The acute respiratory distress syndrome (ARDS) remains a common and highly morbid condition despite advances in the understanding and management of this complex critical illness. Recent work has illuminated the heterogeneity within ARDS and demonstrated the likely impact of heterogeneity on the identification of effective therapeutic interventions. Despite these challenges, new data have also informed the standard of care for ARDS and have resulted in the re-evaluation of previously established therapies, including ventilation strategies, pharmacologic interventions, and rescue therapies. As the field of ARDS continues to evolve, innovative approaches will be needed to further define phenotypes within ARDS and design targeted clinical trials. Despite advances in our understanding of the pathophysiologic cascade that results in ARDS, including key inflammatory mediators and disruption of the normal alveolar-capillary endothelial barrier2, there remain no specific pharmacologic therapies for the condition. Instead, the interventions shown to improve outcomes in ARDS remain clinical management strategies such as lung protective mechanical ventilation and prone positioning. Overall, these interventions have improved outcomes for patients with ARDS, but the burden of lung injury remains significant with a high incidence and risk of both morbidity and mortality. Here, we will review recent advances in the understanding and management of ARDS and discuss ongoing challenges that will require further innovation.Since its initial definition in a case series in 19673, which features three major criteria and changed the categorization of severity. The three criteria defining ARDS are (1) onset within 1 week of known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar/lung collapse, or nodules on chest x-ray or computed tomography; and (3) respiratory failure not fully explained by cardiac failure or fluid overload (requires objective assessment—such as echocardiography—to exclude hydrostatic edema if no ARDS risk factor is present). Other observed clinical features of ARDS include decreased lung compliance and regional heterogeneity of aeration and tissue injury. The Berlin definition also grouped patients with ARDS into categories of mild, moderate, and severe on the basis of the ratio of arterial blood partial pressure of oxygen (PaO2) to the fraction of inspired oxygen (FiO2) (P:F ratio). These categories of severity  were applied to a cohort of over 4,000 patients gathered from clinical and physiologic trials. In this validation cohort, increasing severity corresponded well with increasing mortality. Similarly, severity of lung opacification on chest radiograph based on the Radiographic Assessment of Lung Edema (RALE) score correlated well with severity of illness and mortality as validated in the FACTT trial cohort4. The Kigali modification of the Berlin definition offers alternate criteria, including peripheral capillary oxygen saturation (SpO2)-to-FiO2 ratio and chest ultrasound, which is a useful adaptation in low-resource settings5.ARDS is a syndrome of respiratory failure marked by clinical features of hypoxemia and altered respiratory system mechanics. A consensus definition was refined most recently in 2012 with the Berlin definition6, the estimated annual incidence of acute lung injury is 190,600 cases and the estimated annual mortality is 74,500 patients. This corresponds to a mortality of 38.5% for patients with acute lung injury, which is similar to mortality rates seen in multiple interventional clinical trials in ARDS. More recently, a population-based cohort study7 evaluated trends in ARDS incidence over the course of 8 years. Notably, the incidence of ARDS on admission remained stable, but the incidence of hospital-acquired ARDS fell significantly over the study period, suggesting that changes in care have been effective in preventing cases of iatrogenic ARDS. Additional US studies and study of the global incidence and outcomes of ARDS have shown similar numbers, including an intensive care unit incidence of 10.4% and an unadjusted mortality of 35.3%9. Recent study of a single-center cohort of patients in Rwanda, a lower-resource setting compared with prior studies, revealed an incidence of 4% among all hospital admissions with mortality of 50%; affected patients were younger and ARDS was more frequently associated with trauma compared with the King County cohort5. A secondary analysis of the LUNG SAFE cohort compared ARDS populations between high- and middle-income countries, showing that adjusted in-hospital mortality was higher in the middle-income cohort and that lower gross national product was associated with poorer hospital survival in patients with ARDS10. Overall, these data demonstrate the ongoing burden of ARDS around the world—despite recent advances—and ongoing disparities in outcomes. There is also increasing recognition of significant sequelae in ARDS survivors, including persistent functional deficits and neurocognitive morbidity such as cognitive deficits and post-traumatic stress disorder11.ARDS remains a common and highly morbid condition. In the US, based on a cohort of patients studied in and around King County, WashingtonThe Berlin definition of ARDS uses easily determined clinical parameters for diagnostic criteria and is purposefully inclusive of a heterogeneous cohort with varied etiologies of lung injury and diverse underlying disease states. One benefit of this definition is that patients with acute lung injury are easily identified in the clinical setting, thereby allowing both early intervention and pragmatic clinical trial design with clear enrollment criteria. However, the widely encompassing definition results in a heterogeneous population that necessarily includes patients with both different prognoses and divergent response to specific therapies. The likely impact of this heterogeneity is increasingly recognized, particularly on the background of so many failed clinical trials in ARDS and other critical illness. Although a syndromic definition allows clinical trial enrollment, a study may underestimate the impact of an intervention if there exists significant heterogeneity within the enrolled population. Specifically, a study could have an overall negative result while a small group of high-risk patients actually benefits from the intervention. Conversely, a study could show a benefit to treatment while a small subgroup of patients is actually harmed in the interventional group. If these subgroups are not easily identified, a randomized clinical trial may miss the significance of a therapy. Additionally, if enrollment in two trials includes populations that differ in the representation of these subgroups, they may have conflicting results. Further understanding of the heterogeneity within ARDS and the distinct subgroups within the syndrome is therefore important not only for interpreting completed trials but also for guiding future investigation.12. Only a limited proportion of patients with clinical ARDS had diffuse alveolar damage, the histopathologic hallmark of ARDS. Many other post-mortem diagnoses, including pneumonia, pulmonary edema, cancer infiltration, pulmonary hemorrhage, and even some cases with no pulmonary lesion identified, were made in this cohort. Since biopsies are only infrequently obtained in these patients, there are a number of other methods to identify and define subgroups of patients with ARDS.The heterogeneity of ARDS is highlighted by the diversity of underlying pathologic findings on autopsy of patients with ARDS diagnosed by the Berlin criteria13. ARDS can also be categorized by radiographic appearance, and some studies have correlated a diffuse versus focal pattern of disease with other biomarkers such as inflammatory mediators14. Lastly, a more complex approach focuses on mechanistic difference in ARDS pathobiology, which may represent various endotypes with distinct disease processes. This approach may delineate subgroups that would respond differently to various interventions and further study of these could lead to pathway-targeted therapies.One strategy is to categorize patients by nature of insult. Acute lung injury corresponding with ARDS can be triggered by a variety of insults, including sepsis, pneumonia, trauma, pancreatitis, and toxic inhalation. These can be more broadly categorized into direct insults to the lung  and indirect insults to the lung ; these categories have been shown in retrospective studies to correlate with prognosis15 identified two subphenotypes (a hyperinflammatory group and a hypoinflammatory group) in whom positive end-expiratory pressure (PEEP) interventions had different impacts on outcomes, including mortality, ventilator-free days, and organ failure-free days. In a similar post-hoc analysis of the HARP-2 trial16, which showed no benefit with simvastatin therapy in either ventilator-free days or mortality, the hyperinflammatory subphenotype had a mortality benefit when patients received simvastatin versus placebo. This hyperinflammatory subphenotype was identified by using latent subclass analysis and was defined by several features, including higher values of soluble tumor necrosis factor receptor 1 (sTNFr1) and interleukin-6 (IL-6), lower platelet counts, and more vasopressor use compared with the hypoinflammatory subphenotype group. A post-hoc analysis of the FACCT trial cohort17 identified a hyperinflammatory subphenotype (with higher serum levels of IL-8 and TNFr1 and lower serum bicarbonate levels) in which mortality was lower with the fluid-liberal strategy compared with the fluid-conservative strategy. This is notably different from the primary analysis of these data, which showed no mortality difference between the study arms. Together, these studies demonstrate the importance of understanding subgroups within ARDS in order to explore prior study data and also to inform future study design to identify effective therapies.Although ARDS endotypes have yet to be fully uncovered, recent studies have shown that patients with ARDS can be categorized into subphenotypes based on clinical features and that these subphenotypes can have differing responses to various interventions. One latent class analysis19, there remains a diversity of opinion on optimal management. A foundational study of ARDS demonstrated that mortality was reduced in ARDS when patients received lower tidal volumes  compared with higher tidal volumes 20. This is theorized to be due to a reduction in overdistention of ventilated lung with high regional volumes and pressures—so-called volutrauma and barotrauma—and has become the standard of care. Subsequent studies have shown that the use of an “open lung” ventilation strategy improves lung mechanics, oxygenation, and inflammatory markers21. This strategy relies on the use of optimal lung recruitment to increase the fraction of lung that is aerated with the goal of delivering the set tidal volume to the largest functional lung. Different strategies of titrating PEEP have been studied extensively. PEEP is thought to benefit patients with ARDS by preventing derecruitment of collapsible lung units at the end of exhalation, thus preventing the cyclic opening and closing of alveoli that can promote lung injury via shear stress. However, the ALVEOLI trial22 previously demonstrated no benefit of high PEEP over