Background: The global incidence of pre-obesity and obesity is rising noticeably. Where medical students should be advocating healthy lifestyles, they are actually indulging in unhealthy eating habits and sedentary lifestyle. This is resulting in an increased incidence of obesity in this population.Methods: It was a descriptive cross-sectional study conducted from September 2018 till January 2019 in four medical colleges of Karachi. Sociodemographic profile, body mass index (BMI), food habits, and exercise routine of the students were recorded. Data was entered and analyzed using SPSS version 22 .2 (range: 16.24–38.19). The mean age was 21.345 ± 1.4709 years with a minimum of 17 years and maximum of 25 years. The combined frequency of pre-obese and obese students was 33.2%. Among these, there were more women (60.6%) than men (39.3%). Among these pre-obese and obese students, 55% had breakfast rarely to sometimes, 47.9% took four or more meals per day, 39.3% consumed fast food thrice weekly or more, and 58.1% consumed soft-drinks/juices alternate to every day. Among the students who indulged into binge eating when stressed, 56.9% were pre-obese to obese. The nutritional status of the study sample was significantly associated with female gender, living status with parents, irregular breakfast, infrequent daily meals, increased consumption of fast food and beverages, decreased consumption of red meat, sedentary lifestyle, and altered eating habits when stressed.Results: The mean BMI of the study population was 21.717 ± 4.33 kg/mConclusion: The incidence of pre-obesity and obesity is noticeably escalating among young adults. If this trend continues, obesity-related complications will form the major chunk of medical illnesses in the near future. Strategies are needed to nip this dilemma in the bud by indulging in healthy and clean eating habits and performing regular physical activity. Not a problem of a certain part of the world any longer; obesity has now become a global pandemic. The number of individuals falling prey to this health hazard is multiplying dramatically. In 2016, more than 1.9 billion individuals of age 18 and above were pre-obese and around 650 million of them were obese [Where genetic predisposition cannot be neglected as a contributing factor to obesity; diet, physical activities, and behavioral factors are also crucial risk factors . Morbid In a large cohort, 19.4% of Asians had a body mass index (BMI) of 25 or more. When subcategorized, it was established that 23.9% of South Asians had their BMI of 25 or more. Sixty-seven percent of these South Asians were men, and 33% were women .By default, healthcare providers and facilitators should be presenting themselves as advocates and ambassadors of healthy lifestyle for the general population. However, studies suggest otherwise. Scientific evidence is present which endorse unhealthy eating habits -8, distuIncreased intake of high-calorie foods and a sedentary life have tripled the rate of obesity among the young adults of the developing countries over the last 20 years. Several studies among university students in multiple developing countries including India, Bangladesh, and China suggested a high prevalence of obesity .In a recent study from Lahore, 21% of medical students were of BMI 25 or more. There were 46% men with central obesity and 31.4% were women. The associated risk factors were high-calorie food, studying at private medical colleges, male gender, lack of sports, and no regular exercise [It was a descriptive cross-sectional survey-based study which was conducted in four medical colleges of Karachi. Two public medical colleges and two private medical colleges were included. The maximum combined strength of these colleges is 4,000 students. With an anticipated frequency of 50% and a confidence interval of 95%, the sample size calculated was 351. Non-probability convenient sampling technique was adapted and data was collected from September 1, 2018 till January 31, 2019.Sociodemographic profile of the students was recorded. It included their age, gender, medical college name, year of education, and their living status (living with parents/hostel). Body weight (in kilograms) and body height (in meters) was recorded to calculate their BMI. BMI was categorized as per the standard World Health Organization (WHO) classification . ResponsAlmost three-fourth of the study sample comprised of female students. The mean ± SD age of the study sample was 21.345 ± 1.4709 years with a minimum of 17 years and maximum of 25 years. The sociodemographic profile of the students