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Aims To understand the impact of storage temperature on recovery of Staphylococcus aureus on sampling swabs. Staphylococcus aureus is a common cause of skin and soft tissue infections, but also causes a variety of life-threatening diseases. With a large pool of asymptomatic carriers and transmission that can occur even through indirect contact, mitigation efforts have had limited success. Swab sampling, followed by culturing, is a cornerstone of epidemiological studies, however, S. aureus viability on swabs stored at different temperatures has not been characterized. Methods and Results We determined survival rates on swabs stored at five different temperatures. Samples stored at −70°C had no decay over time while samples stored at higher temperatures showed an exponential decay in viability. Mortality rates were greatest for swabs stored at 37°C. Survival at intermediate temperatures (−20 to 20·5°C) did not differ significantly, however, we observed more variation at higher temperatures. Conclusions To maximize recovery of S. aureus cells, samples should be stored at −70°C or processed for culturing without delay. Significance and Impact of the Study Epidemiological studies of bacterial diseases are typically limited to determination of pathogen presence/absence, yet quantitative assessments of pathogen load and genetic diversity can provide insights into disease progression and severity, likelihood of transmission and adaptive evolutionary potential. For studies of S. aureus where time or access to a microbiology laboratory may delay culturing, deep freezing or timely culturing will maximize the degree to which sampling results reflect source status.

The white spot syndrome virus (WSSV) is lethal in penaeid shrimp. Successful efforts made to develop a natural oil blend formulation (NOBF) by blending Eucalyptus globulus, Pinus sylvestris, and Lavandula latifolia in different compositions with anti-WSSV properties. A bioassay challenge trial conducted using 1 g of specific pathogen-free Penaeus vannamei Boone, 1931 shrimp in 4 replicates of aquaria for each group. The NOBF dose of 0.4 ppm applied throughout the trial period by mixing in the aquarium water daily, starting seven days before the challenge. The efficacy of NOBF against WSSV was measured using the per os method of a challenge. The cumulative mortality in the positive control group reached 100 % after ten days of the challenge. The NOBF was applied in the commercial shrimp ponds to study its efficacy and palatability. A study in 8 ponds in a WSSV prone area in Indonesia, demonstrated no significant difference in crucial water quality parameters, especially on algae as natural food. The NOBF applied ponds had higher productivity (1.28 Kg of shrimp per m3) as compared to control (1.18 Kg of shrimp per m3). The FCR of NOBF group was lower (1.41) than control (1.53). The survival rate of the NOBF group was higher (86.96%) than control (80.36). The trial outcomes show that NOBF is safe and user-friendly, with properties to reduce pathogen load.

Animal waste contains high numbers of microorganisms and therefore can present a potential biological threat to human health. During episodic rainfall events resulting in runoff, microorganisms in the waste and soil may migrate into surface runoff, contaminating surface water resources. A probabilistic human exposure (HE) model was created to determine exposure to faecal indicator bacteria (FIB): total coliforms (TC), E. coli and enterococci following application of bio-based fertiliser (dairy cattle slurry, digestate) to grassland; using a combination of experimental field results and literature-based data. This step was followed by a quantitative microbial risk assessment (QMRA) model for pathogenic E. coli based on a literature-based dose-response model. The results showed that the maximum daily HE (HEdaily) is associated with E. coli for unprocessed slurry (treatment T1) on day 1, the worst-case scenario where the simulated mean HEdaily was calculated as 2.84 CFU day -1. The results indicate that the overall annual probability of risk (Pannual) of illness from E. coli is very low or low based on the WHO safe-limit of Pannual as 10 -6. In the worst-case scenario, a moderate risk was estimated with simulated mean Pannual as 1.0 × 10 -5. Unpasteurised digestate application showed low risk on day 1 and 2 (1.651 × 10 -6, 1.167 × 10 -6, respectively). Pasteurised digestate showed very low risk in all scenarios. These results support the restriction imposed on applying bio-based fertiliser if there is any rain forecast within 48 h from the application time. This study proposes a future extension of the probabilistic model to include time, intensity, discharge, and distance-dependant dilution factor. The information generated from this model can help policymakers ensure the safety of surface water sources through the quality monitoring of FIB levels in bio-based fertiliser. ispartof: JOURNAL OF ENVIRONMENTAL MANAGEMENT vol:299 ispartof: location:England status: published

There is a need for wastewater based epidemiological (WBE) methods that integrate multiple, variously sized surveillance sites across geographic areas. We developed a novel indexing method, Melvin’s Index, that provides a normalized and standardized metric of wastewater pathogen load for qPCR assays that is resilient to surveillance site variation. To demonstrate the utility of Melvin’s Index, we used qRT-PCR to measure SARS-CoV-2 genomic RNA levels in influent wastewater from 19 municipal wastewater treatment facilities (WWTF’s) of varying sizes and served populations across the state of Minnesota during the Summer of 2020. SARS-CoV-2 RNA was detected at each WWTF during the 20-week sampling period at a mean concentration of 8.5 × 104 genome copies/L (range 3.2 × 102–1.2 × 109 genome copies/L). Lag analysis of trends in Melvin’s Index values and clinical COVID-19 cases showed that increases in indexed wastewater SARS-CoV-2 levels precede new clinical cases by 15–17 days at the statewide level and by up to 25 days at the regional/county level. Melvin’s Index is a reliable WBE method and can be applied to both WWTFs that serve a wide range of population sizes and to large regions that are served by multiple WWTFs.