low PEEP in ARDS when low tidal volumes and limitations of plateau pressure (PPlat) were used. More recent studies may help clarify the effect of various open lung ventilation and PEEP titration methods on outcomes.With the lack of effective pharmacologic interventions, initial management has focused on ventilator strategies that stabilize gas exchange while avoiding further injury to lung tissue (ventilator-associated lung injury). Despite many studies on ventilator strategies and guidelines published by various professional societies to promote evidence-based management23. This variable is determined by both the compliance of the respiratory system and the delivered tidal volume and may be better associated with outcomes because in some patients a high PEEP is required to optimize recruitment , which in turn results in a high PPlat (>30 cm H2O) but these patients are not experiencing significant regional overdistention. Conversely, patients may be on very low PEEP and have an “acceptable” PPlat but have a very high distending pressure, reflecting a poorly compliant respiratory system and high regional strain. Data on driving pressure remain limited; it has not yet been the target of a randomized clinical trial. Additionally, in the recent ART trial24 (discussed below), the intervention group had a lower mean driving pressure and higher mortality, suggesting that its predictive value is limited. However, as we await further clinical trial data, the physiologic rationale of optimizing compliance through recruitment and judicious tidal volumes to avoid overdistention, thereby minimizing driving pressure, remains strong.Given the prior association of high airway pressures with poor outcomes in ARDS, conventional management focused on minimizing the PPlat. However, a recent retrospective analysis of patients enrolled in ARDS trials showed that the physiologic parameter best associated with outcomes was, in fact, driving pressure (PPlat – PEEP)24 assessed the effect of a lung recruitment maneuver and PEEP titration to the best respiratory system compliance  on all-cause mortality. In that study, patients in the intervention group underwent a “maximum alveolar recruitment maneuver” followed by a decremental PEEP trial to determine the level at which respiratory system compliance was optimized. Of note, the recruitment maneuver protocol was modified partway through the study after reported serious adverse outcomes, including cardiac arrest. The study authors found a significantly increased 28-day mortality in the intervention group compared with control (55.3% versus 49.3%), raising questions about the safety of such recruitment maneuvers as part of an open lung ventilation strategy. Additionally, the patients in the study cohort did not appear to be very recruitable; although driving pressure was improved in the intervention group, it was not different enough to be expected to significantly affect mortality. It remains unknown whether recruitment maneuvers with lower (and possibly safer) airway pressures in a more recruitable cohort of patients may provide the benefit of alveolar recruitment with less risk. It also remains unknown whether a decremental, physiologically targeted PEEP titration method may have benefit when not combined with such aggressive recruitment.The ART trial25 showed no significant improvement in ARDS outcomes with use of esophageal manometry to target a combination of transpulmonary pressure and FiO2 compared with PEEP titration based on an empirical high PEEP-FiO2 table. Significantly, there was no difference in the primary outcome measure, a composite score of death and days free from mechanical ventilation through day 28. There was also no significant difference in other physiologic variables—including PEEP, driving pressure, and PPlat—between the two groups. There are therefore no data to suggest that routine use of esophageal manometry, in combination with oxygen targets, is necessary to optimize ventilator settings and minimize ventilator-associated lung injury in patients with ARDS. However, clinically, not all physicians use a combination of transpulmonary pressure and oxygen targets in titrating PEEP with an esophageal balloon, which means that this study may not reflect common practice. Additionally, esophageal balloons allow distinction of the two mechanical elements of the respiratory system , which remains useful in complex clinical situations.The placement of an esophageal catheter to measure esophageal pressure  has also been used to guide PEEP titration. This is based on the concept that excessive transpulmonary pressure  is a more accurate indicator of ventilator-induced lung injury than use of airway pressure alone. However, the recent EPVent-2 study28. A large randomized clinical trial, the PROSEVA trial29, did show an impressive mortality benefit  in patients who are placed in the prone position early in illness (<48 hours after ARDS onset) and who are maintained in the prone position for most of the day and until gas exchange is significantly improved. Additionally, this study did not show any increased risk for adverse events in patents placed in the prone position despite being conducted across many trial sites. These data should strongly encourage clinicians to consider prone positioning in patients with moderate to severe ARDS, and prone positioning should be considered as an upfront therapy and not a rescue therapy. However, the comparison group in the PROSEVA trial received relatively low levels of PEEP, which may have biased toward a benefit with prone position since PEEP can provide some of the same physiologic benefits as prone positioning. Therefore, a second large study comparing prone position with higher levels of—or personalized—PEEP would be useful in assessing the necessity of prone positioning in patients with ARDS.Placing patients with ARDS in the prone position has multiple physiologic effects that are potentially beneficial. It has been known for quite some time that the prone position improves oxygenation; this occurs because of both more homogeneous distribution of perfusion to the lung and recruitment of collapsed lung units, which reduces that amount of perfusion that is functional shunt. Additionally, recruitment alone may be beneficial since it increases the functional size of the lung, thereby reducing the risk of volutrauma/barotrauma, and may decrease the risk of atelectrauma. However, clinical trial data on prone positioning in ARDS have been conflicting, and there have been ongoing concerns about potential adverse events, including dislodgement of indwelling lines and the endotracheal tube and pressure ulcers30. There are multiple plausible mechanisms for the benefit of NMB in ARDS, including elimination of patient-ventilator asynchrony, which reduces the risk of volutrauma and barotrauma, and a reduction in end-expiratory derecruitment due to expiratory effort, which thereby reduces atelectrauma. These potential benefits are mirrored by a reduction in circulating inflammatory mediators in patients receiving NMB, which may also be due in part to a direct effect of NMB. However, a recent randomized trial that attempted to replicate the prior positive result showed no benefit to NMB in a similar patient cohort31. Additionally, the group of patients receiving NMB were less mobile and had a greater incidence of serious adverse cardiovascular events. One criticism of that study has been that many patients were excluded because they were already receiving NMB and this may have biased against finding a benefit. Given the current body of evidence, it is reasonable to use NMB in patients in whom ventilator synchrony cannot otherwise be achieved, but there are no data to support routine use in patients with moderate to severe ARDS.Neuromuscular blockade (NMB) has been a common intervention for patients with moderate to severe ARDS after a randomized trial demonstrated a mortality benefit to NMB for 48 hours in patients with a P:F ratio of less than 150 early in their illness32,N-acetylcysteine33, and sivelestat34 (a neutrophil elastase inhibitor). Even inhaled nitric oxide, which can provide transient improvement in oxygenation, has been repeatedly demonstrated to offer no outcome benefit and to increase the incidence of adverse renal outcomes36. Despite the lack of effective drug interventions thus far, there remains significant interest in identifying medications that may improve ARDS outcomes either by modifying the inflammatory process or by promoting the re-establishment of functional lung tissue.Many other pharmacologic therapies have been studied and have failed to demonstrate a clinical benefit. These include surfactant37 evaluated high-dose simvastatin (80 mg) compared with placebo but found no difference in either ventilator-free days or mortality at 28 days. Subgroup analyses did not identify any separate group that may benefit, including patients with sepsis or with elevated C-reactive protein levels. However, as noted above, a more recent post-hoc re-evaluation of the HARP-2 dataset has used latent class analysis to identify a subgroup characterized by a more inflammatory phenotype of ARDS that may benefit from statin therapy.The previously referenced HARP-2 trial38 randomly assigned patients to receive aerosolized albuterol versus placebo but was stopped early for futility with no observed benefit in ventilator-free days at 28 days or mortality at days 60 and 90. In an attempt to gauge whether beta-agonist therapy may reduce inflammation, serum IL-6 and IL-8 levels were measured; however, there was no significant difference found between the two groups in the serum concentration of either of these cytokines. Intravenous delivery of beta-agonist therapy showed some promise of reduced pulmonary edema in early studies. However, the BALTI-2 trial39, which evaluated intravenous salbutamol versus placebo, was terminated early after data showed increased 28-day mortality in the intervention group. There are therefore no data to support the use of either inhaled or intravenous beta-agonist therapy in patients with ARDS.Inhaled beta-agonist therapy has been of interest as a treatment for ARDS after the observation in animal models that beta-agonist therapy enhanced alveolar fluid clearance. The ALTA trial40 found that patients with ARDS for at least 7 days had a similar rate of mortality when given methylprednisolone compared with those receiving placebo. However, those patients who received methylprednisolone at least 14 days after the onset of ARDS had an increased rate of mortality compared with the placebo group, suggesting a detrimental effect of steroids in late ARDS. A more recent meta-analysis41 that included the LaSRS trial found that patients receiving steroids (either methylprednisolone or hydrocortisone) had a higher rate of achieving unassisted breathing by day 28 (80% versus 50%) and had a lower rate of hospital mortality (20% versus 33%) compared with those receiving placebo. A 2019 Cochrane Review on this topic concludes that steroids may reduce 3-month mortality, but the certainty of the evidence is low42. While it is possible (and perhaps likely) that a subgroup of patients with ARDS will benefit from steroids, we do not yet know which patients these are. Additionally, given the heterogeneity of the underlying pathology in patients with diagnosed