Ca. Phytoplasma palmicola,” which is responsible for coconut lethal yellowing disease (CLYD) on the African continent. MLST analysis applied to diseased coconut samples collected in western and eastern African countries also showed the existence of three distinct populations of “Ca. Phytoplasma palmicola” with low intrapopulation diversity. The reasons for the observed strong geographic patterns remain to be established but could result from the lethality of CLYD and the dominance of short-distance insect-mediated transmission.Coconut is an important crop for both industry and small stakeholders in many intertropical countries. Phytoplasma-associated lethal yellowing-like diseases have become one of the major pests that limit coconut cultivation as they have emerged in different parts of the world. We developed a multilocus sequence typing scheme (MLST) for tracking epidemics of “ Candidatus Phytoplasma palmicola” based on eight housekeeping genes. At the continental level, eight different sequence types were identified among 132 “Candidatus Phytoplasma palmicola”-infected coconuts collected in Ghana, Nigeria, and Mozambique, where CLYD epidemics are still very active. “Candidatus Phytoplasma palmicola” appeared to be a bacterium that is subject to strong bottlenecks, reducing the fixation of positively selected beneficial mutations into the bacterial population. This phenomenon, as well as a limited plant host range, might explain the observed country-specific distribution of the eight haplotypes. As an alternative means to increase fitness, bacteria can also undergo genetic exchange; however, no evidence for such recombination events was found for “Candidatus Phytoplasma palmicola.” The implications for CLYD epidemiology and prophylactic control are discussed. The usefulness of seven housekeeping genes to investigate the genetic diversity in the genus “Candidatus Phytoplasma” is underlined.To sustain epidemiological studies on coconut lethal yellowing disease (CLYD), a devastating disease in Africa caused by a phytoplasma, we developed a multilocus sequence typing (MLST) scheme for “IMPORTANCE Coconut is an important crop for both industry and small stakeholders in many intertropical countries. Phytoplasma-associated lethal yellowing-like diseases have become one of the major pests that limit coconut cultivation as they have emerged in different parts of the world. We developed a multilocus sequence typing scheme (MLST) for tracking epidemics of “Ca. Phytoplasma palmicola,” which is responsible for coconut lethal yellowing disease (CLYD) on the African continent. MLST analysis applied to diseased coconut samples collected in western and eastern African countries also showed the existence of three distinct populations of “Ca. Phytoplasma palmicola” with low intrapopulation diversity. The reasons for the observed strong geographic patterns remain to be established but could result from the lethality of CLYD and the dominance of short-distance insect-mediated transmission. Staphylococcus aureus (Mycobacterium abscessus (Mycoplasma pneumoniae (Mollicutes (Molecular epidemiology requires easy-to-use tools that can sufficiently discriminate at a population level. Both multilocus sequence typing (MLST) and multilocus variable number tandem repeat (VNTR) analysis (MLVA) have been developed to fulfill this objective and then were applied to the surveillance of human, animal, and plant pathogens , 2. Nexts aureus , Mycobacbscessus , and Myceumoniae . Mycoplallicutes . Mycoplallicutes , 8.Candidatus Phytoplasma,” and have revealed the horizontal transfer of potential mobile units and effectors , also known as lethal yellowing type syndromes (LYTS), are among the most destructive, with significant economic and social impact . CLYD arnsidered .Candidatus Phytoplasma” species described exclusively on the African continent were identified: “Ca. Phytoplasma cocostanzania” in Kenya and Tanzania  pattern of the central part of its 16S rRNA gene, “16SrXXII  and is dry Coast . A few szambique , 42, whi regions .Ca. Phytoplasma palmicola” depends on its insect vectors, dissemination potential, alternative host plants, and a still unproved seed transmission. In practice, sampling CLYD and insects potentially vectoring the disease is also challenging due to the difficulty in accessing the several-meters-high canopy of coconut trees. Despite years of epidemiological research, the insect vectors are still unidentified. The rationale of this study is based on the assumption that deciphering the genetic structure of “Ca. Phytoplasma palmicola” populations at regional, national, and continental levels and mapping the distribution of its genetic variants will give insights into the origin and pathways of the spread of “Ca. Phytoplasma palmicola.” We therefore developed an eight-gene MLST scheme that follows the general requirements for genotyping bacterial agents and applied this scheme to a set of 132 “Ca. Phytoplasma palmicola” isolates collected from three African countries where active CLYD epidemics had been previously associated with the two “Ca. Phytoplasma palmicola” taxonomic subgroups, 16SrXXII-A and 16SrXXII-B.The propagation of “dnaC, gyrB, leuS, lpd, secA, recA, rsmI, and rplV, were selected to investigate the genetic diversity among 132 samples of “Ca. Phytoplasma palmicola” originating from three of the main African countries affected by this phytoplasma, i.e., Ghana, Nigeria, and Mozambique. The primers designed for this study successfully amplified the respective gene targets for all 132 DNA samples of “Ca. Phytoplasma palmicola” of both the 16SrXXII-A and 16SrXXII-B groups, irrespective of their collection dates or geographic origins. The PCR products observed on agarose gels each showed a unique and clear DNA band from 553 to 983 bp depending on the target gene  was between 26.3% (gyrB) and 30.4% (recA) (The GC content of “% (recA) . The aveaK/sK values, measuring the ratio of the number of nonsynonymous substitutions per nonsynonymous site (aK) to the number of synonymous substitutions per synonymous site (sK), are presented in aK/sK values ranging from 0.01 (gyrB) to 0.17 (lpd) indicated that all the selected genes underwent purifying selection.The The number of haplotypes (H/ST), the number of segregating sites (SS), the nucleotide diversity (π), and the haplotype diversity (Hd) for each housekeeping gene and its concatenated sequence are presented in secA, revealed three haplotypes, HsecA1 to HsecA3, each corresponding to a different country of origin. The more discriminating gene, lpd, differentiated into six haplotypes, Hlpd1 to Hlpd6.The less discriminating gene, The eight genes were concatenated to obtain a sequence of 4,512 bp for each isolate. With 205 segregating nucleotides, the concatenated sequences allowed the differentiation of eight sequence types (STs) ; Fig. 1.Y14175) (KF364359) . Two types of sequences were identified. Seventeen samples corresponding to ST5 to ST8 originating from Nigeria and Mozambique were all identical to the 16S rRNA sequence of the LDN strain from Nigeria (accession number Y14175) , 45 and F364359) . SequencAll the DNA samples were spatially referenced, allowing the mapping of the distribution of their corresponding STs. The geographic distribution of the STs illustrates their aggregation in each country . Each STrsmI from Ghana. Even though they were not significant, the calculated Tajima's D values were negative using most of the Mozambican sequences, probably because ST7 was represented by a unique sample. With the exception of dnaC and rplV, all Tajima's D and Fu and Li's D and F values were significantly positive at the continental level, suggesting bottleneck effects.Tajima's D and Fu and Li's D and F were calculated for each housekeeping gene, and the concatenated sequences were calculated for each country and at the African continent level . Tajima'Ca. Phytoplasma palmicola” populations. From the network, we observed three distinct populations. Multilocus sequence analysis (MLSA)-concatenated sequences of samples from Ghana are similarly distant from the closer Nigerian and Mozambican genotypes, from which they differed by 169 (3.75%) and 171 (3.79%) SNPs, respectively. While from the same 16SrXXII-A subgroup, Mozambican and Nigerian samples differed by at least 64 SNPs (1.42%). The within-country genetic diversity was low, with no SNPs for Nigeria, 6 SNPs for Ghana, and 9 SNPs for Mozambique.An integer neighbor-joining network was calculated using the concatenated sequences of the 132 isolates . Of overWithin the 16Sr subgroups, the genetic diversity was higher for subgroup 16SrXXII-A than for subgroup 16SrXXII-B. In detail, the STs of subgroup 16srXXII-B split into two divergent branches . Only 1 Ca. Phytoplasma palmicola” STs for each housekeeping gene . The split-graph structures observed for lpd and rsmI did not appear to exclude recombination phenomena, and phi test results with P values of 0.3405 and 0.1417, respectively, were not significant irrespective of the gene being considered. The phi test result calculated for the concatenated sequences was also not significant (P = 0.0748), and the Recombination Detection Program  did not identify any recombination signals.Split networks were calculated from “dnaC, gyrB, leuS, lpd, secA, rsmI, and rplV) and 16S rRNA gene were found for 16 of 23 phytoplasma genomes or draft genomes. The 16S rRNA sequences of the corresponding phytoplasmas were used to construct a maximum likelihood (ML) phylogenetic tree after removal of a recombinant region of 155 bp. The ML phylogenetic tree presents a topology apparently similar to that of the housekeeping genes but increased the strength of the phylogenetic tree (Icong) of 1.1039 was not significant (P = 0.35), indicating that the two calculated trees were not more congruent than what is expected by chance  genes would play a role in adapting to different environments  in Tanzania, “ Delgado  and Zamb Delgado  provinceoplasmas , 64.Ca. Phytoplasma palmicola.” However, since phytoplasmas cannot be cultivated and since coconut cannot be grafted, historical “Ca. Phytoplasma palmicola” strains have not been maintained. Until now, we have been unable to find old frozen CLYD plant material or nucleic acids.Genotyping old historical samples would have been the best way to redraw the routes of introduction and the evolution of “Ca. Phytoplasma palmicola” STs are geographically clustered and distributed as small or large foci, suggesting a gradual spread. This observation is consistent with the previous observation of aggregated disease patterns and predominant dissemination within short distances  could have been introduced in one plot in Florida by the planting of St. Augusta grass, on which Haplaxus crudus, identified as an insect vector of this phytoplasma, could achieve part of its life cycle as it does on many grasses  improved the discrimination of Ureaplasma urealyticum strains  by including genes involved in the pathogenicity and insect transmission to the present MLST could increase its resolution. Using eMLST that had been expanded by adding two putative virulence genes  are widely used to investigate monomorphic or poorly diversified bacterial species, such as Bacillus anthracis  were used to select housekeeping genes and to design the corresponding PCR primers.Highly fragmented preliminary genome drafts obtained for one Mozambican isolate of the “Ca. Phytoplasma palmicola” genome drafts were aligned with BioEdit 7.0 software (http://www.mbio.ncsu.edu/BioEdit/bioedit.html) to produce a consensus sequence. Consensus sequences served as the templates to define specific PCR primers with predicted product sizes of 450 to 850 bp using Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast). The seven housekeeping genes selected, primer sequences, annealing temperature, and PCR product sizes are shown