is shown in Table The mean ± SD BMI of the study population was 21.717 ± 4.33 kg/m2 (range: 16.24–38.19). There were 93 students (26.5%) with BMI < 18.5 (underweight), 141 students (40.2%) with BMI between 18.5 and 24.9 , 52 students (14.8%) with BMI between 25 and 29.9 (pre-obesity), 42 students (11.9%) with BMI between 30.0 and 34.9 (obesity class I), and 23 students (6.5%) with BMI between 35.0 and 39.9 (obesity class II).None of the students were beyond BMI 39.9. The pre-obese, obese I, and obese II students 33.2%) were classified according to their sociodemographic profiles as shown in Table % were clOnly 62 of 351 (17.7%) students played sports at college; 45 of these were male students (72.5%). The median sitting time of the students for studying purposes  was 7.4 hours (range 6–12 hours). The median sitting time of the students for non-studying purposes was 3.1 hours (range 1.5–5.5 hours). Only 98 students (27.9%) had a regular habit of standing/strolling while studying. There were 168 out of 351 students (47.8%) who exercised regularly. Their mean time of exercise was 27.15 ± 11.258 min (range: 15–120 min). Only 45 of these 168 students (26.7%) were enrolled at a gym and the remaining did exercise at home.Among 117 pre-obese and obese students, 65 of them (55%) were taking breakfast rarely to sometimes, 56 (47.9%) were taking four or more meals per day, 46 39.3%) were consuming fast food thrice weekly or more than that, 68 (58.1%) were consuming soft drinks/juices alternate to everyday, 79 (67.5%) were consuming red meat thrice or more than that weekly. Among 109 students who indulged into binge eating when stressed, 62 (56.9%) were pre-obese to obese. Among 100 students who rarely ate when stressed, 20 (20%) were pre-obese to obese, and remaining 80 (80%) were underweight to normal healthy. The food and drinks habits, stress eating habits, and exercise routine of the students were recorded. Their correlation with the nutritional status of the students is shown in Table 9.3% wereThis study reports a frequency of 33.2% of pre-obesity and obesity among the medical students of Karachi. This nutritional status was significantly associated with female gender and living status with parents. Unhealthy eating habits including irregular breakfast, infrequent daily meals, increased consumption of fast food and beverages, and decreased consumption of red meat were significantly associated with the nutritional status of these students. Sedentary lifestyle and binge eating/rarely eating when stressed were also significant correlators. Medical college status – private versus public – did not show any significant effect on the nutritional status.It was a multicenter study, with a large sample. It compared the nutritional status of public and private medical college students. It assessed the eating habits of the students in detail including stress eating. However, the study neither calculated the daily calorie consumption nor the calorie expenditure. This balance plays a crucial role in weight gain.In the past, there have been studies to assess the nutritional status of medical students. In 2012, a Karachi based study reported a combined incidence of 27.1% of pre-obesity and obesity which was significantly associated with male gender . In anotIn another Indian study with 21% pre-obese and 3% obese medical students, 62% were physically active . A studyHealthy eating habits and regular physical activity plays a fundamental role in the prevention of obesity and its consequent health-related complications including metabolic and cardiovascular disorders. The rising incidence of “above healthy BMI” in young adults is a global public health concern. These individuals will increase the future burden of non-communicable diseases including dyslipidemia, hypertension, cardiovascular morbidity, subfertility, and others. Hence, preventive strategies are the need of the time. Preventive studies and awareness programs must be conducted on a national scale to highlight this crisis our young generation is going through.The incidence of pre-obesity and obesity is noticeably escalating in the young adult population. At this rate, obesity-related complications will form the major chunk of medical illnesses in the near future. Obesity not only reduces the quality of life but also the lifespan. Strategies are needed to nip this dilemma in the bud, particularly among the healthcare personnel who themselves must be practicing and advocating healthy lifestyles. Students must indulge in healthy and clean eating habits and perform regular physical activity.


