The Need for European Surveillance of CDI.

Since the turn of the millennium, the epidemiology of Clostridioides difficile infection (CDI) has continued to challenge. Changes in clinical presentation, severity of disease, descriptions of new risk factors and the occurrence of outbreaks all emphasised the importance of early diagnosis and standardised surveillance systems. However, a lack of consensus on case definitions, clinical guidelines and optimal laboratory diagnostics across Europe has led to the underestimation of CDI and impeded comparison between countries. These inconsistencies have prevented the true burden of disease from being appreciated.Acceptance that a multi-country CDI surveillance program and optimised diagnostic strategies are required has built the foundations for a more robust, unified surveillance. The concerted efforts of the European Centre for Disease Prevention and Control (ECDC) CDI networks led to the development of the European surveillance protocol and an over-arching long-term CDI surveillance strategy for 2014-2020, which has been followed by the development of surveillance systems in at least 20 European countries. However, surveillance activities in individual countries have slowed during the COVID-19 pandemic as resources were diverted to the global health crisis. A renewed and strengthened focus on CDI surveillance and prevention is therefore urgently needed post COVID-19.

An evaluation of PlayStreets in the South Side neighborhood of Columbus, Ohio.

AIMS: Outdoor play, physical activity, and social cohesion are crucial indicators of community health. PlayStreets, a street play initiative to engage local children and families in outdoor play, physical activity, and social interactions, were implemented in a low-income neighborhood in Columbus, Ohio throughout the summer of 2019. This article aims to describe the implementation of a hospital-sponsored PlayStreets model executed through support from a community health initiative and to assess neighborhood impact through parent and child surveys. METHODS: Approximately 350 children attended the events and 69 surveys were collected. Descriptive statistics were used to analyze survey data. RESULTS: The mean age of children was 7 years, and the majority of children who attended were male. If not for PlayStreets, 55% of caregivers reported that their children would be inside. Event satisfaction levels were high, and 54% of caregivers said that they had more contact with their neighbors because of the events. CONCLUSIONS: Hospital buy-in and community support were crucial to the success of the event. We found that this model can successfully engage the local community while increasing opportunity for childhood outdoor play, physical activity, and neighborhood social interaction.

Designing a mHealth clinical decision support system for Parkinson's disease: a theoretically grounded user needs approach.

BACKGROUND: Despite the established evidence and theoretical advances explaining human judgments under uncertainty, developments of mobile health (mHealth) Clinical Decision Support Systems (CDSS) have not explicitly applied the psychology of decision making to the study of user needs. We report on a user needs approach to develop a prototype of a mHealth CDSS for Parkinson's disease (PD), which is theoretically grounded in the psychological literature about expert decision making and judgement under uncertainty. METHODS: A suite of user needs studies was conducted in 4 European countries (Greece, Italy, Slovenia, the UK) prior to the development of PD_Manager, a mHealth-based CDSS designed for Parkinson's disease, using wireless technology. Study 1 undertook Hierarchical Task Analysis (HTA) including elicitation of user needs, cognitive demands and perceived risks/benefits (ethical considerations) associated with the proposed CDSS, through structured interviews of prescribing clinicians (N = 47). Study 2 carried out computational modelling of prescribing clinicians' (N = 12) decision strategies based on social judgment theory. Study 3 was a vignette study of prescribing clinicians' (N = 18) willingness to change treatment based on either self-reported symptoms data, devices-generated symptoms data or combinations of both. RESULTS: Study 1 indicated that system development should move away from the traditional silos of 'motor' and 'non-motor' symptom evaluations and suggest that presenting data on symptoms according to goal-based domains would be the most beneficial approach, the most important being patients' overall Quality of Life (QoL). The computational modelling in Study 2 extrapolated different factor combinations when making judgements about different questions. Study 3 indicated that the clinicians were equally likely to change the care plan based on information about the change in the patient's condition from the patient's self-report and the wearable devices. CONCLUSIONS: Based on our approach, we could formulate the following principles of mHealth design: 1) enabling shared decision making between the clinician, patient and the carer; 2) flexibility that accounts for diagnostic and treatment variation among clinicians; 3) monitoring of information integration from multiple sources. Our approach highlighted the central importance of the patient-clinician relationship in clinical decision making and the relevance of theoretical as opposed to algorithm (technology)-based modelling of human judgment.

