Winnerless coevolution of hosts and parasites could exhibit Red Queen dynamics, which is characterized by parasite-driven cyclic switching of expressed host phenotypes. We hypothesize that the application of antibiotics to suppress the reproduction of parasites can provide an opportunity for the hosts to escape such winnerless coevolution. Here, we formulate a minimal mathematical model of host–parasite interaction involving multiple host phenotypes that are targeted by adapting parasites. Our model predicts the levels of antibiotic effectiveness that can steer the parasite-driven cyclic switching of host phenotypes (oscillations) to a stable equilibrium of host survival. Our simulations show that uninterrupted application of antibiotic with high-level effectiveness (greater than 85%) is needed to escape the Red Queen dynamics. Interrupted and low level of antibiotic effectiveness are indeed useless to stop host–parasite coevolution. This study can be a guide in designing good practices and protocols to minimize the risk of further progression of parasitic infections. The Ra). For example, the Red Queen dynamics explain coevolution in invertebrate–parasite systems, such as infection of Daphnia magna by bacteria Pasteuria ramosa [The Red Queen dynamics in host–parasite system describes winnerless coevolution between hosts and parasites, realized through perpetual negative frequency-dependent selection . The can2a ramosa ,19.FiguThrough The Looking Glass: ‘it takes all the running you can do’ (dominance switching represented by out-of-phase cycles), ‘to keep in the same place’  [The canonical Red Queen dynamics can be characterized by populations of host types (and parasite types) undergoing cyclic dominance switching in population abundance, where every host (parasite) type has the opportunity to have an abundant frequency for some period of time . Mathemaolution) ,20. Moreolution) ,16,21,22The Red Queen dynamics is akin to the kill-the-winner hypothesis in bacteria–phage systems ,24. The antibiotic; although, our results can be applied beyond bacterial parasites but without side effect to the host.Intermediate level of parasite mortality is essential in maintaining Red Queen dynamics. Low parasite death rate imposes a high degree of parasitism that adversely affects the host populations; while high parasite death rate limits the antagonistic influence of parasites to initiate negative frequency-dependent selection in hosts . If it iOur minimal mathematical model, for the first time, explains a strategy for escaping the Red Queen dynamics via application of antibiotic to reduce reproduction of parasites at the population level. We considered the conditions favourable in steering the Red Queen dynamics as the starting point of our simulations. Then we show the levels of antibiotic effectivity that can stop the cycles. We also show if the interrupted or discontinued application of antibiotic will provide an opportunity for the parasites to recover. Our model assumes that hosts are killed or castrated once infected by the parasite, that is, they cannot recover from disease nor further reproduce. This is often the case in ecological systems with castrating parasites –28.2.r1 = r2 = r), and they have equal death rates (ρ1 = ρ2 = ρ). Both the parasite growth and death rates are also equal, that is, ξ1 = ξ2 = ξ and d1 = d2 = d, respectively. Furthermore, parasitism efficiency is the same for both parasites (α1 = α2 = α), and effects of antibiotic to both parasites are equal (δ1 = δ2 = δ). Lastly, the strength of competition for both H1 and H2 is equal to 1 (γ12 = γ21=1), meaning both hosts have similar competitive abilities. These simplifying assumptions characterize a host–parasite system where the hosts are from the same family of species, and the parasites are of closely similar types except that each parasite strain targets different host phenotype. The specificity of parasites characterizes a system where the hosts are able to evolve or adapt against the infecting parasite strain. Mathematical modelling is very useful in understanding host–parasite interaction and diseases ,29–31. Tr, ρ, ξ and d) conform to the Red Queen dynamics in host–parasite system with Type II functional response [Y) and efficacy (ϕ). Without losing generality, we set δ = ϕY . Effectiveness of antibiotic to fully suppress parasite population growth depends on the value of ξ. The higher the ξ, the higher is the δ possibly needed. With the addition of an antibiotic, we inspect if the host–parasite coevolution will escape the Red Queen dynamics, and how much δ is needed to realize this.The combinations of parameter values in the simulations  isUninterrupted antibiotic application means that the antibiotic is replenished before its effect declines. Here, we also simulate cases where the antibiotic application is interrupted (periodic) and discontinued. We denote 3.The existence of Red Queen dynamics in host–parasite interaction follows from the existence of parasites, and eradication of parasites can lead to the escape from Red Queen dynamics. We started with an initial state where the host–parasite interaction exhibits Red Queen dynamics at the population level. Then we answer the question: what level of antibiotic effectiveness can stop the Red Queen cycles leading to the stable survival of one or more host types? We intend to escape the Red Queen dynamics for two main reasons: (i) to eliminate or minimize parasite infection leading to the winnerless coevolution of hosts and parasites and (ii) to avoid the risk of extinction of hosts (impermanent coexistence), especially when there is demographic stochastic noise in host population growth . The posa) even with the introduction of antibiotic but with low effectiveness. This suggests that the level of antibiotic is inadequate to eradicate parasitism. In the presence of an ineffective antibiotic, cyclic host population abundance can still have high amplitude similar to the host–parasite system without antibiotics (a). One of the positive consequences of this ineffective antibiotic is that it is possible that the time period before reaching impermanent coexistence widens. However, still the hosts are not able to escape the Red Queen dynamics, with impermanent coexistence as a possible associated recurring risk.Based on our simulations, the host–parasite interaction still exhibits Red Queen dynamics a even wiibiotics a. One ofb). This happens when the antibiotic has partial effect on the parasite, driving a decrease in parasite efficiency but only for a limited duration. To totally suppress parasitism and escape the Red Queen dynamics, a greater level of effectiveness of the antibiotic is needed (figure 2c). The suppression of parasitism leads the hosts to a state of equilibrium wherein one or both host types converge to have non-zero population sizes. As shown in figure 2c, both the host types may coexist; however, a specific host could be more dominant than the other host.Increasing the level of effectiveness of the antibiotic may lead to a phenomenon wherein the oscillations become irregular . This is because a high host growth rate is favourable to generate Red Queen cycles [As illustrated in n cycles .a, the growth rate of the parasite (ξ) has a direct relationship with the effectiveness of the antibiotic needed to escape the Red Queen dynamics. The higher the parasite growth rate, the greater the effectiveness of the antibiotic is needed. This is logical since the eradication of parasites is more difficult if they have a high growth rate. The parasite with less growth rate (δ = 1) will be suppressed using a 91.8% effective antibiotic. However, the parasite with high growth rate (δ = 10) will be suppressed using 99.6% effective antibiotic. On the other hand, the parasite death rate (ρ) has an inverse relationship with the effectiveness of the antibiotic (figure 4b). As the death rate of the parasite increases, the lower the antibiotic effectiveness is needed to escape the Red Queen dynamics. This is because high parasite mortality (not due to antibiotic) can hasten the parasite's extermination when antibiotic is introduced. A parasite death rate of d = 1 will result in full suppression of parasitism if we introduce 86.5% effective antibiotic.We investigate the relationship between the parasite growth and death rate, and the effectiveness of the antibiotic. As shown in figure 4τ with other parameters being fixed. Compared to the case where the antibiotic application is uninterrupted (a), the abundances of both host and parasite populations are low when antibiotic is applied periodically . Moreover, the periodic application of the antibiotic may lead to oscillatory population sizes, with the population dynamics of the hosts having a very low amplitude . This interrupted antibiotic application is more adverse to the hosts compared when the antibiotic is less effective but applied continuously. This suggests that unsustainable antibiotic application could result in more potent parasite infection. In figure 5d,e, we show that discontinuing the application of antibiotic, even for a short time, may result in the death of hosts. The parasites could become more potent that drives host extinction. This is worse compared to the Red Queen dynamics without antibiotics, since host populations undergoing Red Queen cycles still have the opportunity to survive through deterministic coevolution. Uninterrupted application of antibiotic is recommended until parasite population is completely eradicated or, possibly, until a level where host immunity can inhibit parasite infection.Our simulations show that uninterrupted application of effective antibiotic could lead to the full suppression of parasitism through the reduction in parasite population growth. However, this result may change when we vary the application periods of antibiotic (i.e. on specific simulation periods only). We investigate the dynamics of the hosts and parasites using the extended model in equation  with varerrupted a, the abodically b,c. Moremplitude b,c. This4.Using mathematical simulations, we show two novel testable hypotheses: (i) for the hosts to escape the Red Queen dynamics, a high level of antibiotic effectiveness is needed and (ii) interrupted or discontinued application of antibiotic could be detrimental to the hosts. Here, the introduced antibiotic suppresses the population growth of the parasites which will then result in parasite eradication. However, if the application of the antibiotic is discontinued, surviving parasites can recover and these parasites can be more potent causing the sudden extinction of hosts. This suggests that for the hosts, the case where antibiotic is introduced but cannot be sustained is riskier compared to the case of Red Queen dynamics without antibiotic. Red Queen dynamics can drive impermanent coexistence in hosts but the hosts have the chance to survive through deterministic cyclic coevolution. Thus, the use of antibiotic should be regulated ensuring its effectiveness even though costly.