How are some individuals surviving infections while others die? The answer lies in how infected individuals invest into controlling pathogen proliferation and mitigating damage, two strategies respectively called resistance and disease tolerance. Pathogen within-host dynamics (WHD), influenced by resistance, and its connection to host survival, determined by tolerance, decide the infection outcome. To grasp these intricate effects of resistance and tolerance, we used a deterministic theoretical model where pathogens interact with the immune system of a host. The model describes the positive and negative regulation of the immune response, consider the way damage accumulate during the infection and predicts WHD. When chronic, infections stabilize at a Set-Point Pathogen Load (SPPL). Our model predicts that this situation can be transient, the SPPL being then a predictor of life span which depends on initial condition (e.g. inoculum). When stable, the SPPL is rather diagnostic of non lethal chronic infections. In lethal infections, hosts die at a Pathogen Load Upon Death (PLUD) which is almost independent from the initial conditions. As the SPPL, the PLUD is affected by both resistance and tolerance but we demonstrate that it can be used in conjunction with mortality measurement to distinguish the effect of disease tolerance from that of resistance. We validate empirically this new approach, using Drosophila, melanogaster and the pathogen Providencia rettgeri.

Detecting and quantifying pathogens with quick, cost-efficient and sensitive methods is needed across disease systems for addressing pertinent epidemiological questions. Typical methods rely on extracting DNA from collected samples. Here we develop and test an extraction-free method from water bath samples that is both sensitive and efficient for 2 major amphibian pathogens—Batrachochytrium dendrobatidis and B. salamandrivorans. We tested mock samples with known pathogen quantities as well as comparatively assessed detection from skin swabs and water baths from field sampled amphibians. Quantitative PCR (qPCR) directly on lyophilized water baths was able to reliably detect low loads of 10 and 1 zoospores for both pathogens, and detection rates were greater than those of swabs from field samples. Further concentration of samples did not improve detection, and collection container type did not influence pathogen load estimates. This method of lyophilization (i.e. freeze-drying) followed by direct qPCR offers an effective and efficient tool from detecting amphibian pathogens, which is crucial for surveillance efforts and estimating shedding rates for robust epidemiological understanding of transmission dynamics. Furthermore, water bath samples have multiple functions and can be used to evaluate mucosal function against pathogens and characterize mucosal components. The multifunctionality of water bath samples and reduced monetary costs and time expenditures make this method an optimal tool for amphibian disease research and may also prove to be useful in other wildlife disease systems.

Healthcare-associated infections (HAIs) contribute to patient morbidity and mortality with an estimated 1.7 million infections and 99,000 deaths costing USD $28–34 billion annually in the United States alone. There is little understanding as to if current environmental surface disinfection practices reduce pathogen load, and subsequently HAIs, in critical care settings. This evidence map includes a systematic review on the efficacy of disinfecting environmental surfaces in healthcare facilities. We screened 17,064 abstracts, 635 full texts, and included 181 articles for data extraction and study quality assessment. We reviewed ten disinfectant types and compared disinfectants with respect to study design, outcome organism, and fourteen indictors of study quality. We found important areas for improvement and gaps in the research related to study design, implementation, and analysis. Implementation of disinfection, a determinant of disinfection outcomes, was not measured in most studies and few studies assessed fungi or viruses. Assessing and comparing disinfection efficacy was impeded by study heterogeneity; however, we catalogued the outcomes and results for each disinfection type. We concluded that guidelines for disinfectant use are primarily based on laboratory data rather than a systematic review of in situ disinfection efficacy. It is critically important for practitioners and researchers to consider system-level efficacy and not just the efficacy of the disinfectant.

Worldwide variation in human stature and limb proportions is widely accepted to reflect thermal adaptation, but the contribution of population history to this variation is unknown. Furthermore, stature and relative lower limb length (LLL) show substantial plastic responses to environmental stressors, e.g., nutrition, pathogen load, which covary with climate. Thus ecogeographic patterns may go beyond temperature-based selection. We analysed global variation in stature, sitting height and absolute and relative LLL using large worldwide samples of published anthropometric data from adult male (n = 571) and female (n = 268) populations in relation to temperature, humidity, and net primary productivity (NPP). Population history was modeled using spatial eigenvector mapping based on geographic distances reflecting the hypothesized pattern for the spread of modern humans out of Africa. Regression models account for ~ 50% of variation in most morphological variables. Population history explains slightly more variation in stature, sitting height and LLL than the environmental/climatic variables. After adjusting for population history, associations between (usually maximum) temperature and LLL are consistent with Allen's "rule" and may drive similar relationships with stature. NPP is a consistent negative predictor of anthropometry, which may reflect the growth-limiting effects of lower environmental resource accessibility (inversely related to NPP) and/or pathogen load.

