting tremendous losses in yield and for which there are few effective control measures. Studies of parasitic plant virulence and host resistance will be greatly facilitated by the recent emergence of genomic resources that include extensive transcriptome sequence datasets spanning all life stages of S. hermonthica. Functional characterization of Striga genes will require detailed analyses of gene expression patterns. Quantitative real-time PCR is a powerful tool for quantifying gene expression, but correct normalization of expression levels requires identification of control genes that have stable expression across tissues and life stages. Since no S. hermonthica housekeeping genes have been established for this purpose, we evaluated the suitability of six candidate housekeeping genes across key life stages of S. hermonthica from seed conditioning to flower initiation using qRT-PCR and high-throughput cDNA sequencing. Based on gene expression analysis by qRT-PCR and RNA-Seq across heterogeneous Striga life stages, we determined that using the combination of three genes, UBQ1, PP2A and TUB1 provides the best normalization for gene expression throughout the parasitic life cycle. The housekeeping genes characterized here provide robust standards that will facilitate powerful descriptions of parasite gene expression patterns.

Off-host aggregation in the non-fed, female brown dog tick, Rhipicephalus sanguineus (Latreille), is induced by tick excreta and enhanced by low relative humidity.

We report that Rhipicephalus sanguineus (Ixodida: Ixodidae) faeces and its main component, guanine, act as assembly pheromones in short-range Petri plate bioassays. Arrestment activity in response to guanine was lower than that in response to natural excreta, indicating the presence of other active ingredients in natural excreta. The selective removal of appendages was used to establish the important roles played by the palps and the front pair of legs in the detection of the pheromone. Reaction to chemically pure guanine at varying concentrations occurred without a dose response; thus only the presence of guanine, not a critical amount, is required to induce assembly. Higher speed and intensity of clustering occurred at 33% relative humidity (RH). We conclude that female adults of R. sanguineus are more prone to assemble under dry conditions that match the arid microhabitats preferred by this species and that this tendency allows this tick to reside in human dwellings and dog kennels that maintain standards of comfort at 30-50% RH. Cleaning or removing tick excreta-covered surfaces on which ticks aggregate from within and around human dwellings may prove useful as a means of interfering with the establishment of off-host clusters of R. sanguineus.

The Cyclin Cln1 Controls Polyploid Titan Cell Formation following a Stress-Induced G2 Arrest in Cryptococcus.

The pathogenic yeast Cryptococcus neoformans produces polyploid titan cells in response to the host lung environment that are critical for host adaptation and subsequent disease. We analyzed the in vivo and in vitro cell cycles to identify key aspects of the C. neoformans cell cycle that are important for the formation of titan cells. We identified unbudded 2C cells, referred to as a G2 arrest, produced both in vivo and in vitro in response to various stresses. Deletion of the nonessential cyclin Cln1 resulted in overproduction of titan cells in vivo and transient morphology defects upon release from stationary phase in vitro. Using a copper-repressible promoter PCTR4-CLN1 strain and a two-step in vitro titan cell formation assay, our in vitro studies revealed Cln1 functions after the G2 arrest. These studies highlight unique cell cycle alterations in C. neoformans that ultimately promote genomic diversity and virulence in this important fungal pathogen. IMPORTANCE Dysregulation of the cell cycle underlies many human genetic diseases and cancers, yet numerous organisms, including microbes, also manipulate the cell cycle to generate both morphologic and genetic diversity as a natural mechanism to enhance their chances for survival. The eukaryotic pathogen Cryptococcus neoformans generates morphologically distinct polyploid titan cells critical for host adaptation and subsequent disease. We analyzed the C. neoformans in vivo and in vitro cell cycles to identify changes required to generate the polyploid titan cells. C. neoformans paused cell cycle progression in response to various environmental stresses after DNA replication and before morphological changes associated with cell division, referred to as a G2 arrest. Release from this G2 arrest was coordinated by the cyclin Cln1. Reduced CLN1 expression after the G2 arrest was associated with polyploid titan cell production. These results demonstrate a mechanism to generate genomic diversity in eukaryotic cells through manipulation of the cell cycle that has broad disease implications.

ATI-2307 Exhibits Equivalent Antifungal Activity in Cryptococcus neoformans Clinical Isolates With High and Low Fluconazole IC50.

