Drosophila it is received through compound eyes, Hofbauer-Buchner eyelet, ocelli, using phospholipase C-dependent phototransduction and by deep brain photoreceptors, like Cryptochrome. Even a single light pulse during early life induces larval-time memory, which synchronizes the circadian clock and maintains daily rhythms in adult flies. In this study we investigated several processes in adult flies after maintaining their embryos, larvae and pupae in constant darkness (DD) until eclosion. We found that the lack of external light during development affects sleep time, by reduction of night sleep, and in effect shift to the daytime. However, disruption of internal CRY- dependent photoreception annuls this effect. We also observed changes in the expression of genes encoding neurotransmitters and their receptors between flies kept in different light regime. In addition, the lack of light during development results in decreasing size of mushroom bodies, involved in sleep regulation. Taking together, our results show that presence of light during early life plays a key role in brain development and affects adult behavior.Light is one of most important factors synchronizing organisms to day/night cycles in the environment. In  Drosophila is a very useful model for sleep/activity research since it exhibits a clear daily activity pattern. Under LD12:12 (12 h of light and 12 h of darkness) conditions it shows two peaks of activity, morning and evening, while in the middle of the day and night it has resting time called siesta and sleep, respectively. This rhythmic behavior is regulated by brain sleep centers, glial cells, as well as by different clusters of clock cells   and wakeDrosophila by the ple gene. Once produced and released into synaptic cleft it can bind to specific receptors on postsynaptic cells in the following structures involved in sleep regulation: Dop1R1 in neurons of mushroom bodies, ellipsoid body and fan-shaped body, Dop1R2 in neurons of mushroom bodies and DD2R in three pairs of dorsal neurons in the ventral nerve cord and sleep promoting protocerebral bridge interneurons . One of its receptor HisCl1 is expressed in photoreceptors and clock neurons, and the other one—Ort receptor—in the lamina and medulla interneurons .Octopamine is synthesized by anterior paired lateral neurons (APL) using tyramine beta hydroxylase (Tbh) and its signal is transmitted to mushroom bodies through Oamb receptor .All these neurotransmitters control activity of specific neurons located in structures called sleep centers. The most important ones are mushroom bodies (MB) and central complex in the central and dorsal brain . The musLight input is received by the visual system: retinal photoreceptors, Hofbauer-Buchner eyelet and ocelli through the rhodopsin-dependent phototransduction pathway . All phoIn larvae light activates CRY located in clock neurons (four LNvs and DN1s) and photoreceptors of the Bolwig organ (BO), which contains rhodopsin 5 or rhodopsin 6  and commDrosophila as a model, we showed that lack of light in early life affects behavior of adult flies.The role of light in the sleep regulation is already well described, however, little is known about the link between light exposure during development and sleep amount in adult life. In this study, using Drosophila melanogaster were used: wild type CantonS, norpA7 mutant , cry01 mutant  regime during development and adulthood (group described as LD/LD), while experimental flies were kept in constant darkness (DD) during development  (group described here as DD/DD). Because sleep assay was measured on the second day of LD12:12 conditions, we added the additional group called DD/LD, in which flies exposed to DD during development were transferred for 2 days to LD12:12.Drosophila Activity Monitoring System  for 5 days in LD12:12 and 6 days in DD. Activity was counted every 1 min and analyzed in Excel using “Befly!” software . Lomb—Scargle normalized periodogram was used to determine rhythmic flies; individuals with period below 10 (confidence level 0.05) were regarded as arrhythmic. Flies, which did not survive until the end of experiments were removed from analyses. Every experiment was repeated three times, at least 60 flies in total were used.Locomotor activity was recorded using Sleep analysis was performed on the second day of LD12:12, and sleep was recorded as at least 5 min of a fly immobility.Males from the strain OK107 > GFP were collected at ZT1 (1 hour after lights-on in LD 12:12) or CT1 (in DD), their heads were fixed in 4% paraformaldehyde and brains were isolated. GFP fluorescence of this strain is strong enough to skip additional staining steps.For TH staining wild type flies were used. Fixed brains were washed in PBS and 0.2% PBST (PBS with Triton X), then unspecific antigens were blocked with Normal Goat Serum (NGS) for 1 h. Samples were incubated with mouse anti-TH antibodies  for 3 days at 4°C. Then, brains were washed three times in 0.2% PBST and incubated for 2 h with goat anti-mouse antibodies conjugated with Cy3 . After washing in 0.2% PBST, brains were mounted in Vectashield medium (Vector) and examined with a Zeiss Meta 510 Laser Scanning Microscope.The size of the brain and GFP-labelled mushroom bodies lobes was measured using ImageJ software (Area measurements). To avoid changes in body size, dependent on overcrowding, the same number of parental flies was used for every group.The TH immunofluorescence intensity was measured for specific dopaminergic cells: PPL1, PAM and PPM3 because these cluster are known to innervate sleep centers. Area of every single cell was selected manually and mean grey value was measured using ImageJ software.Experiments were repeated 3 times and each repetition was composed of 10 brains per group.rpl32. Experiments were repeated 5 times.For gene expression analysis we divided the experimental flies into two groups—first was continuously kept in constant darkness (DD/DD), the second one was moved to LD12:12 for 2 days (DD/LD) to provide light input and synchronize flies. All groups were collected at the same time—ZT1 (1 hour after lights-on for control and flies transferred to LD12:12) and CT1 (for flies kept in DD). At least 20 heads were cut off and the total RNA was isolated using a TriReagent (Invitrogen). 1 μg of the total RNA was used to prepare cDNA with the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) and random primers. cDNA (diluted 1:10) was used for SybrGreen qPCR (KapaBiosystems) analysis. The specific primers (the specificity was controlled with Primer BLAST and gel electrophoresis) used for the reaction are listed in t-test to detect statistically significant differences between groups.GraphPad Prism software was used for statistics and making graphs. Outliers were removed using Grubbs’ test (GraphPad online software). Shapiro-Wilk’s test was used to check normality in distribution. Data were analyzed using one-way ANOVA with post-hoc Tukey’s test or p = 0.0052), activity index was increased (p = 0.0025) and their sleep amount during the night, measured in minutes, was decreased (p = 0.0202), while siesta was not changed (p = 0.0723)  were more active than the control (LD/LD) ( 0.0723) . MoreoveSleep is regulated by several neurotransmitters released by specific neurons and recognized by receptors on target cells. Because we observed increased activity and decreased sleep level in the experimental flies, we expected to see changes in the expression of wake-promoting factors. The most important ones are dopamine, histamine and octopamine, as well as PDF and sNPF which affect the clock network. To investigate how light during development regulates these pathways we decided to check changes in the mRNA level of both, neurotransmitters and their receptors in heads. We compared mRNA level of selected genes in adult flies from three different groups: flies which were maintained during the development and as adults in DD (group DD/DD), flies developing in DD and transferred to LD12:12 as adults, which is similar to the experiment for sleep analysis (group DD/LD) and control flies developing and kept in LD12:12 after eclosion (group LD/LD).p = 0.00159, LD/LD vs. DD/DD p = 0.0286), which suggests that light during development is necessary to maintain high level of dopamine signaling. This result was not expected, as dopamine is wake-promoting factor, and knowing that experimental flies are more active we predicted to observe higher dopamine synthesis. However, the opposite situation was observed for Dop1R1, which was expressed at higher level in the groups DD/LD and DD/DD . The level of Dop1R2 mRNA was increased in the group DD/DD, but it was decreased after moving flies to LD12:12 . Finally, DD2R showed the pattern of expression similar to Dop1R2, but the observed changes were not statistically significant . The obtained results suggest more complex, cell-type specific regulation of dopamine signaling.First, we investigated effect of light during development on dopamine signaling. Our results showed that dopamine synthesis was decreased in flies developing in constant darkness , HisCl1  and ort  in our experiment was similar, with decreased level in flies kept in DD during development  and Oamb , however, flies developing in DD and then transferred to LD had the lowest amount of transcript .Finally, we checked the expression level of two neurotransmitter genes known to be involved directly in the circadian control of sleep: sNPF and PDF and their receptors, sNPF-R and PDFR. All of them had the similar pattern—in flies developing in DD their expression level was decreased, and after transferring adults to LD, the level of mRNA did not change significantly , but activity index was not changed (p = 0.8724). Their pattern of sleep was changed and the total amount of sleep was decreased during the day (p = 0.0001) and night (p = 0.0039) , encoded by the gene  0.0039) .norpA mutants.Having in mind that photoreception through the retina photoreceptors can affect neurotransmission level in the brain we checked gene expression of the previously selected genes in head homogenates from Dop1R1 expression between norpA mutants developing in LD12:12 and DD (p = 0.0317), while tyrosine hydroxylase (p = 0.6905), Dop1R2 (p = 0.0952) and DD2R (p = 0.3095) encoding genes were expressed at the similar level in both groups (Dop1R2 and ple expression in PLC-dependent manner.In dopamine pathway we found changes only in h groups . This