Two QTL with pleiotropic effects on plant height and spike length linked in coupling phase on chromosome 2DS were dissected, and diagnostic marker for each QTL was developed.QPht/Sl.cau-2D.1 and QPht/Sl.cau-2D.2, were separated and characterized. QPht/Sl.cau-2D.1 is a novel QTL located between SNP makers BS00022234_51 and BobWhite_rep_c63957_1472. QPht/Sl.cau-2D.2 is mapped between two SSR markers, SSR-2062 and Xgwm484, which are located on the same genomic interval as Rht8. Moreover, the diagnostic marker tightly linked with each QTL was developed for the haplotype analysis using diverse panels of wheat accessions. The frequency of the height-reduced allele of QPht/Sl.cau-2D.1 is much lower than that of QPht/Sl.cau-2D.2, suggesting that this novel QTL may be an attractive target for genetic improvement. Consistent with a previous study of Rht8, a significant difference in cell length was observed between the NILs of QPht/Sl.cau-2D.2. By contrast, there was no difference in cell length between NILs of QPht/Sl.cau-2D.1, indicating that the underlying molecular mechanism for these two QTL may be different. Collectively, these data provide a new example of QTL dissection, and the developed diagnostic markers will be useful in marker-assisted pyramiding of QPht/Sl.cau-2D.1 and/or QPht/Sl.cau-2D.2 with the other genes in wheat breeding.Plant height (PHT) is a crucial trait related to plant architecture and yield potential, and dissection of its underlying genetic basis would help to improve the efficiency of designed breeding in wheat. Here, two quantitative trait loci (QTL) linked in coupling phase on the short arm of chromosome 2D with pleiotropic effects on PHT and spike length, The online version of this article (10.1007/s00122-019-03318-z) contains supplementary material, which is available to authorized users. Triticum aestivum L.) is an important food crop worldwide, providing calories and proteins consumed by humankind  appropriately reduced PHT and contributed significantly to a worldwide increase in potential grain yield , which is 1.95 cM away from Xgwm261  for PHT linked in coupling phase were mapped on the short arm of chromosome 2D  that carried the heterozygous segment at the genetic region from SSR markers SSR-2212 to SSR-2429 was self-pollinated to produce the F10 generation for further study  without recombinant were selected as NILs (NILY8679 and NILJ411) to validate the presence of the QTL. Two plants that carried heterozygous segments covering the intervals from SSR-2212 to Xwmc503 and from Xcfd53 to SSR-2429 were identified to produce segregate populations  for genetic analysis  and NIL-II .Following our previous study of QTL mapping using the recombinant inbred lines (RILs) of Yumai 8679 (Y8679)/Jing 411 (J411) . These included 1347 hexaploid wheat accessions , and 86 diploid accessions .In total, 1433 wheat accessions with varying ploidy were used to test the allele frequency of two diagnostic markers  and Hebei, and NIL-I and NIL-II were grown in three locations, Beijing, Shanxi and Hebei (Supplementary Table S5). In each environment, two segregating populations were planted with a sowing rate of 20 seeds per row (1.5 m long and 0.3 m apart), while the NILs were planted 25 seeds per line. The irrigation and other management of all sites were in accordance with local standard practices.Populations I and II were tested in Hebei . NILPlant height (PHT), spike length (SL) and spikelet number (SN) of the individuals in populations I and II were measured before harvest. For the NILs, five plants from each line were used for phenotypic evaluation. PHT, SL, SN and length of 5 internodes from the main tillers were measured. For NILs, the mean values over three replications at each site were used for data analysis. Spikelet compactness (SC) was calculated by dividing the SN by the SL.t test.Statistical analysis was performed with IBM SPSS Statistics 20 . Significance analysis was calculated using Student’s http://www.wheatgenome.org/) and Aegilops. tauschii genomic DNA contig sequence (http://aegilops.wheat.ucdavis.edu/ATGSP/) were extracted to design new SSR markers by using BLASTN method with the sequences of markers DG279 and DG371   were considered equivalent and named as suggested by McIntosh et al.   and then, finally, 72 °C for 10 min. Then, 8% PAGE was adopted to separate the length polymorphism.To develop a diagnostic marker for Y8679, NIL-IJ411, NIL-IIY8679 and NIL-IIJ411 plants, then fixed in FAA solution (50% (v/v) ethanol, 5% (v/v) glacial acetic acid and 4% (v/v) formaldehyde) and subjected to vacuum pumping for 30 min. Next, the internodes were dehydrated in a series of ethanol solutions (75% (v/v) ethanol, 85% (v/v) ethanol, 90% (v/v) ethanol, 95% (v/v) ethanol and anhydrous ethanol) and destained in a series of xylene solutions . The internodes were soaked in each ethanol and xylene solution for 1 h and then embedded in paraffin .The medial sections of peduncle at flowering stage were collected from NIL-IGenomic DNA for Y8679 and J411 was used to build paired-end sequencing libraries with insert sizes of approximately 500 bp, according to vendor-provided instructions (Illumina). An average 5 × coverage of the assembled genome, with 150-bp paired-end reads for each accession, was generated with the Illumina HiSeq X Ten platform. All the sequence reads for each parent were mapped to the newly updated genome of Chinese Spring (RefSeq v1.0) in the Burrows–Wheeler Aligner program  with default parameters  were detected on chromosome 2DS by reanalysing the phenotypic data from 191 RIL lines in 9 individual environments and 1 combined analysis (BLUP)  were also detected under all environments, which explained 30.94–40.63 and 31.31–41.95% of the phenotypic variations, respectively. All increasing alleles for PHT and SL came from J411  and spike length (SL) on the short arm of chromosome 2D, 632 SSR markers were developed, among which 17 markers exhibited polymorphism between parents Y8679 and J411 (Supplementary Table S14). The collinearity of these markers with QPht/Sl.cau-2D.1 and QPht/Sl.cau-2D.2 on PHT and SL, one line (RIL171) from the F9 generation of the RIL population was selected for investigation because of its residual heterozygosity at the target QTL genomic regions. First, one pair of NILs (NILY8679 and NILJ411) from the self-pollinated progeny of RIL171 (F10) harbouring QPht/Sl.cau-2D.1 and QPht/Sl.cau-2D.2 were genotyped using the wheat 660 K SNP array (https://wheat.pw.usda.gov/ggpages/topics/Wheat660_SNP_array_developed_by_CAAS.pdf). Of 630517 SNP markers with detected signal, only 434 (0.07%) exhibited polymorphism between NILY8679 and NILJ411, indicating that the genetic backgrounds of these two NILs had very high similarity (Supplementary Table S6). Notably, based on the information of SNP markers with genomic position, 154 of 434 polymorphic SNP markers (35.48%) were located on genomic interval flanking QPht/Sl.cau-2D.1 and QPht/Sl.cau-2D.2 (Supplementary Table S7). Second, the progenies of the above NILs (F11) were used for phenotypic analysis. As shown in Table Y8679 and NILJ411 (P < 0.001) under different environments. By contrast, the differences in length of the second internode under peduncle (2IL), length of the third internode under peduncle (3IL) and length of the fourth internode under peduncle (4IL) were only detected under a specific environment. In addition, no difference was observed for spikelet number (SN). Consequently, PHT of NILJ411 was 12.62 and 15.08% (P < 0.001) higher than that of NILY8679 under two different environments. Correspondingly, spikelet compactness (SC) of NILY8679 was significantly (P < 0.001) higher than that of NILJ411 , eight polymorphic SSR markers between NILJ411 and NILY8679 were used to genotype the segregating populations of RIL 171 (F10). Two plants carrying different heterozygous genomic regions (I and II) were selected for further analysis, corresponding to QPht/Sl.cau-2D.1 and QPht/Sl.cau-2D.2, respectively . As expected, polymorphic SSR markers in each population were significantly (P < 0.001) associated with PHT and SL   had significantly higher values than those of NIL-IY8679 and NIL-IIY8679 (genotype same with Y8679) under all environments, with average differences of 9.97% and 12.62, 16.25 and 19.56%, and 13.43% and 18.11%, respectively  than those of NIL-IIY8679 under all environments (Supplementary Table S9).To further investigate the genetic effect of each QTL (SL Table . To furtII) Fig.  were devQPht/Sl.cau-2D.1, NIL-IY8679 showed no difference in cells length compared to NIL-IJ411 .In wheat, internode elongation is caused by cell division and/or cell elongation  was converted into convenient STARP marker (STARP-2001)  under different environments  than those of the SSR-2433-Y allele (5.10–7.07 and 12.37–15.73%) . By contrast, SSR-2433-Y allele presented in 73 of 181 accessions (40.33%) (Supplementary Table S2). In addition, a diverse panel of 86 accessions of Aegilops tauschii, the D-genome progenitor of T. aestivum, was also analysed. The results exhibited that the STARP-2001-Y allele was not detected in 86 accessions of Aegilops tauschii, but the SSR-2433-Y allele presented in 39 of 86 accessions (45.35%) (Supplementary Table S4).To trace the origin of dwarf genes in the QTL intervals of QPht/Sl.cau-2D.1 and Rht8, we analysed the gene models in the mapping