Prediction model and web-based risk calculator for postoperative ileus after loop ileostomy closure.

BACKGROUND: Postoperative ileus (POI) is a significant complication after loop ileostomy closure given both its frequency and impact on the patient. The purpose of this study was to develop and externally validate a prediction model for POI after loop ileostomy closure. METHODS: The model was developed and validated according to the TRIPOD checklist for prediction model development and validation. The development cohort included consecutive patients who underwent loop ileostomy closure in two teaching hospitals in Montreal, Canada. Candidate variables considered for inclusion in the model were chosen a priori based on subject knowledge. The final prediction model, which modelled the 30-day cumulative incidence of POI using logistic regression, was selected using the highest area under the receiver operating characteristic curve (AUC) criterion. Model calibration was assessed using the Hosmer-Lemeshow goodness-of-fit test. The model was then validated externally in an independent cohort of similar patients from the University of British Columbia. RESULTS: The development cohort included 531 patients, in whom the incidence of POI was 16·8 per cent. The final model included five variables: age, ASA fitness grade, underlying pathology/treatment, interval between ileostomy creation and closure, and duration of surgery for ileostomy closure (AUC 0·68, 95 per cent c.i. 0·61 to 0·74). The model demonstrated good calibration (P = 0·142). The validation cohort consisted of 216 patients, and the incidence of POI was 15·7 per cent. On external validation, the model maintained good discrimination (AUC 0·72, 0·63 to 0·81) and calibration (P = 0·538). CONCLUSION: A prediction model was developed for POI after loop ileostomy closure and included five variables. The model maintained good performance on external validation.

ANTECEDENTES: El íleo postoperatorio (postoperative ileus, POI) es una complicación importante tras el cierre de la ileostomía en asa, dada su frecuencia e impacto en el paciente. El propósito de este estudio fue desarrollar y validar externamente un modelo de predicción para el POI después del cierre de la ileostomía en asa. MÉTODOS: El modelo fue desarrollado y validado de acuerdo con la lista de verificación TRIPOD para el desarrollo y validación de un modelo de predicción. La cohorte de desarrollo incluyó pacientes consecutivos en los que se realizó el cierre de la ileostomía en asa en dos hospitales universitarios en Montreal, Canadá. Las variables candidatas consideradas para su inclusión en el modelo se seleccionaron a priori en función del conocimiento del problema. El modelo de predicción final, que modeló la incidencia acumulada a 30 días de POI mediante regresión logística, se seleccionó según el criterio del área más alta bajo la curva operativa del receptor (area under the receiver operating curve, AUC). La calibración del modelo se evaluó utilizando la prueba de bondad de ajuste de Hosmer-Lemeshow. El modelo fue posteriormente validado externamente en una cohorte independiente de pacientes similares de la Universidad de British Columbia. RESULTADOS: La cohorte de desarrollo incluyó a 531 pacientes, y la incidencia de POI fue de 16,7%. El modelo final incluyó cinco variables: edad, clasificación ASA (American Society of Anaesthesiologists), patología inicial y tratamiento, tiempo entre las dos intervenciones quirúrgicas y tiempo operatorio del cierre de ileostomía (AUC = 0,68; i.c. del 95%: 0,61 a 0,74). El modelo demostró buena calibración (P = 0,142). La cohorte de validación consistió en 216 pacientes, y la incidencia de POI fue de 15,7%. En la validación externa, el modelo mantuvo una buena discriminación (AUC = 0,72; i.c. del 95%: 0,63 a 0,81) y calibración (P = 0,538). CONCLUSIÓN: Se ha desarrollado un modelo de predicción de POI después del cierre de la ileostomía en asa que incluía cinco variables. El modelo mantuvo un buen funcionamiento en la validación externa.

Risk of infection following semi-invasive ultrasound procedures in Scotland, 2010 to 2016: A retrospective cohort study using linked national datasets.