The common house mosquito, Culex pipiens s. l. is part of the morphologically hardly or non-distinguishable Culex pipiens complex. Upcoming molecular methods allowed us to identify members of mosquito populations that are characterized by differences in behavior, physiology, host and habitat preferences and thereof resulting in varying pathogen load and vector potential to deal with. In the last years, urban and surrounding periurban areas were of special interest due to the higher transmission risk of pathogens of medical and veterinary importance. Recently, surveys of underground habitats were performed to fully evaluate the spatial distribution of rare members of the Cx. pipiens complex in Europe. Subterranean environments and their contribution to mosquito-borne pathogen transmission are virtually unknown. Herein, we review the underground community structures of this species complex in Europe, add new data to Germany and provide the first reports of the Cx. pipiens complex and usually rarely found mosquito taxa in underground areas of Luxembourg. Furthermore, we report the first finding of Culiseta glaphyroptera in Luxembourg. Our results highlight the need for molecular specimen identifications to correctly and most comprehensively characterize subterranean mosquito community structures.

Blueberry stunt phytoplasma (BBSP; ‘Candidatus Phytoplasma asteris’) is an insect-vectored plant pathogen that causes severe yield losses in blueberry (Vaccinium corymbosum), which is the most valuable fruit crop in Canada. Rapid, field-based diagnostic assays are desirable tools for the control of BBSP, as part of an integrated, proactive approach to production management termed biovigilance. We designed and validated a chaperonin-60 (cpn60)-targeted LAMP assay for detection of BBSP, providing a rapid, low cost, field-deployable diagnostic option. Our validation demonstrates that the assay is reproducible, with high analytical specificity and improved sensitivity when compared with 16S rRNA nested PCR. We applied the validated LAMP assay to nearly 2000 blueberry samples from Québec and Nova Scotia over three growing seasons (2016–2018). Our surveys revealed that BBSP is present in most sites across both provinces, though detection of the pathogen in individual plants varied in different tissues across sampling dates and across years, and evidence of spread between plants was limited. To quantify pathogen load in select plants, we designed additional qPCR and ddPCR assays, also based on cpn60. We found that pathogen load fluctuates in individual plants, both within and between growing seasons. Finally, we designed an interactive map to visualize the results of our surveys. These results provide a validated diagnostic assay that can be used as part of a biovigilance strategy for detecting and controlling infections caused by BBSP.

A long-standing question in infection biology is why two very similar individuals, with very similar pathogen exposures, may have very different outcomes. Recent experiments have found that even isogenic Drosophila melanogaster hosts, given identical inoculations of some bacterial pathogens at suitable doses, can experience very similar initial bacteria proliferation but then diverge to either a lethal infection or a sustained chronic infection with much lower pathogen load. We hypothesized that divergent infection outcomes are a natural result of mutual negative feedbacks between pathogens and the host immune response. Here, we test this hypothesis in silico by constructing process-based dynamic models for bacterial population growth, host immune induction and the feedbacks between them, based on common mechanisms of immune system response. Mathematical analysis of a minimal conceptual model confirms our qualitative hypothesis that mutual negative feedbacks can magnify small differences among hosts into life-or-death differences in outcome. However, explaining observed features of chronic infections requires an extension of the model to include induced pathogen modifications that shield themselves from host immune responses at the cost of reduced proliferation rate. Our analysis thus generates new, testable predictions about the mechanisms underlying bimodal infection outcomes.