in rplV, coding for the 50S ribosomal protein L22, was included in the MLSA scheme because of its capacity to differentiate two strains in Ghana . Sampling was extensively performed in Ghana during the 2004-2009 period, as the CLYD epidemics were very active. There was no restriction of sampling in any country. Ghana and Nigeria were subjected to extensive surveys in 2009 and 2012, respectively. In Nigeria, where the disease was first reported in 1932, four samples, collected in four different areas, were found positive for “Ca. Phytoplasma palmicola.” In Ivory Coast, “Ca. Phytoplasma palmicola” had not yet been reported at the time of the survey. A survey performed in Togo in 2006 was unsuccessful, and the disease was not reported during the 2004-2013 period. Similar feedback was obtained from Cameroon. In East Africa, LYTS is associated with “Ca. Phytoplasma cocostanzania” except in Mozambique, where both “Ca. Phytoplasma palmicola” and “Ca. Phytoplasma cocostanzania” are associated with distinct disease cases. In Mozambique, a survey in Cabo Delgado Province was done proposedly with systematic sampling of symptomatic coconut trees. Samples from the Zambezia Province of Mozambique were collected during a time-limited survey.In West Africa, Ghana and Nigeria are the only countries where the CLYD epidemics associated with “Ca. Phytoplasma palmicola” DNA samples  plus the rplV gene for the 132 isolates. The rplV gene was amplified with the rpLYF/rpLYR primer pair . The 16S rRNA gene was amplified with the classic P1/P7 (aT) presented in PCR amplification was performed for each of the seven selected housekeeping genes , extremities were trimmed to identical lengths and to start at the first base of a codon, and finally consensus sequences were exported for phylogenetic analyses. Because rpLYF/rpLYR primers amplify both the aK/sK ratio, measuring the level of selection based on the ratio of the number of nonsynonymous substitutions per nonsynonymous site (aK) to the number of synonymous substitutions per synonymous site (sK), was calculated for each housekeeping gene by using MEGA version 7.0.26 (https://www.megasoftware.net). An integer neighbor-joining network (reticulation = 0.5) of the 132 concatenated sequences was constructed using PopART v1.7 software , and average percent cGC were calculated by using DnaSP version 6.10. The software .phi) test (A split network of the sequence types observed for each housekeeping gene was generated by using the neighbor-net method  of Splithi) test . Recombihi) test .recA sequences, the analysis was restricted to the seven other housekeeping genes .To conduct interspecific analysis, a total of 23 full and incomplete phytoplasma chromosomes available in GenBank were screened for the availability of all eight MLST-selected housekeeping genes and the 16SrRNA operon. Since some of the drafts lack complete Ca. Phytoplasma asteris” strain onion yellows OY-M (NC_005303.2), wheat blue dwarf (AVAO00000000.1), chrysanthemum yellows strain CYP (JSWH00000000.1), rice orange leaf phytoplasma (MIEP00000000.1), maize bushy stunt (NZ_CP015149.1), and aster yellow witches broom (NC_007716.1), “Ca. Phytoplasma mali” strain AT (CU469464.1); “Ca. Phytoplasma australiense” strains Australian grapevine yellows (NC_010544.1) and strawberry lethal yellows NZSb11 from New Zealand (NC_021236.1); “Ca. Phytoplasma pruni” strains milkweed yellows-MW1 (AKIL00000000.1), Italian clover phyllody MA1 (AKIM00000000.1), and vaccinium witches'-broom VAC (AKIN00000000.1); 16SrII-A phytoplasma strain peanut witches'-broom phytoplasma NTU2011 (AMWZ00000000.1); “Ca. Phytoplasma solani” strain 284/09 (FO393427.1); “Ca. Phytoplasma pruni” (LHCF00000000.1); and “Ca. Phytoplasma phoenicum” (JPSQ00000000.1). Acholeplasma palmae (NC_022538) was used as an outgroup.The data set included “Icong (http://max2.ese.u-psud.fr/icong/index.help.html).The seven housekeeping gene sequences were aligned using Muscle (Codon) implemented in MEGA version 7.0.26 and then concatenated. The maximum likelihood (ML) phylogenetic tree was constructed by using the best-fitting model option of RDP4 , with reIcong  (http://https://www.ebi.ac.uk/ena/) under project no. PRJEB27044 (study ERP109081). The sequences can be found under accession numbers LR028084 to LR029139. The 16S rRNA sequences were deposited in the ENA under project no. PRJEB27044 with accession numbers LR028043 to LR028083.The 1,056 sequences obtained are available in the European Nucleotide Archive (ENA) (


Rc. Many pigmented rice landraces are still grown by ethnic communities for their nutritional and cultural value. This study assesses the genetic diversity in a collection of pigmented rice accessions from the Philippines.The wild ancestors of domesticated rice had red seed, white rice being the result of a mutation in the rice domestication gene japonica and indica sub-species. No genetic structure was observed in the core collection based on geographic origin. A pairwise comparison of accessions by region indicating that both local and long-distance exchange of rice accessions had occurred. The majority of the genetic variation was within regions (82.38%), rather than between regions (10.23%), with the remaining variation being within rice accession variance (7.39%). The most genetically diverse rice accessions originated from the Cordillera Administrative Region (CAR) in the far north of the Philippines, and in the regions of Davao and Caraga in the southeast. A comparison with pigmented rice accessions from the neighbouring countries Taiwan, Laos, China and India revealed a close relationship between accessions from Taiwan, supporting the hypothesis of southward diffusion of Austronesians from Taiwan to the Philippine. The 14-bp deletion within the gene Rc, known to result in loss of red pigmentation, was found in 30 accessions that still had coloured pericarps. Multi-spectral phenotyping was used to measure seed geometric and colour-appearance traits in 197 accessions from the core collection. The purple and variable purple rice accessions had the lowest values for the seed colour parameters - lightness (L*), intensity, saturation, a*  and b* .We undertook an analysis of the genetic and colour variation in a collection of 696 pigmented rice accessions held at PhilRice in the Philippines. The collection was reduced to 589 genotypes after removal of accessions with limited passport data or with low SNP marker call rates. Removal of duplicate genotypes resulted in a final, core collection of 307 accessions, representing all administrative districts of the Philippines, and composed predominately of These pigmented rice accessions represent a diverse genetic resource of value for further study and nutritional improvement of commercial rice varieties.The online version of this article (10.1186/s12284-019-0281-2) contains supplementary material, which is available to authorized users. Oryza sativa) is a staple food for over half of the world’s population. While rice can provide up to 50% of the daily calorie intake, white rice is a poor source of vitamins and minerals, and is associated with malnutrition among communities where rice is the predominant food  within exon 6 of the Rc gene; a basic Helix-Loop-Helix (bHLH) protein  or with a SNP marker call rate of < 0.90 were removed from the collection, leaving 589 accessions for subsequent analysis, along with 1536 SNP markers that had passed quality control.Cryptic relatedness analyses of the 589 rice accessions, using the 1536 SNP markers that had passed the quality control checks, revealed the presence of duplicate rice genotypes (accession sample genotypes with an estimated Probability of Identity (estimated PI) = 1; Additional file indica and japonica rice types, results being consistent between the model-based clustering analysis implemented in ADMIXTURE  and japonica (He = 0.21) sub-species . A similar pattern was observed between the groups for allele richness (Table Assessment of genetic diversity indicators within the 307 unique Philippine pigmented rice accessions (He = 0.35) revealed a higher level of genetic diversity within the admix group (He = 0.35) compared to the  He = 0.2 sub-spec He = 0.2 sub-specindica and japonica accessions were analyzed separately, the japonica accessions exhibited a lower level of polymorphism than the indica accessions, with a mean He value of 0.18 across regions versus a mean He value of 0.22 for the indica accessions  (He = 0.21) exhibited the least diversity. There was greater variation in genetic diversity across regions in the japonica group, He values ranging from 0.15 in Visayas to 0.2 in Central Luzon, MIMAROPA and Mindanao1 (Northern Mindanao and ARMM).When the indica and japonica, respectively .The inbreeding coefficient values (FIS) were 0.16, 0.91 and 0.95 for admix, FST test was used to assess genetic differentiation between all possible groups based on allele discrepancies . Genetic differentiation was high between the indica and japonica groups (FST = 0.50). High structuralization was also observed between the indica and admix groups (FST = 0.21), and between the japonica and admix groups (FST = 0.21). When considering the geographic origin of the rice accessions  to high .An  FST = 0.0. High sFST: 0.17 to 0.24) from accessions originating from Mindanao 2, Mindanao 3, Visayas, Calabarzon and Central Luzon, and even higher for accessions collected from Mindanao1, MIMAROPA and the Cagayan Valley (FST > 0.28), indicating genetic separation of rice accessions from these regions. A similar observation was reported between rice accessions from NCR versus Mindanao1, Mindanao 2, Mindanao 3, Central Luzon, Calabarzon, MIMAROPA, Cagayan Valley and Visayas (FST: 0.15 to 0.23). However, the average genetic differentiation over all regions was 0.09, showing that a high proportion of the variation was due to within region variation.Accessions from the Ilocos Regions were highly differentiated ), Mindanao 2  and Mindanao 3 (Davao Region/Caraga), and the neighboring regions of Calabarzon and Central Luzon (FST = 0.0099) indicating seed exchange between these regions. However, long distance transfer of rice seed was apparent between Calabarzon and Mindanao 1 (FST = 0.0099), Mindanao 2 (FST = 0.0099) and Mindanao 3 (FST = 0.0077), and between MIMAROPA and Mindanaol 1 (FST = 0.0054). Rice seed movement was also apparent between Bicol and the regions of Central, Eastern and Western Visayas (FST = 0.0084). Seed from Bicol may have found its way to Mindanao 3 (FST = 0.0059), via Visaya (Visayas – Mindanao 3 FST = 0.0024). In addition there was very little genetic differentiation (FST = 0.0006) between rice accessions from the regions of Ilocos and NCR.Low indica group of Philippine pigmented rice accessions, pairwise FST comparisons between regions ranged from little or no genetic variation (FST = − 0.04), to reasonable levels of genetic variation (FST = 0.19). Significant differences were seen between indica accessions from the Ilocos region, and accessions from the MIMAROPA (FST = 0.15) and Mindanao1 regions (FST = 0.17) . Considering only the japonica rice accessions, greatest genetic differentiation was seen between accessions from Visayas and Cagayan Valley  than the average genetic differentiation when all rice accessions were analyzed together, again indicating that most of the genetic variation was due to within region variation.Looking just at the indica/japonica/admix groupings, while within group differences accounted for 46.96% of the variation. Variation within the rice accessions, representing residual heterozygosity, accounted for 5.91% of the total genetic variation. After removing the admix accessions, the between group molecular variance increased to 50.14%, while the within group variation fell to 46.31% . When indica and japonica accessions were analyzed separately, the AMOVA results again