INTRODUCTION: Outbreak reports indicate a risk of cross-infection following medical procedures using semi-invasive ultrasound probes. This study aimed to evaluate the risk of infection, using microbiological reports and antibiotic prescriptions as proxy measures, associated with semi-invasive ultrasound probe procedures, including transoesophageal echocardiography, transvaginal and transrectal ultrasound. METHODS: Patient records from the Electronic Communication of Surveillance in Scotland and the Prescribing Information System were linked with the Scottish Morbidity Records for cases in Scotland between 2010 and 2016. Three retrospective cohorts were created to include inpatients/day-cases and outpatients in the following specialties: Cardiology, Gynaecology and Urology. Cox regression was used to quantify the association between semi-invasive ultrasound probe procedures and the risk of positive microbiological reports and community antibiotic prescriptions in the 30-day period following the procedure. RESULTS: There was a greater hazard ratio of microbiological reports for patients who had undergone transoesophageal echocardiography (HR: 4.92; 95% CI: 3.17-7.63), transvaginal (HR: 1.41; 95% CI: 1.21-1.64) and transrectal ultrasound (HR: 3.40; 95% CI: 2.90-3.99), compared with unexposed cohort members after adjustment for age, co-morbidities, previous hospital admissions and past care home residence. Similarly, there was a greater hazard ratio of antibiotic prescribing for those who had received transvaginal (HR: 1.26; 95% CI: 1.20-1.32) and transrectal (HR: 1.75; 95% CI: 1.66-1.84) ultrasound, compared with unexposed patients. CONCLUSION: Analysis of linked national datasets demonstrated a greater risk of infection within 30 days of undergoing semi-invasive ultrasound probe procedures, using microbiological reports and antibiotic prescriptions as proxy measures of infection.

Trends in mortality following Clostridium difficile infection in Scotland, 2010-2016: a retrospective cohort and case-control study.

BACKGROUND: National surveillance of Clostridium difficile infection (CDI) in Scotland enables the monitoring of trends in incidence rates but not mortality. AIM: To assess factors associated with mortality for all CDI cases aged ≥15 years in Scotland between 2010 and 2016. METHODS: All CDI cases aged ≥15 years in Scotland between 2010 and 2016 were linked to hospital admission and mortality datasets. Logistic regression was used to assess factors associated with mortality (30-day all-cause). A case-control study of a hospitalized subset of cases and matched hospitalized controls assessed the impact of CDI on mortality and length of stay. FINDINGS: Thirty-day all-cause mortality decreased over the seven-year period (from 20.5% to 15.6%; P < 0.001), mainly among healthcare-associated CDI (HA-CDI). Increased age, higher Charlson score, HA-CDI, as well as liver, heart and malignancy comorbidities were associated with higher mortality. No association was observed between polymerase chain reaction ribotype and higher mortality, though 015 and 078 were associated with lower mortality. Adjusted odds ratio (OR) for 30-day mortality in hospitalized CDI cases compared to controls was 2.67 (95% confidence interval (CI): 2.42-2.94; P < 0.001). Whereas mortality declined over time in cases and controls, the trend in ORs remained relatively stable. Having CDI increased additional mean length of stay beyond infection by 22.3% (95% CI: 18.0-26.8%; P < 0.001). CONCLUSION: CDI is associated with an almost three-fold increase in 30-day mortality and places an increased burden on hospital resources by increasing mean LOS beyond the infection date by 22.3%. The decreasing CDI mortality trends may be due to overall improvements in mortality among the general and hospital population of Scotland. Therefore, despite large declines in incidence rates, CDI remains a serious healthcare problem.

The Need for European Surveillance of CDI.

Since the turn of the millennium, the epidemiology of Clostridium difficile infection (CDI) has continued to challenge. Over the last decade there has been a growing awareness that improvements to surveillance are needed. The increasing rate of CDI and emergence of ribotype 027 precipitated the implementation of mandatory national surveillance of CDI in the UK. Changes in clinical presentation, severity of disease, descriptions of new risk factors and the occurrence of outbreaks all emphasised the importance of early diagnosis and surveillance.However a lack of consensus on case definitions, clinical guidelines and optimal laboratory diagnostics across Europe has lead to the underestimation of CDI and impeded comparison between countries. These inconsistencies have prevented the true burden of disease from being appreciated.Acceptance that a multi-country surveillance programme and optimised diagnostic strategies are required not only to detect and control CDI in Europe, but for a better understanding of the epidemiology, has built the foundations for a more robust, unified surveillance. The concerted efforts of the European Centre for Disease Prevention and Control (ECDC) CDI networks, has lead to the development of an over-arching long-term CDI surveillance strategy for 2014-2020. Fulfilment of the ECDC priorities and targets will no doubt be challenging and will require significant investment however the hope is that both a national and Europe-wide picture of CDI will finally be realised.