Significance With increasing antibiotic resistance, there is a pressing need to understand how host resistance naturally influences bacterial virulence and replication rates. We test this in an infection experiment using 55 isolates of a bacterium, which were collected over the course of the epidemic following its natural emergence in a North American songbird. We demonstrate virulence has increased linearly from outbreak to the present day, encompassing >150,000 bacterial generations. Despite this, bacterial replication rate only increased during the initial spread of host resistance but not thereafter. Thus, contrary to common assumptions, virulence and replication rates can evolve independently, particularly after the initial spread of host resistance., Host resistance through immune clearance is predicted to favor pathogens that are able to transmit faster and are hence more virulent. Increasing pathogen virulence is, in turn, typically assumed to be mediated by increasing replication rates. However, experiments designed to test how pathogen virulence and replication rates evolve in response to increasing host resistance, as well as the relationship between the two, are rare and lacking for naturally evolving host–pathogen interactions. We inoculated 55 isolates of Mycoplasma gallisepticum, collected over 20 y from outbreak, into house finches (Haemorhous mexicanus) from disease-unexposed populations, which have not evolved protective immunity to M. gallisepticum. We show using 3 different metrics of virulence (body mass loss, symptom severity, and putative mortality rate) that virulence has increased linearly over >150,000 bacterial generations since outbreak (1994 to 2015). By contrast, while replication rates increased from outbreak to the initial spread of resistance (1994 to 2004), no further increases have occurred subsequently (2007 to 2015). Finally, as a consequence, we found that any potential mediating effect of replication rate on virulence evolution was restricted to the period when host resistance was initially increasing in the population. Taken together, our results show that pathogen virulence and replication rates can evolve independently, particularly after the initial spread of host resistance. We hypothesize that the evolution of pathogen virulence can be driven primarily by processes such as immune manipulation after resistance spreads in host populations.

Farm-to-fork quantitative microbial risk assessments (QMRA) typically start with a preliminary estimate of initial concentration (Cinitial) of microorganism loading at farm level, consisting of an initial estimate of prevalence (P) and the resulting pathogen levels in animal faeces. An average estimation of the initial concentration of pathogens can be achieved by combining P estimates in animal populations and the levels of pathogens in colonised animals' faeces and resulting cumulative levels in herd farmyard manure and slurry (FYM&S). In the present study, 14 years of data were collated and assessed using a Bayesian inference loop to assess the likely P of pathogens. In this regard, historical and current survey data exists on P estimates for a number of pathogens, including Cryptosporidium parvum, Mycobacterium avium subspecies paratuberculosis (MAP), Salmonella spp., Clostridium spp., Campylobacter spp., pathogenic E. coli, and Listeria monocytogenes in several species (cattle, pigs, and sheep) in Ireland. The results revealed that Cryptosporidium spp. has potentially the highest mean P (Pmean) (25.93%), followed by MAP (15.68%) and Campylobacter spp. (8.80%) for cattle. The Pmean of E. coli is highest (7.42%) in pigs, while the Pmean of Clostridium spp. in sheep was estimated to be 7.94%. Cinitial for Cryptosporidium spp., MAP., Salmonella spp., Clostridium spp., and Campylobacter spp. in cattle faeces were derived with an average of 2.69, 4.38, 4.24, 3.46, and 3.84 log10 MPN g -1, respectively. Average Cinitial of Cryptosporidium spp., Salmonella spp., Clostridium spp., and E. coli in pig slurry was estimated as 1.27, 3.12, 3.02, and 4.48 log10 MPN g -1, respectively. It was only possible to calculate the average Cinitial of Listeria monocytogenes in sheep manure as 1.86 log10 MPN g -1. This study creates a basis for future farm-to-fork risk assessment models to base initial pathogen loading values for animal faeces and enhance risk assessment efforts. ispartof: SCIENCE OF THE TOTAL ENVIRONMENT vol:786 ispartof: location:Netherlands status: published

In recent years, the incidence of infected wounds is steadily increasing, and so is the clinical as well as economic interest in effective therapies. These combine reduction of pathogen load in the wound with general wound management to facilitate the healing process. The success of current therapies is challenged by harsh conditions in the wound microenvironment, chronicity, and biofilm formation, thus impeding adequate concentrations of active antimicrobials at the site of infection. Inadequate dosing accuracy of systemically and topically applied antibiotics is prone to promote development of antibiotic resistance, while in the case of antiseptics, cytotoxicity is a major problem. Advanced drug delivery systems have the potential to enable the tailor-made application of antimicrobials to the side of action, resulting in an effective treatment with negligible side effects. This review provides a comprehensive overview of the current state of treatment options for the therapy of infected wounds. In this context, a special focus is set on delivery systems for antimicrobials ranging from semi-solid and liquid formulations over wound dressings to more advanced carriers such as nano-sized particulate systems, vesicular systems, electrospun fibers, and microneedles, which are discussed regarding their potential for effective therapy of wound infections. Further, established and novel models and analytical techniques for preclinical testing are introduced and a future perspective is provided. Graphical abstract