Amycolatopsis are well known for the production of a number of important antimicrobials and other bioactive molecules. In this study, we have sequenced the genomes of five Amycolatopsis strains including Amycolatopsis circi DSM 45561T, Amycolatopsis palatopharyngis DSM 44832T and Amycolatopsis thermalba NRRL B-24845T. The genome sequences were analyzed with 52 other publically available Amycolatopsis genomes, representing 34 species, and 12 representatives from related genera including Saccharomonospora, Saccharopolyspora, Saccharothrix, Pseudonocardia and Thermobispora. Based on the core genome phylogeny, Amycolatopsis strains were subdivided into four major clades and several singletons. The genus Amycolatopsis is homogeneous with only three strains noted to group with other genera. Amycolatopsis halophila YIM93223T is quite distinct from other Amycolatopsis strains, both phylogenetically and taxonomically, and belongs to a distinct genus. In addition, Amycolatopsis palatopharyngis DSM 44832T and Amycolatopsis marina CGMCC4 3568T grouped in a clade with Saccharomonospora strains and showed similar taxogenomic differences to this genus as well as other Amycolatopsis strains. The study found a number of strains, particularly those identified as Amycolatopsis orientalis, whose incorrect identification could be resolved by taxogenomic analyses. Similarly, some unclassified strains could be assigned with species designations. The genome sequences of some strains that were independently sequenced by different laboratories were almost identical (99–100% average nucleotide and amino acid identities) consistent with them being the same strain, and confirming the reproducibility and robustness of genomic data. These analyses further demonstrate that whole genome sequencing can reliably resolve intra- and, inter-generic structures and should be incorporated into prokaryotic systematics.Strains belonging to the genus  Amycolatopsis is well known for the commercial production of multiple antibiotics, including the important broad spectrum antibiotics rifamycin and, vancomycin (1) that may vary in their phenotypic and morphological characteristics  revealed the presence of four major groups of species within the genus, with some singletons . The type strains of Amycolatopsis halophila and Amycolatopsis marina were found to be quite distinct from other Amycolatopsis strains. Although average nucleotide identities based on MUMmer (ANIm) supported the classification of these strains within the genus, the percentage of conserved proteins indicated that A. halophila might belong to a different genus  also indicated that A. halophila is more similar to members of the genus Prauserella than to Amycolatopsis species . This study highlights the subgeneric structure within the genus and supports the separation of A. halophila as a member of a different genus. In addition, it is clear that a number of strains have been misidentified and misclassified using traditional approaches. Some strains sequenced independently by different laboratories grouped together, suggesting that genome based approaches are more reliable and reproducible than techniques such as DNA–DNA hybridisation, which suffers from a lack of reproducibility and compatibility of results between different laboratories  based on seven housekeeping genes  at 28°C for 48 h. Genomic DNA from each strain was extracted from 1.5 ml culture using the UltraClean® Microbial DNA Isolation Kit (MoBio).Five strains, Supplementary Table 1).The genome sequencing was performed on an Illumina MiSeq instrument and the paired-end reads were assembled using SPAdes 3.9.0 . The draAmycolatopsis strains and 12 representative strains of related genera were obtained from GenBank (Supplementary Table 1). Two independent genome assemblies were available for seven Amycolatopsis strains in GenBank and both of them were included to test the reliability and reproducibility of the genomic data.The genome sequences of 52 Thermobispora bispora DSM 43833T. Pairwise percentage of conserved proteins (POCP) were calculated using the scripts, data_file_4.sh (BLAST-based pairwise average nucleotide identity (ANIb) and pairwise fragment similarity scores (fragment size of 500-bp) were calculated from the nucleotide sequences using Jspecies  and GEGEile_4.sh  and runPile_4.sh  that areile_4.sh .Supplementary Table 1). A ML tree generated from concatenated core proteins resolved Amycolatopsis strains, representing 34 species (including 29 type strains), into four major groups and several singletons . These groupings are consistent with both the MLSA based phylogenies albeit with minor exceptions . Amycolatopsis taiwanensis DSM 45107T, Amycolatopsis sacchari DSM 44468T, Amycolatopsis nigrescens CSC17Ta 90T and Amycolatopsis xylanica CPCC202699T are present as singletons within the Amycolatopsis clade . These strains were also recovered as singletons in the MLSA analysis of A. xylanica which was located at the periphery of the strains in our cluster A. Of the other singletons, A. sacchari DSM 44468T is consistently associated with A. dongchuanensis in 16S rRNA gene trees  formed  group J ] is quitAmycolatopsis groups varying from 35.9 ± 18.8 for Group C to 65.1 ± 17.0 for Group D . The mean FBS score between the Amycolatopsis groups varied from 8.6 ± 1.8 to 11.4 ± 2.1 and between Amycolatopsis  and strains of the other genera from 3.9 ± 1.6. Although the group of A. palatopharyngis, and A. marina is closely related to Saccharomonospora strains, the mean FBS score (5.3 ± 1.2) between these taxa was comparable to the score with the other Amycolatopsis strains (5.6 ± 1.1). Similarly, A. halophila was also equidistant from the Amycolatopsis strains (4.1 ± 1.1) and from the other genera .The results of fragmented genome BLAST searches are consistent with the phylogenetic groupings with a mean fragmented BLAST similarity (FBS) within the Figures 2B,C). Average ANIb values within and between the Amycolatopsis groups are >80% (85.94 ± 5.24 - 93.65 ± 3.41) and >75% (75.68 ± 0.69 - 76.97 ± 0.91), respectively. The strains of the other genera and the three anomalous strains shared lower ANIb values when compared with the Amycolatopsis strains . Similarly, average AAI values are >80% (86.69 ± 6.11 - 94.48 ± 3.06) within the Amycolatopsis groups and approximately 70%, or higher (70.99 ± 1.02 - 73.93 ± 1.98) between these groups. Average AAI values between Amycolatopsis strains and the three anomalous strains and the other genera are 67.54 ± 4.27 and 66.45 ± 3.70, respectively.Average nucleotide identity and AAI values also support the phylogenetic groupings . The average dDDH values ranged from 21.4 ± 0.5 to 22.2 ± 0.5 between the Amycolatopsis groups which is comparable to the values between the Amycolatopsis strains and the strains from the other genera (20.1 ± 0.7). This confirms that dDDH is useful in identifying strains belonging to the same species but has limited resolution at the intergeneric level.Pairwise dDDH values were also calculated for the dataset which varied between 36.2 ± 14.4 and 59.5 ± 19.7 within each Amycolatopsis strains  varied between 50.71 and 99.96% . However, POCP could not resolve the status of the anomalous strains as A. marina and A. palatopharyngis type strains showed POCP values of 57.09 ± 2.95 and 58.53 ± 3.13 against the strains in the Amycolatopsis clade and the Saccharomonospora strains, respectively. POCP values between A. halophila YIM93223T, and other Amycolatopsis strain was <50% except for some of the strains in group D, A. marina and A. palatopharyngis (Supplementary Table 2). All of these results suggest that 54 of the 57 Amycolatopsis strains belong to the same genus while A. halophila YIM93223T should be assigned to a different genus. A. palatopharyngis DSM 44832T and A. marina CGMCC4 3568T grouped in the clade with Saccharomonospora strains; however, they showed comparable distances to both Saccharomonospora and Amycolatopsis strains. A larger analysis including more species from the genus Saccharomonospora and other representatives of the family Pseudonocardiaceae is required to clarify the status of these two species. However, it is notable that the recent phylogenomic study of A. marina within the genus Amycolatopsis, although this study did not include A. palatopharyngis.A POCP value of 50% is often used as the genus boundary where two strains with more than 50% conserved proteins are considered to belong to the same genus . ConsistAmycolatopsis strains varied between 7 and 11 Mb with a GC content of 68 – 72 mol% . A. halophila YIM93223T is clearly an outlier with a genome size of 5.6 Mb and a 67.8 mol% GC content. Amycolatopsis salitolerans consistently clusters with A. halophila in 16S rRNA gene trees . Cumulatively, these data are consistent with the recent proposal that A. halophila and A. salitolerans should be reclassified into the genus Haloechinothrix . Although the range of the genome size and GC content is quite broad for Amycolatopsis sensu stricto strains, some patterns are visible at the group level. For instance, the strains in Group A have a genome size of 8.27 – 9.81 Mb, and a GC content of 68.5 – 69 mol%. The genomes of strains in Group B varied between 9.04 and 10.86 Mb with a GC content ranging from 70.8 to 72 mol%.The genome sizes of the ne trees  and thusof 66.4% ; whilst inothrix  as Haloeectively . A. palaA. halophila, A. palatopharyngis and A. marina. The core genome of the entire dataset contained 602 genes (6.1–16.9%). The number of annotated genes among the Amycolatopsis strains ranged between 5,098 and 9,890 after excluding those of other genera (Supplementary Table 1) with 1,382 of them (14.0 – 27.1%) shared by all of the strains, which