interval of the Chinese Spring RefSeq v.1.0 sequence. In rice, WRKY transcription factor Dlf1 and gibberellin-inactivating 2-beta-dioxygenase gene played important roles in the regulation of PHT , TraesCS2D01G051000 and TraesCS2D01G051500 encoded two WRKY transcription factors, and TraesCS2D01G049700 encoded gibberellin 2-beta-dioxygenase, which may be the candidate genes (Supplementary Table S10). DNA sequence analysis showed that there were 5 SNPs and 3 InDels between TraesCS2D01G051500 of parental lines Y8679 and J411, including 3 SNPs in exon 1, 1 InDel in 3′-UTR region, 1 InDel in intron 2 and 2 SNPs and 1 InDel in the upstream region of translation start codon  encoded transcriptional factors belong to the GRAS family  homoeostasis to maintain cellular osmotic potential, which affected PHT in turn . DNA sequence analysis revealed that there were 38 SNPs and 8 InDels between TraesCS2D01G055700 of Y8679 and J411, 3 SNPs and 3 InDels in the exon, and 35 SNPs and 5 InDels in the upstream region of translation start codon. For TraesCS2D01G058700, only 2 SNP variants were found, which were located in intron 4 and the upstream region of translation start codon, respectively  and yield per plant (qYD3) by using two rice near isogenic populations  controlling PHT and SL were mapped on the short arm of chromosome 2D. However, it is difficult to conclude whether QPht/Sl.cau-2D.1 is a shadow or genuine QTL because it is only 7 cM away from QPht/Sl.cau-2D.2  using segregating populations and NILs derived from a residual heterozygous line (RIL171). They were linked in coupling phase on chromosome 2DS, with increased alleles from the same parent (J411). QPht/Sl.cau-2D.1 was located between SNP markers BS00022234_51 and BobWhite_rep_c63957_1472, and QPht/Sl.cau-2D.2 was located between SSR markers SSR-2062 and Xgwm484. The results of NILs indicated that the genetic effect of these two QTL is similar, which could reduce plant height by 4.79–14.32 and 9.49–14.43 cm under different environments (Table QPht/Sl.cau-2D.1 and QPht/Sl.cau-2D.2) is still an area for further elucidation.Neighbouring QTL that are linked in coupling phase are commonly observed in primary QTL analysis  that was tightly linked to QPht/Sl.cau-2D.2/Rht8. Moreover, the SSR-2433 locus only has two allelic variants in the diversity panel of wheat accessions, which may be an ideal diagnostic marker for the QPht/Sl.cau-2D.2/Rht8 gene.Due to the widespread use of Rht1–Rht24) have been catalogued in wheat  QPht/Sl.cau-2D.1 was mapped to a position distal to Rht8 on the short arm of chromosome 2D  and Aegilops tauschii (DD) 0.43 million years ago   encoded GRAS transcription factors. Notably, a 35-bp deletion in the coding region of TraesCS2D01G055700 was found in parental line J411 as compared to Y8679, which resulted in frame shift for translation. Moreover, another two SNP variations between Y8679 and J411 leaded to amino acid substitutions. In addition, 35 SNPs and 5 InDels in the upstream region of translation start codon were detected  in exon 1 that leaded to the substitution of 1 amino acid (I/L) and 2 SNPs and 1 InDel in the upstream region of translation start codon Below is the link to the electronic supplementary material.


Our study demonstrated, for the first time, the beneficial effect of a two-step strategy  for AIDs diagnosis and stressed the possible usefulness of the strategy in anticipation of the development of pan-genomic analyses in routine settings.Monogenic autoinflammatory diseases (AIDs) are caused by variants in genes that regulate innate immunity. The current diagnostic performance of targeted next-generation sequencing (NGS) for AIDs is low. We assessed whether pre-analytic advice from expert clinicians could help improve NGS performance from our 4 years of experience with the sequencing of a panel of 55 AIDs genes. The study included all patients who underwent routine NGS testing between September 2014 and January 2019 at the laboratory of autoinflammatory diseases . Before March 2018, all medical requests for testing were accepted. After this time, we required validation by a reference center before NGS: the positive advice could be obtained after a face-to-face consultation with the patient or presentation of the patient’s case at a multidisciplinary staff meeting. Targeted NGS resulted in an overall 7% genetic confirmation, which is consistent with recent reports. The diagnostic performance before and after implementation of the new pre-requisite increased from 6% to 10% ( Monogenic autoinflammatory diseases (AIDs) are characterized by variants in genes coding proteins involved in innate immunity . InflammSince the discovery in 1997 of the first gene MEFV (Mediterranean fever MIM 608107)), responsible for familial Mediterranean fever (FMF), more than 40 new known genetically defined AIDs have been identified, notably by high-throughput sequencing approaches  1,3,4,5,4,53,4,508107), rHowever, the genetic diagnosis yield of targeted panels for AIDs is still currently low (from 4% to 22% depending on how the identified genotype is interpreted)  8,9,10,,10,11,12Here, we presented the results of our 4 years of experience with targeted analysis of a panel of 55 known AIDs genes as a routine NGS-based strategy and compared its performance  before and after implementation of new pre-requisite, involving approval for this testing from a national reference center for AIDs.Patients referred to us for genetic diagnosis of AIDs and, if necessary, some of their relatives were fully informed and gave written consent for DNA analysis. This study included all consecutive patients who underwent routine genetic screening with the targeted NGS panel between September 2014 and January 2019. Before March 2018, all medical requests for NGS testing were accepted. After this date, because this permissive procedure resulted in unmanageable laboratory work overload and a disappointing rate of diagnosis confirmation, we decided to implement, with the reference centers for rare AIDs, a new decisional tree for genetic testing. For time and cost-saving, individuals with a reasonable clinical suspicion of FMF  ,14,15, dAll coding exons and intronic borders of 55 genes  were scrMEFV:p.(Glu148Gln) or TNFRSF1A:p.(Arg121Gln). The diagnosis was not confirmed or excluded when one rare VOUS was found for which relatives were not available for segregation analysis. We reported heterozygote status in MEFV because of its known dose-effect [Variants were analyzed by using standard in silico tools available on the Alamut pipeline : Align-Grantham variation and Grantham deviation (Align-GVGD), Sorting Intolerant From Tolerant (SIFT), Polyphen2, and MutationTaster for missense substitutions, and MaxEntScan (MES), splice-site analysis (SSF), Human Splice Finder (HSF), and neural network splice (NNSplice) for splicing defect prediction ,19. The e-effect .p < 0.05 was considered statistically significant.We retrospectively reviewed the performance of our NGS panel and whether patients were referred by a clinician from a reference center for AIDs or their case was presented at an MSM. Clinical data were extracted from our request form for genetic diagnosis of AIDs. We used descriptive statistics, including frequencies (percentages) for categorical data and medians (Q1–Q3) for continuous data. Data were compared by two-sample Mann–Whitney rank sum, chi-square, or Fisher exact test as appropriate. MVK:p., MEFV:p., and RNASEH2B:p.(Ala177Thr). At least one variant was reported in one-quarter of patients (158/631). The yield of conclusive genetic diagnosis with this NGS strategy was 7% over this 4-year period (MVK (n = 6), NLRP3 (n = 5), and MEFV (n = 4). r period . The genThe origin of the requests for NGS considerably switched upon the implementation of the new pre-requisite for NGS . Beforep = 0.021) (p = 0.029) or not , which supports the positive effect of the pre-requisite. The lack of significance of the subset of clinicians not from a reference center is most likely due to the considerable reduction in a number of direct requests by clinicians not from reference centers since we imposed their validation by an MSM.The diagnostic performance of our NGS panel almost doubled after March 2018, from 6% to 10% (= 0.021) . We obseNLRP3-associated AID phenotype. Consanguinity was documented in seven of the 10 patients in whom we identified a homozygous pathogenic variant in a gene responsible for a recessive condition. The main symptoms reported are described in SLC29A3-associated histiocytosis-lymphadenopathy–plus syndrome) or sideroblastic anemia , were also reported.The age of onset was pediatric for all but one patient . The 53-year-old patient exhibited a class 4 somatic variant , 24% mosaicism) consistent with her MEFV), p.(Arg121Gln) (TNFRSF1A), or p. (NLRP3), and all known susceptibility factors for inflammation were considered consistent with the diagnosis [Recognizing patients with monogenic AIDs is difficult because their clinical spectrum may range from very mild inflammatory episodes to severe chronic disease. Moreover, the disease features significantly overlap . Our tarr p.Val19Met  and better information on these three diseases because they are among the best-described AIDs [MEFV. If we had included heterozygous carriers of a MEFV pathogenic variant, the genetic diagnosis yield would have improved up to 10%  variant [ADA2:c.-47+2T>C) [We cannot rule out a relative underestimation of the potential of NGS panels for AIDs in general. Indeed, because of our study design, some of the patients or their relatives were not available for NGS or segregation analysis. However, our aim was not to evaluate the exact performance of NGS but to find ways to improve it. In addition to the successful clinical option we demonstrated here, it is easy to predict the upcoming discovery of new genes or molecular mechanisms involved in AIDs that will continue to revolutionize our routine genetic diagnosis ,27. Rece variant ; the lac47+2T>C) ; or larg47+2T>C) . In thosThe role of the reference centers is to determine whether or not the clinical/anamnesis/genealogical tree is compatible with a known AID. If compatibility does not seem to be the case, the role is to evaluate the potential added value to perform the NGS. If this value appears to be low, the reference center recommends that no NGS be conducted in discussion with geneticists. Thus, this discussion decreases the number of individuals whose phenotype is not typically enough.Our results confirmed the currently low performance of the use of NGS panels for the genetic diagnosis of AIDs . Our tar