Objectives: This study aims to determine the contamination Antimicrobial-Resistance Pathogen load and Public Health Risk of Drinking Water in the case of Debre Tabor Town, Northwest Ethiopia.Study design and period: A laboratory-based cross-sectional study design was employed from March to May 2020.Methods: A total of 60 water samples were collected from the household tap and household drinking water storage container by following the standard microbial analysis method. Besides Sanitary survey was conducted for the municipal water supply system. Water samples were analyzed for differences in bacteriological parameters and drug-susceptibility patterns. Descriptive statistics, independent sample t-test, and multiple linear regression models were used to analyze the data.Results: The drinking water was mostly contaminated with multiple antibiotics-resistance waterborne Escherichia coli 35% (95% CI: 31.2%, 46.9%), Salmonella 22.7% (95% CI: 23.2%, 28.7%), and Shigella 15% (95% CI: 11.2%, 20.9%). Approximately 52.78% and 36.11% of the tap and an equal 23.33% of the household storage container water samples were categorized under low and intermediate risks respectively, and the overall health risk index of the water samples showed that 45.83%, 41.67%, and 12.5%, of them, are categorized under low, intermediate and high-risk classes respectively.Conclusion: The contamination of drinking water with antimicrobial-resistant waterborne bacteria in the community could indicate the likelihood of the occurrence of treatment failure and increased mortality. Hence, proper drinking water treatment and strict supervision are needed to prevent the contamination of the water and related consequences.

International audience; Host resistance through immune clearance is predicted to favor pathogens that are able to transmit faster and are hence more virulent. Increasing pathogen virulence is, in turn, typically assumed to be mediated by increasing replication rates. However, experiments designed to test how pathogen virulence and replication rates evolve in response to increasing host resistance, as well as the relationship between the two, are rare and lacking for naturally evolving host–pathogen interactions. We inoculated 55 isolates of Mycoplasma gallisepticum, collected over 20 y from outbreak, into house finches (Haemorhous mexicanus) from disease-unexposed populations, which have not evolved protective immunity to M. gallisepticum. We show using 3 different metrics of virulence (body mass loss, symptom severity, and putative mortality rate) that virulence has increased linearly over >150,000 bacterial generations since outbreak (1994 to 2015). By contrast, while replication rates increased from outbreak to the initial spread of resistance (1994 to 2004), no further increases have occurred subsequently (2007 to 2015). Finally, as a consequence, we found that any potential mediating effect of replication rate on virulence evolution was restricted to the period when host resistance was initially increasing in the population. Taken together, our results show that pathogen virulence and replication rates can evolve independently, particularly after the initial spread of host resistance. We hypothesize that the evolution of pathogen virulence can be driven primarily by processes such as immune manipulation after resistance spreads in host populations.

BackgroundMultiple bacteria, viruses, protists, and helminths cause enteric infections that greatly impact human health and wellbeing. These enteropathogens are transmitted via several pathways through human, animal, and environmental reservoirs. Individual quantitative PCR (qPCR) assays have been extensively used to detect enteropathogens within these types of samples, whereas the TaqMan Array Card (TAC) that allows simultaneous detection of multiple enteropathogens has only previously been validated in human clinical samples.MethodsHere, we performed a comprehensive double-blinded comparison of the performance of a custom TAC relative to standard qPCR for the detection of eight enteric targets, by using spiked samples, wastewater from Melbourne (Australia), and human, animal, and environmental samples from informal settlements in Suva, Fiji.FindingsBoth methods exhibited high and comparable specificity (TAC: 100%, qPCR: 94%), sensitivity (TAC: 92%; qPCR: 100%), and quantitation accuracy (TAC: 91%; qPCR: 99%) in non-inhibited sample matrices. PCR inhibitors substantially impacted detection via TAC, though this issue was alleviated by 10-fold sample dilution. Among samples from informal settlements, the two techniques were comparable for detection (89% agreement) and quantitation (R2= 0.82). The TAC additionally included 38 other targets, enabling detection of diverse faecal pathogens and extensive environmental contamination that would be prohibitively labour intensive to assay by standard qPCR.InterpretationOverall, the two techniques produce comparable results across diverse sample types, with qPCR prioritising greater sensitivity and quantitation accuracy, and TAC trading small reductions in these for a cost-effective larger enteropathogen panel that enables a greater number of enteric pathogens to be analysed concurrently, which is beneficial given the abundance and variety of enteric pathogens in environments such as urban informal settlements. The ability to monitor multiple enteric pathogens across diverse reservoirs in turn allows better resolution of pathogen exposure pathways, and the design and monitoring of interventions to reduce pathogen load.FundingWellcome TrustOur Planet, Our Healthprogram [OPOH grant 205222/Z/16/Z].