showed that most of the variation occurred within regions, with the japonica and indica rice groups showing 87.18% and 84.05% variation, respectively within geographical regions  and Laos (FST = 0.08). However, when compared with pigmented rice accessions from India (FST = 0.16) and China (FST = 0.23) the accessions were quite distinct. The average FST value among these countries was 0.14, indicating that 14% of the genetic diversity can be explained by population sub-structuring . AMOVA indicated that the majority of genetic variation (79.03%) occurred within country populations, with 14.87% of the variation being between populations, and only 6.10% of genetic variation being within rice accessions, representing residual heterozygosity (Table FST = 0.04) and the close phylogenetic relationship between the majority of the accessions from Taiwan with accessions from the Philippines . This deletion is known to result in the loss of red pericarp  was preponderant among the accessions screened. Of the 202 accessions for which a deletion was not observed, 84.16%, 9.41%, and 3.96% of the samples were reported as red, variable purple and purple, respectively, 1.49% of the samples had mixed colour, while 0.99% were white. A total of 29 rice accessions carried the Rc 14-bp deletion but still had pigmented pericarps  accessions)), while two white (japonica) rice accessions did not carry the 14-bp deletion within the Rc locus. We recorded eight accessions, all with red pericarps, with ambiguous profiles, i.e. with more than one distinct band.A total of 241 genotypes  and 1 mix (red and variable purple)) from the 307 Philippine pigmented rice accessions were screened for the presence of the 14-bp deletion in the P < 0.05) for roundness, a*, b*, hue angle, intensity, saturation and lightness, while seed area, length and width showed no significant deviation from normality (P > 0.05) . Hue angle was the less dispersed (CV = 0.07) trait, while saturation showed the highest dispersion (CV = 0.43). The highest lightness, a* and intensity values were reported in the red rice accessions, while the lowest lightness, a* and intensity were reported in the purple rice accessions. The lowest saturation and b* values were detected within variable purple accessions, while the highest values were found in a red accession , intensity, hue angle, saturation, a*  and b* . The Shapiro-Wilk test indicated departure from a normal distribution  seed colour. A few discrepancies were noted; four accessions reported as variable purple on the basis of visual classification now clustered closer to red accessions. The first principal components (PC) explained 45.0% of the phenotypic variation, while PC2 explained 20.5% Fig. a. The va4, 192 = 0.568, p = 0.686), area , roundness  or width , substantiating the weak correlations reported between geometric and colour traits. ANOVA analysis, followed by post-hoc comparisons using the Tukey HSD test, indicated a difference in colour intensity , saturation  and lightness  between rice accessions with different pigmentation  reflecting the distinctiveness and barriers to crossing that normally prevents gene flow between these two sub-species , while the rice accessions from NCR showed the least diversity (He = 0.26). Genetic diversity is considered a function of a population’s ecological and evolutionary history , indica types predominated in CAR (48.9%). Indica rice is normally grown in low altitude and latitude areas, while japonica rice is favored in mountainous regions  is indicative of regular seed movement between local farmers. We would also expect farmers to maintain genetic diversity, choosing to grow genetically diverse varieties. However, low genetic differentiation between rice accessions grouped by region is evidence of seed exchange between regions. Long distance transfer of rice seed was apparent between the regions of Calabarzon and Mindanao 1, Mindanao 2 and Mindanao 3, and between MIMAROPA and Mindanaol 1. Rice seed movement was also apparent between Bicol and the regions of Central, Eastern and Western Visayas. In addition there was evidence for movement of rice accessions between the regions of Ilocos and NCR. Barter trading for goods is still frequent among rural farmers. Likewise, social and cultural practices, including giving rice as a gift, offerings as part of religious and spiritual practices, and during community events such as festivities could all have contributed substantially to movement of rice seeds  in rice pericarp pigmentation  or other genes, that effect pericarp pigmentation  in rc that reverted white rice pericarp to red. The Rc-g mutation was caused by a 1-bp deletion, 20 bps upstream of the rc 14-bp deletion in exon 6, that restores the reading frame to a functional allele. Considerable genetic variation has been found in the Rc gene, with Singh et al. , Cui et al. , redness (a*) and yellowness (b*) values of pigmented rice are strong indicators of bioactive components  . Twenty pigmented rice accessions from Taiwan, including 17 red and 3 purple accessions, were retrieved from the IRRI Genebank and genotyped using the Illumina Infinium 7 K SNP chip  . Genotypic data of red pigmented accessions from China, India and Laos were extracted from the Rice SNP-Seek Database  were retrieved.Pigmented rice accessions, collected from all administrative regions of the Philippines, were provided by the Genebank at PhilRice Fig. . Accessi Thomson . Genotyp Thomson . SubsequControl quality of the genotypic data was performed using SNP & Variation Suite (SVS v8.6.0) . Markers with call rates lower than 0.95  or minor allele frequencies (MAF < 0.05) were removed from the data set of 4551 SNP markers. The remaining SNPs were screened for redundant markers using the software’s default parameters , resulting in a final, usable data set of 1536 evenly distributed markers.Rice accessions with low genotype call rates  were removed from further analysis, leaving 589 rice accessions. The relatedness between rice accessions was also calculated using SNP & Variation Suite. Where rice accessions had similar genotypic SNP marker profiles (i.e. estimated probability of identity = 1) one accession was removed from further analysis (282 accessions removed), leaving 307 distinct rice accessions subsequently referred to as the core collection .The genetic distance between all possible pairs of rice accessions were calculated using the bitwise.dist function implemented in the R package Poppr  using the R package rrBLUP Endelman . PCA wasThe 1536 SNP markers were used to assess genetic diversity among the 307 accessions of the core collection. The following metrics were estimated by rice subspecies and region of origin; the number of alleles (Na), observed heterozygosity (Ho), expected heterozygosity (He), and the inbreeding coefficient (FIS). FIS significance was assessed using a 95% confidence interval and a 1000 bootstrap replicates estimate of genetic diversity. Estimates of genetic diversity were performed with the R package diveRsity using the divBasic function  was performed with the aim of determining the partition of genetic variation among populations and/or groups. These analyses were carried out using the R package Poppr  that utilizes a genetic algorithm  using the following PCR profile: An initial denaturation step at 94 °C for 5 min followed by 35 cycles of, a denaturation step at 94 °C for 1 min, annealing at 72 °C for 5 min, and extension at 72 °C for 2 min, ending with a final extension at 72 °C for 5 min. PCR products were separated in a 8% non-denaturing polyacrylamide gel ran at 100 V for 2.5 h. The gel was stained with SYBR Safe DNA gel stain for 10 min and visualized using an AlphaImager HP gel imaging device . PCR products were scored as binary data, with the presence of the deletion being scored as 0 and the absence of the deletion as 1.The rice gene https://videometer.com), calibrated with respect to intensity and geometric parameters, followed by light set up. Randomly selected seeds, representing the darkest and the lightest rice accessions were used for the light set up. Twenty seed from each accession were placed evenly across a 90 mm Petri dish. The multi-spectral image of each individual seed is captured at 19 different wavelengths from 365 to 970 nm, each with a resolution of 2056 × 2056 pixels  in 1976 and colour-appearance attributes; a* (green to red shade), b* (blue to yellow shade), L , intensity, saturation  and hue  were extracted and quantified, as well as geometric traits; area, length, width and roundness (an estimate of how closely the shape of the seed resembles a circle).Seed of 197 rice accessions from the core collection were dehulled and used for multi-spectral phenotyping  for each accession were calculated and used in further analyses. Descriptive statistics for each trait , standard deviation (SD)) were obtained using the R package pastecs describe function Additional file 2:Table S1. The 307 unique Philippine pigmented rice accessions used to assess genetic diversity. Table S2. Pairewise FST comparison between rice varietal types and regions among the 307 unique accessions. Table S3. Pairewise FST comparison between regions considering only the  indica accessions among the 307 unique accessions. Table S4. Pairewise FST comparison between regions considering only the japonica accessions among the 307 unique accessions. Table S5. Descriptive statistics of colour parameters and geometric-related traits. Table S6. Data: Intensity by Colour. Table S7. Passport information on the 696 Philippine traditional pigmented rice accession collected. Table S8. Accessions from neigbouring countries used in this study. (XLSX 171 kb)Additional file 3:Table S9. Philippine pigmented rice accessions allocated to redefined geographical regions. Table S10. Summary statistics of genetic diversity indicators across regions comparing the Philippine pigmented rice accessions that belong to the indica and japonica accessions. Table S11. Analysis of molecular variance of the Philippine pigmented rice accessions after removing the admix accessions. Table S12. Unique set of SNP markers that distinguishes individual accessions within the 307 core collection of Philippine pigmented rice accessions. Table S13. Summary of Philippine pigmented rice accessions carrying the 14-bp deletion within the rice Rc locus. (DOCX 43 kb)