Pho4 mediates phosphate acquisition in Candida albicans and is vital for stress resistance and metal homeostasis.

During interactions with its mammalian host, the pathogenic yeast Candida albicans is exposed to a range of stresses such as superoxide radicals and cationic fluxes. Unexpectedly, a nonbiased screen of transcription factor deletion mutants revealed that the phosphate-responsive transcription factor Pho4 is vital for the resistance of C. albicans to these diverse stresses. RNA-Seq analysis indicated that Pho4 does not induce stress-protective genes directly. Instead, we show that loss of Pho4 affects metal cation toxicity, accumulation, and bioavailability. We demonstrate that pho4Δ cells are sensitive to metal and nonmetal cations and that Pho4-mediated polyphosphate synthesis mediates manganese resistance. Significantly, we show that Pho4 is important for mediating copper bioavailability to support the activity of the copper/zinc superoxide dismutase Sod1 and that loss of Sod1 activity contributes to the superoxide sensitivity of pho4Δ cells. Consistent with the key role of fungal stress responses in countering host phagocytic defenses, we also report that C. albicans pho4Δ cells are acutely sensitive to macrophage-mediated killing and display attenuated virulence in animal infection models. The novel connections between phosphate metabolism, metal homeostasis, and superoxide stress resistance presented in this study highlight the importance of metabolic adaptation in promoting C. albicans survival in the host.

Meals served to hypertensive and cardiac inpatients in Jordan: comparison with WHO and NIH dietary guidelines.

This study aimed to evaluate the compliance of daily meals served to hypertensive and cardiac inpatients in Jordan according to WHO guidelines and the Therapeutic Lifestyle Changes (TLC) and Dietary Approach to Stop Hypertension (DASH) diets plans. Weekly cycle menus from the food service department of major hospitals in Jordan (n = 16) were analysed using ESHA Food Processor software to obtain data about macroand micronutrient contents and food groups represented. The results showed inappropriate amounts of several nutrients in the menus provided, along with a general noncompliance with the DASH, TLC and WHO guidelines. Meals had higher than recommended sodium content coupled with low potassium content. Fatty acid profiles were often outside the recommended ranges. Meals provided to cardiac inpatients in Jordan need to be revised to meet the guidelines specified for the health conditions of these patients.

Occurrence and distribution of brominated flame retardants and perfluoroalkyl substances in Australian landfill leachate and biosolids.

The levels of perfluroalkyl substances (PFASs), polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecane (HBCDDs) were studied in Australian landfill leachate and biosolids. Leachate was collected from 13 landfill sites and biosolids were collected from 16 wastewater treatment plants (WWTPs), across Australia. Perfluorohexanoate (PFHxA) (12-5700ng/L) was the most abundant investigated persistent, bioaccumulative and toxic (PBT) chemical in leachate. With one exception, mean concentrations of PFASs were higher in leachate of operating landfills compared to closed landfills. Polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecane isomers (HBCDDs) were detected typically at operating landfills in comparatively lower concentrations than the PFASs. Decabromodiphenyl ether (BDE-209) (<0.4-2300ng/g) and perfluoroctanesulfonate (PFOS) (

The good, the bad and the tasty: The many roles of mushrooms.

Fungi are often inconspicuous in nature and this means it is all too easy to overlook their importance. Often referred to as the "Forgotten Kingdom", fungi are key components of life on this planet. The phylum Basidiomycota, considered to contain the most complex and evolutionarily advanced members of this Kingdom, includes some of the most iconic fungal species such as the gilled mushrooms, puffballs and bracket fungi. Basidiomycetes inhabit a wide range of ecological niches, carrying out vital ecosystem roles, particularly in carbon cycling and as symbiotic partners with a range of other organisms. Specifically in the context of human use, the basidiomycetes are a highly valuable food source and are increasingly medicinally important. In this review, seven main categories, or 'roles', for basidiomycetes have been suggested by the authors: as model species, edible species, toxic species, medicinal basidiomycetes, symbionts, decomposers and pathogens, and two species have been chosen as representatives of each category. Although this is in no way an exhaustive discussion of the importance of basidiomycetes, this review aims to give a broad overview of the importance of these organisms, exploring the various ways they can be exploited to the benefit of human society.