Summary Background Multiple bacteria, viruses, protists, and helminths cause enteric infections that greatly impact human health and wellbeing. These enteropathogens are transmited via several pathways through human, animal, and environmental reservoirs. Individual qPCR assays have been extensively used to detect enteropathogens within these types of samples, whereas the TaqMan array card (TAC), which allows simultaneous detection of multiple enteropathogens, has only previously been validated in human clinical samples. Methods In this methodological comparison study, we compared the performance of a custom 48-singleplex TAC relative to standard qPCR. We established the sensitivity and specificity of each method for the detection of eight enteric targets, by using spiked samples with varying levels of PCR inhibition. We then tested the prevalence and abundance of pathogens in wastewater from Melbourne (Australia), and human, animal, and environmental samples from informal settlements in Suva, Fiji using both TAC and qPCR. Findings Both methods exhibited similarly high specificity (TAC 100%, qPCR 94%), sensitivity (TAC 92%, qPCR 100%), and quantitation accuracy (TAC 91%, qPCR 99%) in non-inhibited sample matrices with spiked gene fragments. PCR inhibitors substantially affected detection via TAC, though this issue was alleviated by ten-fold sample dilution. Among samples from informal settlements, the two techniques performed similarly for detection (89% agreement) and quantitation (R2 0·82) for the eight enteropathogen targets. The TAC additionally included 38 other enteric targets, enabling detection of diverse faecal pathogens and extensive environmental contamination that would be prohibitively labour intensive to assay by standard qPCR. Interpretation The two techniques produced similar results across diverse sample types, with qPCR prioritising greater sensitivity and quantitation accuracy, and TAC trading small reductions in these for a cost-effective larger enteropathogen panel enabling a greater number of enteric pathogens to be analysed concurrently, which is beneficial given the abundance and variety of enteric pathogens in environments such as urban informal settlements. The ability to monitor multiple enteric pathogens across diverse reservoirs could allow better resolution of pathogen exposure pathways, and the design and monitoring of interventions to reduce pathogen load. Funding Wellcome Trust Our Planet, Our Health programme.

Bronchoalveolar Lavage in Children: Still the Gold StandardShivanthan Shanthikumar1,2,3 and Sarath C Ranganathan1,2,3Respiratory and Sleep Medicine, Royal Children’s Hospital, Melbourne, AustraliaRespiratory Diseases, Murdoch Children’s Research Institute, Melbourne, AustraliaDepartment of Paediatrics, University of Melbourne, Melbourne, AustraliaCorresponding Author; Dr Shivanthan Shanthikumar; Respiratory Medicine, Royal Children’s Hospital, 50 Flemington Road, Parkville, VIC, 3052, Australia; shivanthan.shanthikumar@rch.org.auAcknowledgements; The authors have no conflicts of interest to declareDear Editor,We read with great interest the recent article by Craven et al , entitled “High levels of inherent variability in microbiological assessment of bronchoalveolar lavage samples from children with persistent bacterial bronchitis and healthy controls .”1 In a small study of 18 children, funded by GlaxoSmithKline, the authors demonstrate variability in the results of bronchoalveolar lavage (BAL) collected from controls and children with protracted bacterial bronchitis (PBB). Specifically, they show that when the BAL was divided and sent to two laboratories the results were discordant in terms of both the organisms isolated and their relative abundance. From these data the authors draw conclusions which include questioning “assumptions about this procedure being the gold standard .” Whilst these data are of interest, there are significant limitations to their value especially when considering existing literature.One of the key findings of the study is the discordant results between laboratories. A lack of detail regarding the methods used at each site is a major limitation. It is recognised that laboratory processes can affect the yield of samples collected from patients with chronic airway infection, and the need for a consistent approach has led to disease specific consensus guidelines on this topic.2 The discordant results seen in the study could result from different laboratory handling of specimens, and hence the findings of this study could purely be explained by a difference in practice between two centres, not least of which was the transport of samples to the second laboratory in STGG. Molecular studies have identified that even media considered sterile can contain numerous organisms albeit in low densities.3 We note that it was laboratory 2 where additional bacteria were cultured from the BAL.Hare et al analysed BAL samples from 655 children collected and analysed at two different sites compared with 18 samples in the study of Craven et al .4 They compared bacterial pathogen load (control, negative, 102 colony forming units per ml (CFU/ml), 103 CFU/ml, 104 CFU/ml, 105 CFU/ml) and inflammatory markers to determine an appropriate definition for infection. They found that a bacterial pathogen load of ≥104 CFU/ml was associated with increased markers of inflammation and hence an appropriate threshold for defining infection. This was in keeping with previous studies.4 Whilst the authors contend the current paper does not support the use of ≥104 CFU/ml, given it only includes 13 children with PBB an explanation of the findings of Hareet al in their considerably larger study and other studies needs explanation.Another key finding of the study was the limited correlation between semiquantitative and quantitative methods of measuring bacterial pathogen load. Whilst there has not been direct comparison of different methods of determining bacterial pathogen load in PBB and other paediatric suppurative disorders, a large amount of data speaks to the validity of using a semiquantitative or qualitative approach. For instance, the previously discussed Hare et al study utilised a semiquantitative approach, and was able to clearly identify a threshold for lower airway infection that was associated with inflammation. In addition, the qualitative approach used by AREST CF (the long running study of CF patients cited in the article) to define infection is supported by the fact that this definition is associated with important clinical outcomes. For example, in a recent AREST-CF study analysing 1161 BAL from 265 children with CF, the presence of early life infection using the AREST-CF definition, was associated with future risk of structural lung disease severity.5Further, we have used molecular studies to assess the microbiome in CF and shown considerable agreement between pathogen-dominated microbiota and routine laboratory bacterial culture even though these samples were assessed by two different techniques, in two laboratories in different continents and analysed two decades apart in time.6Despite the data that contradicts the findings of their study, and while not discussed by the authors themselves, we do contend that use of both quantitative and semi-quantitative microbiologic cultures are likely problematic given that bacterial density is influenced by the dilution from the 0.9% saline used to lavage the target lobe. Dilution further depends on the volume of return retrieved on suctioning. The consensus has been that standardising for this dilution is not required but data supporting this are few.In summary, there are significant issues that limit the value of the key findings of the study by Craven et al . A large amount of published data in PBB and cystic fibrosis support the use of BAL as a biological specimen associated with important clinical outcomes. These studies have been conducted in multiple centres, over many years, and included many children. While the findings of Craven et alhighlight there can be inconsistencies in results, this potentially speaks to the methods used by the laboratories involved in handing the small number of samples. When these findings are compared to the large amount of evidence already generated, they should prompt evaluation of local practices and not just a reconsideration of whether BAL is the gold standard method of sampling the lower airway of children with suppurative lung disease. While we believe that BAL remains the gold standard for the detection of lower respiratory infection we do not believe it is a perfect test and its use and many limitations need to be considered and minimised.Therefore, we agree with the authors that interpretation of microbial culture results utilizing BAL samples can be challenging. However, we disagree that assumptions about this procedure being the “gold standard” fail to take into account its many limitations as despite these BAL remains the best test to detect endobronchial infection that is associated with lower respiratory inflammation especially in CF.