reHdc gene expression) was decreased in norpA mutants growing without light (p = 0.0079), but mRNA levels of HisCl1 (p = 0.8413) and ort genes of histamine receptors (p = 0.4206) were unchanged. However, other neurotransmission pathways checked previously  seem do not to be PLC-dependent, as Tbh (p > 0.9999), Oamb (p = 0.2222.), Pdf (p = 0.8413), Pdfr (p = 0.1508), sNPF (p = 0.0556) and sNPF-R (p > 0.9999) mRNAs had similar level in LD/LD and DD/DD groups in norpA mutants.Histamine synthesis , activity index (p = 0.5335), sleep pattern nor its level  between flies reared in LD and DD , Dop1R1 (p > 0.9999) nor DD2R expression (p = 0.0571), but Dop1R2 mRNA level was increased in flies reared in DD, similarly to wild-type flies (p = 0.0266). Histamine , octopamine , PDF  and sNPF  signaling components were also not changed.Moreover, we did not observe changes in ple , p = 0.4p < 0.0001), while the brain size was not changed (p = 0.4483) . Because we observed the strong effect of light during development on sleep level in adult flies, we wondered whether it can be correlated with MB size changes. We used transgenic cross OK-107-Gal4 with UAS-mCD8::GFP to visualize MB. We reared flies in LD or DD, respectively and collected brains of 2-days old males. We measured brain size as well as mushroom bodies of every single individual and found that flies kept in DD had smaller MB ( 0.4483) .Additionally, we checked tyrosine hydroxylase immunofluorescence in specific cell clusters innervating MB. We found that flies kept in DD during development have lower expression of TH in PPL1, PPM3 and PAM neurons, however, transferring flies to the light:dark conditions restored normal level of the enzyme, but in the case of PPM3 the amount of TH was higher than in the control group .Sleep is one of most important functions of organism. During resting time many processes occur, like memory consolidation, wound healing, etc. Sleep is regulated by internal signals coming from the circadian clock and sleep centers, but it is also responsive to external signals, mostly light inputs. Sleep regulation is very complex and requires multicellular communication and homeostasis. Light is a well-known arousal factor, however, mid-day siesta is promoted during the most intense light exposure.per and tim expression in the pacemaker in the brain —key enzyme in dopamine synthesis, and in effect also dopamine level in the brain most probably was decreased. Since the inhibition of dopamine-releasing neurons causes hyperactivity in response to mechanical startle  and mushroom bodies (PPL1 and PAM), the structures hosting a sleep center  and sleep in the opposite way, acting on specific cells  or the control flies developing in LD12:12 (LD/LD group). On the other hand, ple and Dop1R1 expression seems to be continuously inhibited by the lack of light during development, while DD2R expression level is not affected by light conditions. The results obtained for cry01 and norpA mutants are very interesting, since they show that gene expression of the dopamine pathway in the group DD/LD strictly depends on photoreception—Dop1R1 is CRY-dependent, while Dop1R2 is PLC—dependent, and ple is both CRY and PLC dependent. The fact, that dopamine acts through Cryptochrome, and both of them are required for nocturnal increased activity was shown also by other authors  and octopamine receptor gene Oamb, which indicates that this signaling pathway was decreased in adults. It was previously shown that octopamine regulates activity/sleep cycle in larvae, since the activation of Tdc-expressing neurons decreases sleep time, while Tbh and Oamb mutations or silencing increases sleep time . PDF released from l-LNVs is also responsible for nocturnal activity  and ligh mutants . DecreasThe second neurotransmitter present in the circadian clock neurons, called sNPF is broadly expressed in the brain, including sleep centers  and its DopR1 expression. Changes in the expression of other gene studied seems do not to be connected with the observed behavioral phenotype because we did not observe them in norpA mutants which show nocturnal sleep deficits similar to wild-type flies. However, norpA mutants showed increased total activity time with no changes of activity index, which indicates that they were more and longer active than blind flies reared in LD.Comparing wild-type flies with mutants used in the study we can conclude that the exposure to DD during development affects sleep during the night, reduces its time and increases activity time, with lower locomotor activity during wake. This indicates that experimental flies spent more time being active, but their activity level was similar to that in the control. Observed changes are regulated mostly by increasing per01 and cry01 mutants kept in LD12:12  derived from mushroom bodies neuroblasts, similarly to  LD12:12 . In addi LD12:12 , which w LD12:12 . It has  LD12:12 . One of


Plasmodium vivax have proven diagnostic performance to identify people exposed in the previous 9 months.The landscape of malaria transmission in the Peruvian Amazon is temporally and spatially heterogeneous, presenting different micro-geographies with particular epidemiologies. Most cases are asymptomatic and escape routine malaria surveillance based on light microscopy (LM). Following the implementation of control programs in this region, new approaches to stratify transmission and direct efforts at an individual and community level are needed. Antibody responses to serological exposure markers (SEM) to P. vivax in peri-urban (Iquitos) and riverine (Mazán) communities of Loreto, communities that have seen significant recent reductions in malaria transmission. Socio-demographic, geo-reference, LM and qPCR diagnosis data were collected from two cross-sectional surveys. Spatial and multilevel analyses were implemented to describe the distribution of seropositive cases and the risk factors associated with exposure to P. vivax.We measured antibody responses against 8 SEM to identify recently exposed people and determine the transmission dynamics of P. vivax in Mazán (56.5%) than Iquitos (38.2%). Age and being male were factors associated with high odds of being seropositive in both sites. Higher antibody levels were found in individuals >15 years old. The persistence of long-lived antibodies in these individuals could overestimate the detection of recent exposure. Antibody levels in younger populations (<15 years old) could be a better indicator of recent exposure to P. vivax.Low local transmission was detected by qPCR in both Iquitos (5.3%) and Mazán (2.7%); however, seroprevalence indicated a higher level of (past) exposure to The large number of current and past infections detected by SEMs allows for detailed local epidemiological analyses, in contrast to data from qPCR prevalence surveys which did not produce statistically significant associations. Serological surveillance will be increasingly important in the Peruvian Amazon as malaria transmission is reduced by continued control and elimination efforts. Plasmodium vivax is its relapsing nature that can contribute up to 80% of the P. vivax cases. The detection of this source of transmission remains crucial for focused interventions in settings attempting to achieve elimination. Antibody responses to serological exposure markers to P. vivax have been validated to detect recent exposure and likely hypnozoite carriers in low transmission settings. To evaluate the benefits of adding serology to the detection of infection in areas where malaria transmission is rapidly reduced, we measured the prevalence of P. vivax by qPCR and serology in two sites of the Peruvian Amazon. This study shows that antibody responses to P. vivax antigens can detect more recently exposed people than molecular testing, providing more statistical power for surveillance and critical additional epidemiological knowledge for regions attempting to eliminate malaria transmission.A challenge for eliminating  Peru is considered a low-endemic transmission setting for malaria, with 24,000 cases reported in 2019  and the there was a noteworthy increase in the number of malaria cases [Over the last 20 years, the case incidence of malaria in Peru has fluctuated, influenced largely by the prevailing intervention programs. After the cessation of the Global Fund Malaria Project “PAMAFRO” in 2010, ia cases , with a ia cases . In threia cases . In its Plasmodium vivax causes the majority of malaria cases in Peru. The detection of cases is based on light microscopy (LM) diagnosis; this may substantially underestimate prevalence as greater than 50% of infections that are detectable by qPCR may be missed by microscopy [croscopy . Being bcroscopy  and hypnP. vivax have been developed and validated for their use in low transmission settings [New uses of malaria serological tools have been proposed to inform the impact of such control measures . Ideallysettings . These ssettings .P. vivax through antibody responses to serological markers in the Peruvian Amazon. Whereas previous studies in the Peruvian Amazon only focused on the analyses of antibodies to individual protein such as PvAMA1 [19 [P. vivax: PvMSP119, PvTRAg_2, PvTRAg_28, PvMSP8, PvMSP3.10, PvRAMA, PvRBP2b and PvEBPII. This provided an excellent opportunity to evaluate the benefits of adding serology to infection detection in monitoring and understanding changes in malaria epidemiology in the context of rapidly intensifying elimination efforts.Given that the Malaria Zero program has been implemented in the Peruvian Amazon region, new tools are needed to capture transmission patterns, detecting hot-spots and individuals likely contributing to transmission. In 2018, cross-sectional surveys were executed in two contrasting geographical areas of Loreto: Iquitos (peri-urban communities) and Mazán  to determine the exposure level to s PvAMA1 , PvMSP11AMA1 [19 , PvMSP8 AMA1 [19 , or PvMSAMA1 [19 , the advSamples were collected from consenting individuals through the CAM study (SIDISI 101645/2017) and the ICEMR study (SIDISI 101518/2018); both studies were approved by the Institutional Ethics Committee of Universidad Peruana Cayetano Heredia (UPCH). UPCH also approved the use of the serum samples collected in those studies and the use of Peruvian negative controls at the Institut Pasteur (SIDISI 100873/2017).Two cross-sectional studies were carried out in two districts of Maynas province, Department of Loreto in 2018: Iquitos