ARDS, it remains possible that those patients who respond, in fact, have an undiagnosed inflammatory lung disease such as organizing pneumonia. At present, steroids are not recommended for routine use in ARDS and further study is needed.Perhaps the most studied and discussed pharmacologic intervention for ARDS is steroids. Multiple prior studies have shown mixed results. For example, the LaSRS trial43. This is due in part to the lack of other specific therapies and in part to very promising case reports demonstrating fairly low mortality in patients with severe ARDS who had not responded to other interventions44. However, few randomized trials have evaluated the use of ECMO in the management of ARDS and the ones that have done so do not demonstrate a convincing benefit. The CESAR trial45 randomly assigned patients with severe ARDS to either standard of care at the institution to which they presented or transfer to a specialty center for ECMO therapy. Although there was a significant difference in the primary outcome, a composite endpoint of survival without significant disability, not all patients who were randomly assigned to ECMO actually received the therapy and the patients in the “usual care” arm did not uniformly receive what is now considered standard of care, including low tidal volume ventilation. Therefore, the degree to which the improved outcomes in the intervention arm were due to ECMO alone is unclear. In order to address these ongoing questions, a large randomized trial that compared true standard of care with early ECMO was recently completed. The EOLIA trial46 did not find a significant difference in mortality between the two groups and was stopped early after crossing a futility boundary. Notably, the patients in the conventional management group were allowed to “cross over” and receive ECMO if they failed standard interventions, including prone positioning, paralysis, and inhaled pulmonary vasodilators. It seems unlikely that another large randomized trial of ECMO will be undertaken soon, and although early use of ECMO for all patients with severe ARDS is not supported by the data, it remains an important rescue therapy for patients who are declining despite other interventions. Patients who are likely to have the best outcomes on ECMO are those who have few other organ failures and who are early in their illness (<7 days). Local experience in ECMO cannulation and management is also likely to be an important factor in patient outcomes, and early referral to an ECMO-capable ARDS treatment center is recommended for patients with severe ARDS.Since its development decades ago, extracorporeal membrane oxygenation (ECMO) has been an appealing rescue therapy for patients with severe hypoxemia. Though historically more commonly employed in neonates, the use of veno-venous ECMO in adults with hypoxemic respiratory failure has been on the rise47. The recent identification of populations who have benefitted from prior interventions highlights the likely impact of heterogeneity of treatment effect and signifies the importance of this work in advancing the science of ARDS. Ongoing studies of novel therapy, including medications50 and stem cell therapy53, and replication of prior successful studies of interventions such as prone positioning will also contribute significantly to the field. Additionally, increased understanding of optimal supportive care, including sedation targets and implementation of early mobility, is necessary to improve outcomes in these patients. Lastly, a better understanding of how to prevent ARDS and identify those at most risk may reduce the incidence of this highly morbid disease going forward.Despite improved outcomes and increased understanding of ARDS, many areas remain for future investigation and innovation; only with further progress will clinicians continue to advance care for these critically ill patients. Further delineation of subphenotypes and endotypes is necessary not only to provide a more accurate prognosis to any individual but also to inform and enrich future trials. Precise classification of patients, paired with an understanding of their underlying disease process, may increase the likelihood of identifying beneficial therapies and make real the potential of precision medicine. This may require the identification of novel biomarkers and biologic pathways but could also leverage already-available biomarkers and clinical data, as recent latent class analyses have. Development of easily-available testing of these biomarkers would make classification of patients by subphenotype more clinically feasible


Antibodies directed against malaria parasites are easy and inexpensive to measure but remain an underused surveillance tool because of a lack of consensus on what to measure and how to interpret results. High-throughput screening of antibodies from well-characterized cohorts offers a means to substantially improve existing assays by rationally choosing the most informative sets of responses and analytical methods. Recent data suggest that high-resolution information on malaria exposure can be obtained from a small number of samples by measuring a handful of properly chosen antibody responses. In this review, we discuss how standardized multi-antibody assays can be developed and efficiently integrated into existing surveillance activities, with potential to greatly augment the breadth and quality of information available to direct and monitor malaria control and elimination efforts. Elimination of malaria or effective and sustainable control requires deployment of interventions updated using surveillance data. Contemporary, accurate information on malaria transmission is required to determine which interventions should be deployed where to reduce transmission effectively given resource constraints. The ability to detect changes in transmission is an integral part of evaluating program activities by providing evidence for the effectiveness of interventions and identifying when and where changes are needed. Given the central role of surveillance data in guiding strategy, any tools that could provide data more accurately or cost-effectively would be of great value; the next generation of antibody assays for measuring malaria transmission holds promise for doing both.1 Thus, although the quality of and access to these data are improving, limitations remain, particularly in settings where a small minority of infections get reported through standard surveillance.2One would be hard-pressed to find a malaria program officer, epidemiologist, or funder of interventions who would not prefer a clearer picture of malaria in their target population. Some types of surveillance data, such as detailed entomological measurements or cohort studies, are not feasible for routine surveillance of transmission because they are too resource intensive to be performed extensively. These high-resolution snapshots are extremely valuable for answering research questions in small geographic areas at particular times but are too sparse to provide a contemporary picture of malaria transmission complete enough for programmatic use. The rest of the picture is filled in primarily from two other sources of data—routine passive surveillance and cross-sectional surveys. Passive surveillance data are widely and continuously collected as health systems provide care to those with malaria. The utility of these data depend heavily on the quality of diagnosis, completeness of reporting, and ability to account for important factors regarding catchment populations, care seeking, and clinical immunity.3 Prevalence data, although useful, are limited in the ability to detect changes in malaria transmission where transmission is so high that PR remains high even if exposure decreases considerably, or where it is so low that infeasibly large sample sizes are required to accurately measure changes over time or at fine enough spatial scales to be programmatically useful.4The most widely collected, standardized data currently available are based on surveys that collect blood from a cross-sectional sample of individuals in a population. The primary metric collected in most surveys to date has been the prevalence of parasites or parasite rate (PR), based on light microscopy, rapid diagnostic test, or nucleic acid detection . Data from such surveys form the basis of global maps of malaria.Plasmodium vivax, where people can harbor dormant liver-stage infection without having a concurrent blood-stage infection. It is possible to learn more about malaria exposure from the same blood sample by additionally measuring antibody responses to parasite antigens. This holds the promise that a single sample could add substantially more information to population-level measures of transmission because each antibody response measured provides information about past exposure in addition to current infection status. Obtaining antibody data is quite practical: enzyme-linked immunosorbent assay (ELISA) and some multiplexed assays are inexpensive and can be performed on material extracted from dried blood spots which are simple to collect and transport; point-of-contact tests such as lateral flow and microfluidic assays are also options. The result promises to be a more resolved picture of malaria transmission.Prevalence data are an important mainstay of surveillance, but each sample provides a single piece of information—whether a person has detectable blood-stage infection or not. This is a particular challenge in seasonal transmission environments or with 5 Antibodies have been used to estimate exposure to pathogens, including malaria parasites for over 70 years, but the approach has become more accessible and standardized because of availability of purified recombinant antigens and development of appropriate analytical methods. A commonly used strategy to estimate transmission intensity from serological surveys has been used to analyze seropositivity by age to compute a force of infection or seroconversion rate (SCR), which takes advantage of the fact that individuals in more highly endemic areas are more likely to have been exposed to malaria parasites and thus to have detectable antibody responses. These methods were initially developed for permanently immunizing infections such as measles and yellow fever,7 but adapted for malaria in the last decade.9 By translating age-stratified antibody prevalence data into a metric which reflects overall transmission in a community, the SCR extracts meaningful population information from individuals’ binary, typically long-lived responses to immunogenic Plasmodium proteins  using information contained in the titers of antibodies, instead of reducing them to binary responses; 2) measuring antibodies to multiple antigens with differing kinetics, rather than limiting measurement to one or a few antigens with long half-lives; and 3) developing analytical methods which take advantage of these rich data to provide precise, quantitative estimates of exposure history in populations from more intensive analysis of fewer samples.The SCR provides a useful metric for stable, community-level transmission, but with recent successes in decreasing malaria transmission, it is frequently of interest to measure changes in exposure over time. Signals of abrupt changes in exposure have been detected in the age-stratified antibody prevalence