To assess the efficacy and safety of high-flow nasal cannula oxygen therapyin treating moderate hypercapnic respiratory failure in patients who cannottolerate or have contraindications to noninvasive mechanicalventilation.A prospective observational 13-month study involving subjects admitted to anintensive care unit with or developing moderate hypercapnic respiratoryfailure. Clinical and gas exchange parameters were recorded at regularintervals during the first 24 hours. The endpoints were a oxygen saturationbetween 88 and 92% along with a reduction in breathing effort (respiratoryrate) and pH normalization (≥ 7.35). Subjects were considerednonresponders if they required ventilatory support.versus 24.3± 1.5, p = 0.22), which was apparent in the first four hours oftreatment. The pH improved, although normal levels were only reached after24 hours on high-flow nasal cannula therapy . The rate ofnonresponders was 13.3% (4 subjects), of whom one needed and acceptednoninvasive mechanical ventilation and three required intubation. Intensivecare unit mortality was 3.3% (1 subject), and a patient died after dischargeto the ward .Thirty subjects were treated with high-flow nasal cannula oxygen therapy.They consisted of a mixed population with chronic obstructive pulmonarydisease exacerbation, acute cardiogenic pulmonary edema, and postoperativeand postextubation respiratory failure. A nonsignificant improvement wasobserved in respiratory rate (28.0 ± 0.9 High-flow nasal cannula oxygen therapy is effective for moderate hypercapnicrespiratory failure as it helps normalize clinical and gas exchange levelswith an acceptable rate of nonresponders who require ventilatorysupport. The purpose ofthis treatment is to prevent the development of hypoxemia and the resulting tissuehypoxia. However, oxygen should be administered in a strictly controlled way toprevent its known adverse effects. Complications of oxygen therapy include hypercapnia andacidosis caused by several pathophysiological mechanisms , which may result in the patient requiringventilatory support.The first line of treatment for acute hypercapnic respiratory failure - apart frommeasures for controlling causative and precipitating factors - is oxygentherapy. Oxygen saturation (SpO2)must be continuously monitored by pulse oximetry and maintained within a narrowinterval, ranging between 88 and 92%. In case of hypercapnic acidosis, excessive respiratory work orhypoxemia despite the administration of oxygen > 40%, noninvasive mechanicalventilation (NIV) should be used, unless it cannot be tolerated, is contraindicatedor if the patient needs to be intubated.In these situations, scientific societies recommend administering a specific oxygenconcentration via a high-flow oxygenation system such as Venturi masks.,8 Furthermore, HFNCcan be used in combination with heaters/humidifiers of inspired gas and prongs,which facilitates patient tolerability partly due to the replacement of fitted facemasks. High-flow nasalcannulas have been successfully employed in patients with moderate hypoxemicrespiratory failure. Some ofthe mechanisms of action of HFNC can also be effective in the treatment of acutehypercapnic respiratory failure. High-flow nasal cannula provides a washout effect of the upperairway dead space, which reduces hypercapnia. Additionally, it reduces airwayresistance and, consequently, the work of breathing. Finally, HFNCs deliverexpiratory positive airway pressures (continuous positive airway pressure - CPAPeffect) that can counterbalance intrinsic positive end-expiratory pressure (PEEP),which is present in most of these patients.In recent years, high-flow nasal cannulas, the so-called HFNC therapies, have gainedpopularity, since they deliver high flows (up to 50 - 60L/minute) and accurateconcentrations (21 - 100%) of oxygen. Initially, as with any othertherapy that has not been previously tested in robust clinical trials, HFNC could beused for mild to moderate hypercapnic respiratory failure in settings where patientsafety is preserved and under the supervision of independent research ethicscommittees.For these reasons, our hypothesis is that - in hypoxemic acute respiratory failure-HFNC can be used as an alternative therapy to NIV in patients in which the latteruse is not possible due to intolerance or is contraindicated.The purpose of this study was to assess the efficacy and safety of HFNC oxygentherapy in the treatment of moderate hypercapnic respiratory failure as analternative to NIV in the context of a treatment protocol in patients of anintensive care unit (ICU).This is a prospective, observational study conducted between October 1, 2014 andNovember 30, 2015 involving ICU patients with moderate acute hypercapnic respiratoryfailure who received HFNC therapy as part of a treatment protocol established forthis condition. The study was approved by the Ethics Committee of the hospital thatwaived the need for consent for reviewing medical records. All data weredisaggregated, anonymized and entered into a database.vCO2) > 50mmHg and pH < 7.35 in acompatible clinical context. The criteria for initiating HFNC oxygen therapy inpatients with respiratory failure were persistent SpO2 < 88%, despitethe use of an oxygen face mask with fraction of inspired oxygen (FiO2)set at ≥ 40%. Eligible patients were those who could not tolerate theinterface or had relative or absolute contraindications to NIV. At this point, thechoice of HFNC therapy over NIV, IMV or continuing oxygen face mask was at thephysician's discretion. The exclusion criteria were as follows: pH ≤ 7.25;Glasgow coma scale < 12 points; dysfunction of multiple organs > 2,respiratory included; and clinical and metabolic criteria for shock. This means thatpatients were excluded if they required immediate invasive (or noninvasive)mechanical ventilation (MV). The criteria for discontinuation of HFNC therapy werea) patient improvement with stable SpO2 ≥ 88%, FiO2≤ 0.4 with a flow below 25L/min; and b) worsening of the patient's conditiondue to intolerance to treatment, persistent or worsening dyspnea, persistentabdominal paradox, respiratory rate ≥ 35rpm, systolic blood pressure <90mmHg, SpO2 < 88%, increase in pvCO2 by >10mmHg and/or decrease in pH by > 0.08; and c) patient refusal. All patients admitted to the ICU with a diagnosis of hypercapnic acute respiratoryfailure episode were treated following a treatment protocol previously implementedin clinical practice, which included the following devices: (1) nasal cannula set ata flow of up to 5L/min; (2) Venturi face mask set up to 40%; (3) HFNC oxygentherapy; (4) NIV; and (5) invasive mechanical ventilation (IMV). The treatment wasinitiated with one of these devices according to the clinical status of the patient,then depending on the patient's responsiveness and course of disease, the therapywas maintained or shifted to another device. Complementarily, patients received theconventional therapies indicated for their status according to standard clinicalpractice guidelines . Thediagnostic criteria for hypercapnic acute respiratory failure were carbon dioxidevenous pressure or baseline time, at patient's admission to the ICU with their previous oxygentherapy; (t1) 1 - 4 hours after initiation of HFNC therapy; (t2) 5 to 8 hours; (t3)9 to 12 hours; and (t4) 13 to 24 hours. The null hypothesis was that no significantdifferences would be observed between the study periods concerning the studyvariables. This was tested using ANOVA for repeated measures and a post hoc test(Scheffé's test) between them when statistically significant differences wereobserved. A p < 0,05 was considered significant. The statistical softwareemployed was Statistical Package for Social Science (SPSS), version 15.Follow-up of patients was performed from patient admission to the ICU until hospitaldischarge. Other variables included demographics (age and sex); etiology of therespiratory failure; level of severity as estimated by Acute Physiology and ChronicHealth Evaluation II (APACHE II); time on HFNC; need for NIV or endotrachealintubation; stay in the ICU; and mortality . Patients whoreceived palliative HFNC therapy were excluded from the analysis.® ventilator set at "oxygen therapy" mode, whichconstantly controls FiO2 (0.21 - 1) and flow (2L/min at 50L/min). Optimalgas conditioning was achieved by the use of a MR850 heated humidifier with an RT340dual-heated breathing circuit connected in series to a set of OptiFlow(tm) nasalcannulas (Fisher & Paykel)  .Of the 1,304 patients admitted to the ICU during the study period, 35 received HFNCtherapy for acute or chronic respiratory failure. High-flow nasal cannula oxygentherapy was palliative in five of the 35 patients and was excluded from the study. Apatient flowchart is shown in chronic obstructive pulmonary disease (COPD): 20 (66.6%);congestive heart failure: 5 (16.6%); and sleep-related or obesity hypoventilation: 5subjects (16.6%). Ten episodes were at admission to the ICU and 20 after extubationfrom the IMV . The reasons for not using NIV asthe first choice were as follows: 12 not tolerant to the oronasal mask (anxiety anduncooperative); 10 copious secretions or inability to cough; 3 esophageal or gastricsurgery; 2 mismatch between mask and face and not stated in 3.With regard to the etiologies of respiratory failure, the most common wereversus 24.3± 1.5, p = 0.22) within four hours after initiation of the HFNC therapy.The clinical effects of HFNC included a reduction in respiratory rate  . Posthoc analysis revealed statistically significant differences in pH between baselineand all the other times, as well as between t1 and t4.The effects of HFNC on gas exchange parameters are shown in Of the 30 patients who received HFNC, four subjects (13.3%) required MV, of whomthree (10%) required IMV and one (3.3%) required and accepted NIV. The mean stay was7.3 ± 11.9 days (95%CI 5.1 - 9.5) in the ICU and 15.5 ± 12.8 days(95%CI 13.2 - 17.9) in the hospital. One patient in this group died in the ICU(3.3%), and another subject died in the hospital, which represents a mortality rateof 6.6%.The data obtained demonstrate that HFNC is clinically effective, since it reducesrespiratory rate and reverses respiratory acidosis. The protocol implemented in ourICU in moderate cases, introducing HFNC as an alternative to NIV when there isintolerance to the interface or relative/absolute contraindications to its use, hasbeen shown to be safe in this setting, with an acceptable rate of ventilatorysupport rescue.vCO2, which is consistent with the resultsreported in previous clinical and pathophysiological studies.,14-16 The fact that adecrease in respiratory frequency was not accompanied by an increase inpvCO2 is due to the HFNC washout effect of the upperairway dead space. Conversely, a statistically significant increase was observed inpH, probably caused by the slight decrease in pvCO2 which,although it was not significant, had an effect on the acid-base status.Interestingly, pH values within the first hours after the initiation of thetreatment increased from 7.28 ± 0.18 to 7.31 ± 0.18, which are verysimilar to the increase from 7.27 ± 0.10 to 7.31 ± 0.09 reported byBrochard on his seminal study on NIV. Furthermore, although respiratory work and oxygen cost werenot measured, they must have presumably been reduced as a result of the improvementachieved on ventilatory efficiency. The high flow rates employed produce anexpiratory pharyngeal pressure and hence a CPAP effect, which can counterbalance theintrinsic PEEP and airway inspiratory resistance present in these patients.The use of HFNC was associated with a reduction in respiratory rates, although it hadno effect on p Thus,our rate of intubation was 10% for HFNC (18 - 28% reported for NIV), and thehospital mortality rate was 6.6% (versus 10 - 13% for NIV).The clinical outcomes obtained with the use of HFNC for decompensated chronicrespiratory failure are consistent with the moderate severity of the patients whoreceived this therapy, with lower intubation and