Background: Postural balance and fall efficacy  have been found to be risk factors associated with falls in older adults. Stretching is one intervention that has been investigated to improve balance and therefore reduce fall risk. Various forms of stretching have been evaluated with different outcomes, but there is a lack of knowledge about the effect of stretching (continuous and intermittent) on plantar pressures and balance. Therefore, the aim of the present study was to analyze the effects of stretching (continuous and intermittent) of the bilateral ankle plantar flexors on plantar pressures and static balance. Methods: A randomized clinical trial was carried out. Forty-eight healthy subjects  were recruited in an outpatient clinic. Subjects were randomly assigned to an intermittent stretching group  or a continuous stretching group (2 min of continuous stretching) of the plantar flexors. Plantar pressures and balance using stabilometry were measured before and after stretching. Results: There were significant differences between intermittent and continuous stretching in rearfoot maximum pressure, forefoot surface area, and center of pressure surface area with eyes open. Conclusions: Bilateral intermittent stretching of the ankle plantar flexors was found to be more effective than continuous stretching for the reduction of rearfoot maximum pressure and improved balance. Worldwide, each year an estimated 646,000 individuals die from falls . Fall efTwo of the more common forms of static stretching include continuous and intermittent modalities. Continuous static stretching has been shown to improve range of motion (ROM) more than intermittent stretching . SpecifiStatic stretching has also been shown to cause a decrease in force production of the muscle. The loss of force with stretching occurs following a minimum of 2 min . When coPostural control is maintained by continuous contraction of anti-gravitational musculature throughout the body. Alterations in the position of the body may lead to variations of load due to the acceleration of gravity and may influence the contraction of the lower back/pelvic  and lower limb flexor muscles. Posture has been deeply analyzed by means of electromyography (EMG), Romberg test analysis, pressure platform analysis, and thermography. The lower limb flexor muscles muscle activation pattern has demonstrated a relationship with static and dynamic balance. Therefore, stretching of the flexor muscles may help to improve the muscle functionality ,19,20,21Currently, there is a lack of consensus on the relationship between lower limb stretching and balance. Several systems may be involved for individuals’ postural control, which may include the neuromuscular, vestibular, visual, and somatosensory systems ,7. Behm Thus, there is a lack of knowledge about the effects of static stretching (continuous and intermittent) on plantar pressures and balance. We hypothesized that static intermittent stretching could improve balance effects. Therefore, the aim of the present study was to analyze the effects of stretching (continuous and intermittent) of the bilateral ankle plantar flexors on plantar pressures and static balance.Forty-eight healthy subjects  were recruited for the study. The demographic data of the study participants were as follows: 32.12 ± 7.60 years old, 166.64 ± 8.13 cm height, and 62.72 ± 8.97 kg weight. All demographic data are shown in 2. Before each use, auto-calibration was performed.First, a clinician with ten years of experience performed a baseline balance evaluation to confirm the inclusion and exclusion criteria of each subject using the Balance Evaluation Systems test (BESTest)  in orderThe protocol started with a pre-stretching evaluation. Participants were then randomly divided into two groups (intermittent stretching group or continuous stretching group) by a random number table provided by the clinician. Each group then performed the prescribed stretching protocol. In order to maintain consistency between groups, the stretching methods were standardized. The subjects climbed on a raised platform and placed the forefoot of both feet on the edge of the platform. They next dropped both heels off the platform to the ground without making contact with the ground ,30 and hStabilometry assessment was used and subjects were instructed to stand barefoot on the force platform . The feeStabilometry was measured by displacement of the center of pressures in X and Y with eyes open and closed , center Static plantar pressure was evaluated by means of maximum pressure, medium pressure and surface area of each aspect of the foot .p > 0.05. Descriptive statistical analysis was performed using mean ± SD and a 95% confidence interval. All data were explored for normality using the Shapiro Wilks test, and data were considered normally distributed if t-test was used for parametric variables. A p-value < 0.05 with a confidence interval of 95% was considered statistically significant for all tests .The Mann–Whitney U test was performed to examine differences in non-parametric variables. Student’s p < 0.05), except for socio-demographic characteristics of the sample population. There were no significant differences between groups at baseline  of the bilateral ankle plantar flexors on plantar pressures and static balance.Both stretching groups (intermittent and continuous) did not demonstrate significant differences during pre-intervention testing. Intermittent stretching demonstrated a reduced maximum pressure in the rearfoot compared to the continuous stretching group. There was also an increase of forefoot surface area in the intermittent group compared to the continuous group. Thus, these results suggest that a greater surface area in the forefoot can reduce the maximum pressure on the rearfoot. Ankle equinus generates a large deforming force to the foot and may be considered as a related factor to several foot and ankle conditions, including plantar fasciitis , pes plaStretching exercise leads biomechanical and physiological adaptations related to motor unit excitability improvements, body temperature increases, kinesthetic awareness improvements, and active ROM increases. These biomechanical and neuromuscular modifications may be deeply related to the changes of viscoelastic components of the muscle-tendon units and depressed reflex activity of neural outputs. This phenomenon is called viscoelastic stress relaxation and demonstrates that the muscle–tendon unit is affected during stretching activity . Continuous stretching effects may be also related to a muscle compliance increase, which increases the time and decreases the force for myofibril-unit contraction during muscular elements activation. Continuous stretching may affect the maximal force production and reduce muscle stiffness and muscle activation, as well as generating an acute increase of ROM. These modifications have been related to an increased tolerance to the imposed stretching activity ,29. NeveBoth continuous and intermittent stretching interventions have been related to dynamic balance and postural control. Balance and joint position sense are proprioceptive parameters that depend on contributions from visual, vestibular and peripheral receptors that are found in skin, joints, muscles and ligaments . Proprioception may provide conscious and subconscious responses for body posture and motion, being essential for lower limb joints functioning in order to maintain optimal balance control during daily physical activities. Joint position sense may be considered as an aspect of proprioception that plays an important role in functional dynamic stability of joints through the action of the muscles and ligaments around them, which seems to be influenced by ROM modifications .Therefore, intermittent stretching use has been recommended for pathologies that present an increased heel pressure such as heel pain , ,10,12,41The force platform assessment demonstrated that bilateral intermittent stretching of the ankle plantar flexor muscles seems to be more effective than continuous stretching for the reduction of rearfoot maximum pressure and balance improvement.