Half maximal inhibitory concentrations (IC50) to the experimental drug ATI-2307 and complete inhibition (IC90) of the common clinically used antifungal drug amphotericin B were determined by microbroth dilution assay for a collection of 69 clinical isolates of Cryptococcus neoformans from Uganda that had high fluconazole IC50 values. The majority of the clinical isolates tested had fluconazole IC50 at or above 8 µg/mL, but were susceptible to both amphotericin B (IC90 ≤1 µg/mL) and ATI-2307 (IC50 ≤0.0312 µg/mL). No correlation between increased fluconazole minimum inhibitory concentration (MIC) and ATI-2307 or amphotericin B MIC was observed, suggesting that the cellular changes impacting fluconazole susceptibility did not impact the effectiveness of ATI-2307. Our results suggest that ATI-2307 is a promising new antifungal drug for use in the context of high fluconazole or other antifungal drug MICs and/or in combination drug therapy regimens.

Absence of population-level phenotype matching in an obligate pollination mutualism.

Coevolution is thought to promote evolutionary change between demes that ultimately results in speciation. If this is the case, then we should expect to see similar patterns of trait matching and phenotypic divergence between populations and between species in model systems for coevolution. As measures of divergence are frequently only available at one scale (population level or taxon level), this contention is rarely tested directly. Here, we use the case of co-divergence between different varieties of Joshua tree Yucca brevifolia (Agavaceae) and their obligate pollinators, two yucca moths (Tegeticula spp. Prodoxidae), to test for trait matching between taxa and among populations. Using model selection, we show that there is trait matching between mutualists at the taxon level, but once we account for differences between taxa, there is no indication of trait matching in local populations. This result differs from similar studies in other coevolving systems. We hypothesize that this discrepancy arises because coevolution in obligate mutualisms favours divergence less strongly than coevolution in other systems, such as host­parasite interactions.

Ecological opportunity and the origin of adaptive radiations.

Ecological opportunity--through entry into a new environment, the origin of a key innovation or extinction of antagonists--is widely thought to link ecological population dynamics to evolutionary diversification. The population-level processes arising from ecological opportunity are well documented under the concept of ecological release. However, there is little consensus as to how these processes promote phenotypic diversification, rapid speciation and adaptive radiation. We propose that ecological opportunity could promote adaptive radiation by generating specific changes to the selective regimes acting on natural populations, both by relaxing effective stabilizing selection and by creating conditions that ultimately generate diversifying selection. We assess theoretical and empirical evidence for these effects of ecological opportunity and review emerging phylogenetic approaches that attempt to detect the signature of ecological opportunity across geological time. Finally, we evaluate the evidence for the evolutionary effects of ecological opportunity in the diversification of Caribbean Anolis lizards. Some of the processes that could link ecological opportunity to adaptive radiation are well documented, but others remain unsupported. We suggest that more study is required to characterize the form of natural selection acting on natural populations and to better describe the relationship between ecological opportunity and speciation rates.

Host-associated divergence and incipient speciation in the yucca moth Prodoxus coloradensis (Lepidoptera: Prodoxidae) on three species of host plants.

A wide range of evolutionary processes have been implicated in the diversification of yuccas and yucca moths, which exhibit ecological relationships that extend from obligate plant-pollinator mutualisms to commensalist herbivory. Prodoxus coloradensis (Lepidoptera: Prodoxidae) is a yucca moth, which feeds on the flowering stalks of three Yucca species as larvae, but does not provide pollination service. To test for evidence of host-associated speciation, we examined the genetic structure of P. coloradensis using mitochondrial (cytochrome oxidase I) and nuclear (elongation factor 1 alpha) DNA sequence data. Multilocus coalescent simulations indicate that moths on different host plant species are characterized by recent divergence and low levels of effective migration, with large effective population sizes and considerable retention of shared ancestral polymorphism. Although geographical distance explains a proportion of the mitochondrial and nuclear DNA variation among moths on different species of Yucca, the effect of host specificity on genetic distance remains significant after accounting for spatial isolation. The results of this study indicate that differentiation within P. coloradensis is consistent with the evolution of incipient species affiliated with different host plants, potentially influenced by sex-biased dispersal and female philopatry.