mortality rates than that reportedfor patients with NIV. reported the useof HFNC for the management of a patient with chronic hypercapnic respiratory failurewho did not tolerate NIV. Patient tolerability was achieved due to improvement inpatient comfort and reversion of pathophysiological alterations. Similarly,Díaz-Lobato et al.reported the case of a patient with amyotrophic lateral sclerosis who presented inthe emergency department with acute hypercapnic respiratory failure. Since thepatient did not tolerate NIV and refused intubation, she was successfully treatedwith HFNC as evidenced by the improvement in pH and partial pressure of carbondioxide (PaCO2) achieved, regaining consciousness and being dischargedafter five days of hospitalization. The authors stated that her response to HFNC wassimilar to that expected for NIV. The only case-series study where patients withCOPD were not excluded was that conducted by Rittayamai et al., where the etiology of respiratoryfailure was an exacerbation of COPD in 6 of the 17 patients. Although these patientswere not analyzed separately and the use of HFNC was immediately after extubation(as a preemptive therapy), the effects obtained were similar to those observed inour study. Recently, a retrospective analysis of 33 patients in a medical intensivecare unit with acute hypercapnic respiratory failure and the use of HFNC waspublished. Thus,patients and settings were similar to ours but probably they had less severe diseasebecause the pH was in the normal range. They found a slight decrease inPaCO2 (approximately 4mmHg in the first hour), similar to ourresults. In a more complete physiological study on the effect of HFNC onneuroventilatory drive and work of breathing of 14 patients with hypercapnic failurein the postextubation period, Di Mussi et al. did not find any differences in the breathing pattern andgas exchange compared to those observed with oxygen delivered through a face mask.Again, patients had a pH in the normal range, indicating a less severecondition.The use of HFNC in adult patients with acute on chronic respiratory failure is stillanecdotical, and only a few case-report studies have been published. Thus, Millar etal. Comparedwith unaided breathing, VT increased in patients in the COPD andidiopathic pulmonary fibrosis groups, while it decreased in healthy volunteers. Therespiratory rate and minute volume decreased in all groups. Nilius et al.investigated the effects of HFNC in COPD patients with chronic hypercapnicrespiratory failure; althoughthere was a high interindividual response, in general terms, respiratory rates andPaCO2 were reduced. Chatila et al. observed increased exercise capacity withimproved oxygenation via HFNC compared to spontaneous breathing in patients withCOPD in an unloaded bicycle ergometer test. Okuda et al. used HFNC to improvesleep-related hypoventilation in a patient with COPD. In conclusion, there is emerging evidence thatHFNC is a highly promising treatment for some types of hypercapnic respiratoryfailure. Two protocols for such studies have been recently published.,29Regarding stable patients, a range of studies on the effects of HFNC on ventilatoryparameters support the findings of our study. Bräunlich et al. evaluated theeffect of HFNC in healthy volunteers, COPD patients, and idiopathic pulmonaryfibrosis patients. investigated theeffects of a HFNC system on dyspnea, mouth dryness and overall comfort as measuredby a visual analog scale versus conventional Venturi® masks in thetreatment of 20 ICU patients with acute respiratory failure. The HFNC was associatedwith less dyspnea and mouth dryness and was found to be more comfortable than facemasks. Schwabbauer et al.compared the subjective degree of dyspnea , the generallevel of discomfort and a general evaluation of each type of therapy (Venturi maskversus HFNC versus NIV). The scores werehigher for HFNC in all dimensions than for NIV. The main limitation of both studiesis the short period of observation, which was less than one hour. In our study-where the time on HFNC therapy and follow-up period were longer- and in agreementwith the studies by Carratalá Perales et al. and Tiruvoipati, no remarkable adverse effects were observed, andonly one patient rejected the HFNC system due to discomfort.According to the available evidence, one of the most remarkable aspects of HFNC isthe high patient acceptability and comfort observed, since this system allowspatients to eat, drink, talk, cough and clear secretions. In 2010, Masclans etal. Another limitation of this studyis that venous gases instead of arterial gases were considered for patient controland follow-up, together with the SpO2 and theSpO2/FiO2 ratio. This choice was made toprevent complications from repeated arterial punctures and/or cannulation whenhemodynamic control was not indicated, as well as to reduce the use of invasivemethods and avoid work overload for the nursing staff. It has been demonstrated thata correlation and concordance exist between pH and bicarbonate values in venous andarterial gasometry when novariations occur in cardiac output and carbon dioxide production. Thus, in our study, a goodpH/bicarbonate concordance can be assumed, since patients with shock, hypotension,increased lactic acid levels or who were in a hypermetabolic state wereexcluded.The main limitation of this study is its observational design, which may involve aselection bias. As this is a noncontrolled, pretest/posttest study, it allows us toassess the efficacy of a measure, but we cannot be certain that the improvementsobserved were due to the intervention. The admission policy of our ICU involves theadmission of patients who can be potentially cured and the rejection of those withadvanced illness and a very poor prognosis, although their physiological respiratoryparameters during decompensation may be similar. Although the quantitative criteriafor the initiation of HFNC therapy are clearly established in the protocol, theclinical judgment of the severity of symptoms has a subjective component that mayinfluence the type and timing of therapy initiation. Nonetheless, as the clinicaljudgment of physicians has been demonstrated to be better correlated with the courseand prognosis of disease, it must be taken into account in all protocols.-39 The easy administration andfollow-up of HFNC therapy may provide a false sense of safety. However, this type oftherapy should be administered to selected patients, and follow-up should beperformed by trained personnel who can frequently assess treatment response andperform immediate intubation and MV when necessary.Finally, in accordance with the existing literature, we advise against theindiscriminate use of HFNC.This preliminary study demonstrates that high-flow nasal cannula therapy is effectivein improving clinical and gas exchange parameters in patients with moderatehypercapnic respiratory failure, with an acceptable rate in nonresponders whorequired ventilatory support. These results should be confirmed with rigorousclinical trials before being translated into clinical practice.


Community assembly of crop-associated fungi is thought to be strongly influenced by deterministic selection exerted by the plant host, rather than stochastic processes. Here we use a simple, sorghum system with abundant sampling to show that stochastic forces  act on fungal community assembly in leaves and roots early in host development and when sorghum is drought stressed, conditions when mycobiomes are small. Unexpectedly, we find no signal for stochasticity when drought stress is relieved, likely due to renewed selection by the host. In our experimental system, the host compartment exerts the strongest effects on mycobiome assembly, followed by the timing of plant development and lastly by plant genotype. Using a dissimilarity-overlap approach, we find a universality in the forces of community assembly of the mycobiomes of the different sorghum compartments and in functional guilds of fungi. Fungal community assembly on crop plants is thought to be driven by deterministic selection exerted by the host. Here Gao et al. use a sorghum system to show that stochastic forces act on fungal community assembly in leaves and roots early in host development and when sorghum is drought stressed. Equally well appreciated is their association with dead plants where their role in ecosystem carbon and nitrogen cycling has been highlighted7. In contrast, studies of the total communities of fungi associated with plants are few and far fewer than those of total communities of bacteria1, despite the fact that, in terrestrial environments, fungi account for more biomass than bacteria9. Here we tackle this gap in knowledge of fungal communities associated with plants by adding the fungal mycobiome to studies of the bacterial microbiome11. We use an approach that accounts for the variation in plant compartment , plant development (from seedling emergence to grain maturation), physical environment (irrigation and drought), and host genotype (drought response to either retain or suppress photosynthesis) as summarized in Supplementary Data 13, and will not only directly affect plants and their fungal communities, but also indirectly affect plants through changes in the fungal community14. Positive, indirect effects of fungal communities on plants could best be harnessed by modern agriculture15, if the mechanisms of community assembly were better understood.In nature, the intimate, symbiotic association of fungi with living plants is well appreciated, whether as mycorrhizal partners or parasites16. Selection is wholly deterministic and drift is wholly stochastic17, but both dispersal and diversification have stochastic and deterministic components17. Of the four processes, drift, the stochastic extinction caused by random species abundance fluctuation18, is the most difficult to demonstrate because one must first rule out the other three processes, two of which have stochastic components, although one of these—the evolutionary process of diversification—can reasonably be ignored for fungi over a period as short as a season.The assembly of communities rests on the activity of the four processes that influence constituent species: selection, drift, diversification, and dispersal18. However, neither of these methods of estimation necessarily represent the consequences of ecological drift due to possible undermeasurement of the processes of selection, dispersal and diversification18. Drift is thought to most strongly influence community assembly when (i) communities are small or when (ii) they have recently been released from selection imposed by a stress such as drought20; However, due to the difficulty of detecting drift, even in these two situations its demonstration in nature remains rare23.Ecological drift has been estimated in ecological model fitting as the ‘unexplained’ compositional variation, or has been estimated from empirical data as the dispersion of beta diversity 24, in particular, are the underlying ecological dynamics of microbiomes universal across all communities or unique to individual communities? To address this point, we employed the recently developed, dissimilarity-overlap curve (DOC)24. The DOC approach has been applied to recent studies of human-associated bacterial microbiomes, finding universal ecological dynamics across all communities25. Although many attempts have been made to assess the universality of microbial community assembly, assessment of universality by the DOC method has been applied to just two types of fungal mycobiomes, both of which are arbuscular mycorrhizal (AM) fungal communities. These studies found both universal and unique ecological dynamics depending on ecosystem type  and phosphorus availability (low v. high)27.Adding our study to those already in existence raises the question of universal features of mycobiome assembly and temporal changeSorghum bicolor (L.) Moench] grown in homogenous soil with intensive sampling from thrice-replicated