and Mazán.Iquitos is the capital of Loreto and is the most populated metropolitan area in the Amazon Region. It is accessed only by air or by river and the majority of the population are mestizo (very mixed-race identity). Three peri-urban communities of the district of Iquitos were surveyed for this study: Rumococha (RC), Santo Tomás (ST), and Quistococha (QC). RC is located 8 km from Iquitos city; ST is 11km from Iquitos city and surrounded by the Nanay River. QC is located 12km from Iquitos city and close to the Nauta-Iquitos Road. The most common occupations are commerce, mototaxi and being a housewife .Mazán is a district of Maynas province located 55–60 km northeast of Iquitos. It is comprised of many small communities, and the community of Mazán is surrounded by the Mazán and the Napo Rivers . The only access is by speedboat (1 hour from Iquitos). Gamitanacocha (GC), Libertad (LI), Primero de Enero (PE) and Puerto Alegre (PA) are located on the Mazán River. Salvador (SA), Lago Yuracyacu (LY) and Urco Miraño (UM) are located on the Napo River. The main economic activities are farming, timber extraction and fishing .Malaria transmission in Maynas is perennial, with a peak between February and July . While iP. vivax according to the MINSA reports of 2017, and due to ease of sample transport to the reference laboratory installed in Iquitos city. The enrolled individuals represented a total of 358 households. From the enrolled participants, 895 individuals consented to provide a blood sample for LM and qPCR diagnosis, and 880 individuals accepted donating a plasma sample for serology purpose (In April 2018 (mid transmission season), 933 individuals were enrolled to participate in the Circles of Research on Arboviruses and Malaria (CAM) study executed in the 3 communities of Iquitos mentioned above. In this study, purposive sampling was implemented to the household members presented at the moment of survey. These communities were selected due to their high incidence of  purpose .In the district of Mazán, a representative population-based cross-sectional survey was conducted in July 2018 (end of transmission season), where 1127 individuals were enrolled to participate in the baseline survey by the Amazonia International Center of Excellence in Malaria Research (ICEMR) team . A totalA population survey and geo-referencing of households were implemented in communities from both areas, and data were collected through surveys using tablets and Open Data Kit (ODK) ,21. AxilWhole blood samples (5 mL) were collected by a venous puncture in citrate tubes. Serum was separated by centrifugation and kept at -80°C until processing. Red blood cell pellets were stored at -20°C prior to molecular diagnosis.Thick and thin blood smears were stained for 10 min with a 10% Giemsa solution. A slide was declared negative if no malaria parasite was found after examining 100 microscopy fields . Slides DNA was isolated from parasitized red blood cell pellets using the E.Z.N.A. Blood DNA Mini Kit , according to the manufacturer’s instructions. Subsequent amplification was performed by a real-time quantitative PCR (qPCR) method using PerfeCTa SYBR Green FastMix 1250  as previously described .P. vivax were measured using a Luminex platform, as described elsewhere [P. vivax has been previously validated [19), PVX_096995 , PVX_112670, PVX_097625 , PVX_097720 , PVX_087885 , PVX_094255  and KMZ83376.1d . The additionally proteins screened in this study were: PVX_090240 , PVX_110810 , PVX_000930  and PVX_082700 (merozoite surface protein 7 (MSP7), putative, PvMSP7). The complete IgG dataset generated is available on IgG antibody responses to 12 proteins of lsewhere . The panalidated  and consTo account for inter-plate variation, a standard curve was prepared using a plasma pool from hyper-immune adults from Papua New Guinea. Relative Antibody Units (RAU) or dilutions were obtained extrapolating the Median Fluorescence Intensity (MFI) in a standard curve by a 5-parameter logistic model written in R.χ2 test or Fisher’s exact test when required. Continuous covariates were compared using Mann-Whitney U or T- test when required.Demographic data was collected from the databases of the CAM study and the baseline survey of the cohort executed by the Amazonian ICEMR. Categorical covariates were compared using the P. vivax by a seropositivity classifier based on the combined antibody responses to SEM. The seropositivity classifier was a Random Forests based classification algorithm previously validated in low P. vivax transmission contexts: Thailand (n = 826), Brazil (n = 925), the Solomon Islands (n = 754), and negative controls (n = 274) [https://github.com/MWhite-InstitutPasteur/Pvivax_sero_dx.Participants were classified as seropositive  or seronegative  to n = 274) . The diaP. vivax SEM was estimated as the proportion of individuals classified as seropositive stratified by age. The seropositive rate of individuals per household was estimated to visualise the spatial distribution of seroprevalence to P. vivax. For the spatial data processing and visualisation, QGIS 2.16  was used.The proportion seropositive for P. vivax exposure (binary) and P. vivax parasitaemia (binary) by qPCR were assessed by mixed-effects logistic regression models with random intercepts per households and communities. The following potential risk factors were assessed as fixed effects: age, sex, fever, travels in the last month, outdoor occupation, education level, livestock inside dwelling, indoor spray, and housing type. Factors in final models were selected by manual backward elimination guided by the minimisation of the Akaike information criterion. Three levels were included in these models: individuals within households, households within communities and communities. Intra-class correlation (ICC) was estimated as a measure of correlation and homogeneity at each level [https://www.r-project.org/).The association of epidemiological factors with ch level . The ICCch level . Statistp < 0.001). Outdoor occupations such as logger, farmer and fisher were more common in Mazán (40.9%) than in Iquitos (8.9%) (p < 0.001). Farmer and housewife were the most common economical occupation in adults older than 18 years old in Mazán (38.1%) and in Iquitos (30.8%), respectively. Only 2.5% of participants from Iquitos and 1.2% from Mazán had fever (axillar temperature > 37.5°C) at the moment of survey (p = 0.045).Of the 933 surveyed inhabitants in the CAM study, 880 individuals from 358 households (HH) were included in the analysis Figs  and S1. P. vivax in Mazán [p = 0.004). Having livestock inside dwelling was seen in 24.6% of the HH from Iquitos and 26.6% from Mazán (p = 0.565).Crowded households  were slightly more frequently reported in Mazán than in Iquitos with no significant differences. Open houses have been associated as a high risk factor for in Mazán ,19 and iin Mazán . 16% of P. vivax prevalence by LM in Iquitos was not significantly different from Mazán . Although P. vivax prevalence by qPCR was higher in Iquitos than in Mazán ; Mazán  showed higher seroprevalence than Iquitos  (Tablep mean < 0.001). Participants from Iquitos had 1.2 times higher antibody levels to PvMSP119 than participants from Mazán (p = 0.013).P. vivax by community, we found high heterogeneity within study sites (p = 0.005) and Quistococha  (p < 0.001). Conversely, seroprevalence was higher in Santo Tomás  than Rumococha  (p < 0.001) and Quistococha  (p < 0.001).When exploring the prevalence of dy sites . Both LMdy sites . For betIn Mazán, Libertad had the highest qPCR prevalence among all the communities , followed by Gamitanacocha , Puerto Alegre , Urco Miraño , and Lago Yuracyacu . Salvador and Primero de Enero did not have any positive qPCR sample in the survey (0%). On the other hand, Gamitanacocha was the community with higher seroprevalence , followed by Libertad with 75% (138/184) of seroprevalence, Primero de Enero , Urco Miraño , Puerto Alegre , Salvador  and Lago Yuracyacu .p mean < 0.001) (p = 0.006) (Antibody levels and seropositivity rate increased with age in both study sites  Tables. < 0.001) . There w< 0.001) . This di= 0.006) . Antibod= 0.006) .Within each study area, villages had different seropositive rate-age patterns . The lowIn order to detect spatial patterns of exposure within communities, a seropositivity rate per household was estimated for both areas. The household-level spatial distribution of individuals is represented in In the peri-urban areas of Iquitos, Santo Tomás had the highest proportion of households with seropositive rate ≥ 0.75 (40%), in contrast to Rumococha (20%) and Quistococha (20%) . HoweverA more heterogeneous transmission landscape was found in the communities of Mazán, probably related to the geographical location of households. Mazán district is crossed by two rivers: Mazán River and Napo River. The highest proportions of seropositive individuals at household level were detected in communities along the basin of Mazán River: Gamitanacocha (88%), Libertad (63%) and Primero de Enero (60%). Although also located in the basin of Mazán River, only 31% of the households in Puerto Alegre had ≥ 0.75 seropositive rate. While the proportion of households with high seropositive rate in the communities of the basin of Napo River were the following: Urco Miraño (45%), Salvador (20%) and Lago Yuracyacu (4%) . The expP. vivax parasitaemia output was measured by PCR positivity. The effect of households and community levels on individual outcomes were measured by the intra-class correlation (ICC). The ICC quantifies the size of the general contextual effect, so the more relevant the context is for understanding individual differences in the outcome, the higher the ICC [Due to the multilevel data structure of this study, we implemented multilevel multi-community logistic regression models at three levels: individual, households, and communities. The exposure output was measured by seropositivity to SEM, whereas the  the ICC . The estP. vivax increased with increasing age , being male  and working outside . Having domestic animals was associated with a low risk of being seropositive . There was no association with fever symptom and seropositivity, as well with the travel history in the last month and the type of house . There was an association between being male and being seropositive to P. vivax . Having a high education level (either secondary school or higher) was a protective factor . History of travel in the past month was not associated with seropositivity. The ICC values indicated that the household variable explains considerably more variance of seropositivity to P. vivax. This possibly indicates a heterogeneous individual exposure that could be related to job- related habits rather than to community factors  identified more exposures (both current and previous) than molecular testing in two low transmission regions of the Peruvian Amazon. The measures of P. vivax seroprevalence resulted in greater statistical power for epidemiological analyses than qPCR parasite prevalence in such settings with low transmission rates. P. vivax SEM will be a key tool for serological surveillance as malaria transmission is reduced by continued control and elimination efforts in the Peruvian Amazon.Here we show that IgG antibody responses to 8 P. vivax in the last 9 months. The seroprevalence of P. vivax was higher in the riverine communities of Mazán than in the peri-urban communities of Iquitos. Seroprevalence increased with age in both sites, with a significant increase in participants older than 15 years old, although the distribution by age was more heterogeneous among communities from Mazán. The distribution of seropositive cases did not show a spatial structure in communities of Iquitos but did it in Mazán.We used a Random Forest algorithm that incorporated the combined antibody responses to the 8 SEM to classify surveyed individuals into seropositive or seronegative, i.e., recent exposure to P. vivax seropositivity in both areas, while having higher education level and livestock inside dwelling were low-risk factors in Iquitos and Mazán, respectively. Age is a factor that can be linked to i) lifetime cumulative exposure  or ii) labor exposure . Our previous work showed that antibody levels increased with the recency of infection, the number of blood-stage infections and age [P. vivax prevalence in the community of Urco Miraño [P. vivax seropositivity in Mazán only, which is in line with previous reports of the malaria epidemiology in the area [Age and being male were factors associated with high  and age . However and age . The preo Miraño . Previouo Miraño . This opo Miraño . From outhe area .The high seroprevalence in Iquitos and Mazán is consistent with recent intense exposure in both sites ,19,28. OP. vivax could have a significant impact on the control measures implemented by health authorities. For instance, in population screening studies in Mazán, only 23% of the infections were detected by LM-based active case detection, compared to qPCR [P. vivax in Iquitos and Mazán, respectively. However, our study showed that in areas that after long periods of moderate-to-high transmission rapidly goes to low transmission, antibodies remain at high levels for several years after transmission has been reduced. As a consequence, they are not well suited to measure short-term changes in transmission in areas where populations have established high levels of immunity. This could impact the use of antibodies to measure the impact of interventions [The choice of tools to detect exposure to  to qPCR . Also, o to qPCR . In low  to qPCR . In our ventions ,31. We tSome limitations of this study are the lack of information about recent malaria episodes and time of residence in the study survey to better explain the communities’ transmission patterns and the relationship with seropositvity. The differences in the timing of execution of each study could have influenced the LM and qPCR prevalence in both sites, i.e., in Iquitos, samples were collected during transmission months. In contrast, in Mazán, samples were collected after the transmission peak. The low proportion of participants sampled per household in Iquitos could have overestimated the seropositive rate per household and thus the differences between households detected by our multilevel logistic model. This sampling bias may have also affected the distribution of seropositive cases or maps of seropositivity.Overall, this study confirms that malaria transmission in the Peruvian Amazon is heterogeneous and that geographical, socio-demographic and human behaviour are important factors for the maintenance of disease burden in this region. Our findings have implications for designing appropriate interventions against malaria in the region; these novel serological markers could be deployed as a tool to target individual and community interventions, but care needs to be taken with their use in areas where transmission has been reduced rapidly from high to low transmission.S1 FigCAM: Circles of Research on Arboviruses and Malaria study, ICEMR: Amazonia International Center of Excellence in Malaria Research, HH: Households, Ind: Individuals.(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigP. vivax detected by antibody responses to serological exposure markers.A) Proportion of individuals with ongoing infections detected by Light microscopy (LM). B) Proportion of individuals with ongoing infections detected by qPCR. C) Proportion of individuals exposed to (TIF)Click here for additional data file.S4 FigA) Age curve of antibody titers in Iquitos and Mazán. B) Distribution of antibody titers stratified by seropositivity given by the Random Forests based classification algorithm. Note the controls are grouped together regardless of age.(TIF)Click here for additional data file.S5 FigSeropositive rate per household was estimated as the proportion of seropositive individuals classified per number of household members participating in the study.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.


Major surgery for ovarian cancer is associated with significant morbidity. Recently, guidelines for perioperative care in gynecologic oncology with a structured “Enhanced Recovery after Surgery (ERAS)” program were presented. Our aim was to evaluate if implementation of ERAS reduces postoperative complications in patients undergoing extensive cytoreductive surgery for ovarian cancer.134 patients with ovarian cancer (FIGO I-IV) were included. 47 patients were prospectively studied after implementation of a mandatory ERAS protocol (ERAS group) and compared to 87 patients that were treated before implementation (pre-ERAS group). Primary endpoints of this study were the effects of the ERAS protocol on postoperative complications and length of stay in hospital.p < 0.001), indicating a higher surgical risk in the ERAS group. Total number of postoperative complications , and length of hospital stay (ERAS: 11 (6–23) vs pre-ERAS: 13 (6–50) days; p < 0.001) differed significantly. A lower fraction of patients of the ERAS group (87.2%) needed postoperative admission to the ICU compared to the pre-ERAS group (97.7%), p = 0.022). Mortality within the ERAS group was 0% vs. 3.4% (p = 0.552) in the pre-ERAS group.Preoperative and surgical data were comparable in both groups. Only the POSSUM score was higher in the ERAS group (11.8% vs. 9.3%, The implementation of a mandatory ERAS protocol was associated with a lower rate of postoperative complications and a reduced length of stay in hospital. If ERAS has influence on long-term outcome needs to be further evaluated. The Enhanced Recovery after Surgery (ERAS) “program has been developed based on the principle of fast track surgery in colorectal surgery –6. It thThis study is an analysis of the prospectively collected data of patients scheduled for suspected or diagnosed ovarian cancer or recurrent ovarian cancer scheduled for cytoreductive surgery after implementation of an ERAS program in the year 2018 (ERAS group), and a comparison to retrospectively acquired data of a historical control group of patients treated with the same diagnosis between 2015 and 2017 (pre-ERAS group).ERAS Group: in this group all patients admitted to our department with suspected or diagnosed ovarian cancer and the indication of cytoreductive tumor surgery via laparotomy were included. Preoperative preparation, perioperative management, and postoperative treatment was standardized according to the published ERAS guidelines .The single elements of this patient management, such as renunciation of bowel preparation, preoperative carbohydrate-loading, early mobilization, and principles of fluid- and pain therapy were obligatory settled in written standard operating procedures. Furthermore, all pre-, peri-, and postoperative data were systematically collected according to the requirements of the database of the International ERAS society. Additionally, values of hemoglobin, thrombocytes, and amount of ascites were assessed.Pre-ERAS group: for this group data were acquired from patients who were treated with cytoreductive surgery because of the same oncological indication in our department between 2015 and 2017. All suitable patient charts of this period were screened in detail. Patients were included, if at least 80% of data, which is assessed in the ERAS database, was available.p values were interpreted as descriptive measures.Data were analyzed using R, version 4.0.3 . The twop < 0.05.Normally distributed variables are reported with mean and standard deviation and analyzed with a linear ANOVA. Variables with skewed distributions are reported with median and IQR (interquartile range) and were either transformed using the logarithm function and analyzed with a linear ANOVA where applicable or with Kruskal–Wallis’ rank sum test. Fisher’s exact test was used for categorical variables. Distribution assumptions were made based on histograms and measures of location. Results were considered to be significant if Acquisition of all data was approved by the Ethics Committee of the Medical Board Hamburg (PV190504). Informed consent was obtained from all patients.Between January 1 2018, and December 31, 2019, 47 patients were prospectively included into the ERAS group. For the pre-ERAS cohort, we screened in total medical charts of 153 patients who underwent laparotomy for ovarian cancer in the years 2015–2017. Sixty-six patients were excluded, because of incompleteness of data sets according to the ERAS-database. Finally, 87 patients were analyzed.p < 0.001).Data are given in Table p < 0.001). Patients of the ERAS group received considerably fewer crystalloid fluids during surgery (3500 (2500–4500) vs. 4000 (3000–5000) ml p = 0.036). Other perioperative data did not reveal any relevant differences.Data are summarized in Table p = 0.011). Total LOS was significantly shorter in the ERAS