profile by estimating a change in SCR, reflecting a change transmission intensity at some point in time or with a certain age.14 Some of these models are similar in concept to those used to calculate SCR, but allow for different rates of boost and decay of relative antibody concentrations—data which are often already available from standardized ELISA or multiplex assay readouts. By using more information, these methods should produce more precise estimates of malaria transmission than those derived from binary responses. In parallel, investigators have begun to evaluate what information can be obtained by measuring responses to different antigens.18 Not surprisingly, responses to different antigens appear to provide information of greater or lesser value for answering specific epidemiologic questions in different populations depending on their immunogenicity and other properties. For example, by evaluating SCR to hundreds of proteins using a protein microarray, Baum et al.15 showed that a distinct subset of these targets was more efficient in distinguishing transmission in two areas of highland Kenya. Similarly, Ondigo et al.16 demonstrated that seroconversion and reversion rates to different antigens varied considerably and illustrated how evaluating SCR to a number of antigens could provide more information on the temporal structure of past exposure than looking at responses to a single antigen. To turn these promising findings into reliable, informative antibody assays, we propose a strategy to design Combined Antibodies to Measure Exposure Recency Assays (CAMERAs) with the aim of answering actionable questions across a range of epidemiologic settings.19Methods have been recently developed to extend the analysis of age-stratified antibody prevalence by incorporating antibody titers.Plasmodium exposure (including different species). Initiating prospective studies to define antibody kinetics throughout a representative set of epidemiologic settings would be an enormous endeavor.Obtaining precision surveillance data from antibodies requires a detailed understanding of how antibody responses change over time in response to infection, and how these kinetics are modulated by factors such as age, genetic diversity in parasite and host, and prior 28 The most useful of these have detailed capture of malaria infections through frequent active  and continuous passive surveillance, follow participants for an extended period of time , and have sufficient sample sizes to capture individual heterogeneity . Cohorts in which a documented change in transmission intensity has occurred are of particular interest, as these offer the best opportunity to disentangle recent from cumulative exposure. With these existing studies providing at least a subset of the samples needed, the next step is to identify antibody responses that are most informative about transmission. Although it has been shown that different antigens tend to elicit different qualities of response,30 it is still difficult to predict these a priori. In addition, it is not just the average of any particular metric of response to an antigen  that matters but also biological variation in these qualities across individuals. Therefore, a broad screen of responses provides the highest probability of identifying the optimal set of informative biomarkers.Fortunately, a number of well-characterized cohorts have been performed or are ongoing in malaria-endemic areas, and many have archived appropriate biologic material in the form of serum, plasma, or dried blood spots.31 This technique allows rapid screening of a large number of responses and requires minimal amounts of both sample and antigen. Rapid production of “crude” antigens without much optimization allows a large number of antibody responses to be screened up front with minimal start-up time. One potential downside of this approach is that a subset of antigens may not be properly expressed or folded, but this potential limitation is less of an issue for efforts trying to identify markers of exposure as opposed to characterizing correlates of immunity. Furthermore, this downside is potentially overcome by sheer numbers—responses to certain sets of antigens are likely to provide similar information, and by screening many analytes simultaneously, it is likely that at least one representative member of each set will be successfully evaluated. Other high-throughput approaches, including peptide arrays32 and phage display libraries,33 may allow rapid evaluation of individual linear epitopes, including the ability to capture naturally occurring genetic variation in the target. The effort in producing standardized reagents at scale—ultimately required for final assays—can then be focused on a down selected set of promising antigens followed by iterations of validation to design CAMERAs  chosen based on their combined information content measured at a single time point provided accurate estimates of whether an individual was infected in the last 30, 90, or 365 days and on their incidence of malaria in the prior year. These quantitative, individual-level data rivaled the rich information obtained from expensive cohort studies. For example, estimates of incidence obtained from antibodies accurately reproduced the spatial heterogeneity in transmission within a community detected by 1 year of entomological and clinical cohort data. When aggregated, antibody data from individuals dramatically outperformed parasite prevalence in precision and dynamic range, providing estimates of the incidence of malaria in a community from sample sizes as small as 20  subclasses of antibodies, and evaluating avidity may all in theory provide additional information given the different kinetics of these responses. Antibodies directed against mosquito salivary antigens may provide additional information regarding exposure to important vectors. In the end, consistent empiric data validated across multiple settings with appropriate gold standards will be the best arbiter of what should be included in a CAMERA. A number of platforms are currently available for measuring antibodies  measuring responses to multiple antigens; 2) sampling multiple people in a given area; 3) incorporating knowledge of age–exposure–antibody relationships into data analysis; and 4) tailoring antibody assays to specific age groups and/or transmission intensities. Regarding the latter, although having a universal antibody assay for use in all settings may seem the most straightforward, in reality it is likely that certain sets of responses will have more utility in some settings versus others. For example, antibody responses which provide information about a decline in entomologic inoculation rate from 100 to 10 infectious bites per person year may be different than those which can confirm the absence of recent exposure in an elimination setting. Depending on multiplex capacity, the same standardized laboratory platform may essentially contain multiple “assays,” of which only a subset are actually used to produce outputs for a given setting. Platforms requiring a more parsimonious set of analytes, for example, standard lateral flow assays may be best targeted to a specific epidemiologic setting.3 Estimates of transmission derived from antibodies will also be averaged across a population sample. Thus, while there will be some uncertainty as to, for example, if a particular individual has been infected two versus three times in the past year, precise estimates of transmission can still be derived for the population. As discussed earlier, the increased granularity of information provided by antibody data for each individual will likely result in more accurate estimates from smaller sample sizes despite inherent biological variation. This advantage holds across all transmission intensities but may be particularly salient in areas of very low transmission.The apparent ambiguity of how to interpret a particular antibody response in a particular individual has been a psychological barrier in the widespread dissemination and acceptance of antibody data for surveillance. However, more commonly used metrics, such as parasite prevalence, are subject to similar caveats. Detecting a parasite in an individual’s blood, while seemingly concrete, is still an indirect measure of transmission mediated by the duration and density of infection, which are functions of age, prior exposure, and the limit of detection of the assay used . For this reason, parasite prevalence requires age standardization a reasonably sized sample of the population to obtain a single point estimate.Antibody measurements have great potential for augmenting information obtained in malaria surveillance activities. However, specific questions remain on what should be measured, in whom, and how to best make sense of the data . This wi5 Efficient collection of samples from other convenient venues, for example, from those presenting to health facilities for routine care may provide continuous, low-cost data to augment metrics such as test positivity rates and malaria incidence. The ease of collecting more comprehensive data at low cost via such approaches will need to be balanced with issues surrounding potentially biased sampling and is an area ripe for further investigation.Once CAMERAs are designed and test characteristics are known, for example, among which age groups they are informative in a given setting, appropriate surveillance activities for sample collection and antibody measurement can be determined. For cross-sectional surveys, for example, malaria indicator surveys, school surveys, active and reactive case detection, and other surveillance activities, including those performed in intervention trials, CAMERAs offer a clear opportunity for obtaining additional information. Such measurements will add little incremental cost and, in fact, may offer substantial cost savings as smaller sample sizes may be required to obtain similar information. Incorporating measurement of antibodies in many cases requires no additional field work because many such surveys are already collecting dried blood spot samples. For example, demographic and health surveys (DHS) conducted in more than 50 countries already collect serum samples to measure specific biomarkers.Finally, antibody data generated need to be consistently translated into easily interpretable metrics of transmission. Development and evaluation of analytical methods will be an integral part of assay design, critical for making decisions regarding the number and type of antibody responses which will be measured. Simple point-of-care assays may require straightforward interpretation, for example, a band visible on a lateral flow assay might indicate infection within the past year. However, given the potential increase in the breadth and accuracy of information obtained from combining data from multiple antibody responses, other assays may use more sophisticated algorithms. Such algorithms can be easily implemented in software to provide straightforward interpretation regardless of the complexity of the underlying algorithm.The path for developing high-resolution CAMERAs is clear, and a number of research teams are working toward answering the salient questions outlined previously. As CAMERAs are designed, validated, and improved, their key role in malaria surveillance will come into clear focus.