plots for all possible combinations of two genotypes, four plant compartments, three water treatments, and 17, weekly time points, giving a total of 1026 samples 11, and the sorghum transcriptome28.To thoroughly address the mechanism of community assembly, we combined a simplified environment with one crop species, sorghum  cultivars  for which we collected a total of 1026 samples  and ITS4Fun (AGCCTCCGCTTATTGATATGCTTAART)30. The yields of PCR products were quantified using a Qubit dsDNA HS kit  and 200 ng of DNA from each of the 1026 samples were randomly assigned to four different pools, purified using AMPure magnetic beads , checked for concentration and amplicon size using the Agilent 2100 Bioanalyzer , and sequenced on the Illumina Miseq PE300 sequencing platform  at the Vincent J. Coates Genomics Sequencing Laboratory .DNA was extracted from leaf, root, rhizosphere, and soil samples using the MoBio PowerSoil DNA kit . DNA concentration was measured using a Qubit dsDNA HS kit . Fungal internal transcribed spacer 2 (ITS2) was PCR-amplified from DNAs diluted to 5 ng/μl with ddH11. Briefly, raw fastq sequences were subjected to quality evaluation using FastQC v0.11.576, removal of primers using cutadapter v1.9.177, merging of forward and reverse reads, control of quality, clustering of OTUs, and global search using USEARCH v8.078, to generate a table of 1026 samples × 1293 OTUs . The representative sequence of each OTU was identified by a BLAST search against the curated, fungal specific UNITE database79 and the NCBI database. A total 1070 OTUs were identified as fungal , and 223 OTUs were non-fungal . Fungal OTUs were assigned into functional guilds using the FUNGuild v1.180.Detailed description of bioinformatic analysis can be found in our previous publication81. Analysis of qPCR mixtures was accomplished using a Real-Time PCR Detection System  containing 1 μl of 5 ng/μl genomic DNA, 10 μl iTAQ SYBR Green Supermix with ROX , 0.2 μl of 100 mg ml−1 BSA, 0.15 μl of each 50 μM primer and water to 20 μl82. Thermal cycling conditions consisted of an initial denaturation at 95 °C for 3 min, followed by 40 cycles of 15 s of denaturation at 95 °C and 1 min of annealing and extension at 60 °C, finishing with a dissociation stage of 95 °C for 15 s, 60 °C for 30 s, and 95 °C for 15 s82. Standard curves were developed using a series of 10-fold dilutions of plasmids containing a fragment of an insert of the 18S gene of Penicillium purpurogenum82.Fungal biomass was estimated by quantitative PCR (qPCR) of the fungal small subunit rRNA (SSU or 18S) using the FF2 (GGTTCTATTTTGTTGGTTTCTA) and FR1 (CTCTCAATCTGTCAATCCTTATT) primers28. Using the BBsplit script in BBmap83, fungal transcripts were mined from these transcriptomes, by referencing fungal genomes in MycoCosm84 of Cladosporium fulvum v1.0, Paraphoma chrysanthemicola PD 92/468 v1.0, Acremonium strictum DS1bioAY4a v1.0, Alternaria alternata 133aPRJ v1.0, Fusarium fujikuroi IMI 58289, Fusarium verticillioides 7600 v2, Fusarium oxysporum f. sp. lycopersici 4287 v2, Chaetomium globosum v1.0, Talaromyces marneffei ATCC 18224, Cryptococcus vishniacii v1.0, Ustilago maydis 521 v2.0, and Sporobolomyces roseus v1.0.Alternatively, fungal relative abundance was estimated by the percent of fungal transcripts in transcriptomes containing both sorghum plants and their fungi generated from 198 root samples and 197 leaf samplesIn 2017, thirteen passive air samplers (empty petri dishes) were installed following a nested design . Fungal OTUs distributions among leaf, root, rhizosphere and soil (both unplanted and planted) were visualized by Ternary plots using the ggtern package85 in R v3.5.186. Indicator species for selected sampling times were identified and their threshold values were calculated using threshold indicator species analyses (TITAN) in the TITAN2 package87 in R. Manhattan plots were constructed in the ggplot2 package88 in R to visualize the P-values for the pairwise comparison of fungal OTUs between cultivar BTx642 and cultivar RTx430, and between control and either pre- or post- flowering drought. The most important age-discriminant fungal OTUs were identified from a random subset of 50% control samples using the random forest (RF) model in the RandomForest package89 in R, and then visualized in a heatmap plot using the pheatmap package90 in R. The sparse RF model of these age-discriminant fungal OTUs was used to predict the ages of drought samples and another subset of 50% control samples; and the discrepancies between predicted ages of control and drought samples were used to assess the extent of drought effect. To visualize the relative abundances of fungal functional guilds, fungal phyla, and common fungal OTUs, bar plots were constructed using the ggplot2 package88 in R.The functional affiliation of fungal genera and their relative abundances of both unplanted soil and sorghum mycobiomes were depicted by Krona charts constructed using the ktImportText command of the KronaTool v2.7 , we rarefied the number of fungal sequences per sample to 362  using the rrarefy command in package vegan91. Detail about the compositional method (dataset 2) can be found at our previous publication11. Briefly, the raw read data is transformed by zeros imputation and centered log-ratio (CLR) conversion using the zCompositions package92 and the CoDaSeq package (https://github.com/ggloor/CoDaSeq)31. Direct comparison of the two approaches is possible with permutational analysis of variance (PERM ANOVA), but not for other analyses because the statistical methods for compositional datasets are different from those for traditional count datasets, e.g., Bray–Curtis dissimilarity for counts v. Aitchison distance for compositional, and principal coordinate (PCo) analysis for counts v. principal component (PC) analysis for compositional31. For most of our analyses, methods are not yet available for compositional datasets, e.g., the DOC24, the Simpson metric93, the RCI54, and the βNTI53.Recent recognition that microbiome data from HTS represents a random sample of the DNA molecules in an environment and not absolute counts of the molecules argues that the data be treated as compositional and not as counts91 in R, and Aitchison distances were calculated for dataset 231. PERM ANOVA was carried out to assess the effect of compartment , time period (weeks 1–17), cultivar  and drought treatment  on the fungal community variation either detected by Bray–Curtis dissimilarities or Aitchison distances using the adonis command in vegan package91 in R. To visualize the variations in fungal community compositions, the Bray–Curtis dissimilarity was subjected to principal coordinate analysis using the pcoa command in the Ape package94, and the Aitchison distance was subjected to principal component analysis in base package86 in R. Euclidean dissimilarities were calculated to construct distance matrices of geographic and temporal distances in the vegan package91 in R. Mantel tests were carried out to explore the correlations between geographic, temporal and community composition distance matrices in the vegan package91 in R. To remove the effect of richness variation on fungal community composition, the Simpson metric of community dissimilarity was calculated based on presence/absence data using the beta.pair command in the betapart package95 in R, and then ordinated by the principal coordinate analysis in the Ape package94 in R. To test the homogeneity of the fungal community during succession96, beta dispersions of Bray–Curtis, Jaccard and Simpson dissimilarities were explored by the betadisper function in the vegan package91 in R. To explore the impact of richness on bacterial community compositional variances, we used betadispesion analysis of the Jaccard and Simpson dissimilarities for the rice datasets35.Bray–Curtis dissimilarities were calculated for dataset 1 to construct distance matrices of the fungal community (Hellinger transformed) using the vegdist command in vegan package54 and Stegen et al.53 in R. Stochasticity was recognized from the proportions of community pairs that fell within |RCI| < 0.95 and |βNTI| < 2. Our calculation of βNTI from 1070 fungal ITS2 OTUs relied on a fungal phylogeny based on 18S + 28S rDNA sequences . To investigate potential links between stochasticity and ecological drift, relationships of the proportion of |RCI| < 0.95 and |βNTI| < 2 were compared with the size of fungal communities as estimated from: (i) the percent of fungal reads as compared to the total fungal and plant reads in ITS2 amplicon and Illumina sequencing, (ii) abundance of fungal SSU detected by qPCR and (iii) the percent of fungal transcripts as compared to the total fungal and plant transcripts in sorghum root and leaf transcriptomes. Meanwhile, we also explored the relationships between fungal richness and the proportion of |RCI| < 0.95 and |βNTI| < 2, to explore the link between stochasticity and ecological drift. These comparisons of stochasticity and fungal abundance and richness were explored by linear mixed-effects models, that included random effects of compartment type using the lme command in the lme4 package97 in R. The variance explained  by the mixed effect models was calculated by the r.squaredGLMM function in the MuMIn Package98 in R. To depict the compositional variation in fungal communities, Fst measures were calculated using the script of Gilbert and Levine18 in R.To assess stochasticity in fungal community assembly and temporal change, we calculated the Raup-Crick index (RCI) and the beta Nearest Taxon Index (βNTI) using the scripts of Chase et al.https://github.com/Russel88/DOC) in R. The DOC emerges by plotting, for each possible pair of communities, the dissimilarity on the y axis  against the fraction of taxa that overlap on the x axis. Where the DOC dips as the overlap grows, universality is supported and the level of support is proportional to the fraction of pairwise comparisons under the graph where the DOC slope is negative . For the smoothed curve of a given DOC, the initiation of negative slope represents the median of initiation of negative slopes calculated from DOCs of 1000 bootstrapped data sets. To explore which OTUs tend to co-occur, a co-abundance network was constructed using the igraph package99 in R. To avoid type I error of multiple comparisons, all statistical significance measures were corrected using the Bonferroni method100.To assess the universality in ecological dynamics across fungal communities, we used the DOC approach by constructing the curves using the DOC package (Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Software 1Reporting Summary