Sentinel community Clostridium difficile infection (CDI) surveillance in Scotland, April 2013 to March 2014.

Surveillance of Clostridium difficile infection (CDI) in Scotland does not currently distinguish between CDI cases from hospitals and the community. Therefore, the incidence of CDI in the community is unknown, and the burden of disease and the relationship with the hospital/healthcare setting is not well understood. A one-year sentinel community surveillance programme was initiated in collaboration with five Scottish health boards in 2013 (representing 36% of all CDI cases reported in Scotland). Inclusion criteria were all cases aged ≥15 years with a CDI diagnosis in the community or within 48 h following admission to hospital. CDI cases were categorised according to definitions used by the European Centre for Disease Prevention and Control. 256 CDI cases met the inclusion criteria, of which 158 (62%) were community-associated cases (CA-CDI). This represented 26% of all cases reported during the surveillance period by the participating health boards (n = 614). The overall CA-CDI incidence rate was 9.9 per 100 000 population per year. CA-CDI cases were more likely to be female and younger, compared to hospital acquired cases (HA-CDI). The total proportion of cases that had onset in the community was 27%. Ribotypes 015, 002, 078 and 005 were the most common types isolated from both CA-CDI and HA-CDI cases. There were no statistically significant differences between the proportion of types that were either CA-CDI or HA-CDI. Of the CA-CDI cases, 37% had not received antibiotics in the 12 weeks preceding CDI diagnosis, 4% were resident in care homes, and the case-fatality rate for CA-CDI cases was 5.6% (with a 30-day mortality rate for CA-CDI of 0.44 per 100 000 population per year). This study has shown that a substantial proportion of CDI cases reported in Scotland are community associated and that there are close links between the community and healthcare settings. It is therefore essential to monitor the trends in CDI in the community at a national level. The study also provides evidence for the need to examine the feasibility for development of interventions to reduce the burden in the community in addition to hospitals.

Genetic Networks Required to Coordinate Chromosome Replication by DNA Polymerases α, δ, and ε in Saccharomyces cerevisiae.

Three major DNA polymerases replicate the linear eukaryotic chromosomes. DNA polymerase α-primase (Pol α) and DNA polymerase δ (Pol δ) replicate the lagging-strand and Pol α and DNA polymerase ε (Pol ε) the leading-strand. To identify factors affecting coordination of DNA replication, we have performed genome-wide quantitative fitness analyses of budding yeast cells containing defective polymerases. We combined temperature-sensitive mutations affecting the three replicative polymerases, Pol α, Pol δ, and Pol ε with genome-wide collections of null and reduced function mutations. We identify large numbers of genetic interactions that inform about the roles that specific genes play to help Pol α, Pol δ, and Pol ε function. Surprisingly, the overlap between the genetic networks affecting the three DNA polymerases does not represent the majority of the genetic interactions identified. Instead our data support a model for division of labor between the different DNA polymerases during DNA replication. For example, our genetic interaction data are consistent with biochemical data showing that Pol ε is more important to the Pre-Loading complex than either Pol α or Pol δ. We also observed distinct patterns of genetic interactions between leading- and lagging-strand DNA polymerases, with particular genes being important for coupling proliferating cell nuclear antigen loading/unloading (Ctf18, Elg1) with nucleosome assembly (chromatin assembly factor 1, histone regulatory HIR complex). Overall our data reveal specialized genetic networks that affect different aspects of leading- and lagging-strand DNA replication. To help others to engage with these data we have generated two novel, interactive visualization tools, DIXY and Profilyzer.

Quantitative Fitness Analysis Identifies exo1∆ and Other Suppressors or Enhancers of Telomere Defects in Schizosaccharomyces pombe.

Synthetic genetic array (SGA) has been successfully used to identify genetic interactions in S. cerevisiae and S. pombe. In S. pombe, SGA methods use either cycloheximide (C) or heat shock (HS) to select double mutants before measuring colony size as a surrogate for fitness. Quantitative Fitness Analysis (QFA) is a different method for determining fitness of microbial strains. In QFA, liquid cultures are spotted onto solid agar and growth curves determined for each spot by photography and model fitting. Here, we compared the two S. pombe SGA methods and found that the HS method was more reproducible for us. We also developed a QFA procedure for S. pombe. We used QFA to identify genetic interactions affecting two temperature sensitive, telomere associated query mutations (taz1Δ and pot1-1). We identify exo1∆ and other gene deletions as suppressors or enhancers of S. pombe telomere defects. Our study identifies known and novel gene deletions affecting the fitness of strains with telomere defects. The interactions we identify may be relevant in human cells.