Hygro-thermo-chemo-mechanical modelling of time-dependent concrete behavior requires the accurate determination of a large set of parameters. In this paper, the parameters of a multiphase model are calibrated based on a comprehensive set of experiments for a particular concrete of grade C30/37. The experiments include a calorimetry test, tests for age-dependent mechanical properties, tests for determining the water desorption isotherm, shrinkage tests, and compressive creep tests. The latter two were performed on sealed and unsealed specimens with accompanying mass water content measurements. The multiphase model is based on an effective stress formulation. It features a porosity-dependent desorption isotherm, taking into account the time-dependency of the desorption properties. The multiphase model is shown to yield excellent results for the evolutions of the mechanical parameters. The evolution of the autogenous shrinkage strain and evolutions of the creep compliances for loading at concrete ages of 2 days, 7 days, and 28 days are well predicted together with the respective mass water content evolution. This also holds for the evolution of the drying shrinkage strain, at least for moderate drying up to one year. However, it will be demonstrated that for longer drying times further conceptual thoughts concerning the coupled representation of shrinkage and creep are required. Shrinkage and creep are important phenomena affecting the load bearing capacity, durability and serviceability of plain and reinforced concrete structures. Drying of concrete structures commonly results in restraint shrinkage and will therefore cause compressive as well as tensile stresses in a structure. The tensile stresses, often associated with near-surface regions, can cause material degradation and are for this reason potentially harmful to the integrity of the concrete cover. The material degradation can result in an increased susceptibility to chloride ingress and carbonation, and may therefore cause a significant reduction in durability. Creep may induce significant long-term deformations in concrete structures resulting in prestress losses of prestressed concrete members. This may affect the safety as well as the serviceability of these structures. If concrete structures are loaded at early age, either mechanically or hygrally (by drying), the time-dependency of the material behavior, governed by cement hydration, is also of great importance. The chemical reaction rate in the hydration process depends not only on time but on the hygral and thermal state as well, which can be quite inhomogeneous in concrete structures.Proper consideration of such coupled processes in numerical simulations requires the application of a coupled hygro-thermo-chemo-mechanical multiphase model ,2. An exThe use of such a comprehensive multiphase model is both a blessing and a curse. The capability of considering all the different phenomena comes at the price of a large number of balance and constitutive equations with many parameters to be calibrated. The required experiments include calorimetry tests, tests on mechanical and hygral parameters as well as sorption, shrinkage, and creep tests. Since many of these properties are age-dependent, experiments have to be performed not only once but several times at different ages and/or for long durations. Furthermore, they have to be carried out in different ambient conditions.A major contribution of this paper is the simultaneous calibration of all parameters of a fully coupled hygro-thermo-chemo-mechanical multiphase model for a single concrete mixture, summarized in The number of different experiments is considerably more comprehensive than the tests used, for instance, for calibrating the material parameters of a multiphase model for shotcrete . MoreoveA further key aspect of the present paper is the proposition of a porosity-dependent desorption isotherm based on an analogy to a deformation-dependent soil water retention curve developed in the context of geomechanics . Due to The paper is structured as follows. The hygro-thermo-chemo-mechanical model is governed by a set of balance equations for momentum, mass, and enthalpy, see ,14,15, fThe balance of momentum for the multiphase continuum can be stated for quasi-static conditions using the total stress n, the density of the solid phase The balance of mass of the solid phase is given asfined in . The terBased on the passive air phase assumption, and neglecting gravity, the balance of mass of the water vapor phase readsT:In  ρgw and t. As in ,17, for The exponents turation ,18. The 0−5 m2/s  will be K, the dynamic viscosity of water Parameters for the mass flux of liquid water relative to the solid phase are the intrinsic permeability of concrete er vapor  and liquer vapor  finally To sum up, the mass balance of the water phase  takes inThe balance of enthalpy for the multiphase mixture is given asCpeffρeff side of , EquatioCpeffρeffAll time derivatives are evaluated with respect to an observer fixed in the material configuration of the solid phase. The approach used here takes into account full coupling between the hygral and mechanical problem. Nevertheless, the observed back-coupling from mechanical deformation to the hygral problem is weak.The chemical reactions, characterizing the hydration process, are modelled taking into account chemo-hygro-thermal phenomena at a macroscopic level. For that purpose, the reactions are lumped into a single reaction model for a normalized degree of hydration, as also done in ,5. The eCompressive strength evolution was modelled as in ,20 usingThe exponent init=0.1 ,21. The In hygral equilibrium and isothermal conditions, the relation between the mass water content lace lawφ=exp−pc·lace lawφ=exp−pc·In the present contribution, the desorption isotherm is included in a constitutive equation for the degree of water saturation. Its general form is assumed as:Shrinkage is modelled following the approach of ,2. AssumThis formulation adopts the generalized saturation-dependent Bishop parameter The constitutive relation describing the mechanical behavior is assumed to be governed by the effective stress rate, i.e.,ε˙−ε˙th−εTherefore, from  and 18)18), the A decomposition of the creep strain rate into a viscoelastic part The thermal strain rate is proportional to the rate of temperature, i.e., The effective elastic-viscoelastic compliance In the formulation of  adopted The parameters are the asymptotic elastic compliance Eve−1 in  is the rThe microscopic creep compliance function of the solidified matter is independent of the degree of hydration and is assumed to be of log-power law type  with a cFor the implementation, the microscopic creep compliance is expanded into a Dirichlet series , consisttep. See  for how S evolves according to [c, Here, rding to 27)S˙+cS˙+cKeff=The total amount of chemically bound water per unit volume quations  and 6)  Δmw in tete mass . The masg-sensor  obtainedg-sensor . The conFollowing , the stoan Mills , see alsan Mills , as(29)n in the balance equations of n and For the evolution of the porosity rding to ,3:(30)n=The parameters in this equation are In a first step, the porosity er phase . It is aSince according to the mass balance Equations  and 6)  6) the wThe final mass of solid on the left side of this equation is equal to the mass of the solid phase at the time the solid forms plus the amount of water bound in the solid in the subsequent chemical reaction. The density of the solid phase In combination with  one obtaFrom the latter and the value for rding to , the paro d1−nTherefore, For the present model, the parameters of n∞ in  is closent paste , as propnt paste . AccordiBy analogy to , the usuThe parameters for the concrete mixture are then obtained by multiplying The final value of the porosity is reasonably close to the value obtained in . HoweverInformation on the early-age reaction kinetics is obtained from temperature measurements on insulated cubic concrete specimens. For these tests, a cubic specimen with the edge length of 200 mm was enclosed in an extruded polystyrene foam (XPS) formwork with a thickness of 100 mm. A first test is used for calibrating the heat conductivity of the insulation. In this test, a matured specimen, preheated to 47 In a further test, fresh concrete was cast in the insulated box, which again was stored at the constant ambient temperature of 20 his test , can be pression  togetherpression  with theters cf.  and the ters cf. .From a third test, performed on a matured cylindrical specimen, the convective heat transfer coefficient The parameters for the evolutions of the compressive strength and the elastic modulus are determined from tests on cylindrical and prismatic concrete specimens, respectively. The cylinders used in the compressive strength tests had a diameter of 150 mm and a height of 450 mm. The prims used in the elastic modulus tests were characterized by dimensions of 100 mm × 100 mm × 400 mm. For the computations, the specimens are assumed to be sealed until the beginning of testing at concrete ages of 2 days, 7 days, 28 days, 56 days, 112 