Citrus sinensis) are highly susceptible to HLB, and trifoliate orange  is widely considered resistant or highly tolerant to HLB. In this study, an intergeneric F1 population of sweet orange and trifoliate orange was genotyped by Genotyping-by-Sequencing, and high-density SNP-based genetic maps were constructed separately for trifoliate orange and sweet orange. The two genetic maps exhibited high synteny and high coverage of the citrus genome. Progenies of the F1 population and their parents were planted in a replicated field trial, exposed to intense HLB pressure for 3 years, and then evaluated for susceptibility to HLB over 2 years. The F1 population exhibited a wide range in severity of HLB foliar symptom and canopy damage. Genome-wide QTL analysis based on the phenotypic data of foliar symptom and canopy damage in 2 years identified three clusters of repeatable QTLs in trifoliate orange linkage groups LG-t6, LG-t8 and LG-t9. Co-localization of QTLs for two traits was observed within all three regions. Additionally, one cluster of QTLs in sweet orange (linkage group LG-s7) was also detected. The majority of the identified QTLs each explained 18–30% of the phenotypic variation, indicating their major role in determining HLB responses. These results show, for the first time, a quantitative genetic nature yet the presence of major loci for the HLB tolerance in trifoliate orange. The results suggest that sweet orange also contains useful genetic factor(s) for improving HLB tolerance in commercial citrus varieties. Findings from this study should be very valuable and timely to researchers worldwide as they are hastily searching for genetic solutions to the devastating HLB crisis through breeding, genetic engineering, or genome editing.Huanglongbing (HLB), or citrus greening, is the most devastating disease in citrus worldwide. Commercial citrus varieties including sweet orange ( Since the first identification of the disease in Florida in 2005, HLB has spread throughout the state, and is now found in most states in the United States where citrus is grown. It is rapidly spreading throughout many of the world’s production areas, as well. Due to widespread infection and lack of effective management strategy, Florida’s nearly $11 billion citrus industry has experienced a rapid and continuous decline. The production of sweet orange dropped from 240 million boxes in 2004 to 45 million in 2018  is the most widespread and virulent species and is the only species reported in the citrus industry of United States. CLas is a heat-tolerant Gram-negative bacterium, resides only in the phloem of plant hosts, and is vectored by the sap-sucking Asian citrus psyllid   . As an od genera . Most cod genera ,b. Withid genera .CLas  to identify QTLs associated with citrus HLB infection and responses separately in trifoliate orange and sweet orange genetic maps.This study inaugurates investigation of HLB infection in citrus by repeated phenotyping of segregating populations and QTL mapping. The objectives of this study were: (1) to evaluate severity level of HLB disease among a field population exposed to intense HLB pressure using two phenotypic traits, foliar symptoms and canopy damage; (2) to construct high-density genetic maps separately for trifoliate orange and sweet orange in an F1 population of 170 individuals derived from mixed intergeneric crosses between two sweet oranges (Citrus sinensis ‘Sanford’ and ‘Succari’) and two trifoliate oranges (Poncirus trifoliata ‘Argentina’ and ‘Flying Dragon’), including 79 progenies of ‘Sanford’ × ‘Argentina’, 40 progenies of ‘Succari’ × ‘Flying Dragon’, and 51 progenies of ‘Flying Dragon’ × ‘Succari’. Of the genotyping population, 86 individuals were randomly chosen as a phenotyping population, including 47 progenies of ‘Sanford’ × ‘Argentina,’ 14 progenies of ‘Succari’ × ‘Flying Dragon’, and 25 progenies of ‘Flying Dragon’ × ‘Succari’. In addition, two sweet orange varieties (‘Navel’ grafted on ‘Swingle’ and ‘Hamlin’ grafted on ‘C-35’), six trifoliate orange varieties , and seedlings of ‘Volkamer’ lemon (Citrus volkameriana) were included as controls in the phenotyping. All progenies were clonally propagated by grafting on ‘Volkamer’ lemon in the greenhouse of the USDA/ARS in Fort Pierce, FL in 2010. Except for ‘Volkamer’ lemon seedings that had 16 replicate trees, each of the progenies and control varieties had eight clonal replicate trees. A completely randomized experiment of 768 trees was established in a field trial at the USDA-ARS in Fort Pierce in 2011. The planting consisted of eight rows oriented south-north, with 4 m × 1.5 m planting distances. Guard citrus trees not analyzed in this study were planted at the ends of each row. The field population trees were irrigated and fertilized with professional practices, but pesticides were not applied during the study, to encourage psyllid population increase, feeding, colonization, and inoculation of CLas to the trees. Maintenance of the field trees was as described for a nearby experiment on the same farm  values of qPCR for direct CLas detection were normalized with Ct values of the corresponding host plant gene. Trees with Ct value under 33 were considered to be HLB-positive  and the period of the most evident disease symptoms. At each time of disease evaluation, the visual evaluation of foliar symptom and canopy damage was conducted twice per tree on each side along the tree rows. All evaluations were carried out by the same individual researcher. Foliar disease symptom severity was assessed on a 6-point scale visual rating for comprehensive typical HLB symptom including mottled rugose leaves and yellow shoots: 0 = no symptom for whole tree, 1 = isolated (less than 1/10 of tree) slight symptom, 2 = partly (approximately 1/3 of tree) slight symptom or isolated severe symptom, 3 = mostly (approximately 2/3 of tree) slight symptom or partly severe symptom, 4 = mostly severe symptom, 5 = severe symptom for whole tree. The slight foliar symptom rating refers to trees with leaves that are slightly mottled, pale or leathery, while the severe symptom rating refers to severely blotchy mottled, rugose or leathery. Canopy damage severity was also assessed on a 6-point scale visual rating basing on dieback, defoliation or stunting: 0 = full tree canopy without apparent dieback or defoliation or stunting, 1 = full tree canopy with isolated short dieback or very slight defoliation, 2 = partly short dieback or isolated long dieback or slight defoliation or stunting, 3 = mostly short dieback or partly long dieback or moderate defoliation or stunting, 4 = mostly long dieback or main branch dieback or severe defoliation or stunting, 5 = trunk dieback or whole tree dead. The mean rating score for each genotype in each year was the averaged rating score of all replicate trees at the two times of evaluation.Evaluations of HLB disease symptoms were performed twice per year from 2015 to 2016 in September and October, which is the optimal time for HLB disease evaluation considering both the growth condition of citrus  Statistics 17.0. Significant differences between phenotypic data were declared with 2 as a reference. Genotypes at each locus were determined using the VCF Popgen Pipeline version 4.0 to generate a customized VCF 4.1  database with parameters set as follows: minimum allele frequency for genotyping 0.075, minimum Phred score 15, minimum depth of sequencing coverage of 12, and minimum 75% of individuals with the specific genotype call. The marker configuration codes “lm × ll” and “nn × np” were used to represent markers that were heterozygous only in one of the parents, and code “hk × hk” to represent markers that heterozygous in both parents.Genomic DNA of each citrange hybrid and parent was extracted using a modified CTAB method , Genomic1 progenies from different crosses between the two genera were considered as a single family, after excluding the inconsistent marker loci between parental varieties. To ensure high quality of linkage mapping for each parent, SNP markers segregating from only one of the parents (with configuration codes “lm × ll” and “nn × np”) were selected to construct the linkage maps; markers with code “hk × hk” were not considered in this study. SNP markers matching the following criteria were excluded from linkage analysis: (1) had a missing genotype in more than 10% of progenies; (2) had a missing genotype for one of the parents; (3) had inconsistent genotypes among different parental sweet oranges or trifoliate oranges; (4) had homozygous genotypes for both parents; (5) had heterozygous genotype for both parents; (6) had genotypes in F1 progeny not expected for the parental genotypes; (7) had no segregation in F1 progeny. Segregation distortion was tested by χ2 conformity tests against the Mendelian segregation ratio of 1:1. Because the method of linkage analysis was based upon a test for independence of logarithm of the odds (LOD) scores that is not affected by segregation distortion, markers with certain skewed segregation were included in linkage analysis referring to previous studies on citrus , molecular polymorphism among sweet orange cultivars is very rare ; likewisn citrus . Linkagen citrus . Linkagen citrus  with twon citrus .QTL mapping was carried out on two parental maps separately using MapQTL 6 under the backcross model by composite interval mapping (CIM) . Phenoty1 progenies were processed for RAD sequencing on the NGS Illumina platform. Except one progeny, all had acceptable quality of sequencing reads. The average number of reads was 8.14E6 and 7.37E6 for trifoliate orange and sweet orange, respectively. The read counts for the 169 F1 progenies ranged from 1.05E6 to 14.91E6, with an average of 4.53E6 per progeny. After alignment with the reference genome, the average number of read clusters was 1.83E5 and 1.47E5 for trifoliate oranges and sweet oranges, respectively, and the numbers of read clusters for the F1 progenies ranged from 0.32E5 to 2.79E5 and averaged to be 1.03E5  and 170 F1 progenies from different crosses were combined as a single family. After stringent filtering, a total of 3,861 high quality SNP markers with configuration code “lm × ll” or “nn × np” were retained for genetic map construction, of which 1,408 SNP markers were heterozygous in trifoliate orange and 2,453 SNP markers were heterozygous in sweet orange.In total, 51,687 putative SNP loci were determined, of which 55.6% were transitions and 44.4% were transversions. Excluding SNP loci without calls, 96.7% of the genotyped SNP loci were identical between ‘Argentina’ and ‘Flying Dragon’ trifoliate orange, and 98.2% were identical between ‘Sanford’ and ‘Succari’ sweet orange , which might indicate partial resistance to CLas infection. The results indicate that all trees of the phenotyping population were adequately inoculated with CLas under continuous reinfection  were also significantly lower than those in sweet oranges (ranged from 3.7 to 4.5). For F1 progenies, the ratings of canopy damage ranged from 0.9 to 4.1, and the average rating was 2.7 in 2015 and 2.6 in 2016. The frequency distributions of foliar symptom and canopy damage ratings among the F1 progenies are illustrated in Figure The evaluation of HLB disease among the phenotyping population was conducted in 2015 and 2016 through repeated rating of the foliar symptom and canopy damage separately (Table R2) ranging from 13.9 to 29.9%. In sweet orange, two QTLs associated with foliar symptom were identified in both 2015 and 2016, but no significant QTL associated with canopy damage was identified in either of the 2 years. The maximum LOD score for each of the QTLs ranged from 3.1 to 5.5, explaining an estimated phenotypic variation (R2) ranging from 17.3 to 29.1%. None of the QTLs alone could explain a majority of the phenotypic variation, but they collectively explained a major part. The graphics of QTL mapping are available in Supplementary Figure QTLs associated with the two phenotypic traits were detected separately in two parental genetic maps for each year Table . In trifBased on the confidence intervals of the QTLs on the genetic maps, most of the QTLs are repeatable between the 2 years Figures , 6. For In this study, we constructed two separate parental genetic maps, which, to our knowledge, are the highest density genetic maps to date for trifoliate orange and sweet orange. The former highest density genetic map of trifoliate orange consisted of 146 SNP markers and 74 SSR markers, spanning a total genetic length of 937.1 cM and having an average density of 0.23 marker/cM . Our tri1 population with only 124 individuals. By contrast, 1 population with 278 individuals from crossing of tangor and sweet orange, but only 661 non-redundant SNP-based DArTseq markers were finally mapped on the integrated map. The differences indicate that the use of intraspecific populations tends to achieve much higher marker density in the construction of genetic maps than interspecific populations, even though the population size is much smaller. In our study, the mapping population is neither interspecific nor intraspecific hybrids, but intergeneric hybrids, which had made it more difficult to achieve high-density genetic maps. The potential karyotypic and genomic divergences between trifoliate orange and sweet orange can prevent normal meiotic pairing and