group (ERAS: 11 (9–12) vs pre-ERAS: 13 (11–16) days; p < 0.001)). LOS on ICU did not differ in both groups. However, in the ERAS group, 12.8% of patients were not admitted to ICU at all, whereas this fraction was only 2.3% in the pre-ERAS group (p = 0.022). Patients in the ERAS group were mobilized out of bed relevantly earlier . In particular, the number of urinary tract infections (ERAS group: 3 (6.3%) vs. pre-ERAS group: 17 (19.5%); p = 0.045), and of paralytic Ileus (ERAS group: 0 (0%) vs. pre-ERAS group: 10 (11.5%); p = 0.015) was considerably lower.Data on postoperative management is given in Table The results of this study give evidence that implementation of an ERAS protocol for patients undergoing extended cytoreductive surgery for ovarian cancer leads to a reduction of postoperative complications and a reduction of LOS in hospital. In contrast to earlier investigations by Bisch et al.  and DickSince 2015, single Fast-Track elements, such as renunciation of routine bowel preparation, fluid restriction, early start of food intake and mobilization, early removal of urinary drainage, and the use of epidural catheters had been implemented into our ovarian cancer surgery program. However, those aspects, although recommended, were not obligatorily established. The here presented comparison of the ERAS group with the historical control group (pre-ERAS) from 2015 to 2017 strengthens the hypothesis that a consistent implementation and strict adherence to an ERAS protocol bundling all single components, is a further step toward improvement of outcome in this patient population.Additional aspects that were added to our patient management, and most importantly that were also clearly compulsory regulated by written standard operating procedures, comprised extended preoperative patient education on ERAS, the avoidance of preoperative bowel preparation, the reduction of preoperative fasting time, preoperative metabolic optimization by carbohydrate loading, early postoperative enteral feeding, avoidance or early abandoning of wound drains and feeding tubes, strict peri- and postoperative pain therapy using epidural anesthesia and opioid sparing medication, and early mobilization out of bed after surgery.Historically, the ERAS protocol for ovarian cancer surgery has been developed on the basis of the protocol for colorectal surgery. It is common sense that in colorectal surgery, the most important singular aspect for beneficial effects is the use of laparoscopy as surgical technique leading to a significant reduction of the surgical trauma –26. Thisp < 0.001). This change in clinical practice was grounded on the findings of the LION study, which demonstrated less postoperative complications, when avoiding this surgical procedure [p = 0.054). Both of these factors might have contributed to the reduced complication rate of the ERAS group. On the other hand, patients in the ERAS group had a higher POSSUM score (11.9 (ERAS group) vs. 9.4 (pre-ERAS group); p < 0.001), reflecting a relevantly higher surgical risk. It is remarkable that even so, less patients of the ERAS group needed postoperative ICU admission (12.8% (ERAS group) vs. 2.3% (pre-ERAS group); p = 0.022).A limitation of our study is the comparison to a historical control group. Even if the baseline variables are comparable, it cannot be excluded that differences regarding the outcome are caused by time effects regarding the treatment strategies. However, our data are in many aspects well comparable to the recently published PROFAST study . In thisrocedure . Furtherp = 0.015) is remarkable. This is in contrast to a recent data analysis by Kalogera et al., who reported less nausea in the ERAS cohort, however, no reduction in the occurrence of paralytical ileus [p = 0.002). Also, the pre-surgery use of carbohydrate loading (5.7% (pre-ERAS group) vs. 89.4% (ERAS group); p < 0.001), and the increased use of PONV prophylaxis, (33.3% (ERAS group) vs. 57.4% (pre-ERAS group); p = 0.014) might have contributed here. It is a frequently cited clinical notion that epidural anesthesia contributes to prolonged postoperative gut dysfunction [p < 0.001). Additionally, we confirm the report by Bergstrom and colleagues regarding postoperative pain [p < 0.001), and this even with a reduced fraction of patients with the need of strong opioids (36.2% (ERAS group) vs. 55.1% . We speculate that early mobilization, which is only possible with an optimized pain therapy, is one main factor for that reduction in postoperative gastrointestinal dysfunction.Further, the significantly lower incidence of gastrointestinal complications, and here in particular of paralytical ileus in the ERAS group . The clinical effect is reflected in the reduced number of urinary tract infections in patients of the ERAS group (3 (6.3%) (ERAS group) vs. 17 (19.5%) .The ERAS protocol demands early removal of any kind of drains: this effect can be clearly recognized in our data, as the use of Foley catheters was significantly shorter in the ERAS group (2 (ERAS group) vs. 3 nights (pre-ERAS group); p = 0.450) despite a 100% compliance of postoperative anticoagulation in both groups. Since analysis of our data, we changed our clinical practice, and we now give preoperative low molecular heparin in prophylactic dosage on a routine basis.After analysis of our data, we became skeptical concerning the non-existing recommendation for pre-operative pharmacological thrombosis prophylaxis in the current ERAS protocol. Following this protocol, our patients did not receive preoperative anticoagulation, whereas this was performed in 39.1% of patients in the pre-ERAS group. We recognized a signal of increased occurrence of pulmonary embolisms in the ERAS group (8.5% (ERAS group) vs. 4.6% (pre-ERAS group); Muallem et al. observed that the implementation of ERAS elements in gynecologic oncology in Germany is still not satisfying as only half of the departments are nowadays able to apply at least 70% of the ERAS elements. We agree with their conclusion that it is of high clinical and scientific relevance to consistently build up a structured database for gynecological oncology units on national and international level to facilitate and monitor further implementation and standardization of the ERAS protocol .In conclusion, our data strongly support the use of a consistent multimodal ERAS treatment program in patients undergoing cytoreductive surgery for ovarian cancer. It led to a reduction of postoperative morbidity, and in particular to a reduced rate of infectious and gastrointestinal complications, which allowed earlier discharge from hospital. These patients generally require subsequent oncological chemotherapy after surgery. Since there is evidence that the timespan between surgery and initiation of subsequent chemotherapy is relevant for long-term survival , ERAS mi


The serving size recommendations for legume consumption varied widely, and there was no consistency in the suggested frequency of consumption. Median bean and legume consumption for countries with FBDG ranged from 1.2 g/d (Norway) to 122.7 g/d (Afghanistan). Classification of legumes into food groups varied, with 38% of countries categorising legumes in the protein-rich food group, 20% were in a group on their own and 15% were in the starchy staples group. In countries where legumes were together with either nuts or seeds had the greatest range in intake (11.6–122.7 g/day), followed by those that grouped legumes together with protein-rich foods (4.0–104.7 g/day), while countries that grouped legumes into two food groups, in an attempt to promote consumption, tended to have a lower consumption. Greater emphasis and perhaps repositioning of legumes in dietary guidelines may be required to encourage consumption for health, environmental and economic benefits.Despite the well-known human and planetary health benefits of legumes, consumption is often low. This scoping review aimed to evaluate the inclusion of legumes in global food-based dietary guidelines (FBDG), and to review consumption data against global food group classifications for legumes. The review of FBDG from 94 countries identified legume-based key messaging, the key terms used to define legumes, recommended serving size and frequency of consumption and the classification of legumes into food groups as depicted by food guides. The 2018 Global Dietary Database isolated consumption data of legumes and beans using individual-level, nationally representative dietary survey data for matched countries. Food-based dietary guidelines from 40/94 countries most often identified legumes utilising the term legumes, followed by beans ( Legume, from the Fabacae (or Leguminosae) botanical family, is the more inclusive classification for non-oil seed (pulse) and oil-seed crops (peanuts and soybeans) and includes both fresh and dried forms . Common National food-based dietary guidelines (“FBDG”) provide advice on foods, food groups, and dietary patterns for populations to promote overall health . AlthougWhile the classification of legumes as both a vegetable and a protein source in the ADGs was intended to encourage consumption, such placement may be confusing for consumers and nutrition professionals due to their distinct nutritional profile . It has ® Excel® spreadsheet  for analysis. The following data were extracted for each country: Region, as defined by the FAO , the official name of the dietary guidelines, date of most recent revision, verbatim legume-based key messages, the specific terminology used to define legumes, the recommended dietary intake of legumes, serving size suggestions and the classification of legumes into food group/s using the provided food guides. Food guides are a visual representation of the dietary guidelines and depict the recommended food groups, and often frequency of consumption for a nutritious diet [To review legume-based messaging within National Dietary Guidelines, the FAO online repository of FBDG was utilised to source data for countries with dietary recommendations . Only thous diet . VerbatiThe pictorial food guides for each country were visually examined to determine the classification of legumes into one of six food group/s: legumes (own group), fruits/vegetables, vegetables only, starchy staples , protein-rich foods , legumes, nuts and seeds (own group), or a combination of the above mentioned. The precise terminology used to define legumes in the guidelines was also noted.