It has been known for centuries that floral and extra-floral nectar secreted by plants attracts and rewards animals. Extra-floral nectar is involved in so-called indirect defense by attracting animals  that prey on herbivores, or by discouraging herbivores from feeding on the plant. Floral nectar is presented inside the flower close to the reproductive organs and rewards animals that perform pollination while visiting the flower. In both cases nectar is a source of carbon and nitrogen compounds that feed animals, the most abundant solutes being sugars and amino acids. Plant–animal relationships involving the two types of nectar have therefore been used for a long time as text-book examples of symmetric mutualism: services provided by animals to plants in exchange for food provided by plants to animals. Cheating (or deception or exploitation), namely obtaining the reward/service without returning any counterpart, is however, well-known in mutualistic relationships, since the interacting partners have conflicting interests and selection may favor cheating strategies. A more subtle way of exploiting mutualism was recently highlighted. It implies the evolution of strategies to maximize the benefits obtained by one partner while still providing the reward/service to the other partner. Several substances other than sugars and amino acids have been found in nectar and some affect the foraging behavior of insects and potentially increase the benefits to the plant. Such substances can be considered plant cues to exploit mutualism. Recent evidence motivated some authors to use the term “manipulation” of animals by plants in nectar-mediated mutualistic relationships. This review highlights the recent background of the “manipulation” hypothesis, discussing it in the framework of new ecological and evolutionary scenarios in plant–animal interactions, as a stimulus for future research. Mutualistic inter-species relationships, i.e., relationships in which interacting species reciprocate benefits received, are very common in all kingdoms of living organisms since virtually every species is involved in one or more such relationships. Mutualism has a pivotal role in the functioning of all current ecosystems and in key events of the evolutionary history of life on our planet, such as the evolution of eukaryotic cells, colonization of land by plants and the radiation of angiosperms .Mutualisms have often been reported as evidence of the ancient classical theory of “balance of nature” that is rooted in Greek philosophy and mythology. According to this theory natural systems tend to remain in a stable equilibrium where natural forces prevent species from becoming too abundant or becoming extinct . MutualiPlants are involved in a myriad of mutualistic interactions with very diverse organisms such as bacteria, fungi and animals. Mutualisms with bacteria (nitrogen-fixing bacteria) and fungi (mycorrhiza) increase nutrient uptake by plants as well as providing organic matter and a suitable ecological niche to the heterotrophic counterpart. Since plants are anchored to the ground and have limited possibility of movements, the benefits they receive in mutualistic interactions with animals, and especially insects, arise from insects’ ability to cover long distances. The animal’s ability to move is involved in two processes invaluable for plant survival: dispersal of propagules, mainly pollen and seeds, and indirect defense against herbivory .Pollen, the male gametophyte of seed plants containing the male gametes, needs to be transported from the anther to the stigma of a compatible carpel, a process called pollen dispersal or pollination that is the first step toward fertilization in all seed plants. According to a recent global estimate, 87.5% of all angiosperms are pollinated by animals  and a siBeing sessile and having limited movements, plants have developed an array of defense strategies against predation by herbivores . Direct Figure 1) since it is produced by organs (nectaries), usually inside the flower close to the reproductive organs, whereas nectar involved in indirect defense is generally offered in the vegetative part of the plant and is known as extra-floral nectar . Most insect pollinated angiosperms produce FN as the main primary floral attractant and their floral nectaries vary widely in position, shape and structure  exploit the EFN of Leonardoxa africana without protecting the plants from herbivores  sometimes rob nectar from flowers with long tubular corollas or spurs where the nectar is inaccessible . RobbingAllomerus cf. demerarae and Crematogaster nigriceps “castrate” their host plants, the former removing flowers from Cordia nodosa and the latter pruning axillary shoots bearing the inflorescences of Acacia drepanolobium  between plants, and ultimately plant population genetic structure .The behavior of foraging insects involves decisions when encountering a food resource according to variability of nectar traits (such as volume and concentration) and their spatial distribution . The abuPlants may be under selection to produce variable nectar resources so as to economize investment in nectar production while increasing the possibility of cross-pollination. At population level, the nectar standing crop generally has a patchy distribution: one or more highly productive plants are neighbors to others that produce less . The samStanding crop structure  affects both the duration of visits and distance between successive visits, since pollinators move quickly to more distant patches, individuals or flowers when they encounter nectarless or nectar-poor specimens . Short vPlants may exert control over nectar standing crop by providing highly variable nectar production that in turn affects the foraging behavior of pollinators. This outcome supports the idea that plants may “manipulate” the foraging behavior of pollinators to optimize pollen flow between individuals. In this framework a manipulation hypothesis was first elaborated by Nonetheless, nectar standing crop is affected by two orders of variability: variability in nectar production controlled by plants, on which further variability generated by the foraging activity of pollinators is superimposed . PollinaIt follows that plant control of pollinator behavior through modulation of variable nectar production is possible but seems quite weak. Plants may, however, use other tools to influence the feeding behavior of pollinators.A nectar trait quite recently considered when studying the effect of nectar on insect feeding behavior is its chemical composition. Floral and EFN is largely composed of sugars, usually together with other primary metabolites, such as amino acids, lipids, and proteins . SecondaBoth primary and secondary metabolites can have effects on insect behavior.Sugars and amino acids are the most abundant primary metabolites and are an important source of energy and nitrogen, respectively . Both suSucrose is the most common and abundant nectar sugar  and is pAll twenty amino acids commonly found in proteins have been identified in various plant nectars. Proline seems to be of special importance for insects. It not only contributes a taste preferred by insects , but alsThe link between sucrose and amino acids in affecting feeding behavior was also revealed by experiments testing how nutritional state affected the taste of specific amino acids  and associative learning of honeybees . ResultsPlant secondary metabolites (SMs) can be defined as “compounds that do not occur universally but are restricted to specific plant taxa, or occur in certain plant taxa at much higher concentrations than in others, and have no (apparent) role in primary metabolism” . Plants Nicotiana attenuata, where it increases the number of flowers visited and reduces the volume of nectar consumed by hummingbirds and moth pollinators  is a typical insect-repelling alkaloid and is found in the FN of linators . The unplinators . Shorterlinators . Plants Other nectar SMs may have phagostimulatory activity, although this function seems restricted to species adapted to feed on plants with a high content of SMs . Note thThe feeding deterrent function of nectar SMs is due to the unpalatable taste of alkaloids, especially nicotine, that is perceived by insects as soon as their proboscis contacts the nectar. The mouth parts of insects have contact chemoreceptors with neurons responding to sugars, salts, acid, water and non-nutrient compounds .Table 1). It was recently reported that honeybees rewarded with solutions containing caffeine  at concentrations similar to that occurring naturally in the FN of Coffea and Citrus species, remembered the learned floral scent better than honeybees rewarded with sucrose alone (Table 1). Caffeine, an adenosine-receptor antagonist, affected Kenyon cells’ activity, potentiating the response of honeybee brain mushroom body neurons that are involved in olfactory learning and memory formation . Bumblebees were only deterred by unnaturally high nicotine concentrations (50 ppm) and this deterrence disappeared or became attraction at lower nectar-like concentrations (1 and 2.5 ppm)  . The sam2.5 ppm) . AlthougTable 1). Those more common in nectar, i.e., γ-aminobutyric acid (GABA) and β-alanine, are important insect nervous system neuromodulators , i.e., amino acids that are not used by organisms to build proteins, are potentially involved in modulating insect behavior . In particular, when ingested, the alkaloids altered locomotion, memory, olfactory perception and reactions to stimuli in the Myrmica sabuleti ant model (Table 1). Whether any of these or other neuroactive compounds could be components of EFN, and their effects on attending ants at concentrations plausible for EFN, are not known. In this context, it is worth noting that ants are subject to manipulation by other organisms  can have significant effects on many aspects of ant physiology and behavior ; Table 1rganisms . A recenAcacia cornigera and the mutualist ant Pseudomyrmex ferrugineus is therefore not surprising (Pseudomyrmex ferrugineus ants were believed to lack invertase (a sucrose hydrolysing enzyme) in their digestive tract, a physiological trait compensated by the plant through secretion of sucrose-free EFN (Table 1), a dominant EFN protein that has a primary function in defense against nectar-dwelling pathogenic fungi . Clear effects of nectar-mediated manipulation are known for pollinating insects and are mainly based on secondary metabolites in FN . Although detailed studies are only available for caffeine and nicotine . On the animal side, although improved recall can be positive for efficient foraging activity, it also has a negative counterpart since bees tend to return to the source of caffeinated nectar when it is no longer available . Microorganisms such as yeasts and bacteria are very common in FN where they are inoculated by pollinators and can be considered a third partnership in nectar-mediated plant–pollinator interactions . Complexity similar to that of FN-mediated interactions is also likely for EFN but has not yet been investigated (Secondary metabolites in EFN and their possible interactions with tending ants (and other insects) have not been the subject of much research (stigated . Since tstigated .Plants modulating the concentration of SMs in their tissues and secretions evolved strategies to deter herbivores (high concentrations), while attracting and manipulating mutualists (low concentrations) to maximize the benefits they obtained. When such strategies evolved is hard to say. The oldest plant–insect relationship is predation of plants by herbivores and plants underwent natural selection on the basis of chemical defenses (secondary metabolites) evolved against herbivores. When mutualistic insects evolved (defenders and pollinators) they presumably drove plant selection toward optimal (low) concentrations of SMs (and other substances) in secretions they fed on, while plants probably started to manipulate insect behavior pharmacologically, improving their own fitness. Most “modern” mutualist insects  radiated in the interval 125–90 Mya , simultaneously with angiosperms . NectarsConcluding, since conflicts also arise in cooperative partnerships, nectar-mediated partner manipulations may be more frequent than previously thought in plant–insect interactions conventionally regarded as mutualistic. This may provide new evidence supporting the idea that elements of coercion/manipulation are not necessarily linked to parasitic habits but may be functional for stabilizing certain insect–plant mutualisms , openingMN, DG, and SM conceived the idea of the article and wrote the final version of the paper. MN designed the outline and wrote the draft of the paper. DG and SM commented on the draft.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The etiology of inflammatory bowel disease (IBD) remains elusive but it is believed to result from incompletely understood interactions between environmental triggers in a potentially genetically susceptible host and a subsequent aberrant immune response. Its incidence is increasing worldwide at an unprecedented rate, outpacing what genetic influences alone could instigate. The increasingly integral role played by eating in social life has led patients to gravitate to diet and food in their consultations with physicians and other health care professionals, in an attempt to improve, control, or even “cure” IBD through diet. Diet is a modifiable factor, and both patients and healthcare professionals have fuelled resurgent interest in the role of diet in maintaining IBD remission. Despite significant and increasing interest, there is a lack of credible evidence to support dietary modification or restrictions to prevent relapse of IBD. However, recent studies have shown that more than half of the patients believe that diet plays an important role in triggering relapse, leading to self-imposed dietary restrictions, some of which can have adverse consequences. This underpins the need for physicians and health care professionals to have a better understanding of dietary practices, in triggering, perpetuating, and improving IBD. This review examines and discusses the evidence behind this. The inflammatory bowel diseases (IBDs), comprising Crohn’s disease (CD) and ulcerative colitis (UC), are relapsing-remitting immune disorders of the gastrointestinal tract, hypothesized to occur from the combined effects of environmental factors in a genetically susceptible host , 2. An uIBD prevalence is highest in northern America, northern Europe and Australia increasing globally, particularly in Asia with industrialization and westernization of lifestyles and diet , 13, 14.The influence of diet as a risk factor for developing IBD has been studied in a number of epidemiological, observational, prospective, and retrospective case-control studies –24. A coA causal association between increased fat intake and IBD incidence was first suggested through the temporal association between granulomatous ileitis and the introduction of margarine early in the twentieth century . A JapanIn the EPIC study, a higher consumption of linoleic acid  was associated with a higher risk of developing UC whereas higher consumption of ω-3 PUFA was associated with a lower risk of UC . Eating Dietary fat comprises saturated and unsaturated fats, the latter consisting of polyunsaturated fatty acids (PUFA) and monounsaturated fatty acids (MUFA) . The ess2S), which in turn has detrimental inflammatory effects ., 68.4, 6While the prospect of “rewinding the clock” with dietary practices advocated by the Paleolithic diet, when these diseases (possibly) did not exist, may seem attractive, the lack of a mechanistic explanation defining its role in triggering and perpetuating gut inflammation and disease limits our ability to make evidence-based recommendations presently.The unprecedented global rise in incidence in IBD has clearly outpaced what genetic predisposition alone could instigate placing emphasis on environmental triggers in the etiology of the disease. Compelling evidence from laboratory and clinical studies implicates dietary changes as a factor within the complex “exposome” of IBD. Dietary research in IBD is fraught with challenges from ranging the complexity of inflammation biology and its myriad triggers but also by the multiple putative mechanisms by which food components affect the gut and induce symptoms. Strong patient interest will serve as an appropriate impetus and driver for more meaningful research, which has also been outpaced by popularized diets, some with and others without credible evidence leaving our patients and healthcare professionals vulnerable from unfounded literature. It is imperative for healthcare professionals advising patients with IBD to be conversant with the strengths and limitations of current literature to ensure credibility. Meanwhile, the evidence for exclusive enteral nutrition in reducing symptoms and mucosal inflammation holds promise for the therapeutic benefit for certain diets and derived supplements. These observations will serve to optimize clinical studies through identification of nocebo effects and pro-inflammatory food groups, enabling deeper mechanistic insights into the effects of food on the microbiota and the immune system. It is plausible then, that in unraveling the mysteries of the microbiome, diet, and immune interactions, we may discover who “we are” through what we eat.