homology recombination during meiosis. Some genomic divergences between the two species can be obviously observed from the conservation of synteny of the genetic linkage maps and the reference genome, e.g., the divergences on LG-4, LG-7 and LG-8. In addition to parental genotype, differential fitness of gamete genotypes, crossing direction, and regulatory gene interactions likely also contribute to the high level of segregation distortion in citrus , and a few trees were even HLB-positive . It is important to note that at least eight replicate trees were clonally propagated for each genotype and they all were used throughout the phenotyping, which ensured high accuracy and reliability of the phenotypic results for each genotype. Thus, we believed that the trifoliate orange was probably infected by CLas, but the CLas titer was held at relatively low levels. Our results on HLB disease evaluation also support this interpretation. The trifoliate oranges were not completely healthy under the intense HLB pressure, but mostly displayed slight foliar disease symptoms and slight canopy damage. The results further suggest that trifoliate oranges are not immune to CLas infection, but can inhibit growth of CLas and show low symptom levels when they are infected. It is noteworthy that obviously different degrees of tolerance to HLB was observed among the F1 progenies, and a few progenies showed similar results of CLas diagnosis and disease evaluation as trifoliate oranges, indicating the HLB tolerance is inheritable. Moreover, some other hybrids containing the germplasm of trifoliate orange were also found with certain tolerance to HLB (Unlike CTV, CiLV, ABS and citrus nematode for which strong resistance is available within the citrus gene pool, the suppression of HLB in trifoliate orange may be best described as tolerance. Trifoliate orange had been reported as resistant to nfection , but recnfection . In our e to HLB , probablAlthough high-density genetic maps were constructed for both trifoliate orange and sweet orange, the outcome of QTL mapping was still not fine enough for anchoring specific genomic regions or specifying candidate genes associated with citrus responses to HLB. The four anchored genomic regions spanned a total genomic length of approximately 44.6 Mb, consisting of thousands of genes. These genomic regions involved 57 markers from three regions of the trifoliate orange map and 54 markers from one region of the sweet orange map, indicating the marker density of our genetic maps is sufficient for fine mapping. One possible explanation for such phenomenon could be that the quality of the constructed genetic maps is still insufficient for fine mapping of QTLs. The characteristics of plant materials themselves and the utilization of mixed intergeneric populations for genotyping likewise may restrict the construction of genetic maps with sufficient resolution and accuracy. All the linkage groups in trifoliate orange where the QTLs were detected were constructed with fewer markers, especially within the regions of some QTLs. Another possible explanation could be that the size of the phenotyping population is not large enough to yield fine phenotypic data with wide variation. Due to the space requirements for trees and test costs, only 86 of the progenies were selected for phenotyping in our study, accounting for only a half of the genotyping population. Moreover, it is also possible that each of these regions consist of multiple QTLs and they generally co-segregated in the population. As presented in the graphs of QTLs, many QTLs have more than one peak within the LOD score profile (Supplementary Figure 1 population of 170 progenies, we constructed two high-density genome-wide genetic maps for trifoliate orange and sweet orange. Each of the genetic maps contained nine firm linkage groups corresponding to the haploid chromosome number of the species and exhibited high synteny and high coverage of the reported citrus genome. The minor discrepancies among the genetic maps and genome assemblies may represent possible structural rearrangements among citrus species, or alternatively errors in the previous genome assembly. In the replicated field evaluation over 2 years, trifoliate orange and sweet orange showed significant differences in response to CLas infection and HLB, and their progenies exhibited an obvious continuous distribution for two phenotypic traits. Four clusters of QTLs were identified for HLB-incited foliar and canopy responses, respectively, located on LG-t6, LG-t8 and LG-t9 of the trifoliate orange genetic map and LG-s7 of the sweet orange genetic map. These QTLs collectively explained a major part of the phenotypic variation in response to HLB disease. Our results suggest that multiple QTLs are involved in the genetic control of HLB response in citrus. This work provides a starting point for future studies of the underlying genetic architecture of resistance or tolerance to HLB. These QTLs need to be confirmed further to facilitate breeding for resistance or tolerance to HLB in citrus. In addition, the corresponding genomic regions need to be refined if the objective is to discover and characterize candidate genes related to the host response to disease. The final identified QTLs and genes could be good targets for citrus breeding to support long-term solutions to this devastating disease.Based on Genotyping-by-Sequencing of an intergeneric FFG, ES, MR, ZD, and MH conceived the study. FG and MR developed the mapping population. MR, MH, and QY conducted the work of genotyping. MH, QY, DD, YY, and YZ conducted the work of phenotyping. MH and MR analyzed the genotypic data. MH analyzed the phenotypic data, performed the QTL mapping, and drafted the manuscript. All authors read and approved the final 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 handling Editor declared a shared affiliation, though no other collaboration, with several of the authors MH, QY, DD, YY, YZ, and FG.


Appropriate infant and young child feeding practices are critical for optimal child growth and development, but in Ethiopia, complementary feeding (CF) practices are very poor. Alive & Thrive (A&T) provided intensive behavior change interventions through 4 platforms: interpersonal communication (IPC), nutrition-sensitive agricultural activities (AG), community mobilization (CM), and mass media (MM).The aim of this study was to evaluate the impact of A&T intensive compared with nonintensive interventions  on CF practices and knowledge and child anthropometric outcomes.n = 2646 at baseline (2015) and n = 2720 at endline (2017)]. We derived difference-in-difference impact estimates (DDEs) and conducted dose–response and path analyses to document plausibility of impacts.We used a cluster-randomized evaluation design with cross-sectional surveys among households with children aged 6–23.9 mo [P < 0.05) in children aged 6–23.9 mo, decreasing from 36.3% to 22.8% in the intensive group. Dose–response analyses showed higher odds of minimum dietary diversity  and minimum meal frequency  and higher height-for-age z score (HAZ)  among women exposed to 3 or 4 platforms. Path analyses showed a strong relation between AG and egg consumption, which led to increased child dietary diversity and HAZ.At endline, exposure to IPC was 17.8–32.3%, exposure to AG was 22.7–36.0%, exposure to CM was 18.6–54.3%, and exposure to MM was 35.4% in the intensive group. Minimum dietary diversity and minimum acceptable diet increased significantly in the intensive group but remained low at endline . Significant differential declines in stunting prevalence were observed (DDE: −5.6 percentage points; Delivery of social and behavior change interventions using multiple platforms was feasible and effective, resulting in improvements in CF practices and child stunting within a 2-y period. There is a need for continued efforts, however, to expand intervention coverage and to improve CF practices in Ethiopia. This trial was registered at clinicaltrials.gov as NCT02775552. Ethiopia has made considerable progress in reducing infant, child, and maternal mortality during the past decade by expanding primary health care services and improving the quality of health service provision . Child uAppropriate infant and young child feeding (IYCF) practices, which include exclusive breastfeeding (EBF) until 6 mo of age and the age-appropriate provision of safe and nutritious foods in sufficient quantity, in addition to breast milk, from 6 to 23 mo of age, are important for optimal child growth and development . In EthiThe evidence base on impacts of different combinations of interventions to achieve these recommended practices is growing , 4, 8–12In Ethiopia, increased EBF, early initiation of BF, and multiple CF practices were observed in intervention areas , althougLinking nutrition with agriculture by making agricultural programs nutrition-sensitive  has been suggested to reinforce the reach of nutrition-specific interventions and to create enabling conditions for children to grow and develop , 20. An Given the evidence of poor CF practices in Ethiopia and that combined or integrated interventions work, Alive & Thrive (A&T) leveraged multiple platforms to deliver IYCF-related social and behavior change interventions. This article reports on findings from a cluster-randomized impact evaluation of A&T interventions. We hypothesized that the A&T intervention package would have positive impacts on CF practices and knowledge and anthropometric outcomes among children aged 6–23.9 mo.A&T is an initiative to save lives, prevent illness, and contribute to healthy growth and development through improving IYCF practices. In phase I (2009–2014), A&T operated in Bangladesh, Ethiopia, and Vietnam, reaching millions of children <2 y old through large-scale social and behavior change communication interventions and achieving substantial gains in IYCF practices . The focIn 3 western zones of Amhara, A&T with Save the Children as its implementing partner worked with government health extension workers (HEWs), health development team leaders , and agricultural extension workers to deliver IYCF messages through interpersonal communication (IPC) and promote nutrition-sensitive agricultural activities (AG) to benefit children <2 y old. In intensive intervention areas, HEWs provided IYCF-focused counseling during health post visits and home visits and conducted food demonstrations, HDTLs provided IYCF-focused messaging during home visits, and agricultural extension workers promoted AG activities such as designating a chicken whose eggs are prioritized for a child <2 y old in the household and prioritizing vegetables from home gardens for those children (no inputs were provided as part of the program). The Ethiopian Orthodox Church priests and leaders delivered community mobilization (CM) activities such as sermons about adequate child feeding during religious fasting periods, which are common and extensive in the region, and enhanced community conversations about IYCF were led by community-based organizations. In nonintensive areas, HEWs and HDTLs provided standard nutrition counseling and food demonstrations as feasible, without additional implementation support from A&T; agricultural extension workers provided standard agricultural services; and little or no IYCF-focused CM activities were held. However, there was some spillover of activities, as A&T tools and materials were being adopted by government, nongovernmental organizations, and other stakeholders in the country. The mass media (MM) component, implemented in both intensive and nonintensive areas, consisted of a regional broadcast of radio drama called “Sebat Mela” (translated as “Seven Wisdoms”), which included 12 episodes with stories that aligned with A&T's IYCF messages, associated jingles, and testimonials of model mothers. In intensive areas with limited access to radio, supplemental activities were conducted, including broadcasting the radio drama through mobile