A mathematical model is developed to describe the dynamics of the spread of a waterborne disease among communities located along a flowing waterway. The model is formulated as a system of reaction–diffusion–advection partial differential equations in this spatial setting. The compartments of the model consist of susceptible, infected, and recovered individuals in the communities along the waterway, together with a term representing the pathogen load in each community and a term representing the spatial concentration of pathogens flowing along the waterway. The model is applied to the cholera outbreak in Haiti in 2010.

Final peer-reviewed article: https://doi.org/10.1016/j.jhin.2020.08.005Background: In pandemic situations such as COVID-19, shortages of proper protective equipment are common. One solution may be to decontaminate equipment such as facemasks for reuse.Aim: The aim of this review was to collect and synthesize existing information on decontamination of N95 filtering facepiece respirators (FFRs) using microwave and heat-based treatments, with special attention to impact on mask function (aerosol penetration, airflow resistance) and fit.Methods: A systematic review (PROSPERO ID pending) of literature available on Medline, Embase, Global Health, JISRP and JEFF was conducted. Records were screened independently by two reviewers, and data was extracted and analyzed from studies that reported on the effects of microwave- or heat-based decontamination on N95 FFR performance and/or microbial load. Results: All interventions successfully destroyed viral/bacterial contaminants. Other than autoclaving, which significantly increased aerosol penetration, moist and dry microwave and heat conditions did not significantly impact functional parameters or fit. However, several conditions caused physical damage to at least one N95 model. Conclusions: Microwave irradiation and heat provides safe and effective decontamination options for N95 FFR reuse during critical shortages. However, autoclaving masks is not recommended by the evidence in this review. Any mask disinfected using these methods should be inspected for physical degradation before reuse.NOTE: The experiments summarized in this manuscript are performed under specialized laboratory conditions. Household appliances should not be used for any purposes that are not indicated in their manufacturer-supplied guidelines, including mask decontamination. Doing so may lead to damage or injury.

Seed treatments help reduce the pathogen load and thus improve the condition of plants from their earliest developmental stages, but they can have impacts beyond their basic fungicide protection role. In this study, we investigated how seven spring barley seed treatments affected the plants&rsquo, physiological state. The tested seed treatments differed significantly in their impacts on the vigor parameters of barley seeds and on the physiological state of seedlings under drought stress and after regeneration. Seed treatments based on substances from the succinate-dehydrogenase-inhibitors (SDHI) group did not cause inhibition of seedling growth and also display by the highest vigor index values. Using the analysis of photosynthesis-related parameters, we showed that seed treatments from the SDHI group provided a superior tolerance of the imposed drought in spring barley than other treatments. In addition to protection against abiotic stress, SDHI treatments also rendered a higher efficiency of photochemical reactions in the treated plants.