Accurate estimation of microbial concentrations is necessary to inform many important environmental science and public health decisions and regulations. Critically, widespread misconceptions about laboratory-reported microbial non-detects have led to their erroneous description and handling as “censored” values. This ultimately compromises their interpretation and undermines efforts to describe and model microbial concentrations accurately. Herein, these misconceptions are dispelled by (1) discussing the critical differences between discrete microbial observations and continuous data acquired using analytical chemistry methodologies and (2) demonstrating the bias introduced by statistical approaches tailored for chemistry data and misapplied to discrete microbial data. Notably, these approaches especially preclude the accurate representation of low concentrations and those estimated using microbial methods with low or variable analytical recovery, which can be expected to result in non-detects. Techniques that account for the probabilistic relationship between observed data and underlying microbial concentrations have been widely demonstrated, and their necessity for handling non-detects (in a way which is consistent with the handling of positive observations) is underscored herein. Habitual reporting of raw microbial observations and sample sizes is proposed to facilitate accurate estimation and analysis of microbial concentrations. Whether describing pathogens in water or the density of red blood cells, the concentration of discrete objects cannot be measured directly. In these cases, concentration is estimated by enumerating or detecting the objects in finite sample portions ; such approaches are used extensively in health, food, and water applications. These estimates are required for decision making, during which they are typically evaluated against concentration-based criteria or targets  or low counts are widely perceived to be more uncertain and less reliable than those based on higher counts. This has often led to a desire to quantify enough of these objects by modifying the enumerated sample portion so that the count falls in a range that is deemed acceptable  result. With a series of repeated presence/absence tests  and assumed Poisson-distributed numbers of microorganisms in each test , the most probable number (MPN) approach yields a maximum likelihood estimate (MLE) of concentration .Although many of the aforementioned microbial enumeration methods have been standardized, protocols for the representation, reporting, and analysis of resulting data remain largely inconsistent. Standard microbiological methods, such as those stipulated within de facto concentration for statistical analyses and regulatory compliance—despite the recently stipulated caveat that MDLs are inapplicable to “methods that do not produce results with a continuous distribution such as […] presence/absence methods, and microbiological methods that involve counting colonies” . In fact, merely 50 ng of lead in a liter of water  is comprised of more than 1.45 × 1014 lead atoms due to the magnitude of Avogadro's number. In chemistry, random sampling errors associated with specific numbers of analyte particles in a well-mixed sample is largely insignificant compared to errors introduced through the application of the analytical method itself—the accuracy of the measurement is limited by the precision of the measurement instrument. The construct of the MDL is intended to reflect these method-specific errors to facilitate comparisons of data generated using different analytical methods for the same analyte at the lower end of concentration ranges.To understand the widespread convention of reporting NDs as values below MDLs in microbiology, it is important to understand the origin and motivation behind the concept of an MDL. The MDL  was developed as a performance criterion for chemical analyses  by applying appropriate statistical approaches  ; however, this approach is insufficient for fitting concentration distributions and quantifying data reliability or uncertainty in the calculated mean cyst analyses are 0.100 and 100 L, respectively. Intuitively, the probability of an ND observation from a single sample increases with decreasing concentration and analytical sample size. In practice, the occurrence of random NDs can be reduced by increasing sample size.Figure Building upon the previous example, Figure It may be useful to consider the concentration beyond which NDs become improbable  when comparing alternative methods, choosing a target sample volume, or determining the appropriateness of a method for a particular application. We propose that this threshold may be called a method sensitivity limit (MSL) because sensitivity is the probability of detection when the target microorganisms are actually present in the source. Considering the examples in Figure The statistical analysis of inappropriately censored microbial data ultimately leads to erroneous concentration estimates and subsequent interpretations. Bayesian techniques  implies a count of zero within the associated analytical sample volume. A positive test result can be construed as an inherently censored count of at least one microorganism because the method cannot reveal the exact number of microorganisms leading to detection. For a series of presence-absence tests, MPN approaches implicitly reflect censored data analysis by using the cumulative probability of all non-zero counts  to represent a positive test result in the likelihood function.replace TNTC results. We suggest that TNTC results should be retained in subsequent statistical analyses by using likelihood-based methods that allow inference from both the TNTC result and the count obtained through re-analysis of the sample. This would enable more accurate description of knowledge about the concentration by harnessing all of the available information rather than omitting inconvenient data.When using culture-based methods, counts beyond an upper limit are conventionally reported as TNTC [e.g., 150 or 200 colony forming units (cfu) for spread plates, 80 cfu for membrane filtration, and 300 cfu for pour plates, American Society for Testing and Materials Many environmental science and public health decision-making and regulatory frameworks rely upon the accurate evaluation of microbial concentrations and comparison with concentration-based criteria. For example, evaluation of source water pathogen concentrations is used to determine minimum treatment infrastructure requirements in the provision of safe drinking water   . The impact of small statistical sample sizes on concentration distribution parameter estimates is exacerbated when all of the data available are NDs, in which case statistical analysis is not possible without strongly subjective priors.Given sufficient and suitable information, the MLE approach incorporating random sampling error (Approach E) can be extended to account for analytical recovery. However, model fitting by MLE becomes more difficult with increasing model complexity—numerical integration required for evaluating the resulting likelihood function becomes practically intractable in many cases. Bayesian methods can be used to fit more complex probabilistic models to data, but also suffer from substantial parametric uncertainty where insufficient data and/or data that are relatively uninformative about model parameters are available Gleit, . Indeed,Giardia cyst concentrations estimated from a handful of samples, these data may exemplify monitoring results from utilities that draw upon high quality source waters. Such systems, especially those that have limited treatment, operational, and/or monitoring capacity, are particularly vulnerable to the implications associated with overestimated mean concentrations. As demonstrated in this analysis, concentration estimates may be biased high by a factor of two or more just by handling NDs as censored. This could lead to operational and maintenance costs/adjustments  (Cotton et al., Although treatment requirements are not typically determined based on mean Non-detect microbial detection and enumeration data are fundamentally not censored data and should not be reported or analyzed as such.Method detection limits are not intended to be used for, and have therefore been misapplied in, detection- and enumeration-based methods that count discrete microorganisms.The convention of reporting non-detects as censored values relative to a method detection limit is misleading when using enumeration-based methods and has resulted in the misuse of censored data statistical approaches for microbial data analysis.It is inconsistent to consider the uncertainty in non-detects by representing them as censored data while ignoring the inherent uncertainty in all non-zero counts.Censored data approaches should be reserved for data correctly interpreted as being censored, such as too numerous to count plate counts where the actual count is known only to exceed a specified threshold.This work re-emphasizes that raw microbial data must be reported to facilitate proper statistical analysis approaches that account for measurement error.PS, ME, and AC designed the work. AC, PS, and ME analyzed the data and prepared the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The exacerbation of a clinical condition or the occurrence of negative symptoms after an inert substance dispensation or a sham treatment is known as “nocebo effect.” Nocebo is the psychobiological effect due to the negative psychosocial context that accompanies a therapy, and it is a direct consequence of negative expectations by the patients and their own personal characteristics. Although the clinical relevance of the phenomenon is now recognized, a small number of studies have tried to ascertain its neural underpinnings (that it means nocebo responses). Moreover, there is no consensus on the brain networks involved in nocebo processes in humans. In particular, nocebo hyperalgesia has attracted almost no research attention. We conducted a mini-review on the few experimental pain functional magnetic resonance imaging (fMRI) studies of nocebo responses to discuss how negative expectancies and conditioning effects engage brain networks to modulate pain experiences. Moreover, we present possible clinical implications considering Alzheimer’s disease and behavioral frontotemporal dementia, in which the existence of a hypothetically disrupted neurocognitive anticipatory network—secondary to an endogenous pain modulatory system damage—may be responsible for pain processing alterations. Understanding nocebo responses is important for both clinicians and neuroscientists, first of all, because it is substantial across disorders and could be associated with objective pathology and survival. Moreover, research on nocebo provides a way to investigate how the brain systems implicated in the processing of contextual information influence physiology and clinically relevant outcomes.via inert substances and negative verbal suggestions when hyperalgesic stimuli are not dispensed. In such kind of circumstances, the experimenter presents the “supposed incoming pain” in such a believable way to induce the expectation of a “real painful stimulation.” Subjects’ psychobiological responses may be recorded in functional magnetic resonance imaging (fMRI) paradigms where the anticipatory phase of pain—considered as the time that elapses between the beginning of the neuroimaging acquisition and the beginning of the painful stimulation—is acquired.In the last two decades, functional neuroimaging studies analyzed the association among negative expectation, pain