vans with speakers and utilizing traveling performers to enact parts of the drama. Thus, A&T used 4 different platforms—that is, IPC, AG, CM, and MM—to deliver behavior change interventions to targeted beneficiaries. The intensive group received all the interventions; the nonintensive group received standard IPC and AG and less intensive CM and MM. These platforms and the specific interventions were developed based on the experiences and lessons from A&T phase I in Ethiopia.We used a cluster-randomized, nonblinded impact evaluation design with repeated cross-sectional surveys to assess the impact of the A&T intensive intervention package compared to a nonintensive program. A cross-sectional household survey was conducted at baseline (2015) and exactly 2 years later (2017) in the same communities in households with children <2 y old. This article presents findings on the primary outcomes for the evaluation  and the secondary outcomes of maternal knowledge about CF and stunting prevalence among children aged 6–23.9 mo.Sample size was calculated to detect differences in the primary outcomes of CF practices for children aged 6–23.9 mo, between the 2 intervention groups, considering an α of 0.05, a power of 0.80, and an intraclass correlation of 0.02, and estimated baseline prevalence of the primary outcomes. Assuming a baseline prevalence of 5.1% for minimum dietary diversity, we estimated that a total sample of 2700 children aged 6–23.9 mo (1350/group) was sufficient to detect a minimum of 6-pp difference in the proportion of children achieving minimum dietary diversity. With a baseline prevalence of 55.8% for minimum meal frequency, this total sample size was also sufficient to detect a minimum of 10-pp difference in the proportion of children achieving minimum meal frequency.woreda (district). Among the total of 41 woredas in the 3 western zones of Amhara, Save the Children selected 20 woredas as possible A&T intensive areas on the basis of being first- or second-level agriculturally productive areas, not participating in the Productive Safety Net Program , geographic proximity, size, and other operational aspects to ensure homogeneity across the sample. We stratified randomization by zone, and the woredas were randomly assigned to either the intensive (10 woredas) or the nonintensive (10 woredas) intervention by use of computer-generated pseudo-random numbers. All communities within an allocated woreda received the same intensive or nonintensive interventions. Households in the intensive and nonintensive areas were not explicitly informed about the results of the randomization. There was no blinding of the intervention at the level of service delivery.A cluster was defined as a rural 1) minimum dietary diversity (defined as the consumption of foods from ≥4 of 7 food groups in the previous 24 h); 2) minimum meal frequency (defined as the frequency of consuming foods as appropriate for age and BF status); 3) minimum acceptable diet ; 4) consumption of iron-rich or iron-fortified foods; and 5) timely introduction of solid, semisolid, or soft foods , weight-for-age z scores (WAZs), and weight-for-height z scores (WHZs), according to the WHO child growth standards  was defined by measures of exposure to individual interventions as follows: IPC exposure measured by mothers’ report of receiving IYCF messages during HEW home visit, health post visit, or HDTL home visit in the last 3 months; AG exposure measured by receipt of messages about raising a “baby's chicken” or a “baby's vegetable garden”; CM exposure measured by participation in a food demonstration, enhanced community conversation, or a priest sermon about child feeding and fasting; and MM exposure measured by hearing of the Sebat Mela radio program. Exposure to multiple platforms was defined as any combination of platforms exposed, with a range of 0–4 platforms.Differences in sample characteristics at baseline and endline between the 2 intervention groups were tested using linear regression models (for continuous variables) or logit regression models , accounting for geographic clustering . For anaApproval for the study was obtained from the institutional review boards of the Ministry of Science and Technology in Ethiopia and the International Food Policy Research Institute. All mothers of study children were provided with detailed information about the study at recruitment. Verbal informed consent was obtained from mothers prior to their participation in the survey.There were no evaluation clusters lost to follow-up at endline, and there was little variation in cluster size over time (The baseline and endline characteristics of households with children aged 6–23.9 mo by intervention group are shown in P < 0.001) in both intensive and nonintensive groups, but the increases were marginally higher in the intensive group  (P = 0.22).Among all core WHO CF indicators  in both the pure intention-to-treat and fully adjusted models  or at the health post visit (32.3% compared with 17.9%) and HDTLs during home visits (17.8% compared with 4.2%) (P = 0.09). For HAZ, exposure to information about raising a baby's chicken and to enhanced community conversations was associated with 0.1–0.2 greater HAZ, and exposure to an increasing number of platforms was significantly associated with increased HAZ .We observed a significant dose–response association between exposure to the number of intervention platforms and improved CF practices, CF knowledge, and stunting and HAZ  and stunting in comparison with changes observed with the less intensive interventions within a short 2-y period. Although minimum meal frequency increased significantly in the intensive areas, there was no differential impact. In relation to other anthropometric outcomes, we observed significant declines in underweight and wasting and improvements in HAZ, WAZ, and WHZ in both intensive and nonintensive areas over time, but no differential impacts.Our findings of impacts on minimum dietary diversity were primarily explained by differential improvement in the consumption of vitamin A-rich fruits and vegetables, which was specifically promoted by program messages during IPC and during food demonstrations. In turn, we also observed differential improvements in maternal knowledge about CF, particularly knowledge about foods for enriching a child's porridge . Regarding other foods specifically promoted by the interventions , we did not observe any significant change in dairy consumption, but there was a significant increase in egg consumption in both intensive and nonintensive areas. This may have resulted from spillover of interventions, as observed in the exposure results in nonintensive areas, including nearly 10% exposure to messages about baby's chicken. Furthermore, some communities within 3 nonintensive woredas and in 4 intensive woredas were involved in the United States Agency for International Development's Empowering the New Generation to Improve Nutrition and Economic Opportunities (ENGINE) project (2011–2016), which implemented interventions similar to those of A&T; thus, some spillover was anticipated. Analyses comparing ENGINE and non-ENGINE woredas, however, showed no major differences in outcomes. In relation to other CF practices such as timely introduction of foods and minimum meal frequency, we observed increases in the intensive group and declines in the nonintensive group. Still, most CF practices remained poor at endline, with 24.9% for minimum dietary diversity, 18.2% for minimum acceptable diet, and 4.5% for consumption of iron-rich foods in the intensive areas. Thus, there is a need for continued efforts to improve these practices.Although 80% of mothers in the intensive group were exposed to at least 1 of the 4 intervention platforms, exposure across the various IYCF-focused interventions was moderate at endline: IPC was 17.8–32.3%, AG was 22.7–36.0%, CM was 18.6–54.3%, and MM was 35.4%. Thus, no single platform achieved high coverage or >60% among the target beneficiaries. The challenge of achieving high reach of health services and interventions into the communities and target households has been documented in other studies in Ethiopia , 32. AltDespite relatively moderate exposure to the interventions, we observed positive effects of these interventions on CF practices and linear growth. CF practices were associated positively with exposure to certain single interventions, but the largest significant associations were with exposure to multiple intervention platforms. Although exposure to IPC from HDTL only was significantly associated with lower odds of stunting, we observed a pattern of lower stunting among those exposed to a greater number of intervention platforms; this pattern was significant for HAZ, confirming the dose–response relation between intensity of intervention exposure and linear growth in our study. Furthermore, exposure to message about raising a baby's chicken was associated with several outcomes. Results from the path analyses corroborated this, observed by a strong association between raising a baby's chicken and HAZ indirectly via egg consumption and minimum dietary diversity, lending support for including this nutrition-sensitive AG intervention as part of the multisectoral approach for improving child nutrition.The most remarkable finding of our study is the large and significant impact on stunting. The differential decline of −5.6 pp in stunting prevalence in children aged 5–23.9 mo in 2 y (−2.8 pp per year) is double the secular trend (−1.2 pp in stunting in children aged <5 y) . StuntinThere were some limitations to our study. First, an evaluation using cross-sectional surveys was used rather than tracking individual children over time. This design allowed us to sample households with children who are the target population for interventions and had the potential of being exposed throughout the study period, but we were precluded from linking child-level exposures to outcomes for the same children. Second, given the focus on CF practices among children aged 6–23.9 mo, children <6 mo of age were not included in both arms of the study sample, so we are uncertain of differences in this younger age group between intervention groups. It is possible that changes in the earlier growth period may have contributed to the differential improvements in stunting at 6–23.9 mo. Third, the SDs for our HAZ measurements were large and exceed the WHO cutoff for high risk of measurement error (SD > 1.3), likely due to error in height measurement or child age estimate that was based on recorded/reported birthdate, particularly in the baseline survey and among the youngest subgroup of children aged 6–11.9 mo. Measurement error would have reduced our ability to detect differences. Last, the study location was confined to 3 western zones in Amhara, which were considered to be more food secure than other zones in the region. The level of food insecurity in these areas, however, was still ∼40%, so they may not be markedly different from other communities throughout other regions.In conclusion, our study contributes to the evidence that delivering social and behavior change interventions using multiple platforms in Ethiopia is feasible and effective. Intensive interventions delivered through multiple platforms achieved improvements in complementary feeding practices and child stunting within a 2-y period. Given the positive short-term impacts on children's nutrition outcomes, evaluation over longer periods may reveal greater impacts on child nutrition. There is a need for continued efforts, however, to expand intervention coverage and to improve complementary feeding practices in Ethiopia. Behavior change interventions aimed at improving child feeding practices through various delivery platforms involving different sectors may be adapted in other contexts in which such platforms exist and poor practices persist without the constraint of extreme food insecurity.nxz087_Supplemental_FilesClick here for additional data file.