days, and 365 days. This corresponds to the test conditions [Compressive strength evolution is modelled according to . The strent data ,8 in FigThe excellent computed results for the calorimetry test, shown in the previous subsection, indicate that the parameters for the early-age evolution of the degree of hydration are well calibrated. The good agreement with the experimental data visible in The elastic modulus measured in experiments depends on the concrete age as well as on the duration of the measurement period. Therefore, a model for describing the mechanical response of hardening concrete has to take into account such effects. A promising approach to meet these requirements is the use of a multiphase creep model  based onD denoting dimensionless fitting parameters and Gallipoli et al.  suggestei et al.  for soilThe application of a modified air entry value for taking into account changes in the microstructure can also be found in other contexts. For instance, in  a temperD and For the present model, the parameters pbcn∞ in  are caliUsing  this equFurthermore, a  mass water content at Since the desorption isotherm is porosity-dependent, the respective degrees of hydration, at which the water contents were measured, must be estimated. For this purpose, simulations of drying of water saturated thin concrete slices , stored From the calibrated porosity-dependent desorption isotherm representative curves can be obtained for different degrees of hydration as shown in n and the relative humidity In Let us at first focus on the curve, representing the water content evolution resulting from the simulation of a sealed sample. In the first part of this curve the water content mainly decreases by a reduction of porosity while relative humidity remains high. This corresponds to the steep slope of the desorption isotherm close to full saturation for high levels of porosity/low degrees of hydration which can also be seen in The water content evolution resulting from the simulation of a sealed sample in n-n-For all tests the 18% from  is visibK. The former corresponds to a reduction factor between zero and one reducing water permeability in partially saturated states, the latter is a degree-of-hydration-dependent material parameter of the porous solid.According to , the traThe relative permeability is assumed to be governed by a van Genuchten–Mualem based approach ,34:(48)kThe dimensionless parameter d bfS in  based onIn addition, water transport is also studied in cylindrical concrete specimens with 150 mm diameter and 450 mm height for determining the intrinsic permeability. To this end, the mass water content, measured by a multi-ring-sensor embedded at the center of the cylindrical specimens, is exploited .Since transport processes are considered in hardening concrete, for which the structure of the pore system continuously changes due to hydration, the effect of hydration on the intrinsic permeability needs to be considered in the model. Therefore, according to Gawin et al. , the intThe dimensionless parameter ages cf. .The constants nd bχ in , which cFrom al value  and, thuThe fitting parameter ry value  was initThe predicted values of the relative pore humidity and of the normalized degree of hydration after one year are The corresponding porosity after one year is The parameters for the viscous creep model are calibrated from experiments on cylindrical specimens of the same shape and size as used in the previous paragraph. They were loaded at concrete ages of 2 days, 7 days, and 28 days to The parameters are identified based on measured basic creep and drying creep compliances. The former are obtained from sealed specimens. For the latter, the specimens are only sealed at the beginning and the sealing of the perimetral surface is removed upon load application. The measured compliances are obtained as the difference between measured total strain (obtained from loaded specimens) and measured shrinkage strain (obtained from companion shrinkage tests on load-free samples), normalized by the applied load. The computed compliances are obtained as the difference between computed total strain (obtained from loaded specimens) and computed shrinkage strain , normalized by the applied load. Since calibration is performed by means of the creep compliances only, shrinkage does not affect the calibration of the viscous creep parameters. Therefore, the viscous creep parameters do not depend on the actual calibration of the shrinkage formulation. However, in the creep formulation in terms of the effective stress, this is not true in the reverse direction. The calibration of the viscous creep parameters does affect the shrinkage parameter calibration.S in  the measured mass water content evolution for thin concrete slices dried at different ambient relative humidities, (ii) the mass water content evolution measured at the center of cylindrical concrete specimens exposed to drying. Calibrate the hydration-dependency of the intrinsic permeability  by the mCalibrate the parameters for the Bishop parameter  on the bPerform a simulation of the autogenous shrinkage test using the previously calibrated Bishop and desorption law parameters. If necessary, recalibrate the parameter defining the dependence of the desorption isotherm on the porosity in  and recaEvaluate the compliances of sealed and unsealed specimens loaded in compression at different concrete ages and use the results for calibrating the viscous creep parameters in the microprestress formulation  and 27)27). SincThe steps of the calibration procedure for the multiphase concrete model are summarized as follows:The complete calibration of a multiphase concrete model is based on a comprehensive data set from a particular concrete mixture. It includes results from calorimetry tests, results from tests for age-dependent mechanical properties, mass water content and corresponding shrinkage measurements, and results from compressive creep tests on sealed and unsealed specimens loaded at different concrete ages. This thorough calibration allows to fully exploit the capabilities of the coupled multiphase approach.A new desorption isotherm is presented with a porosity-dependent  air entry value and an extra parameter for the improved approximation of the mass water content at low levels of relative humidity and a suitable calibration procedure is deduced. Compared to existing approaches, the proposed desorption isotherm enables considerably improved predictions of early-age autogenous shrinkage strain.n-Visualizations of A connection between the evolutions of the porosity and of the mass water content in a sealed hardening concrete sample is established. Thereby, a relation between porosity and degree of hydration is established, based on measured mass water contents and using the mass balances of the water and solid phase, neglecting thermal and mechanical deformation. It is consistent with the applied multiphase model.The viscous, long-term creep parameters of the multiphase model are calibrated by creep compliance data only. The added value of this procedure is especially prominent for a creep formulation in terms of the effective stress, for which shrinkage is coupled to creep while the modelled creep compliance is independent of the predicted shrinkage behavior. The short-term elastic-viscoelastic creep parameters are fitted independently using data from tests for the elastic modulus at different concrete ages.A further highlight of the paper is the use of separate experiments for calibrating and confirming the parameters of the shrinkage formulation in terms of the effective stress. The former is based on experiments on thin concrete slices, whereas the latter is based on tests on cylindrical concrete specimens. The added value of confirming identified parameters is further enhanced by simultaneously assessing the quality of the predictions for both the strain and the accompanying mass water content.In this paper, a calibration procedure for the numerous parameters of a multiphase model describing the coupled hygro-thermo-chemical-mechanical behavior of concrete at early-age is presented. The major contributions of this work are:The comparisons of measured and computed evolutions of shrinkage strains reveal limitations of the model. It appears that the model in the current form does not work well for predicting the drying shrinkage strain at the lowest level of investigated ambient relative humidity of 43%. However, the model works well for the investigated higher levels of relative humidity, which correspond to the range usually encountered in concrete structures in central Europe. Although a decent approximation of the drying shrinkage strain is obtained for concrete ages up to one year, the drying shrinkage strain is overestimated for longer drying periods. This observation applies to the simulations of both drying concrete slices and drying concrete cylinders. This problem will be further investigated in a forthcoming paper. Therein, an alternative to the shrinkage and creep formulation in terms of the effective stress will be proposed as a remedy for the mentioned problems.