anticipation, and nocebo responses in humans. Brain imaging techniques have been invaluable to explore the neurobiology of pain anticipatory mechanisms occurring Within this research framework, this mini-review first addresses the theme of neurofunctional pain anticipation mechanisms. The results of the only meta-analysis in the literature will be presented in order to summarize data from the literature in human pain experimental models using fMRI activation foci, and it will be used as an explanatory cue to interpret nocebo responses . Indeed,Pavlovian conditioning). The repeated association among the clinical context around the patient , the alleged pharmacological principle (the substance contained in the syringe), and an increased level of experienced pain usually induce a conditioned response, so after repeated associations, only the sight of the syringe or of the doctor will be sufficient to induce hyperalgesia.This mini-review also analyzes how negative expectancies and nocebo conditioning effect engage brain networks to modulate pain experiences in the few fMRI paradigms evaluating nocebo responses. In particular, the proposed fMRI studies not only analyzed the cortical–subcortical and spinal neuronal substrate associated with the conscious influence of negative expectancies in pain anticipation paradigms but also took into consideration the conditioned nocebo response. A model derived from a series of studies  describeFinally, we present possible clinical implications considering Alzheimer’s disease (AD) and frontotemporal dementia (FTD), where a failure of functional recruitment of cortical network corroborates an altered response to pain and a poor response to analgesic treatments .http://www.ncbi.nlm.nih.gov/pubmed). We used a single set of query terms: nocebo AND fMRI [ALL]. With this aim, we reviewed the relevant literature in order to ensure the following: 1) the use of only inert substances or verbal suggestion (not studies on nocebo effects in the context of drug application); 2) the pain anticipation paradigm included a nocebo condition to study related functional activity; 3) the studies reported cerebral activation and deactivation changes, as assessed by blood-oxygen-level-dependent (BOLD) imaging; and 4) the experimental population was composed of healthy subjects. While we were in the selection phase, we found few studies that analyzed nocebo-related responses in the context of fMRI protocols, allowing us only to conduct a descriptive mini-review. The articles selected for the present mini-review are indicated in A systematic search strategy was implemented to identify relevant studies on nocebo responses, published until 31 December 2018, across the online database most frequently used in the international literature (Medline database with PubMed literature search: activation likelihood estimation (ALE), which determines the convergence of foci reported from different experiments , anterior insular cortex (AIC), and lateral and medial prefrontal cortices   and (cognitive expectancy effect (a flexible adaptive response modified by predominant conscious context)  and  cognitivcontext) . Considecontext) . The autcontext) . Specificontext) .To conclude, the nocebo hyperalgesic effect may also envisage a pain-facilitating spinal cord mechanism at a very early stage of pain processing, well before cortical processing . In thisWhen considering pain processing in neurodegenerative disorders—especially in Alzheimer’s disease (AD) and behavioral frontotemporal dementia (bv-FTD)—a reduction in the motivational and affective components of pain is often described . Indeed,If we consider the neuropathogenic changes related to the AD and bv-FTD onset, it is possible to notice that they involve the neural underpinnings of pain processing : In partAlthough no studies exist concerning pain anticipation in AD and bv-FTD due to ethical constraints, it is significant to report a study on pain anticipation in mild traumatic brain injury (TBI) . When coThe neurobiological basis of the nocebo effect is only now beginning to be disentangled. One of the most productive models to better understand the neurobiology of the nocebo effect is pain. Pain anticipation and nocebo response neural correlates have been studied with fMRI neuroimaging techniques in the last decade. A host of cortical and subcortical regions can be activated by various negative verbal instructions and contexts. Those areas identified a set of core sites related with an affective-cognitive pain pathway where the prefrontal, cingulate, insular, and orbital cortices play an important role. Other areas less reported in the literature include the brainstem, hippocampus, amygdala, superior temporal gyrus, parietal operculum, and superior parietal lobule. Nocebo effects are clinically significant but are often underrecognized in clinical practice . MoreoveIndeed, the current clinical approach is to maximize the placebo effect and minimize the nocebo effect to provide the greatest possible benefit to the patient . These sStudies in the field of pain have previously proposed three different mechanisms to explain the nocebo effect: expectation, conditioning, and anticipatory anxiety . Taking For example, doctors usually warn patients of the painful nature of an impending procedure (which means “pain anticipation” in the patients’ mind). This may have an iatrogenic effect and may increase the pain subjectively experienced by the patient at the time of treatment administration. Indeed, it has been demonstrated that lower pain scores occur if the message was focused on the treatment’s beneficial effects rather than it being painful .There is no doubt that “only with attention to the empirical findings from programmatic research of specific and nonspecific effects and their interaction is it possible to improve the outcomes of treatment beyond the status quo” .In our mini-review, we consider the systems-level neurobiology that underlies nocebo effects; we focus primarily on pain, which has been most extensively studied. The issues of nocebo responses basically encompass both the site and the mode of brain function. It can be considered as the initial stage in the genesis of pain perception and conscious suffering. A better understanding of the nocebo-related responses is crucial to relieve its impact on clinical practice and to enhance the therapeutic outcome. The above statements have relevance for neurodegenerative disorders and chronic pain states, for which the functioning of essential brain regions related with nocebo responses may amplify negative outcome to treatments through an activation of a neural cognitive-affective network.MA conceived the content of the review, wrote the first draft of the manuscript, supervised subsequent changes, and took part in critique processes. SP wrote the manuscript, produced images and tables, and took part in the review and critique processes.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.This work was supported by the fund GEMG_RILO_18.


The founding tenet of evidence-based medicine is to combine best evidence with clinical expertise. As David Sackett opined ‘Without clinical expertise, practice risks becoming tyrannised by evidence’. Rigid protocols and mandates, based on an inconclusive and low-level evidence base, cannot suit the physiological, biochemical and biological heterogeneity displayed by the individual septic patient. Indeed, clear proof of outcome benefit through adoption of an inflexible management approach is lacking and will certainly be detrimental to some. Therapy thus needs to be tailored to meet the individual patient’s needs. The same principle should be applied to clinical trials; the continued disappointments of multiple investigational strategies trialled over three decades, despite (often) a sound biological rationale, suggest a repeated methodological failure that does not account for the marked heterogeneity within the septic patient’s biological phenotype and thus marked variation in their host response. The increasing availability of rapid point-of-care diagnostics and theranostics should facilitate better patient selection and titrated optimization of the therapeutic intervention. The history of intensive care has been littered with too many false dawns. Old management dogma, now derided, have been replaced by new and equally resolute convictions, many of which will, in time, undoubtedly follow a similar course. Retired bundles of care have been replaced by new bundles driven by clinical evangelists and government diktat. Yet these too lack a strong evidence base and can be readily challenged. Homogenizing treatment given to a highly heterogenous patient population that fall under the wide syndromic umbrella of sepsis may offer a minimum standard of care though at the potential cost of compromising outcomes in an individual patient. Likewise, the repeated multicentre trial failures of novel interventional strategies over many decades highlight the need to focus more precisely on the individual’s biological phenotype and adopt a personalized treatment approach. This article will briefly cover these issues and offer directions for the future.‘Good doctors use both individual clinical expertise and the best available external evidence, and neither alone is enough. Without clinical expertise, practice risks becoming tyrannised by evidence, for even excellent external evidence may be inapplicable to or inappropriate for an individual patient.’‘Evidence based medicine is not “cookbook” medicine. Because it requires a bottom up approach that integrates the best external evidence with individual clinical expertise and patients’ choice, it cannot result in slavish, cookbook approaches to individual patient care. External clinical evidence can inform, but can never replace, individual clinical expertise, and it is this expertise that decides whether the external evidence applies to the individual patient at all and, if so, how it should be integrated into a clinical decision. Similarly, any external guideline must be integrated with individual clinical expertise in deciding whether and how it matches the patient’s clinical state, predicament, and preferences, and thus whether it should be applied. Clinicians who fear top down cookbooks will find the advocates of evidence based medicine joining them at the barricades.’Evidence-based medicine is intended to offer best treatment and represents a worthy aim to raise standards and homogenize care. It offers a framework when basic expertise is lacking. An excellent example is the Advanced Trauma Life Support course, established by an orthopaedic surgeon (James Styner) after a plane crash in rural Nebraska in 1976 on finding medical care for his injured children was disorganized and suboptimal. However, one size does not, and should not, fit all. While providing structure is clearly important, especially for less-experienced healthcare providers, it must be tailored to the individual patient. To cite David Sackett, one of the ‘godfathers’ of evidence-based medicine )As an example, those with a strong pro-inflammatory phenotype may benefit from an appropriate degree of suppression . HoweverThe advent of rapid point-of-care diagnostics will facilitate appropriate selection of biologically suitable patients into studies, and optimization of drug dosing . There is no guarantee of success as the derangement in biomarker level may be epiphenomenal rather than integral to a causative pathway, or may even be part of an adaptive process. However, the theranostic targeting of a specific biomarker can be tested a priori for proof-of-principle in appropriate animal models .