BRCA1 and BRCA2 is essential to the formulation of clinical management strategies, and in Brazil, there is limited access to these services, mainly due to the costs/availability of genetic testing. Aiming at the identification of recurrent mutations that could be included in a low-cost mutation panel, used as a first screening approach, we compiled the testing reports of 649 probands with pathogenic/likely pathogenic variants referred to 28 public and private health care centers distributed across 11 Brazilian States. Overall, 126 and 103 distinct mutations were identified in BRCA1 and BRCA2, respectively. Twenty-six novel variants were reported from both genes, and BRCA2 showed higher mutational heterogeneity. Some recurrent mutations were reported exclusively in certain geographic regions, suggesting a founder effect. Our findings confirm that there is significant molecular heterogeneity in these genes among Brazilian carriers, while also suggesting that this heterogeneity precludes the use of screening protocols that include recurrent mutation testing only. This is the first study to show that profiles of recurrent mutations may be unique to different Brazilian regions. These data should be explored in larger regional cohorts to determine if screening with a panel of recurrent mutations would be effective.The detection of germline mutations in  BRCA1 and BRCA2 are tumor suppressor genes and their protein products play an important role in the repair of DNA double-strand breaks through homologous recombination (HR)1. Individuals harboring germline pathogenic variants in BRCA1 and BRCA2 (BRCA) are strongly predisposed to the development of breast  and ovarian cancers 2 as well as other solid tumors3. As bona fide tumor-suppressor genes, the wild-type allele is frequently lost (mainly through loss of heterozygosity) during tumorigenesis, thereby becoming completely inactivated in the tumor4. Tumors arising within the context of a complete loss of BRCA function are amenable to treatment with agents targeting HR DNA repair deficiency, such as poly(ADP) ribose polymerase (PARP) inhibitors (PARPi) and platinum-based chemotherapy5. In the past few years, these drugs have shown to be effective in the treatment of advanced ovarian cancer in patients harboring somatic and/or germline BRCA mutations7, and more recently, the FDA extended the approval of PARPi in the BC BRCA-related metastatic clinical setting9. These approvals will benefit many patients since BRCA mutations are identified in 8–13% of all ovarian cancer cases10, and approximately 10% of breast cancers11, which is the most common cancer type among women worldwide and also in Brazil13.BRCA mutation is of paramount importance not only for providing appropriate genetic counseling and discussing risk-reducing interventions, but also for determining treatment options in patients with metastatic disease. A challenge in the identification of carriers in Latin America, however, is the limited availability of cancer risk evaluation programs and genetic testing per se. In Brazil, 70–80% of the population relies on the public health care system14, which does not provide genetic testing. Consequently, the mutational profile of BRCA remains largely unknown. Systematic testing of at-risk individuals brings knowledge of the genetic background of a population, and may enable the identification of multiple recurrent and/or founder mutations, which, in turn, would support the use of mutation panels as a first-line screening tool.Considering this scenario, the identification of a BRCA germline mutations in Brazil and investigate if the use of a panel of recurrent mutations would be useful in this population.In this study, we aimed to describe the landscape of A total of 649 reports of pathogenic/likely pathogenic variants were retrieved from 28 centers in 11 different Brazilian States. As shown in Fig. BRCA1 c.4185G>A and the BRCA2 c.9117G>A. Large genomic rearrangements (LGR) were present in 4.9% of all cases, and among them, the BRCA2 c.156_157insAlu corresponded to 34.3% of all LGRs. The frequency of each type of variant and their molecular consequences are depicted in Fig. The most common types of pathogenic variants identified in both genes were small deletions and single nucleotide variants (SNVs), and are predicted to result in frameshift and non-sense alterations in the protein sequence Fig. . SynonymBRCA1 variants were identified among 441 probands, corresponding to 68% (441/649) of all reported mutations. Among these, a subset of 33 distinct BRCA1 mutations corresponded to 73.4% of all variants identified in this gene. The nine most prevalent BRCA1 mutations accounted for 50.3% of all BRCA1 reported mutations, and among these, the European founder mutation c.5266dupC (formerly known as 5382insC) was the most common, corresponding to 20.2% of all variants found in BRCA1. In BRCA2, 103 distinct variants were identified in 208 probands, corresponding to 32% of all individuals tested  were more common (35.1%) than in BRCA1 (15.4%). Moreover, a higher frequency of novel variants was identified in BRCA2 (17/103) when compared to BRCA1 (9/126). Figures BRCA1 and BRCA2 mutations, respectively, including LGR in both genes. Detailed information about BRCA1 and BRCA2 mutations  are summarized in the Supplementary Dataset.As shown in Table BRCA1 c.5266dupC, was reported in all geographical regions, some recurrent BRCA1 mutations  seem to be unique to a particular Brazilian State. The variants c.188 T>A, c.2405_2406delTG, c.3916_3917delTT, c.689_692delAGAC, c.4287C>A, and c.5123C>A were reported exclusively among individuals recruited from the State of São Paulo (Southeastern region). In addition, the c.1039_1040delCT and c.1039delC variants were reported exclusively in the State of Pará (Northern region), while c.3598C>T and c.5177_5180delGAAA were only reported in pathogenic mutation carriers from the State of Rio Grande do Sul (Southern Region). No similar trends were observed among BRCA2 recurrent mutations.Although the most common mutation, BRCA1 and BRCA2 mutations seen in three or more individuals, a subset of 51 variants (33 in BRCA1 and 18 in BRCA2), accounted for 67% of all reports. In a more stringent scenario, mutations seen in four or more individuals, totaling 30 variants (23 in BRCA1 and 7 in BRCA2) corresponded to 57.3% of all mutations.When considering all BRCA mutation. There are several clinical management options for individuals harboring BRCA mutations, including risk reducing surgeries 15, chemoprevention16 and intensive surveillance with annual breast magnetic resonance imaging17. Several studies have demonstrated that, after identifying a BRCA-mutation carrier, genetic counseling and testing of at-risk individuals results in increased surveillance and use of risk-reduction strategies ultimately leading to primary or secondary prevention of cancer and improved outcomes in carriers18. Despite these benefits there is limited availability of genetic testing in Latin American countries, including Brazil19.Many factors affect the probability of developing breast or ovarian cancer, but no predictor is as determinant and prevalent as the inheritance of a BRCA pathogenic variants (e.g. Ashkenazi Jewish Panel) have been used in certain countries/populations as an initial approach to overcome technical and economical restrictions that still exist for comprehensive BRCA1 and BRCA2 testing. Most of the populations where this strategy is used show few mutations occurring at a high frequency, often due to founder effects20. Thus, the development of such panels depends on a deep knowledge of the mutational spectrum of the target population and the presence of a relatively small number of recurrent mutations explaining a significant proportion of cases. This strategy has been proposed, for instance, for Hispanic breast and/or ovarian cancer families (with predominantly Mexican origin) where nine recurrent variants account for 53% of all detected BRCA mutations21. For this population, a low-cost multiplex PCR-based panel (HISPANEL) was developed and subsequently estimated to identify up to 75% of all true Mexican BRCA mutations. The pattern of highly recurrent mutations is also seen in other Latin American countries: Bahamas , Colombia  and Peru 19. However, this striking pattern of recurrent mutations seen in several Latin American countries may not be observed in all countries, and specific mutations maybe be shared only by a few populations. In fact, a recent Brazilian study showed that the use of a single screening panel for different Latin American populations will likely not be effective22, because there does not seem to be a significant overlap of recurrent mutations among different Latin American populations23. These results are not surprising due to vary distinct population migration waves and therefore genetic admixture background of Brazil in comparison with the other Latin American countries24.Low cost screening panels including recurrent BRCA mutation testing  to date29, corresponding to only 1,041 individuals tested, among a Brazilian population of over 207 million people30. Most studies have focused on specific mutations, or screened only a few regions of BRCA1 and/or BRCA2 (summarized in the Supplementary Dataset). To our knowledge, this is the largest comprehensive description of the spectrum of germline BRCA mutations in different geographical Brazilian regions.Knowledge about the germline mutational spectrum among Brazilian HBOC patients is limited. Only five studies have performed comprehensive BRCA1 ins6Kb rearrangement, which was reported by Esteves et al.31 in seven carriers, five of whom were from Rio Grande do Sul State. However, in our cohort we did not identify this rearrangement in any patient, even considering that Rio Grande do Sul was the second State in terms of the number of reported carriers (N = 107). The BRCA1 6 kb insertion can be detected by routine LGR testing through MLPA, and although we cannot assure that all probands were subjected to MLPA analysis, we can expect that most of them with negative sequencing results were also investigated for LGRs, since patients from the private healthcare setting and also those enrolled in research studies are routinely tested for LGR by MLPA. Indeed, a recent study from Alemar et al. reported LGR data from 351 HBOC probands from the same Brazilian State where the previous cases harboring the 6 kb insertion were reported originally, and the BRCA1 6 kb insertion was not detected, suggesting a very low frequency of this LGR in probands with the HBOC phenotype29.Most of the mutations reported previously in smaller Brazilian studies, involving the analysis of only certain gene regions, have also been identified in our cohort, but it is noteworthy that some of the previously reported recurrent mutations are completely absent in this dataset. The most striking example is the Among all distinct mutations identified 11.8% were novel, corresponding to 4.6% of all carriers and highlighting the heterogeneity of our population. The identification of novel mutations linked to HBOC is a vital information that should be shared with established