Although poor complementary feeding is associated with poor child growth, nutrition interventions only have modest impact on child growth, due to high burden of infections. We aimed to assess the association of malaria with linear growth, hemoglobin, iron status, and development in children aged 6–18 months in a setting of high malaria and undernutrition prevalence.Prospective cohort study, conducted in Mangochi district, Malawi. We enrolled six-months-old infants and collected weekly data for ‘presumed’ malaria, diarrhea, and acute respiratory infections (ARI) until age 18 months. Change in length-for-age z-scores (LAZ), stunting, hemoglobin, iron status, and development were assessed at age 18 months. We used ordinary least squares regression for continuous outcomes and modified Poisson regression for categorical outcomes.p = 0.023), anemia  and better socio-emotional scores , but not with change in LAZ, haemoglobin, iron status or other developmental outcomes. Diarrhea incidence was associated with change in LAZ , stunting , and slower motor development. ARI incidence was not associated with any outcome except for poorer socio-emotional scores.Of the 2723 children enrolled, 2016 (74.0%) had complete measurements. The mean (standard deviation) incidences of ‘presumed’ malaria, diarrhea, and ARI, respectively were: 1.4 (2.0), 4.6 (10.1), and 8.3 (5.0) episodes/child year. Prevalence of stunting increased from 27.4 to 41.5% from 6 to 18 months. ‘Presumed’ malaria incidence was associated with higher risk of stunting  and NCT01239693 . Although poor complementary feeding is associated with poor child growth, many interventions designed to improve complementary foods only have modest impact on growth , possiblLongitudinal studies have reported a significant inverse association of diarrhea with growth –9. HowevThe International Lipid-based Nutrient Supplements (iLiNS) Project DOSE and DYAD-M studies were randomized controlled trials conducted in Malawi to study the impact of lipid-based nutrient supplements (LNS) on growth of children , 16. TheThe iLiNS-DOSE and iLiNS-DYAD-M studies were conducted in one public district hospital (Mangochi), one mission hospital (St Martins), and two rural public health centers (Lungwena and Namwera) in Mangochi District, Southern Malawi. The total catchment population of 180,000 largely subsisted on farming and fishing. In Malawian children aged < 5 years, the prevalence of reported fever , diarrhea and ARI was 29, 22 and 5%, respectively, with seasonal fluctuations . MalariaIn the iLiNS-DOSE study, 6-mo old children were randomly allocated to one of five intervention groups provided with different doses or formulations of LNS or to a control group that did not receive LNS during the 12-mo study period, between November 2009 and May 2012. In the iLiNS-DYAD-M study, pregnant women < 20 weeks’ gestation were randomly allocated to one of three groups to receive iron and folic acid (IFA), multiple micronutrients (MMN) or a small-quantity (20 g) of LNS daily. After delivery, women in the IFA group received placebo tablets, while MMN and LNS supplementation was continued up to 6 mo postpartum. Children of mothers in the LNS group also received LNS 10 g twice daily from age 6 to 18 mo. This study was conducted from February 2011 to April 2015. Details of study design, randomization and enrolment for the two studies were explained in the main outcome papers .In both studies, research assistants visited the children’s homes every week from age 6 to 18 mo to interview the guardians about the child’s health in the previous 7 days using a structured questionnaire. The information was complemented by a picture calendar filled out by the guardians daily to aid memory of their children’s morbidity status. The use of maternal interviews as a means of collecting data on child morbidity has been validated in previous studies , 20. TheAnthropometric measurements were taken at age 6 mo and 18 mo. Study anthropometrists measured the infant’s length with a high-quality length board  and recorded it to the nearest 1 mm. They weighed unclothed infants with electronic infant weighing scale , recording to the nearest 10 g. The anthropometrists were trained and their measurement reliability was verified at the start of the study and at 6-mo intervals thereafter with methods adapted from the procedures used in the WHO Multicentre Growth Reference Study . The antWe assessed iron status at age 6 mo and 18 mo by measuring the zinc protoporphyrin (ZPP) concentration in unwashed venous blood sample using a hematofluorometer . About 5–7 ml of blood was collected by venepuncture using a 23-gauge needle into 7.5 ml evacuated, trace element-free polyethylene tubes containing lithium heparin . The samples were kept covered in aluminium and away from light, in a refrigerator or on ice, and processed within 2 h of collection. We measured blood hemoglobin (Hb) concentration at age 6 mo and 18 mo from a drop of blood taken from a finger prick and collected in a microcuvette. Hb analysis was conducted on-site using a Hemo-Cue instrument .We assessed fine and gross motor development at age 18 mo using the Kilifi Developmental Inventory (KDI) developed in Kenya . LanguagWe used a presumptive diagnosis of malaria derived from episodes of fever during the previous week, reported by the guardians. To ensure the diagnoses were mutually exclusive, we created an algorithm whereby any fever with a diarrhea episode (three or more loose stools in 24 h) was categorized as diarrhea; any fever in the presence of any respiratory symptoms  was categorized as ARI. ‘Presumed’ malaria was defined as any fever episode in the absence of diarrhea and respiratory symptoms.An episode of ‘presumed’ malaria, ARI or diarrhea was defined as the period starting from the day the child had the symptoms when preceded by at least 2 days of no symptoms or no data. The episode ended on the last day the child had the symptoms which was then followed by at least 2 symptom-free days. Incidence of ‘presumed’ malaria, ARI or diarrhea for each child from age 6 to 18 mo was calculated as total episodes / total follow up years at risk.Longitudinal prevalences of common childhood symptoms  from age 6 to 18 mo were defined as the number of days with the symptom divided by the total number of days of observation for each child .We calculated age- and sex-standardized anthropometric indices  based on the WHO Child Growth Standards  and consIron deficiency at age 6 mo and 18 mo was defined as whole blood ZPP > 70 μmol/mole heme . Anemia From the child development data at age 18 mo, fine motor scores were calculated as the sum of 34 KDI fine motor items, each scored 0 or 1, gross motor scores were calculated as the sum of 35 KDI gross motor items, each scored 0 or 1  and vocaWe included in the analysis children who had outcomes measured at age 18 mo. For all continuous outcomes, we used ordinary least squares regression to assess the association between malaria incidence and each outcome; and for all binary outcomes, we used modified Poisson regression (with a robust variance estimator) .We first assessed whether the relationship between the predictor and each outcome differed between the two studies. However, the interaction term was not statistically significant indicating that this relationship was not different between the two studies therefore we pooled data from the two cohorts. We then constructed multivariate models to determine which variables independently predicted the outcomes. We included all theoretically relevant variables, regardless of whether they were statistically significant or not after the bivariate analysis. The following variables collected at age 6 mo were included in the models: child sex, LAZ, WLZ, Hb, iron status, maternal education and household food insecurity access (HFIA) score generated by summing the value of responses to nine questions regarding food insecurity . We alsoWe assessed collinearity among the variables (e.g. LAZ vs WLZ at age 6 mo). If the variables were highly collinear (> 0.5), we dropped the one that was less strongly associated with the outcomes. We accounted for intracluster correlation due to twins using generalised estimating equations .We also performed exploratory analyses by using frequency of malaria episodes (from age 6 to 18 mo) as a categorical variable . In addition, we conducted stratified analyses by stunting at age 6 mo. Although we performed bivariate analyses for each individual variable, we will only report the results from multivariate analysis.We used Stata version 14  for all the analyses.Of the 2723 children enrolled in the two study cohorts, 2016 (74.0%) had length measured both at age 6 mo and 18 mo (1417 children from the iLiNS DOSE study and 599 children from the iLiNS DYAD-M study). These were included in the final analysis  [SD] duration of follow up was 298 (61) days / child. A total of 24,024 morbidity episodes were reported during the home visits. Of these, 9.7% (2324/24024) were episodes of ‘presumed’ malaria. The rest of the morbidity episodes were due to: acute respiratory infections (ARI), 55.6% ; diarrhea, 33.6% (8083/24024); and minor conditions, 1.1% (257/24024).Overall, the mean (SD) incidence of all illnesses combined was 14.8 (6.8) episodes per child year. The mean (SD) incidence of ‘presumed’ malaria was 1.4 (2.0) episodes per child year. The mean (SD) incidence of ARI was 8.3 (5.0) episodes per child year and the mean (SD) incidence of diarrhea was 4.6 (10.1) episodes per child year. The longitudinal prevalences of common childhood symptoms  from age 6 to 18 mo were: 7.5%; 3.4%; and 11.7%, respectively.During the 12-mo follow up period, 39.0% (787/2016) of the children did not report any episode of ‘presumed’ malaria, 30.3% (611/2016) reported one episode and 30.7% (618/2016) reported > 1 malaria episodes. The children who reported > 1 malaria episodes were responsible for 73.7% (1713/2324) of all ‘presumed’ malaria episodes reported in the two studies.At age 18 mo, the mean (SD) length-for-age z-scores (LAZ), weight-for-age z-scores (WAZ) and weight-for-length (WLZ) scores were − 1.8 (1.1), − 1.0 (1.1) and − 0.2 (1.1) respectively. The proportions of children who were stunted, underweight and wasted were 41.5, 16.6 and 5.0%, respectively. The median  zinc protoporphyrin (ZPP) concentration was 74  μmole/mole heme and the proportion with iron deficiency was 54.1%. The mean (SD) hemogobin (Hb) concentration was 108.5 (15.1) g/L and the proportion with anemia was 50.5%. The mean (SD) scores for fine motor, gross motor, language and Profile of Social and Emotional Development (PSED) were 20.9 (2.2), 17.3 (2.6), 26.2 (5.0) and 16.2 (5.4) respectively.p = 0.069) (Table The mean (SD) change in LAZ from age 6 to 18 mo was − 0.44 (0.77). In multivariate analysis, there was no association between the incidence of ‘presumed’ malaria and change in LAZ from age 6 to 18 mo, adjusted for LAZ at age 6 mo  (Table p = 0.002).The proportion of children who were stunted increased from 27.4% at age 6 mo to 41.5% at age 18 mo. In multivariate analysis, the incidence of ‘presumed’ malaria was associated with higher risk of stunting at age 18 mo, adjusted for stunting at age 6 mo  but not with hemoglobin or iron deficiency at age 18 mo , but not for the other domains of child development, adjusted for the covariates listed in the footnotes to Tables 5.The association of incidence of ‘presumed’ malaria from age 6 to 18 mo with child development was significant for PSED scores , higher risk of stunting at age 18 mo  (Table p < 0.001), and higher risk of gross motor delay  and fine motor delay  at age 18 mo , and PSED delay  . Higher incidence of ‘presumed’ malaria was also associated with lower socio-emotional scores , suggesting that children with higher malaria incidence tended to have fewer socio-emotional problems, possibly because malaria causes lethargy and inactivity which may manifest as fewer behavioral problems.Our study had several strengths: the weekly home morbidity data collection for 1 year provided comprehensive data covering periods of both high and low malaria transmission; the longitudinal design made it possible to correlate the malaria exposure with the outcomes and interpret the directionality of association; and pooling of data from two studies helped us draw conclusions from a large sample. Although we did not calculate post-hoc power for this analysis, with a large sample size of 2016 children and narrow confidence intervals obtained for most of the morbidity outcomes, we believe the study was powered to detect clinically meaningful associations.Our results should be interpreted with caution because we excluded children who did not have the outcomes measured at age 18 mo (26% of the sample), resulting in possible survival bias. However, this attrition rate is similar to that of other studies with a long follow up period , 10. MorAnother possible cause of bias is the presumptive diagnosis of malaria, which could lead to misclassification. There is overlap in the symptoms of malaria, diarrhea and ARI  which maOur research assistants referred the children suspected of malaria to a clinic for treatment; the active surveillance and early treatment may have helped improve the study outcomes, which may have resulted in underestimation of the associations.p-values.It is also possible that the association of malaria with stunting, anemia, and PSED scores was significant by chance due to multiple testing . HoweverThe available evidence on the association of malaria with growth and other outcomes is inconclusive. Some studies have reported significant associations of malaria with stunting, hemoglobin concentration, iron status and child development , 41–44. In the studies cited above, different exposure and outcome measures were used, which may also explain the inconsistency in the findings. For example, we defined linear growth as change in LAZ and stunting. Change in LAZ indicates the growth rate between two time-points and therefore provides more information about linear growth faltering than stunting status assessed at one time-point , 52. TheThe association of diarrhea with growth has been reported in previous studies –9. FrequWe conclude that in this population of children aged 6–18 mo living in a malaria-endemic setting, with active surveillance and early treatment, ‘presumed’ malaria is not associated with change in LAZ, hemoglobin or iron status, but could be associated with stunting and anemia. In this population, diarrhoea was more consistently associated with growth than was malaria or ARI. These findings may be different in contexts where there is no active case finding and treatment for malaria is not promptly administered.