Background Fighting and controlling epidemic and endemic diseases represents a considerable cost to livestock production. Much research is dedicated to breeding disease resilient livestock, but this is not yet a common objective in practical breeding programs. In this paper, we investigate how future breeding programs may benefit from recent research on disease resilience. Main body We define disease resilience in terms of its component traits resistance (R: the ability of a host animal to limit within-host pathogen load (PL)) and tolerance (T: the ability of an infected host to limit the damage caused by a given PL), and model the host's production performance as a reaction norm on PL, depending on R and T. Based on this, we derive equations for the economic values of resilience and its component traits. A case study on porcine respiratory and reproductive syndrome (PRRS) in pigs illustrates that the economic value of increasing production in infectious conditions through selection for R and T can be more than three times higher than by selection for production in disease-free conditions. Although this reaction norm model of resilience is helpful for quantifying its relationship to its component traits, its parameters are difficult and expensive to quantify. We consider the consequences of ignoring R and T in breeding programs that measure resilience as production in infectious conditions with unknown PL—particularly, the risk that the genetic correlation between R and T is unfavourable (antagonistic) and that a trade-off between them neutralizes the resilience improvement. We describe four approaches to avoid such antagonisms: (1) by producing sufficient PL records to estimate this correlation and check for antagonisms—if found, continue routine PL recording, and if not found, shift to cheaper proxies for PL; (2) by selection on quantitative trait loci (QTL) known to influence both R and T in favourable ways; (3) by rapidly modifying towards near-complete resistance or tolerance, (4) by re-defining resilience as the animal's capacity to resist (or recover from) the perturbation caused by an infection, measured as temporal deviations of production traits in within-host longitudinal data series. Conclusions All four alternatives offer promising options for genetic improvement of disease resilience, and most rely on technological and methodological developments and innovation in automated data generation.

The effects of global change on semi-natural and agro-ecosystem functioning have been studied extensively. However, less well understood is how global change will influence fungal diseases, especially in a natural ecosystem. We use data from a 6-yr factorial experiment with warming (simulated using infrared heaters) and altered precipitation treatments in a natural Tibetan alpine meadow ecosystem, from which we tested global change effects on foliar fungal diseases at the population and community levels, and evaluated the importance of direct effects of the treatments and community-mediated (indirect) effects (through changes in plant community composition and competence) of global change on community pathogen load. At the population level, we found warming significantly increased fungal diseases for nine plant species. At the community level, we found that warming significantly increased pathogen load of entire host communities, whereas no significant effect of altered precipitation on community pathogen load was detected. We concluded that warming influences fungal disease prevalence more than precipitation does in a Tibetan alpine meadow. Moreover, our study provides new experimental evidence that increases in disease burden on some plant species and for entire host communities is primarily the direct effects of warming, rather than community-mediated (indirect) effects.

Overwinter colony mortality is an ongoing challenge for North American beekeepers. During winter, honey bee colonies rely on stored honey and beebread, which is frequently contaminated with the neonicotinoid insecticides clothianidin and thiamethoxam. To determine whether neonicotinoid exposure affects overwinter survival of Apis mellifera L., we chronically exposed overwintering field colonies and winter workers in the laboratory to thiamethoxam or clothianidin at different concentrations and monitored survival and feed consumption. We also investigated the sublethal effects of chronic thiamethoxam exposure on colony pathogen load, queen quality, and colony temperature regulation. Under field conditions, high doses of thiamethoxam significantly increased overwinter mortality compared to controls, with field-realistic doses of thiamethoxam showing no significant effect on colony overwinter survival. Under laboratory conditions, chronic neonicotinoid exposure significantly decreased survival of winter workers relative to negative control at all doses tested. Chronic high-dose thiamethoxam exposure was not shown to impact pathogen load or queen quality, and field-realistic concentrations of thiamethoxam did not affect colony temperature homeostasis. Taken together, these results demonstrate that chronic environmental neonicotinoid exposure significantly decreases survival of winter workers in the laboratory, but only chronic high-dose thiamethoxam significantly decreases overwinter survival of colonies in the field.

Predators may be limited in their ability to kill prey (i.e., have type II or III functional responses), an insight that has had far-reaching consequences in the ecological literature. With few exceptions, however, this possibility has not been extended to the behaviour of immune cells, which kill pathogens much as predators kill their prey. Rather, models of the within-host environment have tended to tacitly assume that immune cells have an unlimited ability to target and kill pathogens (i.e., a type I functional response). Here we explore the effects of changing this assumption on infection outcomes (i.e., pathogen loads). We incorporate immune cell handling time into an ecological model of the within-host environment that considers both the predatory nature of the pathogen-immune cell interaction as well as competition between immune cells and pathogens for host resources. Unless pathogens can preempt immune cells for host resources, adding an immune cell handling time increases equilibrium pathogen load. We find that the shape of the relationship between energy intake and pathogen load can change: with a type I functional response, pathogen load is maximised at intermediate inputs, while for a type II or III functional response, pathogen load is solely increasing. With a type II functional response, pathogen load can fluctuate rather than settling to an equilibrium, a phenomenon unobserved with type I or III functional responses. Our work adds to a growing literature highlighting the role of resource availability in host-parasite interactions. Implications of our results for adaptive anorexia are discussed.