mutation databases, in order to become useful for interpreting further tests and to answer questions about the association between a variant and phenotype.BRCA1 and BRCA2 mutations identified, and a similar profile of type and molecular consequence of pathogenic variants in both genes. In addition, BRCA1 mutations were more frequent than BRCA2 mutations, which is in agreement with previous data showing this same proportion of mutations between both genes among women from different ethnicities, except Asians32. Also, similar to previous report33, the rate of large genomic rearrangements did not exceed 5% of all mutations. However, it is remarkable that 34.3% of all LGR reported here correspond to the Portuguese founder mutation BRCA2 c.156_157insAlu34. Although significant, this frequency may still be an underestimation, since until very recently the detection of this particular mutation, which requires a specific PCR reaction, was not carried out by most commercial laboratories. Recently (July 2016), MRC-Holland included an extra probe that detects the wild-type sequence of this region in its BRCA2 MLPA kits (P090 version B1 and P45 version C1), allowing the detection of this variant during MLPA testing. This simple modification is expected to increase significantly the detection rate of c.156_157insAlu in Brazilian HBOC patients. The probands with c.156_157insAlu identified here were from the States of Minas Gerais (1), Rio de Janeiro (3) Rio Grande do Sul (2) and São Paulo (5) but screening for this LGR should be done for patients regardless of State of origin.Overall our data show a significant molecular heterogeneity among the BRCA variant detected. It is widely known that synonymous substitutions can alter splicing accuracy, creating or destroying a native donor or acceptor splice site, but they can also can modify translation fidelity, mRNA structure and protein folding35. Indeed, both pathogenic synonymous variants identified in this study disrupt splice donor sites, leading to exon skipping. The G nucleotide of BRCA1 c.4185G>A represents the last nucleotide of exon 11 , and is conserved in 86% of the splice sites in mammals36. This variant lead to an aberrant transcript lacking exon 1237. Similarly, the BRCA2 c.9117G>A leads to a complete deletion of exon 2338 and produces a frameshift effect similar to other deleterious mutations39. The process of evaluating variant significance should include multiple databases, as four mutations reported here were not described in ClinVar, although classified as clearly pathogenic in other databases. Finally, even variants described in one database should have their significance confirmed in other databases, especially if classified as variants of uncertain significance (VUS). As an example, using an ex vivo assay based on a splicing reporter minigene, Brandão et al.40 demonstrated that the BRCA1 c.4987-3C>G variant leads to the skipping of exon 17. However, it remains classified as VUS in ClinVar and it is not reported in other databases.In the current HBOC genetic testing landscape, where most laboratories have migrated to next generation sequencing (NGS), analysis workflows allow the filtering of many types of mutations, including the exclusion of synonymous variants. In our study, we have identified two pathogenic synonymous mutations, and this finding highlights the importance of careful evaluation of each BRCA1 and BRCA2 variants identified in the main Genetic Cancer Risk Assessment centers in Brazil. Although this report in fact is the most comprehensive to date, both in number of mutations reported, as well as in number of centers/regions of the country included, many limitations must be considered when analyzing the results. We were unable to obtain information on the birthplace for most of the carriers, which would have been more informative than the center where the genetic test was performed. Therefore, data on geographical location should be interpreted with caution. In fact, among some of the individuals tested in the State of São Paulo we were able to identify residents from the Midwest States. This is not unexpected since the paucity of clinical and laboratory personnel trained in clinical cancer genetics in Brazil, results in a pattern of patients with suspected hereditary cancer being refered to testing from different parts of the country to only a few reference centers very distant from their residence place. Moreover, it might be possible that the inclusion of data from point mutation analysis could increase the detection and, consequently, the reporting of a few specific mutations. However, our data regarding the most frequently reported mutations is in agreement with previous Brazilian studies that performed full BRCA1 and BRCA2 sequencing and MLPA. These studies show, for example, that the BRCA1 c.5266dupC is the most prevalent mutation across distinct regions of Brazil, which was also the case in our study29.In this study, we have attempted to compile pathogenic and likely pathogenic BRCA1 and BRCA2 genes among Brazilian carriers. Although our findings suggest that this heterogeneity precludes the use of screening protocols that include recurrent mutation testing only, our results also show that certain mutations occur at a high frequency in some Brazilian regions and not others. These variations could be due to mutation founder effects, which have been described for other genes in Brazil. These findings should be explored in larger cohorts from specific Brazilian regions to assess whether in these areas, screening with a panel of recurrent mutations would be effective.We confirm that there is significant molecular heterogeneity in the BRCA1 and BRCA2 testing showing pathogenic or likely pathogenic germline mutations were compiled from 28 public and private health care offices located in 11 Brazilian states, including the main reference centers for Genetic Cancer Risk Assessment (GCRA) in Brazil. Not all probands were subjected to a comprehensive BRCA testing . The genetic testing was performed using distinct methodologies, including full gene analysis by Sanger or next generation sequencing, point mutation analysis by Sanger or genotyping methods (as HISPANEL), and MLPA for analysis of large genomic rearrangements. Most data came from institutions participating in the Brazilian Hereditary Cancer Network (BHCN), convened by the Brazilian National Cancer Institute  and partially supported by public funding from the National Council for Scientific and Technical Development (CNPq)41. These centers, mostly public hospitals, are established in the Cities/States of Belém/Pará , Salvador/Bahia (in the Northeastern region), Vitória/Espírito Santo, Rio de Janeiro/Rio de Janeiro, São Paulo/São Paulo, Ribeirão Preto/São Paulo, Barretos/São Paulo (in the Southeastern region) and Porto Alegre/Rio Grande do Sul (in Southern Brazil). In addition, public or private health care offices from the States of Amazonas (Northern region and the Amazon basin), Minas Gerais (Southeastern region), Rio Grande do Norte, Ceará (Northeastern region) and Santa Catarina (Southern region) also contributed with molecular data from their patients  and all individuals provided written or verbal consent for BRCA testing. All methods were performed in accordance with the relevant guidelines and regulations, and all data supporting the results are shown in the Supplementary Dataset.Laboratory reports of http://varnomen.hgvs.org/) were used to annotate identified variants and the ClinVar database (www.ncbi.nlm.nih.gov/clinvar/) was used to determine the biological significance of all reported variants. For novel variants, BRCA Share , LOVD (http://www.lovd.nl/3.0/home), ARUP (http://arup.utah.edu/database/BRCA/) and BRCA Exchange (http://brcaexchange.org/) databases were also checked. Current ACMG46 guidelines were also used for further classification. BRCA1 and BRCA2 domains were defined using the boundaries in the Pfam database (http://pfam.xfam.org).The Human Genome Variation Society (HGVS) nomenclature guidelines (Supplementary dataset 1


In winter 2018, schools in Hong Kong were closed 1 week before the scheduled Chinese New Year holiday to mitigate an influenza B virus epidemic. The intervention occurred after the epidemic peak and reduced overall incidence by ≈4.2%. School-based vaccination programs should be implemented to more effectively reduce influenza illnesses. Hong Kong, China, located on the coast south of Guangdong Province, has a subtropical climate and a population of 7.3 million. In Hong Kong, influenza epidemics occur during winter every year and sometimes during other seasons  activity in Hong Kong to infer the effect of school closures on community transmission.As in previous studies, we used ILI surveillance data to indicate the incidence of influenza virus infections in the community , panel At, which represents the average number of secondary infections that result from a primary case of infection at time t  before the start of the school closure and 0.87 (95% CI 0.54–1.21) during the closure week, corresponding to a 16% (95% CI 10%–26%) reduction in transmissibility  and school closure , we first fitted a multivariable log-linear regression model for Rt with th and tC as explanatory variables  for a susceptible-exposed-infected-recovered compartmental model to simulate incidence over time. To simulate incidence under no school closure, we set tC to 0 for the period February 8–14 ; the incidence was reduced to 0.505 (95% CI 0.494–0.519) when simulated with closures , panel CIn early 2018, schools were closed an extra week before a holiday in Hong Kong to mitigate an influenza epidemic. Closure after the epidemic peak had a small effect on transmission; we estimated a 4.2% reduction in overall incidence of infections. By the end of the 2017–18 winter season, ≈400 laboratory-confirmed influenza deaths had occurred among the local population of 7.2 million, lower than the rate in the contemporaneous influenza A(H3N2) epidemic in the United States but still a rate of moderate-to-high impact. A reduction in incidence of infections by 4.2% might have reduced hospitalizations and deaths by a similar percentage, with the caveat that hospitalizations and deaths would probably not have been equally distributed among age groups because most infections occur in children and most deaths in older adults.t appeared to increase (The school closures were announced <24 hours before they began. We presume that the school closures were disruptive to parents’ schedules, potentially forcing some parents to stay home from work, and that many children stayed home during closures (increase , panel BWe assumed that the ILI+ proxy for influenza B was linearly correlated with the incidence of infections (Influenza vaccination is considered the best preventive measure against influenza. However, >10 years after introduction of the influenza vaccination subsidy scheme for children, influenza vaccination coverage is still low in Hong Kong: ≈10% overall and ≈15% in children for the 2016–17 and 2017–18 winter seasons (Description of methods, sensitivity analysis, and incidence of influenza B virus and influenza-like illnesses.