To investigate the association between different obesity phenotypes and the risk of incident hypertension among both genders.n = 1540), aged ≥20 years free of hypertension at baseline. Participants were classified into six categories of body mass index (BMI)-metabolic health status, in which unhealthy metabolic status was defined based on the presence of > 1 component of metabolic syndrome (MetS) using the joint interim statement (JIS) criteria or the presence of insulin resistance (IR). The association between different obesity phenotypes and incident hypertension was assessed using multivariate Cox’s proportional hazard models adjusted for age, current smoking, low physical activity, diabetes mellitus, family history of premature cardiovascular disease, estimated glomerular filtration rate, phase of recruitment, BMI and systolic blood pressure, considering metabolically healthy normal weight group as the reference.The study population included 3659 Iranians (meAfter a median follow-up of 11.6 years 1122 participants (men = 493) experienced hypertension. Using JIS criteria, a significant higher risk of hypertension was observed among metabolically healthy obese and well as metabolically unhealthy groups among men in the age adjusted model; however, a significant higher risk in the fully adjusted model was seen among women in the metabolically healthy obese [hazard ratio (HR) 95% confidence interval (CI) 1.96(1.16–3.32)] as well as metabolically unhealthy normal weight [1.98(1.37–2.86)], overweight [2.08(1.49–2.90)] and obese [2.06(1.27–3.30)] groups. Using insulin sensitive normal weight group as the reference, among men, being overweight or obese with and without IR was significant predictors of incident hypertension in the age adjusted model; however, among women, insulin resistant overweight [1.46(1.06–2.02)] and obese groups, [1.63(1.01–2.62)] showed significant risk in the fully adjusted model.We concluded that first, there was significant difference between genders in the associations between obesity phenotypes and incident hypertension. Second, in general, metabolic status defined by MetS components as compared to IR could do better in identifying high risk women for hypertension. Third, women populations who are metabolically healthy obese using MetS definition or those with either > 1 component of metabolic syndrome or overweight/obese ones with IR should be prioritized for implementing urgent preventive strategies against hypertension focusing on lifestyle changes.The online version of this article (10.1186/s12986-019-0340-0) contains supplementary material, which is available to authorized users. Obesity is a serious public health concern worldwide . Both geDifferent subtypes of obesity have been defined based on combinations of obesity and metabolic components . A sub-gConsidering the high prevalence and incidence of obesity  and hypeThe TLGS is an ongoing prospective longitudinal population-based study conducted on a representative sample of Tehran, the capital city of Iran. The main aim of the study was to investigate the risk factors and outcomes of non-communicable disease. TLGS recruitment was conducted in two phases, i.e. the first, (1999–2001) and the second (2002–2005) phases and is planned to continue for at least 20 years with follow-up exams with an approximately 3-year intervals; i.e. the third phase: 2005–2008, fourth phase: 2009–2011 and the fifth phase: 2012–2015. Study protocol has been described in more detail elsewhere .n = 1024), prevalent CVD (n = 126), women who were pregnant during any examination (n = 55) and those with body mass index (BMI) < 18.5 kg/m2 (n = 108), 4141 participants were enrolled the current study. Moreover, other exclusions were those with missing data of BMI (n = 105), hypertension status (n = 76), MetS components (n = 98) and other covariates including smoking, family history of premature CVD, serum creatinine level, physical activity status (n = 139), and those without any follow-up data (n = 64), leaving 3659 participants for the final analysis. The study was approved by the ethics committee of the Research Institute for Endocrine Sciences and the ethical principles of the Helsinki Declaration were followed.For the present study, we considered the data of 5454 participants, aged ≥20 years, who had measurements of insulin level at baseline. After excluding subjects with prevalent hypertension at baseline (2). Waist circumference (WC) was measured at the umbilical level to the nearest 0.1 cm over light clothing, using a tape meter, with no pressure on body surface. Blood pressure was taken twice after 15-min resting in sitting position, using a standardized mercury sphygmomanometer on the right arm, and mean of two measurements was considered as the subject’s blood pressure. Physical activity level was assessed with the Lipid Research Clinic (LRC) questionnaire in the first phase of the TLGS  of continuous variables and mean differences in the prevalence [95% CI] of each category of categorical variables were estimated to compare respondents with non-respondents i.e. missing data of covariates at the baseline or those without any follow up.Differences of baseline characteristics between groups were analyzed using one-way ANOVA for continuous variables  and Chi-squared test for categorical ones. Event date was considered as the time of incident hypertension. Censoring time was defined as time point of leaving the residential area, death, loss to follow-up or end of study (19 January 2015), whichever occurred first. The event date for participants with incident hypertension was defined as mid-time between the date of the follow-up appointment at which hypertension was detected for the first time and the last follow-up visit before the diagnosis; for those with a negative event (censored subjects), the survival time was defined as the difference between the first and the last observation dates.Cox’s proportional hazard models were used to evaluate associations between different obesity phenotypes and development of incident hypertension, considering metabolically healthy normal weight as reference groups. Furthermore, these associations were evaluated using insulin sensitive normal weight individuals as reference groups.Associations were evaluated in 3 models: Model 1, included age; Model 2 was further adjusted for current smoking status, low physical activity, diabetes mellitus, family history of premature CVD, eGFR, phase of recruitment and SBP and model 3; model 2 plus BMI.P = 0.001), metabolically healthy overweight (P = 0.04), metabolically unhealthy overweight (P = 0.05) and metabolically unhealthy obese (P = 0.04) groups. Therefore, we stratified our analysis by gender. However, to make our finding comparable with those of other studies in this field, the analysis was also performed in the pooled sample which included sex as a covariate. Statistical significance was defined as a p-value of less than 0.05 in a two-sided manner. Statistical analyses were conducted using Stata version 12.0 .In the multivariate analysis, the effect modifications of gender on the associations between different obesity phenotypes and incident hypertension were tested by entering the interaction terms (obesity phenotype × gender) in the model. There were significant effect modifications of gender on the metabolically healthy normal weight  mm-Hg and 74.26(8.22) mm-Hg, respectively; corresponding values among women were 38.20(11.65) years, 26.95(4.44) kg/m2, 110.71(11.54) mm-Hg and 73.80(7.97) mm-Hg, respectively. Compared to metabolically healthy normal weight participants, generally, subjects with other obesity phenotypes in both sexes were older and had more unfavorable metabolic profiles, excluding similar low physical activity status among women.A total of 3659 participants men = 1540) were evaluated. Baseline characteristics of respondent and non-respondent groups are compared in Additional file 540 were During a median follow-up of 11.65  years, hypertension was observed in 493 men and 629 women ; corresponding incidence rates were 32.09 and 28.09 per 1000 person- years, respectively.Table Using IR to define the unhealthy metabolic status, higher risk of hypertension was seen among overweight and obese men with and without insulin resistance in the age adjusted model; furthermore, being obese with or without insulin resistance was associated with 46 and 62% higher risk for development of hypertension after adjustment with covariates in model 2 respectively, while all risks reached to null after further adjustment for BMI. Focusing on the women population, the adjusted HRs (95% CI) for incident hypertension were 1.79(1.32–2.43) for insulin sensitive obese, 1.63(1.24–2.14) for insulin resistant overweight and 2.09(1.61–2.74) for insulin resistant obese groups, respectively considering covariates in model 2. These risks remained statistically significant in the insulin resistant overweight and obese groups in the fully adjusted model .As shown in Additional file In this cohort study during over a decade long follow-up, for the first time, we examined the association of BMI and metabolic health status with incident hypertension separately among men and women, using different definitions of unhealthy metabolic status. We showed that considering JIS criteria to define metabolic health, only among women, the metabolically healthy obesity as well as the metabolically unhealthy groups were associated with > 95% greater risk for incident hypertension in the presence of important confounders, i.e. current smoking status, low physical activity, diabetes mellitus, family history of premature CVD, eGFR, phase of recruitment, SBP and BMI. Furthermore, considering the presence of insulin resistance for defining unhealthy status, only women in the insulin resistant overweight and obese groups showed significant and independent risk of hypertension.The association between different obesity phenotypes and incident hypertension has been described in some previous studies, mostly conducted in East Asia –13; neveOur study findings regarding the association between different obesity phenotypes defined by MetS components and consequent hypertension among the pooled sample were generally in line with those of other sex-adjusted prospective studies; the first study in this field conducted among Korean adults , concludIn another study conducted only among Korean men , metabolTo the best of our knowledge, two studies have examined the impact of combinations of BMI-metabolic health status on incident hypertension, using insulin resistance to define unhealthy metabolic status among adults without sex stratification , 12. OneRegarding the sex specific impact of IR on incident hypertension we have previously shown that among an Iranian population, after adjusting for potential confounders only women in the highest quartile of HOMA-IR had a significantly higher incidence of hypertension, compared with those in the lowest quartile [HR: 1.80 Additional file 2:Table S2. Risk for hypertension based on BMI and metabolic status as defined by MetS components or insulin resistance among the pooled sample: Tehran lipid and Glucose study. (DOCX 18 kb)