Recent studies have demonstrated that breast milk contains a population of cells displaying many of the properties typical of stem cells. This review outlines progress made in this newly emerging field of stem cell biology and provides an analysis of the available data on purification, propagation and differentiation of certain types of progenitor cells from breast milk. The possible fates of breast milk cells, including microchimerism caused by their transmission to the distant organs of the infant, are also discussed. Unique properties of breast milk-derived stem cells, such as their unusually low tumorigenic potential and their negligible ability to form teratomas, are highlighted as obvious advantages for using these cells in regenerative therapy. Breast-feeding is an indispensable post-parturition stage in the process of mammalian reproduction that is crucial for neonatal survival during critical periods of their postnatal development. Each mammalian species has a unique composition of milk that has developed during evolution to fulfill the requirements of their newborns and infants. In humans, the natural reproduction process includes a long period when all nutrients and other chemicals required for successful physical and intellectual development of a child come from milk provided by a lactating mother, emphasizing the importance of this biological product. For this reason, human breast milk has been intensively studied for many years , 2.It is now well known that in addition to specially balanced essential nutrients, breast milk contains functionally distinct bioactive components that are involved in remodeling of the immune system of neonates and infants, and alter their susceptibility to different types of infection .Recent studies have revealed the complex supplementation mechanisms that involve a direct supply of mother’s mature immune cells as well as trafficking of other cell types supporting the development and functional regulation of both the innate and adaptive immune systems of infants –8. It haBifidobacterium breve, B. adolescentis, B. longum, B. bifidum, B. dentium and others [One of the most remarkable recent findings is the demonstration that infections or other changes in the status of the nursed infant trigger a rapid response in secretion, composition and cell contents of consumed breast milk. The exact mechanisms of this sophisticated feedback that support the dynamic relationship between infant’s homeostasis and efficient fulfilling of its immediate needs remain unknown –16. The d others –19.7–108 when around 800 ml of milk is consumed daily [Although study of probiotic bacteria in human milk is a very recent and clearly underdeveloped field of research, there is already experimental evidence that selected bacteria from the gastrointestinal microbiota of nursing females are able to access the mammary gland via the entero-mammary pathway. This transduction occurs by dendritic cells and CD18 + cells carrying nonpathogenic bacteria from the gut lumen to the lactating mammary gland . It cameed daily , 21. Thied daily .Breast milk is also a potential source of some previously unrecognized biologically active entities. One recent and very exciting finding is the demonstration that the exosomes purified from breast milk are able to promote intestinal epithelial cell growth in infants even when they are formula feeding . The stiNew methods for the identification and separation of cell suspensions, such as multicolor flow cytometry, allow for the accurate assessment and quantification of the cell composition of biological fluids. Implementation of these methods has already significantly advanced our current knowledge about various cell populations present in breast milk. Cells of eukaryotic origin  found in breast milk can be pooled in to two major groups: blood-derived and breast-derived cells, and in both these pools small groups of progenitor or stem cells have been identified –29.+ luminal epithelial cells and beta-casein-positive lactocytes that synthesize milk proteins. In human milk produced by healthy nursing females feeding healthy infants luminal and myoepithelial cells together could constitute up to 98% of all cells [Not surprisingly, the largest proportion of total cell counts in breast milk is CK18ll cells . Howeverll cells . One of ll cells –34. Normll cells .+), a mammary stem cell marker. In the lactating mammary gland, CK5+ cells usually present in the alveoli and ducts of the epithelium and most probably they represent the source of CK5+ cells in cultures obtained from donor milk. However, the source of these cells and their possible role in milk is still enigmatic [The committed stem cell progeny are seen as an important source of human stem cells for therapeutic purposes –38. Thesnigmatic .+ luminal cells, or CK14+ myoepithelial cells [Other cells with characteristics typical for stem cells were also found in cultures established from cells present in breast milk. These include cells expressing α6 integrin (CD49f), a mammary stem cell marker, and an epithelial progenitor marker p63 , 42, 43.al cells , 42.These groundbreaking reports have boosted further research on the cellular components of breast milk. Remarkably, all the main findings reported in the original publications have been successfully confirmed using colony-forming assays carried out by other laboratories and our own unpublished data , 27. MorSo far, no differentiation techniques specific for cultivated milk cells into various cell lineages have been developed but methods and factors commonly used for differentiation of progenitor cells of other origin linage have been successfully employed. Sani et al.  cultivatIn another study, the hBmSCs were found positively labeled with the stage-specific embryonic antigen 4 marker (SSEA4) as well as TRA-1-60 and TRA-1-81 antigens, both markers for undifferentiated pluripotent human stem cells. Furthermore, the ability of cultured hBmSCs to differentiate under certain conditions towards different epithelial lineages , and to form mini-mammary glands secreting milk proteins has been demonstrated . PerhapsThe presence of a novel stem cell population with multilineage differentiation potential has also been recently identified in bovine milk. This cells isolated and characterized by Pipino et al. were able to grow as a plastic-adherent culture and differentiate into osteogenic, chondrogenic, and adipogenic lineages . This heIn a recent study of cell populations that exhibit a stem cell phenotype, Briere et al. compared breast milk samples obtained from feeding mothers of preterm infants and full-term infants and found obvious differences in the proportion of stem-like cells in these two types of samples. Further confirmation of this observation has been demonstrated by showing distinct patterns of expression of stem cell-specific genetic markers in theses two samples . The lat® blue for flow cytometry analysis of breast cells in human milk. This novel and reliable approach allowed to identify and quantify subpopulation of CD11b+ monocytes as well as putative stem cells positive for cell surface markers TRA-1-81 and SSEA-4 [+ putative stem cells the high variations were detected in samples without significant differences between the gestational age groups.Due to the physico-chemical properties of breast milk, purification of its cellular components with a quality sufficient for the immediate analysis by flow cytometry methods that would be sensitive enough to detect the pool of cells displaying pluripotency markers is a challenging task. The main problem is the ability to discriminate between cells and noncellular components. Keller et al. have developed an improved protocol that use a recently developed staining agents DRAQ5™ far red and SYTOXd SSEA-4 . InteresTaken together, the available experimental evidence suggests that hBmSCs are able to differentiate into all three germ layers Fig.  and that+ hematopoietic stem/progenitor cells has been demonstrated in colostrum and breast milk and it is highly probable that they originate from the maternal bloodstream [So far, no published data provide strong enough experimental evidence for the origin of hBmSCs. Nevertheless, Hassiotou and Hartmann have recently suggested that at least some subpopulations of hBmSCs could have the same origin as hematopoietic stem cells . The preodstream , 54. How+) were used for fostering and breastfeeding wild-type newborn pups. The maternal GFP+ cells in the suckling’s blood and in the brain have been identified by fluorescence-activated cell sorting, polymerase chain reaction and immunohistochemistry. Moreover, the authors have provided experimental evidence that GFP+ cells have differentiated into both neuronal and glial cell types in the brain after being foster fed by GFP+ female mice. These data strongly suggest that GFP+ cells were delivered to offspring by breast milk and were able to differentiate into different types of cells [Transmission of cells from mother to fetus and from fetus to mother via the placenta, and from feeding mothers to nursing infants via breast milk has been studied for several decades and is well documented , 55–60. of cells .Research on microchimerism generated by consumed breast milk cells has firmly supported the important physiological role of this process in the efficient maturation of the infant immune system , 68. ForThe discovery of hBmSCs and identification of cells differentiated from hBmSCs in various organs of recipients paved the way to the idea that breast milk is a novel promising source of transplantable stem cells for therapeutic use. An important reason why hBmSCs are included in the list of prospective sources of human pluripotent cells for use in regenerative medicine is their unusually low tumorigenic potential and the negligible ability to form teratomas . AnotherSo far, stroke therapy seems to be the most attractive application for hBmSC transplantation. In a comprehensive report that compared various stem cell types for their potential efficacy to sequester stroke-induced neuroinflammation, feasibility as translational clinical cell sources, safety and suitability as a transplantable material, hBmSCs have been suggested as a promising source of stem cells for treatment of a stroke-associated pathology . This coOther recently developed protocols allow in vitro differentiation of hBmSCs into various types of neuronal and glial cells, including pentraxin3 producing astrocytes , 75. SinThe current major limitation for clinical applicability of breast-milk-derived stem cells for treating stroke is the lack of knowledge about their therapeutic potential in vivo, particularly about their restorative effects, efficacy and safety . For theIn conclusion, there are more questions than answers in the story of breast milk stem cells but this field is rapidly gaining momentum and might bring surprising results and unexpected benefits.