Antiviral efficacy of entecavir in nucleos(t)ide-naïve patients of Black/African descent with chronic hepatitis B.

This single-arm, open-label, descriptive study assessed the efficacy and safety of entecavir (ETV) in nucleos(t)ide-naïve Black/African American patients with chronic hepatitis B (CHB), a patient population underrepresented in ETV registration trials. Forty patients with HBeAg(+) or HBeAg(-) compensated CHB of self-described Black/African American race received ETV 0.5 mg daily for 52 weeks; 37 patients completed 52 weeks of treatment. At Week 48, 29/40 (72.5%, noncompleter = failure) patients achieved the primary endpoint of HBV DNA <50 IU/mL. Rates for HBeAg loss (11/22; 50%) and HBeAg seroconversion (9/22; 41%) were high, possibly due to the high HBV genotype A prevalence (70%). No patient experienced virological breakthrough. Samples for resistance testing were available in 6/8 patients with HBV DNA >50 IU/mL at Week 48 or last on-treatment visit. No ETV resistance was detected. The safety profile of ETV was consistent with that observed in ETV registration trials. This study shows that in Black/African American patients with CHB, ETV was well tolerated and demonstrated comparable antiviral efficacy to that observed in White and Asian patients in ETV Phase III studies.

Adductor longus ruptures in elite sportsmen--pitfalls of surgical repair: a report of two cases.

Insertional tendinopathy of the adductor longus is a common and problematic condition in elite athletes and may lead to rupture. Previous literature has reported good outcomes in these patients when these ruptures are treated with both surgical and non-operative management. This paper will discuss the available literature on management of adductor tears and describe two case reports of deep infection following surgical repair of ruptures in soccer players and the effects this had on their profession. We conclude that, given the overall evidence of conservative management of adductor tears and possible complications associated with the surgery that these cases highlight, acute adductor tears in the majority should be treated conservatively.

Pentose phosphate pathway function affects tolerance to the G-quadruplex binder TMPyP4.

G-quadruplexes form in guanine-rich regions of DNA and the presence of these structures at telomeres prevents the activity of telomerase in vitro. Ligands such as the cationic porphyrin TMPyP4 stabilise G-quadruplexes and are therefore under investigation for their potential use as anti-cancer drugs. In order to investigate the mechanism of action of TMPyP4 in vivo, we carried out a genome-wide screen in the budding yeast Saccharomyces cerevisiae. We found that deletion of key pentose phosphate pathway (PPP) genes increased the sensitivity of yeast to the presence of TMPyP4. The PPP plays an important role in the oxidative stress response and sensitivity to TMPyP4 also increased when genes involved in the oxidative stress response, CCS1 and YAP1, were deleted. For comparison we also report genome wide-screens using hydrogen peroxide, which causes oxidative stress, RHPS4, another G-quadruplex binder and hydroxyurea, an S phase poison. We found that a number of TMPyP4-sensitive strains are also sensitive to hydrogen peroxide in a genome-wide screen. Overall our results suggest that treatment with TMPyP4 results in light-dependent oxidative stress response in budding yeast, and that this, rather than G-quadruplex binding, is the major route to cytotoxicity. Our results have implications for the usefulness and mechanism of action of TMPyP4.

A quantitative fitness analysis workflow.