Mycobacterium bovis, remains a major livestock and public health problem in both high and low-income countries. With the current absence of an effective vaccine, control in cattle populations is reliant on regular testing and removal of positive animals. However, surveillance and control are hampered by imperfect diagnostic tests that have poorly described properties in naturally infected populations. Recent research in cattle co-infected with the temperate liver fluke, Fasciola hepatica, has raised concerns about the performance of the intradermal skin test in high fluke incidence areas. Further, recent studies of parasitic co-infections have demonstrated their impact on Th1 and Th2 responses, concurrent disease pathology and susceptibility to mycobacterial infections. Here we report for the first time the association of co-infection with the tropical liver fluke, Fasciola gigantica, with the presence of bTB-like lesions and the IFN-γ response in naturally infected African cattle. After adjusting for age and sex we observed a complex interaction between fluke status and breed. Fulani cattle had a higher risk of having bTB-like lesions than the mixed breed group. The risk of bTB-like lesions increased in the mixed breed group if they had concurrent evidence of fluke pathology but was less clear in the coinfected Fulani breed. Further, we observed a slight decline in the IFN-γ levels in fluke infected animals. Finally we explored factors associated with IFN-γ false negative results compared to the presence of bTB-like lesions. Fulani cattle had a higher risk of having a false negative result compared to the mixed breed group. Further, the mixed breed cattle had an increased risk of being false negative if also co-infected with fluke. Interesting, as with the risk of bTB-like lesions, this association was less clear in the Fulani cattle with weak evidence of a slight decrease in risk of having a false negative test result when fluke pathology positive. This interesting interaction where different breeds appear to have different responses to co-infections is intriguing but further work is needed to confirm and understand more clearly the possible confounding effects of different other co-infections not measured here, breed, management or exposure risks.Bovine tuberculosis (bTB), caused by  Mycobacterium bovis infection has been studied in isolation until relatively recently. Co-infections with Fasiola hepatica have been implicated as a potential reason for poor bTB diagnostic test performance and disagreement between tests (F. hepatica have been shown to down-regulate the Th1 responses (with a resultant dampening of the IFN-γ response), with subsequent predominance of Th2 responses, in order for the parasite to survive and reproduce . In many high-income countries, such as the United Kingdom and New Zealand, compulsory bTB “test and slaughter” programs coupled with compensation have been successful in reducing transmission of M. bovis in livestock populations (M. bovis (Bovine tuberculosis (bTB), caused by the bacterium zoonosis , 10 causzoonosis  amountinulations –14. Diagulations , to remoM. bovis . HoweverM. bovis  resultinF. hepatica co-infection is associated with a reduced Th1 immune response  and a single retropharyngeal lymph node per animal collected for culture as controls.Data were collected at the Ngaoundere municipal abattoir in the Adamawa Region, a major cattle-producing area of Cameroon Figure . The detTissue samples (lesioned lymph nodes) were stored in the vapor phase in liquid nitrogen dry shippers (Taylor-Wharton) and shipped to the Tuberculosis Reference Laboratory (TBRL) Bamenda. Upon arrival at the TBRL the samples were stored at -80°C until processed. Heparinised blood samples were stored in a coolbox at the abattoir (ranging between 10°C to 26°C) and then taken to the lab and kept at room temperature prior to being stimulated in the IFN-γ assay (Bovigam®) described below. Animal data recorded on paper in the abattoir, was transferred to a relational Microsoft Access database where the results could be linked back to individual animals.F. gigantica infection by MINEPIA meat inspectors. The meat inspectors examined the liver systematically to identify gross pathology associated with Fasciola infection by slicing down the common bile duct with an additional 1–2 slices through the liver parenchyma. Once an animal was identified to have gross fasciolosis related pathology, the liver was graded by a member of the research team and scored 0–3  or absence (negative) of F. gigantica pathology for subsequent analysis.All carcases were inspected for evidence of ored 0–3 . A scoreThe tissue samples were prepared and cultured as previously described  followinhttp://www.theincubatorshop.co.uk) run in the field. Following incubation samples were centrifuged at 300g for 10 minutes, the plasma was aliquotted and stored at −20°C in a portable travel freezer (Waeco CF50 12V/240 fridge freezer)). Plasma samples were transported at −20°C to the LEID and the IFN-γ ELISA was conducted as per the published protocol. The acceptable averaged negative bovine OD value was < 0.130 and positive bovine control was >0.700. Animals with a bovine stimulated sample optical density of ≥0.1 above that of the avian PPD sample were classified as test positive and interpreted as the animal being infected with M. bovis.The IFN-γ ELISA (Bovigam®) was conducted as per published protocol , 38. BriFasciola pathology and one of the bTB outcomes .The proportions of cattle with bTB-like lesions, a positive IFN-γ result and liver fluke pathology were calculated and various co-infection definitions were explored using M. bovis culture results. Animal-level explanatory variables  were always included in the models as fixed effects to control for confounding. Model selection was based on the AIC and the best model was selected using the lowest AIC and ΔAIC  models were developed to explore the association between fluke infection and bTB status using a number of different definitions including IFN-γ, bTB-like lesion and and ΔAIC . MLR mod package  with AIC package . PredictSome variables were simplified due to small numbers of observations in some categories. The dentition score (DS) was simplified from number of permanent teeth to a binary age category based on the approximate relationship between age and eruption of permanent incisors in cattle. DS <2 was categorized as “ <3 years old” and DS of ≥2 was categorized as “≥3 years old.” The breed variable was simplified to “mixed”  or “Fulani” (collapsing Red and White Fulani to a single category).During a 4 week period in August 2013, 935 cattle were examined at the Ngaoundere abattoir. The details of bacterial culture results have been presented elsewhere . A total2 test p-value <0.001) but the odds ratios did not change across pathology scores above zero so the effect is captured by collapsing the fluke score into a binary variable  of animals had visible bTB-like lesions observed. The proportion of animals with evidence of liver fluke infection was 49.6% (363/732) and the proportion of animals positive by the IFN-γ assay was 6.6% (48/732). The distribution of the prevalances of bTB-like lesion, liver fluke pathology and IFN-γ based on animal dentition (as a measurable proxy for age) are given in Figure  of animae Figure .F. gigantica pathology status with the probability of observing visible TB-like lesions in cattle in this setting. Fulani cattle had a higher risk of having observable bTB-like lesions than the mixed breed group. However, the risk in the mixed breed group increased if they also had fluke pathology. This association ith fluke pathology was less clear in Fulani cattle. For example, an adult, female, mixed breed animal, that had no fluke pathology had a ~5.2% (95% CI: 2.5–7.8) probability of having a TB-like lesion compared to ~27.7% (95% CI: 16.8–38.4) for a Fulani animal  model of the association between visible lesion and fluke status was developed Table . This sul Figure . The preF. gigantica pathology status was investigated. The raw PPD-B minus PPD-A difference in ELISA OD readings were explored in the subset of animals which were confirmed M. bovis culture positive (n = 53). The raw difference is plotted, stratified by F. gigantica pathology status in Figure p < 0.001, n = 52).The association between IFN-γ result and F. gigantica pathology status, with fluke pathology positive cattle more likely to have a lower OD value difference and to be IFN-γ negative. The association using the binary test results suggest an adult, female, mixed breed cow had a ~5.6% (95%: 3.1–8.1) probability of testing positive which dropped to ~3.5% (95%: 1.7-5.6) if infected with liver fluke compared to a Fulani, adult, female animal which had a ~16.3% (95%: 8.7–24.0) of testing positive which dropped to ~11.1% (95%: 5.9–16.2) if fluke pathology positive  and after accounting for age, sex and breed there was a small mean decrease in the difference of −0.02 (−0.04 to 0.00) in OD value in the fluke pathology positive animals Table . Both the Figure .n = 684) a new variable was generated where a IFN-γ test result was classified as a false negative if there was an observed bTB-like lesion (n = 54) and true negative if there was no lesion observed (n = 630). A multivariable logistic regression analysis to explore the association with fluke pathology was conducted including sex, age and breed as confounders. In addition, for this model the presence or absence of non-tubercular mycobacteria (NTM), based on the Haines typing from cultured samples from lesions, was also included as a known potential confounder for being lesion positive but test negative. The final model is given in Table Using the subset of results where the IFN-γ test result was negative n = 68 a new vaF. hepatica have been shown in the United Kingdom  IFN-γ response in a naturally F. gigantica infected cattle population in Africa.It is well recognized, though still poorly studied, that co-infecting pathogens can have a range of synergistic or antagonist effects. For example,  showed A Kingdom , 3 but lnfection . Here weM. tuberculosis have higher bacterial loads and TB lesion pathology when co-infected with the trematode S. mansoni . Alternatively, the breed association may be due to confounding by other unobserved variables such as differing management between breeds or differences in exposures interacting with different genotypes of M. bovis infections as part of control programs often in combination with the SICCT (F. hepatica co-infection can down-regulate IFN-γ responses to M. bovis infection (The IFN-γ assay is particularly useful to detect early (1-4 weeks post infection) he SICCT , 47. Prenfection , 22. In M. bovis and that a number of these animals with lesions had NTMs based on culture results . Using visible bTB-like lesions as the comparison test, which we know from culture results from these cattle is reasonably specific (sp = 69.7%) and sensitive (95.8%),  or being able to incorporate the likely impact on test performance may improve ante-mortem diagnostic test sensitivity within cattle or wildlife populations.It is clear that co-infections can have