® Excel® , description statistics were applied to explore how legumes were classified and encouraged within dietary guidelines.Using Microsoftcovariates to supplement individual level dietary intake data including food and nutrient availability data and food product sales [Global bean and legume consumption data were extracted from the 2018 Global Dietary Database (“GDD 2018”) in June 2022 . The GDDct sales . The firct sales . Accordict sales .® Excel® . Intake data for 185 countries were available; however, only those with FBDG were used in the present analysis (n = 94). Following extraction of data, countries were recategorised according to the FAO regional categorisation scheme as previously defined  to determine regional-level findings. Intake data were analysed for individuals aged 20 years and over (both sexes), from all education levels , medium (6.01–12 years) and high (12.01+ years) and all residences .Median legume intake data (in grams/day) and 95% confidence interval was extracted and analysed in MicrosoftCountry and regional-level consumption data were compared to a daily target of 50 g of cooked legumes, as this target was most commonly utilised in the literature ,12.n = 33), 31% were in LAC (n = 29), 18% were from the APAC region (n = 17), 7% were in Africa (n = 7), 6% were in the Near East (n = 6) and 2% in North America (n = 2).A total of 94 countries with FBDG were included for review to explore how legumes were classified and encouraged within dietary guidelines (excludes Cambodia as the FBDG only related to school children aged 6 to 17 years). Of the 94 included countries, 35% were in Europe (n = 91); however, there was great variability in the language used and the detail provided (n = 4/7), while countries in the APAC region more often group legumes together with other protein sources (n = 10/17).Most countries had at least one key message related to legumes  and highlighted specific serving frequencies, such as daily consumption, e.g., “Eat legumes daily” (Qatar) or encouraged intake at every meal, e.g., “Integrate grains and legumes, other grain and potatoes—in every main meal” (Albania).Of the 91 countries with at least one legume-based key message, 42 utilised qualitative-based messaging and often used descriptors such as regularly, frequently or often to encourage consumption, e.g., “Consume legumes frequently” (Greece). Less than a quarter of countries outlined quantitative-based messages (n = 40/94), followed by ‘Beans’ , ‘Pulses’ , ‘Peas and Beans’ , ‘Beans, Peas and Lentils  and ‘Pulses and Beans’   . Other tOverall, 39 countries provided a recommended serving frequency and 25 provided a recommended serving size for legumes . Frequenn = 33), together with lean meats, poultry, fish, eggs, tofu and/or nuts and seeds. Legumes were a separate food group in 20% of countries (n = 17) and 13 countries grouped legumes together with other starchy staples  (n = 4) and vegetable food group only . Overall, five FBDG grouped legumes into two foods groups. In Australia and the United States, legumes were considered both a vegetable and a meat alternative in the protein-rich food group. In Albania, legumes were classified in the protein-rich food group and the starchy staples group, while in Poland and Sweden, legumes featured in the “eat more” food group and the “replace/switch” food group  . Seven Fod group .n = 93/94) (n = 6/11).Consumption data were available for 98.9% of countries with FBDG (= 93/94)  and of w= 93/94) , followe= 93/94) , Europe = 93/94) , Near Ea= 93/94) , Africa = 93/94)  and Nort= 93/94) . Only 11n = 33) fell below 50 g of legumes per day, except Israel, and 36% (12/33) fell below 10 g/day indicating that Europe had the lowest level of reported consumption overall. In APAC, 65% of countries fell below 50 g/day (n = 11/17) but only two countries fell below 25 g/day. No country in Africa met the 50 g daily target, and 4/7 consumed below 25 g/day. More than two-thirds of countries in LAC (89%) fell below the consumption target of 50 g/day (n = 25/28), although most countries (n = 19/28) consumed above 25 g/day. Most countries in the Near East consumed above 25 g/day (n = 5/6), with just one country, Saudi Arabia, exceeding the 50 g/day target. Neither of the two countries representing North America met the 50 g target.All countries in the European region , Vietnam (104.7 g/day), Sri Lanka (89.6 g/day), Brazil (83.0 g/day) and Israel (75.3 g/day), while Norway (1.2 g/day), Switzerland (2.9 g/day), Poland (3.0 g/day), Argentina (3.7 g/day) and Malta (4.0 g/day) reported the lowest consumption overall. In Australia and New Zealand, median legume intake was 26.1 and 68.0 g/day, respectively, and lower intakes were reported in Canada (20.8 g/day) and the United States (21.8 g/day). The UK reported a median intake of 44.8 g/day.Median bean and legume consumption was compared to the legume food group classification for 81 countries with matched data. This excludes seven countries that did not have a food guide, five countries where the food group classification could not be determined and one country where consumption data was not available. In Albania, where legumes were classified as both a starchy staple and as a meat alternative in the protein-rich food group, median consumption was 42.4 g/day, higher than any other food group classification, although it should be noted that only one country grouped legumes as both a starchy staple and alternative to meat. Countries that placed legumes together with either nuts or seeds had the greatest range in intake (11.6–122.7 g/day), followed by those that grouped legumes together with protein-rich foods (4.0–104.7 g/day), which also had the greatest number of countries exceeding the 50 g/day target (5/33). More than two-thirds of those that classified legumes in their own food group (71%) consumed more than 25 g/day.Food-based dietary guidelines reflect a country’s particular pattern of eating while trying to optimise the health of the general population . This glThis review points to a particular opportunity for Australia to refer more specifically to ‘Beans, peas and lentils’, as opposed to ‘legumes/beans’ with the suggestion that the general terms, legumes and pulses, may not be familiar to consumers. This was reflected in a recent study involving 505 Australian respondents, in which legumes were most commonly associated with foods such as “beans” (72%), “lentils” (55%), “chickpeas” (42%), and “peas” (24%) . InteresGlobal bean and legume consumption drawn from the GDD 2018 was lower than suggested targets, with only 11 of those with FBDG reporting a median intake greater than 50 g/day. Although countries in the APAC region reported the highest level of consumption, this region also had the greatest variability (9.7–122.7 g/day), whereas surveys from North America had very little variation (20.8–21.8 g/day). Variability in consumption may be due to differences in cultural dietary patterns, where there may be a larger number of pulse consumers. For example, the highest level of consumption was reported in Afghanistan (122.7 g/day), where legumes are commonly incorporated into vegetarian and meat dishes as a thickener; however, this country was also one of few that presented the most explicit recommendations for the inclusion of legumes in the dietary pattern . As a reIn Australia, low consumption may be due to unfamiliarity with legumes and the tendency to use them as an ingredient rather than as a traditional pattern of eating or being central to the dish . It has Over time, traditional legume consumption may have been displaced through changes in dietary patterns and availability of alternate protein sources. A historical reflection from Germany in 1850 to 1970 reported a decrease in legume consumption and a rise in meat and fat intake, a trend that may be continuing today in some countries . In SpaiAlmost all food guides visually represented legumes, which were most often categorised as a meat alternative within the protein-rich food group (38%) or within their own group (20%). In contrast, when comparing placement of legumes in written guidelines with the relevant consumption data, higher consumption appeared aligned with placement of legumes in their own category or together with nuts and seeds . This is closely followed by countries where legumes were recognised as a protein food (33 countries) and together with starchy staples. Australia and the US tended to have lower reported consumption, although legumes were classified in two groups which is based on their nutritional profile ,19; grouWhile FBDG are built on the available scientific evidence supporting foods and dietary patterns, the statements within guidelines are designed to specifically suit the population. Similar to the terminology used to define the category, it was clear there was variation in the way guidelines were expressed based on culturally based dietary patterns. Countries such as Mexico, Qatar, Dominican Republic, Albania, Guatemala and Malta encouraged legume intake by categorising legumes as their own food group or provide detailed information on the culinary use of legumes to encourage consumption . Other iThis analysis represents the most comprehensive review of global FBDG and consumption data for legumes, however, should be viewed in light of the limitations. Full FBDG documents could not be reviewed for 49 countries, as they were not in English language which limits the scope the present study. The GDD 2018 dietary intake data are based on estimates using a combination of various sources, particularly self-reported dietary intake data. As such, there needs to be some consideration for the self-reported nature of dietary data collection, although this is true of all large population dietary data sets. In comparison to the GDD 2018, FAO estimates have shown to underestimate legume consumption by 50% , providiDietary guidelines aim to facilitate easy translation to dietary patterns specific to a population. Legumes are potentially difficult to position due to their nutrient composition, being high in dietary fibre and plant-based protein. With greater emphasis on sustainable eating patterns, novel placement of legumes or at least repositioning of legumes may be a consideration within FBDG. Future research should test messaging in dietary guidelines, particularly in countries where increased consumption is desired and where legumes have been added to multiple food groups such as in Australia.