Antibiotics are essential to treat for many childhood bacterial infections; however inappropriate antibiotic use contributes to antimicrobial resistance. For childhood diarrhea, empiric antibiotic use is recommended for dysentery (bloody diarrhea) for which first-line therapy is ciprofloxacin. We assessed inappropriate antibiotic prescription for childhood diarrhea in two primary healthcare facilities in Kenya.We analyzed data from the Kenya Population Based Infectious Disease Surveillance system in Asembo  and Kibera . We examined records of children aged 2–59 months with diarrhea (≥3 loose stools in 24 h) presenting for care from August 21, 2009 to May 3, 2016, excluding visits with non-diarrheal indications for antibiotics. We examined the frequency of antibiotic over-prescription (antibiotic prescription for non-dysentery), under-prescription (no antibiotic prescription for dysentery), and inappropriate antibiotic selection (non-recommended antibiotic). We examined factors associated with over-prescription and under-prescription using multivariate logistic regression with generalized estimating equations.Of 2808 clinic visits with diarrhea in Asembo, 2685 (95.6%) were non-dysentery visits and antibiotic over-prescription occurred in 52.5%. Of 4697 clinic visits with diarrhea in Kibera, 4518 (96.2%) were non-dysentery and antibiotic over-prescription occurred in 20.0%. Antibiotic under-prescription was noted in 26.8 and 73.7% of dysentery cases in Asembo and Kibera, respectively. Ciprofloxacin was used for 11% of dysentery visits in Asembo and 0% in Kibera. Factors associated with over- and under-prescription varied by site. In Asembo a discharge diagnosis of gastroenteritis was associated with over-prescription , while malaria diagnosis was negatively associated with antibiotic over-prescription  but positively associated with antibiotic under-prescription . In Kibera, over-prescription was more common among visits with concurrent signs of respiratory infection  and less common among children aged < 1 year .Inappropriate antibiotic prescription was common in childhood diarrhea management and efforts are needed to promote rational antibiotic use. Interventions to improve antibiotic use for diarrhea should consider the influence of malaria diagnosis on clinical decision-making and address both over-prescription, under-prescription, and inappropriate antibiotic selection.The online version of this article (10.1186/s12889-019-6771-8) contains supplementary material, which is available to authorized users. Inappropriate antibiotic use, which includes prescribing unnecessary antibiotics, prescribing incorrect antibiotics, and not prescribing antibiotics when needed, is a major public health problem . OveruseAntibiotics are frequently prescribed for common clinical syndromes, yet are not recommended for the majority of diarrheal episodes. For childhood diarrhea in resource-poor settings, WHO guidelines for the Integrated Management of Childhood Illness (IMCI) recommend empirical use of antibiotics only for dysentery, or bloody diarrhea , includiShigella bacteria has been frequently isolated in patients with dysentery, 66% for toddlers and 78% for children [Diarrheal disease remains a major cause of morbidity and mortality among children under 5 years in Kenya , and Shichildren . The Minchildren . Althougchildren , little We used data from the ongoing Population-Based Infectious Disease Surveillance (PBIDS) platform of the Kenya Medical Research Institute (KEMRI) and U.S. Centers for Disease Control and Prevention (CDC) in Kenya, described elsewhere . In brieAt the surveillance clinics, a structured questionnaire is administered by trained study staff to collect information on medical history including use of prophylactic cotrimoxazole and antiretroviral medication, and reported clinical symptoms, followed by detailed history taking and physical examinations performed by clinical officers. All data are captured in an electronic medical record system, including clinical diagnoses, any medications prescribed and if the patient illness met the case definitions of one of the four syndromes under surveillance  . PatientWe identified surveillance records of clinic visits of PBIDS participants aged 2–59 months presenting with diarrhea from August 21, 2009 to May 3, 2016. We then excluded records in which there was a non-diarrheal indication for antibiotics per IMCI guidelines, including those meeting criteria for possible serious bacterial infection, severe pneumonia or very severe disease, pneumonia, very severe febrile disease, and acute ear infection . AlthougThe remaining diarrheal cases were categorized into dysentery and non-dysentery, based on parental report and/or clinician observation of blood in the stool. We reviewed treatment data for any antibiotics prescribed during the clinical visit encounter. Antibiotic over-prescription was defined as an antibiotic prescription for non-dysentery cases and under-prescription as no antibiotic prescription for dysentery cases. Inappropriate antibiotic selection was defined as prescription of non-recommended antibiotic for dysentery cases by the guidelines , 17.t-test for continuous variables. Multivariate logistic regression was used to identify factors associated with antibiotic over-prescription and under-prescription for each site. For multivariate analyses, variables were initially selected using a cut-off p-value of 0.2 based on univariate analyses and final multivariate models were constructed using the least absolute shrinkage and selection operator regression [Statistical analyses were performed separately for each site due to heterogeneity between the two settings. Comparison between groups were performed by Pearson chi-squared or Fisher’s exact test for categorical variables and Student’s gression . Interacgression . All staOf 78,602 clinic visits of children aged 2–59 months in the study period, 11,159 (14.2%) were clinic visits of patients with diarrhea  and factors associated with over-prescription varied between the sites , but antibiotic under-prescription was more common in Kibera than Asembo   guidelines are shown on Table S1. Data on multivariate analysis for factors associated with antibiotic over-prescription among diarrheal children under 5 years old without dysentery in Western Kenya, Asembo are shown on Table S2. (DOCX 17 kb)


This study aimed to investigate the suture length to wound length ratio (SL:WL) in an in vitro model of abdominal wall closure. Effects of the surgeon’s experience level on the SL:WL ratio were evaluated, hypothesizing that small animal surgeons do not spontaneously apply SL:WL ratios equal to or larger than 4:1.Three groups of surgeons with varying levels of experience performed 4 simple continuous sutures before (3 sutures) and after (1 suture) being educated on principles of the SL:WL ratio. All sutures were evaluated for their gaping, number of stitches, stitch intervals, tissue bite size and suture length.No significant differences in suture parameters or SL:WL ratios were found among the 3 groups, and 60.5% of control sutures and 77.0% of test sutures had SL:WL ratios above 4:1. There was a significant improvement in the mean ratio after the information was provided (p = 0.003). Overall, the SL:WL ratios ranged from 1.54:1 to 6.81:1, with 36.3% falling between 4:1 and 5:1 . A significant negative correlation was observed between the SL:WL ratio and the stitch interval to tissue bite ratio (r = -0.886). Forty-nine of 120 sutures fulfilled the current recommendations for abdominal wall closure with a mean SL:WL ratio of 4.1:1.A SL:WL ratio larger than 4:1 was achieved in 60% of the control sutures and in 77% of test sutures. Additional animal studies are necessary to evaluate the SL/WL ratio in small animal surgery. Incisional hernias are a frequent and unpredictable complication after midline laparotomy surgeries on humans; depending on the definition, their incidence rate in humans ranges from 4–23%. The mostDuring the early period post-surgery, the closed wound relies on mechanical support until mechanically stable scar tissue is formed and is held together by only suture., 6 HypopBecause incisional hernias are a frequent complication after midline surgical laparotomies in humans, many studies have focused on minimizing their risk of occurrence. In 1976,A high SL:WL ratio has a positive effect on not only the incidence of incisional hernias but also on the ultrastructural composition of the regenerative tissue. FurthermThe SL:WL ratio depends on 3 parameters: suture tension, stitch interval and tissue bite size. Even though suture tension influences the SL:WL ratio, sutures that are too tight result in tissue ischemia, and those that are too loose result in wound gaping.Höer presumed a widespread tendency among surgeons to apply a high suture tension, which noDrop-in tissue perfusion occurs within the first 4 hours after abdominal wall closure even if the wound is closed with moderate tension, most likely due to wound edema. In conclThus, the SL:WL ratio mainly depends on the stitch length , a parameter mainly affected by the size of the tissue bite but also by the stitch interval. A short A small stitch interval reduces the pressure on the tissue exerted by the suture by distributing the tension of the suture more evenly throughout the length of the incision., 20 ThisAlthough current textbooks recommend using tissue bites 4 to 10 mm wide and stitch intervals 5 to 10 mm long for small animal abdominal wall closures, depending on the animal size,, 22 no cAlthough many human studies on SL:WL ratios were conducted on animal models , we are aware of only one study on equine cadavers dealing with the SL:WL ratio in veterinary medicine., 25 In tThis study aimed to investigate the SL:WL ratio in an in vitro model of abdominal wall closure. How the surgeon’s level of experience affected the suturing technique and how the surgeon’s techniques changed after being educated on the SL:WL ratio were assessed. Furthermore, differences among sutures applied using SL:WL ratios below and above 4:1 using the current textbook recommendations were evaluated.We hypothesized that small animal surgeons would apply SL:WL ratios of less than 4:1 when using their normal suturing technique and that educating the surgeons on the SL:WL ratio would significantly improve their SL:WL ratios.Three groups of ten participants each were assembled: group A , group B  and group C .A verbal consent was obtained from each participant of the study. Each was offered to voluntarily participate or decline participation in this study, however, no specific details about why they were asked to perform sutures were discussed as to not influence the participants and their suturing behavior. A number was randomly assigned to each participant of each group and statistical analysis was performed anonymously without identification of the participants. An official ethical approval was not obtained as the authors were not aware of this requirement by the time this study was conducted.To simulate the abdominal wall, suture dummies were built by covering wooden frames (20x13 cm) with cellular rubber , which was then coated with latex  for reinforcement. The latex surface was dusted with talcum powder to ensure easy and smooth penetration of the needle. No artificial muscle, fat, epidermal or dermal tissues were added to the dummies to simplify the evaluation of the sutures .The dummies were marked with a three-digit