complex impacts on test diagnostics and pathogen invasion and this may have important implications for control programmes. As one bacillus is sufficient to establish n a host , leavingF. gigantica on the pathology and detection of M. bovis infections in a natural ecological setting in African cattle. We have shown a complex association between the presence of visible bTB-like lesions in carcasses and the presence of concurrent Fasciola infections which appears to be also affected by breed. Furthermore, we have shown that the IFN-γ response may be slightly dampened down in F. gigantica infected cattle although further data are needed to confirm this. However, it does appear to be sufficient to increase the false negative risk in the mixed breed group at lest in this population. The reduction in sensitivity of the IFN-γ assay by Fasciola spp. co-infection could have profound effects on bTB control and eradication programs as Fasciola spp. are present worldwide  Act, 1986) (ERC No: OS02-13).BB, RK, LN, VT, KM, RN, MS, and NE: conceived and designed the study; RK, SM, NV, NE, and AM: performed the field work; RK, BB, IH, SM, and RC: analyzed the data; RK, BB, IH, SM, MS, and DW: contributed reagents, expertise, materials, analysis tools; RK and BB: wrote the first draft paper; All authors read and contributed to the final draft of the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The management of acute esophageal variceal bleeding remains a clinical challenge. Band ligation is the main therapeutic option, but it may be technically difficult to perform in active bleeders. This may necessitate an alternative therapy for this group of patients. This study was conducted to assess the safety and efficacy of sclerotherapy versus cyanoacrylate injection for management of actively bleeding esophageal varices in cirrhotic patients.This prospective study included 113 cirrhotic patients with actively bleeding esophageal varices. They were randomly treated by endoscopic sclerotherapy or cyanoacrylate injection as banding was not suitable for those patients due to profuse bleeding making unclear endoscopic visual field. Primary outcome was incidence of active bleeding control and secondary outcomes were incidence of six weeks rebleeding, complications, and mortality among the studied patients.P = 0.007). No significant differences between sclerotherapy and cyanoacrylate groups regarding rebleeding , complications, hospital stay or mortality rate were observed.Initial bleeding control was significantly higher in cyanoacrylate versus sclerotherapy groups (98.25, 83.93% respectively, Based on this single-center prospective study, both of these therapies appear to have relatively favorable outcomes, although cyanoacrylate injection may be superior to sclerotherapy for initial control of active bleeding.NCT03388125]–Date of registration: January 2, 2018 “Retrospectively registered”.[ClinicalTrials.gov Identifier: Acute esophageal variceal bleeding is a fatal complication of portal hypertension and one of the main causes of death in cirrhotic patients . The morEndoscopic band ligation is recommended as the main therapeutic modality for bleeding esophageal varices , howeverN-butyl-2-cyanoacrylate is considered the optimal treatment for acute gastric variceal bleeding, but its safety and efficacy in management of bleeding esophageal varices have not been clearly investigated . MoreoveThis prospective study was conducted in endoscopy units of Internal Medicine Department, Tanta University Hospital -Nile delta, Egypt - during the period from January 2016 to August 2017.Our hospital represented as the main tertiary referral hospital that received a large number of upper gastrointestinal bleeding (UGIB) cases from many hospitals in the surrounding cities (about 1500–2000 cases/year). One hundred thirteen cirrhotic patients presented with actively bleeding (spurting or oozing blood) esophageal varices were included in this study . Patients with any of the following: other sources of UGIB than esophageal varices, hepatic encephalopathy or hepatocellular carcinoma (HCC) were excluded from the study.Patients participating in the study were randomly divided into 2 groups (using consecutively numbered envelopes containing the treatment applied); Sclerotherapy group: included 56 patients who treated by endoscopic sclerotherapy using 5% ethanolamine oleate and Cyanoacrylate group: included 57 patients who treated by endoscopic N-butyl-2-cyanoacrylate injection. The study protocol was done in accordance to the ethical guidelines of the 1975 Helsinki Declaration and was approved by research ethics committee of quality assurance unit in faculty of medicine, Tanta University. A written informed consent was obtained from all included patients in this study.All patients were subjected to the following: full medical history taking, thorough clinical examination, laboratory investigations including , abdominal ultrasonography and upper gastrointestinal endoscopy. The severity of liver cirrhosis was assessed using Child-Pugh score .9/L. [Before endoscopy, patients were resuscitated. Blood transfusion was done if a hemoglobin level was < 8 g/dl . Platele9/L.  Ceftriax9/L. , 12.Upper endoscopy was done once the patient’s hemodynamic permitted. After identification of bleeding source , endoscopic haemostatic procedure either by intravariceal injection sclerotherapy (using 5% ethanolamine oleate) or by cyanoacrylate injection was done. In our opinion, inspite of hardly identifying the bleeding source, use of injection sclerotherapy or cyanoacrylate injection has several advantages over band ligation: first, the injection can be performed immediately, i.e. without the need to withdraw the scope, load the banding device, and then reintroduce the scope again. Second, and for the aforementioned reasons, it saves time in this situation where time was precious. Third, the field of view is not narrowed by the attachment of a banding device.Standardized cyanoacrylate injection technique: N-butyl-2-cyanoacrylate ampoule (0.5 mL)  diluted with 0.8 mL of lipiodol® was injected using Pentax video-endoscopy and a 23-gauge disposable injection needle , immediately followed by 1- 2Ml distilled water to flush out the remaining cyanoacrylate from the dead space of the catheter into the injected varix then the needle was retracted. If the bleeding persisted after the first injection, a second ampoule was injected with the same modality . During Esophageal varices were divided (according to their shape and size) into the following grades: F1: small straight veins, F2: slightly enlarged tortuous veins occupying < 1/3 of the esophageal lumen, F3: large coil- shaped varices that occupied > 1/3 of the esophageal lumen .Follow up: clinical assessment and endoscopic follow up with band ligation were performed every two weeks for six weeks. Primary outcome was incidence of control of active bleeding and secondary outcomes were incidence of rebleeding, complications, and mortality among the studied patients.Control of active bleeding: was considered when there were no hematemesis, stable hemodynamic status, and stable hemoglobin concentration without blood transfusion for 24-h interval from endoscopy .Rebleeding: was defined as the occurrence of a new episode of hematemesis or melena that associated with hemodynamic instability or a drop in hemoglobin concentration more than 2 g% per day in a previously stable patient . Early rp value was significant when < 0.05. . A sample size of 100 patients (50 patients in each treatment group) was calculated to detect a 20% difference in control of bleeding with 80% power at a significance level of P-value less than 0.05. The analysis was intention to treat and involved all patients who were assigned randomly.Patients’ data were tabulated and processed using Statistical Program for Social Science (SPSS) version 20.0. Quantitative data were expressed as mean and standard deviation (SD) and were analyzed using unpaired t-test. Qualitative data were expressed as frequency and percent and were analyzed using Chi-square test. In all tests, This prospective study was conducted in endoscopy units of Internal Medicine Department, Tanta University Hospital - Nile delta, Egypt - during the period from January 2016 to August 2017.There were no significant differences between sclerotherapy and cyanoacrylate groups as regards the clinical and endoscopic data as shown in Table With regard to laboratory investigations, there were no significant differences between the studied groups as shown in Table P = 0.007 as shown in Table Control of active bleeding was significantly higher in cyanoacrylate (98.25%) compared to sclerotherapy 83.93%) groups, .93% grouThe amount of ethanolamine oleate that was used for each patient in sclerotherapy group ranged from 3 to 9 mL, with mean (5.96 ± 1.72). In cyanoacrylate group; the dose of injected cyanoacrylate in each patient ranged from 0.5-1 mL, with mean (0.66 ± 0.235).P = 0.344 as shown in Table Rebleeding in sclerotherapy group (26.79%) was higher than that in cyanoacrylate group 19.30%), but without significant difference, 0%, but wP = 0.067 as shown in Table There were no significant differences between the studied groups as regards the incidence of post-endoscopy complications in the form of retrosternal pain, dysphagia, fever or spontaneous bacterial peritonitis (SBP). Regarding hospital stay, there was no significant difference between sclerotherapy (4.79 ± 2.85) and cyanoacrylate (3.95 ± 1.88) groups, P = 0.592 as shown in Table As regards mortality rate, there was no significant difference between sclerotherapy (19.64%) and cyanoacrylate (15.79%) groups, In the present study, 113 out of 520 patients (21.73%) with bleeding esophageal varices had active bleeding at emergency endoscopy. This massive bleeding represented a technical difficulty in identifying and banding the bleeding source, therefore injection sclerotherapy or cyanoacrylate injection was performed as an alternative therapeutic modality. Esmat, et al.  reportedP = 0.007).Regarding control of active bleeding, the current study showed that the initial hemostasis was significantly higher in cyanoacrylate versus sclerotherapy groups . The higher rate of rebleeding in sclerothearpy group could be attributed to development of esophageal ulcers in many cases due to large volume of ethanolamine oleate needed to arrest active variceal bleeding. Maluf-Filho, et al. [P = 0.01. Maluf-Filho, et al. [In this study, rebleeding rate was higher in sclerotherapy group (26.79%) than that in cyanoacrylate group (19.30%), but without significant difference  and cyanoacrylate (15.79%) groups, Esmat, et al.  reportedIn this study, cyanoacrylate injection was superior to sclerotherapy for control of active bleeding despite smaller volume used. Perhaps this was because cyanoacrylate polymerized and hardened within 20 s upon contact with blood owing to the presence of ions and proteins, resulting in immediate control of hemorrhage . Most ofThere were some limitations to this work; it was carried out in a single center, small sample size and short follow up period. Another limitation was exclusion of the patients with hepatic encephalopathy as they would be at high risk of aspiration during endoscopy that necessitates endotracheal intubation to protect their airways. Meanwhile, in this study we used only conscious sedation during performing endoscopy without need to intubation.Based on this single-center prospective study, both of these therapies appear to have relatively favorable outcomes, although cyanoacrylate injection may be superior to sclerotherapy for initial control of active bleeding. We recommended that both injection sclerotherapy and cyanoacrylate injection should be considered in patients with actively bleeding esophageal varices when band ligation is technically difficult.