compares well with the 1,212 core genes identified by A. halophila strain from the dataset and only slightly to 1,739 genes (17.6 – 25%) after exclusion of the genomes of the A. palatopharyngis and A. marina strains. Therefore, the proportion of genes in the core genome may not be reliable for separating members of different genera due to overlap in values within a genus and when strain(s) from other genera are included . As expected, the number of core genes is much higher within each Amycolatopsis group (43.1 – 69.5%).In order to establish whether the size of the core genome can help identify intergeneric boundaries, core genes were calculated for the entire dataset followed by a sequential removal of the other genera and the outlier strains Amycolatopsis groups including nine type strains , many of which are known to produce different antibiotics and other bioactive molecules . This analysis identified a potential case of misidentification of the industrial vancomycin producer A. orientalis strain HCCB10007  and shows a dDDH of 72% and ANIb of 96.4% with A. keratiniphila FH1893T. Therefore, this strain should be reclassified as A. keratiniphila. Two strains, Amycolatopsis sp. CB00013 and Amycolatopsis sp. MJM252 could be assigned to A. japonica based on the dDDH cut-off value of 70%, as noted previously  and all of them were included to check the reproducibility of the sequence data between different laboratories and the robustness of the approach. Pairwise FBS, ANIb, AAI, and dDDH values between these assembles were >99.7%, confirming the authenticity of these strains albeit with a minor exception. The dDDH value between the genome sequences of A. keratiniphila FH1893T  was 97.7%. These assemblies were submitted by two different laboratories and this variation may reflect the quality of the sequences. Other taxogenomic values are consistent with them being associated with the same strain (Supplementary Tables 3A–D).Multiple assemblies were available in GenBank for four of the type strains in Group A . The mean FBS (55.74 ± 21.36), ANIb (90.45 ± 4.72) and, AAI scores (91.23 ± 4.40) for this group are comparable to the values observed for Group A. All of the pairwise matrices support the presence of nine species including Amycolatopsis australiensis, Amycolatopsis balhimycina, Amycolatopsis kentuckyensis, Amycolatopsis lexingtonensis, Amycolatopsis mediterranei, Amycolatopsis pretoriensis, Amycolatopsis rifamycinica, Amycolatopsis tolypomycina, and Amycolatopsis vancoresmycina in the group, which is consistent with their taxonomic assignment . While most of the members of this clade are known to produce antibiotics and/or bioactive molecules . This group also includes seven A. mediterranei isolates that are grouped together with >97.7% dDDH, and 99.8–100% ANIb and AAI similarities, indicating it to be a highly homogeneous species with very limited genomic diversity.Group B is also quite diverse encompassing 16 of the 57 olecules , the A. thogenic . A. vancSupplementary Tables 3A–D). Based on the taxogenomic matrices, eight strains within this group can be assigned to seven species . The assignment of five of these species, A. circi, Amycolatopsis rubida, Amycolatopsis benzoatilytica, Amycolatopsis jejuensis and Amycolatopsis saalfeldensis is consistent with the literature . These strains were assigned to four species based on the pairwise dDDH values including Amycolatopsis methanolica, A. thermalba, Amycolatopsis thermoflava and a potentially novel species with Amycolatopsis sp. strain 75iv2 (Supplementary Table 3D). A. ruanii strain 49.3a probably should be reclassified as A. thermalba due to its high dDDH 93.3%, FBS 92.78 ± 0.12, ANIb 99.19 ± 0.04, and AAI value of 99.23 with the type strain of latter. Both genome sequences of the A. methanolica type strain show ≥99.9% ANIb and AAI similarities but their dDDH is slightly lower (98.3%). Similar to A. keratiniphila, this may be due to minor variations in the quality of genome sequences generated in different laboratories. Although, Amycolatopsis sp. strain 75iv2 and A. thermoflava strain N1165T can be assigned to two different species based on the dDDH values, they share ANIb (95.08%) and AAI (96.42%) with each other that marginally overlap using the recommended cut-off for defining species . However, the type strains of A. palatopharyngis and A. marina clustered more closely to Saccharomonospora and the type strain of A. halophila formed a single member clade . The taxogenomic matrices are in agreement with the phylogenomic groupings  with minor exceptions. For example, the FBS and ANI values between A. taiwanensis strain DSM 45107T and the other Amycolatopsis strains are slightly below the suggested cut-off values ; however, this strains clustered close to group D within the Amycolatopsis clade and clearly belongs to this genus. Therefore, the