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel(1,2,3,4). QFA can be applied to focused observations of single cultures but is most useful for genome-wide genetic interaction or drug screens investigating up to thousands of independent cultures. The central experimental method is the inoculation of independent, dilute liquid microbial cultures onto solid agar plates which are incubated and regularly photographed. Photographs from each time-point are analyzed, producing quantitative cell density estimates, which are used to construct growth curves, allowing quantitative fitness measures to be derived. Culture fitnesses can be compared to quantify and rank genetic interaction strengths or drug sensitivities. The effect on culture fitness of any treatments added into substrate agar (e.g. small molecules, antibiotics or nutrients) or applied to plates externally (e.g. UV irradiation, temperature) can be quantified by QFA. The QFA workflow produces growth rate estimates analogous to those obtained by spectrophotometric measurement of parallel liquid cultures in 96-well or 200-well plate readers. Importantly, QFA has significantly higher throughput compared with such methods. QFA cultures grow on a solid agar surface and are therefore well aerated during growth without the need for stirring or shaking. QFA throughput is not as high as that of some Synthetic Genetic Array (SGA) screening methods(5,6). However, since QFA cultures are heavily diluted before being inoculated onto agar, QFA can capture more complete growth curves, including exponential and saturation phases(3). For example, growth curve observations allow culture doubling times to be estimated directly with high precision, as discussed previously(1). Here we present a specific QFA protocol applied to thousands of S. cerevisiae cultures which are automatically handled by robots during inoculation, incubation and imaging. Any of these automated steps can be replaced by an equivalent, manual procedure, with an associated reduction in throughput, and we also present a lower throughput manual protocol. The same QFA software tools can be applied to images captured in either workflow. We have extensive experience applying QFA to cultures of the budding yeast S. cerevisiae but we expect that QFA will prove equally useful for examining cultures of the fission yeast S. pombe and bacterial cultures.

Quantitative fitness analysis shows that NMD proteins and many other protein complexes suppress or enhance distinct telomere cap defects.

To better understand telomere biology in budding yeast, we have performed systematic suppressor/enhancer analyses on yeast strains containing a point mutation in the essential telomere capping gene CDC13 (cdc13-1) or containing a null mutation in the DNA damage response and telomere capping gene YKU70 (yku70Δ). We performed Quantitative Fitness Analysis (QFA) on thousands of yeast strains containing mutations affecting telomere-capping proteins in combination with a library of systematic gene deletion mutations. To perform QFA, we typically inoculate 384 separate cultures onto solid agar plates and monitor growth of each culture by photography over time. The data are fitted to a logistic population growth model; and growth parameters, such as maximum growth rate and maximum doubling potential, are deduced. QFA reveals that as many as 5% of systematic gene deletions, affecting numerous functional classes, strongly interact with telomere capping defects. We show that, while Cdc13 and Yku70 perform complementary roles in telomere capping, their genetic interaction profiles differ significantly. At least 19 different classes of functionally or physically related proteins can be identified as interacting with cdc13-1, yku70Δ, or both. Each specific genetic interaction informs the roles of individual gene products in telomere biology. One striking example is with genes of the nonsense-mediated RNA decay (NMD) pathway which, when disabled, suppress the conditional cdc13-1 mutation but enhance the null yku70Δ mutation. We show that the suppressing/enhancing role of the NMD pathway at uncapped telomeres is mediated through the levels of Stn1, an essential telomere capping protein, which interacts with Cdc13 and recruitment of telomerase to telomeres. We show that increased Stn1 levels affect growth of cells with telomere capping defects due to cdc13-1 and yku70Δ. QFA is a sensitive, high-throughput method that will also be useful to understand other aspects of microbial cell biology.

The epidemiology of Clostridium difficile in Scotland.

The objective of this study was to characterise the epidemiology of Clostridium difficile in Scotland by determining the distribution of PCR ribotypes and antimicrobial susceptibility in 1613 isolates collected from all healthboard areas of Scotland in the period November 2007-December 2009. Three PCR ribotypes predominated amongst the Scottish isolates of C. difficile; ribotype 106 (29.4%), ribotype 001 (22%) and ribotype 027 (12.6%) followed by the less prevalent ribotypes including 002, 015, 014, 078, 005, 023 and 020. The distribution of ribotypes varied between healthboard areas. Ribotype 106 or 001 was the predominant ribotype in 10 healthboard areas, while ribotype 027 was the predominant type in two neighbouring areas. Antimicrobial susceptibility testing of C. difficile isolates showed high frequencies of resistance to moxifloxacin, levofloxacin, erythromycin and cefotaxime in the epidemic C. difficile ribotypes 001, 027 and 106 compared to other less common ribotypes. Furthermore, reduced susceptibility to metronidazole was found only in the epidemic strains. These findings are compatible with the hypothesis that fluoroquinolones, macrolides and cephalosporins may play a role in the spread of C. difficile in Scotland (while the role of metronidazole needs further investigations), and highlights the role of antimicrobial stewardship in preventing and controlling C. difficile infection (CDI).