code . Two transverse markings were made 10 cm apart to mark the incision length .A set of instruments consisting of Adson-Brown forceps, a Mayo-Hegar needle holder, a Mathieu needle holder and a pair of Mayo scissors was provided. Biosyn 2–0  was used for all sutures. Prior to testing, each participant received the same oral introduction: First, 3 sutures were to be performed, each one on a new suture dummy. The participants were asked to suture in the same manner as that used to close an abdominal fascia on a medium sized dog. A continuous suture pattern was required, and the knots were required to be placed outside of the 2 transverse markings. For each closure, use of a new suture material package was required. The suture dummies where incised right before the suturing. Each participant was given 60 minutes to perform 4 sutures. The first 3 closures were referred to as control sutures. Each participant was then educated on the SL:WL ratio principle and asked to attempt achieving an SL:WL ratio of 4:1 or higher on the 4th suture. For assistance, the incision length (10 cm) and total length of the suture material (75 cm) were disclosed, but no other information regarding how to achieve an SL:WL ratio of 4:1 or higher was provided. The 4th closure was referred to as the test suture.The sutures were evaluated immediately after suturing. Each dummy was photographed with a scale for documentation. .The following parameters were documented:Direction of suturing and use of equipment–right to left or left to right, type of needle holderGaping–minor gaping (0.5–1 mm), medium gaping (1–3 mm), and severe gaping (> 3 mm) were measured by applying minimal lateral tension to the suturesNumber of stitches–within the 10 cm incision lineSuture length–suture material was cut on both ends above the transverse markings and removed from the suture dummy and the effective length of suture material without the knot was then measured in 1 mm incrementsStitch interval and tissue bites–measured for each stitch for far and near wound edges with an accuracy of 0.1 mmSL:WL ratio–calculated by dividing the effective length of suture material by the incision length (10 cm)Stitch interval to tissue bite ratio–calculated by dividing the suture’s mean stitch interval by its mean tissue biteSuture tension was not evaluated specifically, as no viable method to asses suture tension could be found under the study’s settings.All sutures that gaped more than 1 mm were excluded from statistical analysis (18 of 120 sutures). All sutures showing stitch intervals of 5 to 10 mm and tissue bites of 4 to 10 mm were said to follow the current textbook recommendations. Statistical Package for the Social Sciences  software was used for all statistical analyses. Descriptive statistics were created for all groups combined as well as for each group separately for control and test sutures as well as for sutures below and above the SL:WL ratio of 4:1. Furthermore, descriptive statistics were created for all sutures fulfilling the current textbook recommendations for abdominal wall closure in small animals. Significant differences between the groups as well as those within the groups were tested with variance analysis and t-tests. U-tests were used if the parameters were not normally distributed. Assumptions of normal distribution were tested using the Kolmogorov-Smirnov test.Significant differences between the combined groups for control and test sutures as well as for sutures above and below the SL:WL ratio of 4:1 were assessed with t-tests.Correlations among the suturing parameters, the SL:WL ratio and the stitch interval to tissue bite ratio as well as correlations between the SL:WL ratio and suturing parameters were calculated using Pearson’s correlation coefficients.For all statistical analyses, p-values below 0.05 were deemed significant.In total, 120 sutures were analyzed, and 8,747 measurements were taken.Of the 30 participants, 50% sutured from left to right, and 50% sutured from right to left. Only two of the 30 participants chose to use the Mathieu needle holder.Forty of the 120 sutures showed gaping; 63.3% showed minor gaping, 22.4% showed medium gaping, and 14.3% showed severe gaping. Medium and severe gaping occurred independently from the SL:WL ratios above or below 4:1 regardless of whether the participant was educated on the SL:WL ratio and independently from the group. No significant correlations were found.No significant differences in the suturing techniques or SL:WL ratios between the individual groups or between the control and test sutures were observed .60.5% of the control sutures and 77% of the test sutures showed SL:WL ratios higher than 4:1 see . For theWhen trying to achieve an SL:WL ratio of 4:1 or higher, group A participants increased their mean tissue bite size by 1.4 mm and their mean stitch interval by 0.31 mm. Group B participants increased their tissue bite size by 0.71 mm but decreased their stitch interval by 0.44 mm, while group C participants increased their tissue bite size by 0.22 mm but decreased their stitch interval by 0.69 mm.Altogether, the surgeons tended to decrease their stitch interval, thus increasing the number of stitches and the size of the tissue bites.Overall the SL:WL ratios ranged from 1.54:1 to 6.81:1, with most sutures lying between 4:1 and 5:1 (36.3%). Only three sutures had a ratio below 2:1 . SuturesSutures between 4:1 and 5:1 had a mean stitch interval of 5.17 ± 1.47 mm (3.09–8.42 mm) and a mean tissue bite size of 5.52 ± 1.75 mm (3.27–10.55 mm).A significant negative correlation (rs = -0.886) was found between the stitch interval to tissue bite ratio and the SL:WL ratio (p < 0.001) , which rIn total, 49 sutures (48.0%) fulfilled the current textbook recommendations. The mean SL:WL ratio was 4.10:1 (range 1.54:1–5.98:1), but only 27 (26.5%) sutures showed ratios above 4:1 .A high negative correlation was found between the number of stitches and the stitch interval (rs = -0.986). Medium correlations were found between tissue bite size and the number of stitches (rs = -0.561) and the stitch interval (rs = 0.573). Finally, small correlations were found between the SL:WL ratio and the number of stitches (rs = 0.391), the stitch interval (rs = -0.396) and the tissue bite size (rs = 0.383).To our knowledge, this is The hypothesis that small animal surgeons would not apply an SL:WL ratio of 4:1 or higher with their normal suturing technique was not proven. However, educating surgeons on the SL:WL ratio did significantly improve their ratios. In total, 60.5% of the control sutures showed an SL:WL ratio above 4:1, demonstrating that most surgeons achieved this ratio using their normal suturing behavior. No significant differences in the SL:WL ratio could be found between the groups. However, as nearly 70% of the control sutures made by groups A and B had ratios above 4:1 and almost 90% of their test sutures met this ratio, a trend that more experienced surgeons were more likely to achieve a ratio higher than 4:1 than novices was observed. Additionally, group A participants achieved the highest improvement in the SL:WL ratio after being educated on its principles, suggesting that more experienced surgeons can better utilize the information and adapt their suturing behavior.No significant differences regarding the sutures parameters were observed between the groups nor between the control and test sutures. This result could be due to the small group sizes and the wide variation within the groups. Even in the group of diplomates, who all had excellent surgical experience, wide ranges of stitch intervals and tissue bite sizes were observed. Thus, suturing techniques appear to be influenced by personal preference and the surgeons’ habits.When sutures with SL:WL ratios above 4:1 were compared to those with ratios below 4:1, significant differences were observed in the tissue bite size and, even more dramatically, in the stitch interval . This shows that the main difference between sutures with high and low ratios is the stitch interval.Additionally, we found a moderate correlation between the stitch intervals and tissue bite size.23 and Milbourn10.High SL:WL ratios are therefore most likely achieved in 2 ways: using small stitch intervals in combination with small tissue bites  or using large tissue bites with large stitch intervals . This finding resembles those reported by CengizSutures fulfilling the current textbook recommendations for abdominal wall closure  showed a mean SL:WL ratio of 4.1:1, showing that achieving this ratio following those recommendations is possible. However, these sutures also showed an extremely wide range of SL:WL ratios (1.54:1–5.98:1). With the average patient in small animal surgery being smaller than a human, using wider tissue bites and stitch intervals than those suggested for human subjects seems unnecessary.Together, the results of this study suggest that stitch intervals from 4 to 5.5 mm and tissue bites from 4.5 to 6 mm should give a reasonable range, which should result in an SL:WL ratio between 4:1 and 5:1 if followed.In this study, a relationship between the stitch interval to tissue bite ratio and the SL:WL ratio was elucidated, which was a major finding. For sutures with low SL:WL ratios, the stitch interval was shown to be bigger than the tissue bites, and in sutures with higher SL:WL ratios, the stitch interval gradually became smaller than the tissue bites. For SL:WL ratios between 4:1 and 5:1, the mean stitch interval was 95% of the mean tissue bite size. Due to its high significant correlation with the SL:WL ratio, the stitch interval to tissue bite ratio might be a feasible method for predicting suture SL:WL ratios in a clinical setting. This would provide easy and reliable guidance for acquiring a desirable SL:WL ratio in any suture.13 showed that even introducing new suture material in a clinical setting altered the suturing behavior of surgeons, indicating that surgeons might achieve different SL:WL ratios when performing abdominal wall closures on real patients. In the literature, suture parameters are usually obtained by counting the number of stitches after the suture is finished and measuring the length of the closed incision. The suture length, mean stitch length and mean stitch interval can then be obtained mathematically. In this study, artificial models were used to create the same conditions for all surgeons. Furthermore the model made it possible to remove the thread from an exact incision length and to separately measure each stitch interval and tissue bite for the far and near wound edges without worrying distortion were possible.Notably, this study also has a few limitations. First, the tests were performed on an in vitro model. Even though the homemade suture dummies were sufficient, they might not sufficiently compete with commercially available suture dummies. Furthermore, an artificial model can never reproduce real conditions. IsraelssonAnother limitation was the small group size. Ten surgeons per group is sufficient for providing a decent overview for surgeons having different experience levels, but the results must be interpreted with caution due to high within-groups variations in combination with the small group size.By following the current textbook recommendations and applying a stitch interval close to the tissue bite size, an SL:WL ratio equal to or larger than 4:1 can be achieved using an in vitro laparotomy closure model.S1 File(XLS)Click here for additional data file.