B mode ultrasound (US) and shear wave elastography (SWE) are easily accessible imaging tools for idiopathic inflammatory myopathies (IIM) but require further validation against standard diagnostic procedures such as MRI and muscle biopsy.In this prospective cross-sectional study we compared US findings to MRI and muscle biopsy findings in a group of 18 patients  with active IIM  who had one or both procedures on the same muscle. US domains  in the deltoid and vastus lateralis were compared to MRI domains  and muscle biopsy findings . A composite index score (1–4) was also used as an arbitrary indicator of overall muscle pathology in biopsies.p = 0.047) in the vastus lateralis, and showed a non-significant association with muscle inflammation, myonecrosis, fibrosis and fatty infiltration/atrophy (p > 0.333) Severe echogenicity also had a non-significant association with higher composite biopsy index score in the vastus lateralis (p = 0.380). SWS and US measures of fascial thickness and muscle bulk showed poor discrimination in differentiating between pathologies on MRI or muscle biopsy. Power Doppler measures of vascularity correlated poorly with the presence of oedema on MRI, or with inflammation or fatty infiltration on biopsy. Overall, US was sensitive in detecting the presence of muscle pathology shown on MRI (67–100%) but showed poorer specificity (13–100%). Increased echogenicity showed good sensitivity when detecting muscle pathology (100%) but lacked specificity in differentiating muscle pathologies (0%). Most study participants rated US as the preferred imaging modality.Increased echogenicity correlated with the presence of fatty infiltration/atrophy on MRI (Our findings show that US, in particular muscle echogenicity, has a high sensitivity, but low specificity, for detecting muscle pathology in IIM. Traditional visual grading scores are not IIM-specific and require further modification and validation. Future studies should continue to focus on developing a feasible scoring system, which is reliable and allows translation to clinical practice.The online version contains supplementary material available at 10.1186/s41927-022-00276-w. Idiopathic inflammatory myopathies (IIM) are a heterogeneous group of rare autoimmune diseases that carry a significant morbidity from musculoskeletal complications, as well as potentially serious systemic manifestations in some cases . Early dThe gold standard test for the diagnosis of IIM currently remains the muscle biopsy. Varying combinations of muscle inflammation, myofibre necrosis, degeneration, atrophy, and fatty infiltration/fibrosis are typically reported biopsy findings in the IIMs , 3 Less Clinical led point-of-care ultrasound (US) has rapidly progressed to become a useful imaging outcome tool for various rheumatological conditions , 7. HoweIn this study, we have attempted to correlate changes in muscle ultrasound domains with MRI and muscle biopsy findings in patients with different forms of IIM. The primary objective of the study was to assess the construct and criterion validity of different muscle US domains  against (i) MRI findings of muscle oedema, fatty infiltration/atrophy; and/or (ii) muscle biopsy findings  in a mixed group of IIM patients. A secondary objective of the study was to assess intra-observer reliability testing and patient satisfaction with US compared with MRI.Twenty-five patients with a new diagnosis of IIM were recruited sequentially from the rheumatology clinics at Fiona Stanley Hospital and the myositis clinic at the Institute for Immunology and Infectious Diseases at Murdoch University between June 2019 and May 2021. All participants gave written informed consent.Human Research Ethics Committee (HREC) approvals were granted by the South Metropolitan Health Service (HREC) (RGS0000003714) and reciprocal ethics were obtained from Murdoch University (2018/237). Patients were required to be ≥ 18 years old, able and willing to provide written informed consent and attend a clinical study visit at a neuromuscular centre. IIM patients were eligible for the study if they satisfied the 2017 European League Against Rheumatism (EULAR) classification of IIM  and/or 1All participants n = 25) completed patient-reported questionnaires, including a health assessment questionnaire (HAQ), a partially validated tool looking at the functional disability index . A highe5 completUS and SWE were performed by the same clinician (SP), who had 6 months of supervised training in The ultrasound studies were performed in a controlled temperature air-conditioned room. Patients rested for a period of ~ 15 min before the procedure and had been instructed not to perform recreational exercise on the day of the procedure or the preceding day.B mode US was done using a Siemens machine with 750PRF and 14 MHz. The probe position for imaging the deltoid was at a point one-third of the distance from the acromion to the lateral epicondyle. The arm was flexed in a 90° position held at rest on a pillow. The probe position for imaging the vastus lateralis was at a point one-quarter of the distance from the anterior superior iliac spine (ASIS) to the superior border of the patella . The vasFascial thickness was measured in transverse position using the calliper function, with a total of 6 readings in 2 frames with the callipers placed 0.25 cm apart at the most homogenous area of the muscle and averaged . Fascia Muscle bulk was measured at the broadest point of the muscle belly, from the deep peripheral fascia to the deep intramuscular fascia using the calliper function  Additio: Fig. S2Echogenicity, using the Heckmatt visual grading scale  , which iThe B mode features scored were:PD was assessed at rest in both the transverse and longitudinal planes. The PD signal was graded as: ‘normal’, ‘mild’, ‘severe’  interstitial lymphocytic inflammatory infiltrates; (ii) myofibre necrosis/regeneration; (iii) interstitial fibrosis; and (iv) fatty infiltration/atrophy, collectively being indicators of the extent, severity and chronicity of damage in individual muscles. In addition, to enable comparisons between ultrasound findings and overall muscle pathology, an arbitrary composite pathology index score (1–4) was calculated for each biopsy based on how many of the above four pathology domains were present in individual biopsies.All participants were asked to fill in a patient satisfaction survey and were required to answer seven questions on their level of satisfaction with US and MRI, respectively. The overall satisfaction with US and MRI were rated separately on a 5-point Likert Scale . An additional question was tabulated regarding preference for imaging modality, where there were 3 options  available.The echogenicity and power Doppler US domains in 15 of the participants were re-assessed by the first author for intra-observer reliability at an interval of 8 months after recruitment of the last patient.t-test   or reported descriptively. The Mann Whitney U test was used for the comparison between ultrasound and MRI domains. Intra-observer reliability was reported with a kappa value. Patient preference for US was described as the overall percentage.Mean (M), standard deviation (SD), median (Mdn) and interquartile ranges (IQR) were used for continuous data. Categorical data were presented using frequency and percent (%). For statistical analysis, echogenicity was considered increased when it showed a grade of 2–4 according to the Heckmatt visual grading scale. PD was considered present when it showed a grade of 1–4. The sensitivity and specificity of US were estimated using MRI and muscle biopsy as the gold-standard comparators. Differences in the categorical variables were explored using Fischer’s exact test (echogenicity and PD) or a Paired-Samples Following an initial screening, 20 of the 25 patients recruited were considered eligible for inclusion in the study. The 5 patients excluded were initially thought to have a clinical diagnosis of IBM, but after investigation, they did not fulfil the diagnostic criteria for the disease. The median age was 64.0 (IQR: 54.0–70.0) with more females n = 11, 55.0%) than males . Six 30%) participants had a diagnosis of DM, 7 (35%) of NAM, 6 (30%) of IBM, and one of OM (5%). The mean serum CK level at baseline was 4518 U/L (SD = 6036.04). The mean MMT score in the deltoid was 9 (SD = 2.69) and in the vastus lateralis was 7 (SD = 2.77). Eleven participants (55.0%) had both an MRI and muscle biopsy, five (25.0%) had only an MRI, 2 (10.0%) had only a muscle biopsy, and 2 (10.0%) had neither. The median time interval between US and MRI is 53 days (IQR: 15–109) and the median time interval between the US and muscle biopsy is 41 days (21–116). Of the 16 patients who had MRI, 12 had the vastus lateralis imaged, 2 had both the deltoid and vastus lateralis imaged, and 2 had the deltoid only. Of the 13 patients who had a biopsy, 12 had the vastus lateralis biopsied, and 1 had the deltoid biopsied. The two patients who did not have either MRI or muscle biopsy were elderly individuals with IBM who had a typical pattern of muscle weakness and other laboratory findings  and their data (including US data) were included in the demographic analysis but not the comparative analysis. , median muscle bulk was 1.54 cm (IQR: 1.19–1.86), median SWS transverse 2.52 m/s (IQR 2.11–2.99), median SWS long m/s 2.70 (IQR: 2.46–2.93). Eleven  participants showed increased echogenicity and 11  showed increased vascularity on PD.In the vastus lateralis, the median fascial thickness was 0.15 (IQR: 0.70–0.90), median muscle bulk was 1.25 cm (IQR: 0.76–1.86), median SWS transverse 2.45 m/s (IQR1.78–3.04) and median SWS long 2.44 m/s (IQR: 1.98–3.22). Eighteen  showed increased echogenicity and 11  showed increased vascularity on PD.p > 0.400) or vastus lateralis (p > 0.370). In the vastus lateralis, there was a non-significant association between increased muscle bulk on US and muscle oedema on MRI (p = 0.240), and reduced muscle bulk on US and fatty infiltration/atrophy on MRI (p = 0.223) . However, high-grade echogenicity with associated with fatty infiltration and atrophy in the vastus lateralis (p-0.047). There was a poor association between PD signal intensity and the presence of oedema reported on MRI in both the deltoid and vastus lateralis  indicating muscle damage on MRI, in either the deltoid p > 0.00 or vasp > 0.120) , necrosis (p = 1.000), fibrosis (p = 0.406) and fatty infiltration/atrophy (p = 0.333) in biopsies. Similarly, although not reaching statistical significance, it is interesting to note that higher grade echogenicity was associated with more pathologies on biopsy (p = 0.380). There was no association between vascularity on PD and the various pathologies on muscle biopsy (p > 0.318)  in the deltoid and a kappa of 0.881 in the vastus lateralis. For PD, the intra-observer reliability reported a kappa of 0.714 (important agreement) in the deltoid and a kappa of 0.746  in the vastus lateralis.Eighteen of the 20 participants (90%) responded to the participation satisfaction questionnaire. Seventeen participants (94.4%) overall satisfaction with ultrasound and MRI as very good and one (6%) as good. Eleven participants  rated the US as the preferred imaging modality, 4  liked either (US or MRI), with none  choosing MRI.Before B-mode ultrasound can be accepted as a non-invasive diagnostic procedure of choice for IIMs, changes in individual US domains must be validated against current gold standard diagnostic techniques such as MRI and histopathological findings in the muscle examined. Our study has attempted to do this in a mixed clinic cohort of IIM patients, which included all the primary forms of autoimmune myositis , except polymyositis, whose existence as a separate entity is controversial and rarely diagnosed . The priThe median age of the participants in the study was 64 years, which is reflective of the inclusion of specific IIM subtypes such as IBM, which usually presents over the period of 50 . ParticiAlthough our findings indicated a correlation between increased echogenicity on the US and fatty infiltration and atrophy on MRI, the degree of echogenicity did not reliably differentiate between inflammation and other features of muscle damage, which brings into question the US grading scale used. The Heckmatt grading scale , or modiBecause of our small sample sizes and heterogeneity of biopsy findings in participants with different forms of IIM, the comparison of US findings with histopathological features placed more emphasis on descriptive observations. Nevertheless, a non-significant association between high echogenicity and histological features of inflammation, myonecrosis and fatty infiltration/atrophy was observed in the vastus lateralis. This is interesting, as previous reports have suggested that hypoechogenicity is associated with the presence of oedema on MRI, particularly in early, more active forms of myositis . Our finIncreased power Doppler signal, indicative of increased vascularity, has previously been associated with muscle oedema and early, more active myositis , 19. HowThere are several limitations to the present study. Firstly, the small sample size and heterogeneity of the IIM cohort are potential limitations that could affect the significance and generalisability of the findings. Due to the low prevalence of IIMs, future studies will need to consider multi-site data collection or extend the data collection period to overcome this issue. Secondly, ultrasound remains operator-dependant, and the lack of standardised protocols and consensus agreements on what constitutes features of inflammation and muscle damage may influence the reliability of the findings. Thirdly, the MRI and muscle biopsies were not all prospective and coincident in time. Biopsies were not MRI-guided, which may have reduced the strength of some of the associations found between the imaging and pathology domains. The MRI was scored dichotomously in this study, but other ways to achieve pathology on MRI also warrant further investigation.Ultrasound has shown high sensitivity in detecting muscle pathology in IIM, but the discrimination of pathology domains and the utility of different US domains are still contentious. To further improve specificity and reliability, definitions of muscle inflammation and damage on US would need to be refined and scoring systems matched against gold standard techniques to interrogate the validity of the findings in a larger IIM patient cohort having all three procedures. Despite this, our study suggests that ultrasound is a valuable and feasible screening imaging tool in IIM, which patients favourably receive, and may also help identify a suitable muscle for biopsy. Moreover, muscle ultrasound may also have utility in other situations, such as detecting muscle involvement in clinically amyopathic dermatomyositis, detecting active myositis in patients with the treated disease, and differentiating steroid myopathy from persisting active myositis.Additional file 1. Supplementary Figure 1. Study design.Additional file 2. Supplementary Figure 2. Fascial thickness and muscle thickness measurements in a deltoid muscle.Additional file 3. Supplementary Table 1. Demographic of the continuous variables.Additional file 4. Supplementary Table 2. Ultrasound domains (continuous data) against MRI domains in the vastus lateralis and deltoid.Additional file 5. Supplementary Table 3. Ultrasound domains  against MRI domains in the deltoid and vastus lateralis.Additional file 6. Supplementary Table 4. Ultrasound domains (continuous data) against muscle biopsy domains in the Vastus Lateralis.Additional file 7. Supplementary Table 5. Ultrasound domains  against muscle biopsy in the Vastus Lateralis.