Low-type anorectal malformations (ARMs) are considered benign; however, in China, data regarding such conditions are limited. Thus, this study aimed to assess the middle-term bowel functions and quality of life (QOL) among children with low-type ARM.Children > 3 years of age who underwent treatment for low-type ARM  during 2013 and healthy children were included. The children were interviewed during their outpatient visits. The primary outcome measures were bowel function, as assessed using the Baylor Continence Scale (BCS), and QOL, as measured using the Pediatric Quality of Life Inventory (PedsQL 4.0).P < 0.001) and PF  groups. However, there were no differences in PedsQL 4.0 scores among the groups. The patients scored the lowest for emotional function and the highest for physical function. Nearly all measurements of QOL significantly decreased with increased BCS score.A total of 82 patients responded; mean patient age was 6.8  years. Mean BCS score in the control group (7.94 ± 4.74) was significantly lower than that in the VF (18.69 ± 11.11, Children with low-type ARM can achieve good bowel control and QOL. However, although ARMs are benign, several children with this condition suffer from anal function problems that affect QOL. Redo operations, mislocated anus, and incorrect constipation treatment are the iatrogenic causes of fecal incontinence. Most pediatric surgeons use the term “low-type” anorectal malformations (ARMs) in cases of rectoperineal or rectovestibular fistula. Low-type ARMs comprise an important subset of the distinct types of anomalies in both sexes, accounting for approximately half of all ARMs . The surTraditionally, the long-term results of low-type ARMs have been favorable in most patients –4. Thus,One-hundred children who underwent surgery for ARM in 2013 at the Guangzhou Women and Children’s Medical Center (GWCMC) were invited to participate in the study. The children were diagnosed via clinical examination. Children younger than 3 years and those with hypothyroidism, hypokalemia, hypomagnesemia, hypophosphatemia, hypercalcemia, spina bifida, myelomeningocele, and tethered cord were excluded. The control group comprised 86 healthy children without bowel dysfunction (52 boys and 34 girls), with a median age of 6.0  years, from a primary school during the same period.For ARM with a rectoperineal fistula, we preferred cutback anoplasty if the rectum was located inside the voluntary sphincter funnel  and the Pediatric Quality of Life Inventory (PedsQL 4.0). We compared the bowel function and QOL between patients with VF and perineal fistula (PF) ARMs. Data about age, sex, associated anomalies, and surgical methods were collected from the medical records. The primary outcome measures were bowel function score and QOL that were assessed using BCS and PedsQL 4.0, respectively.The study package contained two questionnaires: the BCS for bowel function and PedsQL 4.0 for QOL , 6. The P < 0.05 was considered statistically significant. Categorical variables were presented as n (%) and were compared using the chi-square test. Continuous variables were presented as mean and standard deviation or median (interquartile range) and were compared using t-test or analysis of variance. The correlation between the characteristics of the patients as well as bowel function and QOL were assessed using the Pearson’s correlation coefficient. Linear regression analysis was carried out to assess the association between BCS score and QOL (measured using PedsQL 4.0 and each dimension score). The models were adjusted for sex, current age, age during surgery, weight during surgery, body mass index (BMI), surgical procedure, and presence or absence of comorbidities, and 95% confidence intervals (95% CIs) were calculated.Data were analyzed using the Statistical Package for the Social Sciences software version 19.0  and GraphPad Prism version 5.0 . A two-sided P = 0.356) and BMI (P = 0.667). Children with VF were older during surgery (P = 0.001); thus, they had higher body weights during the time of surgery (P = 0.013) than those with PF. All patients with VF underwent reconstruction via PSARP, whereas only 6 (14.3%) patients with PF underwent reconstruction via PSARP. Moreover, 15 (35.7%) patients underwent reconstruction via B and 21 (50.0%) via C (P < 0.001). The proportions of associated anomalies were not significant between the PF and VF groups (P = 0.425). Two patients underwent anal reconstruction and the pull-through procedure for secondary megacolon.Of the 100 patients, four presented with hypokalemia and one with spina bifida. A total of 13 patients were lost to follow-up, and the remaining 82 patients were included in the case group. Table P = 0.049). The median scores of children with VF, those with PF, and controls were 13 , 12 , and 7.0 , respectively. The percentile distribution of individual scores has also been presented. Half of the patients (53.6%) had a BCS score > 12, which is the median BCS score in children with PF. In fact, 27 (69.2%) children with VF, 17 (39.5%) children with PF, and 14 (16.3%) healthy children had a BCS score > 12 (P < 0.001). Four children in the VF group and one child in the PF group had a BCS score > 40, whereas none of the children in the control group had a BCS score > 40   Fig. .Fig. 4CThe correlation analysis showed that BCS score was not correlated to current age, weight during surgery, and age during surgery. In addition, no correlations were found between the characteristics of the patients and total PedsQL score Table . The relThis study aimed to identify the bowel function and middle-term QOL of patients with low-type ARM, which is traditionally considered a benign abnormality . AlthougThe validated BCS tool was used to assess bowel function, and it can differentiate between normal children and those with various comorbidities. Lower scores indicate better social continence. Normal children without anatomic or functional continence problems had a mean BCS score of 11.5 , whereasThe mean BCS score was higher in patients with VF than in those with PF, indicating that the bowel function of patients with PF was better than that of patients with VF. Intergroup differences can be explained by three facts. First, innervation may be involved; thus, all patients with VF (100%) and > 70% of patients with PF showed varying degrees of innervation abnormalities . Second,QOL has been an important endpoint in the medical care of patients with ARM who have psychological, behavioral, or developmental problems and who experience significantly lower QOL than children without such problems. Poor outcomes have been correlated to neurological damage and mental retardation  or insufMoreover, this study showed that functional scores were significantly correlated to QOL in emotional, social, and school areas. More severe functional situations resulted in a lower QOL. Bad bowel function, particularly fecal incontinence, is highly correlated to QOL. Most adult patients with ARM had no social problems in occupational or student life if they gained good bowel control. Grano has investigated how fecal incontinence may influence the different aspects of QOL in children and adolescents with ARMs . In our The present study had some limitations. In this single children’s medical center in South China, 398 low-type ARM cases were recorded between 2010 and 2013. However, only 21.5% of the patients with VF and 26.7% with PF were included, thereby indicating that > 70% of the affected patients could not be contacted. This may be caused by discrepancies in the postoperative follow-up system, and the charts of the patients cannot be changed to include patient contact information. However, if the patients visit surgeons regularly, their chart can be updated any time. Unfortunately, the situations may not always be ideal, and most of our patients’ parents consider poor bowel control a normal outcome, which does not require any special care. Thus, they were more likely not to visit their surgeons for follow-up after constructive surgery. A higher non-response rate may indicate that the data in this study cannot accurately convey treatment prognosis. In addition, as children grow older, their long-term bowel function and QOL must be evaluated. In future studies, the use of a matched healthy sample from a local population would be optimal for a more direct comparison between the ARM study population and healthy controls.Despite the limitations, this study is part of a new direction for middle-term bowel function and QOL evaluation of children who have undergone surgical correction for low-type ARM in China. Patients with low-type ARM can achieve good bowel control and QOL, and good postoperative anal function can lead to better QOL. Although children present with ARM with favorable outcomes, numerous children with low-type ARM still have persistent anal function problems that affect their QOL. Redo operations, mislocated anus, and incorrect treatment of constipation are the iatrogenic causes of fecal incontinence.