Corticosteroid-induced osteoporosis (CIO) is the most common type of secondary osteoporosis, leading to fractures, and increased morbidity and mortality.Pubmed, EMBASE, Scopus and Google Scholar databases.Prolonged glucocorticoids administration leads to secondary osteoporosis.The optimal management for CIO is controversial.The present study compared bone mineral density, fractures and adverse events in patients undergoing treatment with risedronate, alendronate, zoledronate, denosumab or etidronate for CIO.For selected patients with CIO, alendronate performed better overall. These results must be interpreted within the limitations of the present study.I, Bayesian network meta-analysis of randomized clinical trials. Osteoporosis impacts negatively on bone turnover,,Prolonged glucocorticoids administration leads to secondary osteoporosis.This Bayesian network meta-analysis followed the PRISMA extension statement for reporting of systematic reviews incorporating network meta-analyses of health care interventions.P (population): CIO;I (intervention): pharmacological therapy;C (comparison): risedronate, alendronate, zoledronate, denosumab, etidronate;O (outcomes): BMD, fragility fractures, and adverse events.Two authors (F.M. & G.C.) independently performed the literature search in April 2022. The search was firstly performed on Pubmed database. Subsequently, EMBASE, Scopus and Google Scholar databases were used to identify further articles. No time constraint was adopted for the literature search. The following keywords were used in this schema: osteoporosis [All Fields] AND glucocorticoid-induced [All Fields] COMBINED WITH bone loss [All Fields], BMD [All Fields], glucocorticoid [All Fields], steroids [All Fields], vertebral [All Fields], spine [All Fields], fracture [All Fields], hip [All Fields], femur [All Fields], bisphosphonate [All Fields], alendronate [All Fields], residronate, zoledronate [All Fields], denosumab [All Fields], PTH [All Fields], Teriparatide [All Fields], vitamin D [All Fields], calcium [All Fields]. The resulting publications were independently inspected by the same authors, and, if related to the topic at hand, they were considered for inclusion. The bibliographies of the full-text articles were also screened by hand to identify other articles eligible for inclusion.All randomized clinical trials (RCTs) comparing treatments for CIO were considered. Given the authors’ language capabilities, articles in English, German, Italian, French and Spanish were eligible. Only peer reviewed RCTs of level I evidence, according to Oxford Centre of Evidence-Based Medicine,Two independent authors (F.M. & G.C.) performed data extraction. Generalities of the included RCTs were retrieved . Patient baseline data were also extracted , vitamin D daily supplement (UI), type of treatment, route of administration, number of samples, mean age and percentage of women, BMD). In case of studies which several articles with different length of the follow-up were published, we collected demographic data for baseline assessment from the pivotal trial, while data at the latest follow-up were extracted for the network analyses. The following data at the latest follow-up were retrieved: (1) BMD of femoral neck, hip and spine, (2) fractures  and (3) adverse events . The endpoint serious adverse events gather those necessitating hospital admission .The methodological quality assessment was performed by two authors independently (F.M. & G.C.). The risk of bias summary tool of the Review Manager Software  was used. The risk to incur in the following biases were evaluated: selection, detection, attrition, and other source of bias.P values > 0.1 considered satisfactory. Median and interquartile were evaluated for non-parametric data. The baseline comparability was assessed by the Kruskal–Wallis test, with P values > 0.1 considered satisfactory. A Bayesian hierarchical random-effects model analysis was used for the network comparisons. The inverse variance method was used for analysis. Continuous variables were analyzed using the standardized mean difference (SMD) effect measure, and the log odd ratio (LOR) was adopted for binary data. To obtain most reliable results, given the heterogeneous daily supplementation of calcium and vitamin D, the placebo group was not included in the network comparisons. Rather, all the variables were compared in the network analyses against a fictitious control group (no event). For continuous variables, the maximum score value was considered, while no event was considered for dichotomic outcomes. The overall inconsistency was evaluated through the equation for global linearity using the Wald test. If the P value was > 0.5, the null hypothesis could not be rejected, and the consistency assumption could be accepted at the overall level of each treatment. Both confidence (CI) and percentile (PrI) intervals were set at 95%. Edge plots were performed to display the number of direct comparisons, while interval plots were used to rank the treatment according to their effect size. Funnel plots were obtained to evaluate the risk of publication bias for each comparison.The main author (F.M.) performed the statistical analyses. The STATA Software/MP version 16.1  was used for statistical analyses. The Shapiro–Wilk test has been performed to investigate whether data have a normal distribution. Mean and standard deviation were evaluated for parametric data. The baseline comparability was assessed using analysis of variance (ANOVA), with N = 77), non-clinical studies (N = 100), primary osteoporosis (N = 70), use of adjuvant(s) (N = 40), language limitations (N = 15) or uncertain results (N = 4). Another 22 articles were rejected because of missing quantitative data under the outcomes of interests. This left 11 RCTs for the present study. The literature search is shown in The literature search produced 447 articles, 108 of which were excluded because of duplication. An additional 328 articles were excluded because of: nature of the study  were also low. In conclusion, the quality of the methodological assessment was very good. The Cochrane bias of summary tool is shown in 2. The ANOVA test found comparability among the groups in terms of gender (P > 0.5), age (P > 0.5) and BMD (P > 0.5). Of note, in almost all included studies, patients were treated with additional daily supplements of calcium (mean 900.0 ± 223.6 mg) and vitamin D (mean 600.0 ± 230.9 units). Demographic data are reported in Data from 4157 patients were collected. There were 2409/4157 (58%) women. The mean follow-up was 14.7 ± 4.9 months. The mean age was 58.4 ± 4.1 years. The mean BMD was 0.94 ± 0.06 g/cmP = 0.8, P = 0.8, P = 0.6, respectively).At the end of the observation period, femoral neck BMD  and hip BMD  were greatest in the alendronate group . ConcernP = 0.7, P = 0.6, respectively).Non-spine related fractures  were redP = 0.6, P = 0.8, P = 0.9, respectively). The overall results of the network comparisons concerning adverse events are shown in Serious adverse events were lowest in the alendronate group . The alendronate group also reported the lowest rate of serious adverse events that required discontinuation of a study . Regarding these comparisons, the test for global linearity resulted non-significant for inconsistency . Further studies are required to overcome current limitations and to reach more solid indications according to patient individualities and the underlying pathology peculiarities. The findings of the present investigation are derived from the results of randomized controlled clinical trials. They carry not only the advantages of RCT sourced data but also the limitations inherent in extrapolating them to routine clinical practice. The performance of drugs in RCTs, which are often of limited duration and involve limited numbers of selected patients, can differ significantly from their performance in routine practice. This is particularly relevant with essentially non-symptomatic diseases such as osteoporosis, where non adherence can be a major factor. Potentially important treatment complications can also fail to show up within the confines of a RCT. The concern about accelerated bone loss and a possible increase in vertebral fractures with cessation of denosumab therapy is such an example.Alendronate, risedonate, zoledronate and denosumab are all effective in increasing bone density in the spine and reducing vertebral fractures in patients taking corticosteroids. Alendronate, zoledronate and denosumab increased BMD in the hip. Alendronate produced increased femoral neck and hip BMDs, reduced incidence of novel fractures, and lower incidence of serious adverse events, specifically those leading to study discontinuation. These results must be interpreted within the limitations of the present study.The authors declare that they have no conflicts of interest.No external source of funding was used.This article does not contain any studies with human participants or animals performed by any of the authors.For this type of study informed consent is not required.The data underlying this article are available in the article and in its online supplementary material.