suggested cut-off values should be considered a guide and used in combination with genome based phylogenies . As noted above, further analysis of a larger dataset is required not only to clarify the taxonomic status of A. marina and A. palatopharyngis but also that of the effectively named species Amycolatopsis flava which groups with them in 16S rRNA trees . These results are consistent with the classification of A. halophila into a different genus . However, no clear correlation to core conserved proteins and intergeneric boundary could be identified from this analysis.The percentage of conserved proteins (POCP) has been used to define strains at the genus level with ≥50% proteins between a pair of strains with at least 50% alignable region and 40% sequence identity considered to indicate membership of the same genus . HoweverNA trees . In agre genomes , less thnt genus . We alsoAmycolatopsis strains clustered into four robust groups based on the phylogenomic and taxogenomic analyses . The pan-genomic analyses identified some genes that are conserved within each group but absent in other Amycolatopsis strains (Supplementary Tables S4A–D). 147 genes were specific to Group A, 114 genes to Group B, 54 genes to Group C and 244 genes to group D. A large proportion of these genes (42–59%) encode hypothetical proteins; however, some genes are annotated as transcriptional regulators, Sigma factors and some genes potentially belong to different biosynthetic gene clusters. Indeed, the biosynthetic potential to produce secondary metabolites varied between the Amycolatopsis groups with a strong correlation to the number of biosynthetic gene clusters (The majority of the clusters .Amycolatopsis is a homogeneous genus where most strains conform to the phylogenomic and taxogenomic indices defined for intra-generic boundaries. In contrast, A. palatopharyngis DSM 44832T and A. marina CGMCC4 3568T formed a clade closer to Saccharomonospora strains with comparable taxogenomic distances between them and the other Amycolatopsis strains. We also show that genomic data are robust and reproducible between different laboratories and can help resolve cases of misclassification and misidentification. Some strains identified as A. orientalis should either be assigned to other species or to presumptive novel species. Genomic analyses also assigned some undefined strains to known species. These results provide further evidence that matrices derived from the whole genome sequencing data can provide a robust framework for prokaryotic systematics.Supplementary Table S1.The genome sequence data from this study has been submitted to GenBank/DDBJ/EMBL databases and are publically available with the accession numbers given in VS, MG, IS, and H-PK have designed the study. VS and GT carried out the experimental work. VS and JB analyzed the data. VS drafted the manuscript. All the authors provided intellectual inputs and approved the final version.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.


Intestinal parasites are present throughout the world in varying degrees of prevalence due to many factors. The aim of this study was to determine the 5-year trend prevalence of intestinal prevalence among patients who had been suspected for intestinal parasite infections. A retrospective study was conducted from 2009 to 2013 at Poly Health Center Gondar, Northwest Ethiopia. Samples were examined using direct saline wet mount methods. Statistical analysis was done using SPSS version 20 software and a P-value of < 0.05 was considered statistically significant. The results were presented in tables and graphs.Entamoeba histolytica/dispar (16.8%) being the predominant parasite followed by Giardia lamblia (11.4%) and Ascaris lumbricoides (6.7%). Both males (49%) and females (51%) were equally affected (P = 0.14). The intestinal parasite was reported in all age groups in the area but the highest and the lowest prevalence were reported in age groups of 20–29 years and 40–49 years, respectively (26.5% vs 6.4%) (P < 0.001).During the study period, a total of 13,329 stool samples were requested for intestinal parasite diagnose and 5510 (41.3%) laboratory-confirmed cases were reported with a fluctuating trend. Ten different parasites were reported in each year with  Intestinal parasites are present throughout the world in varying degrees of prevalence and tend to be most prevalent in developing countries . This isAmong African countries, intestinal parasites have been widely distributed in Ethiopia due to poverty, lack of environmental sanitation and ignorance of simple health promotion practice. According to the Ethiopian Ministry of Health, the prevalence rate of infection with intestinal parasite is as high as 70% and there is also a high rate of multiple infections reported from many urban and rural areas –19. But A retrospective study was conducted from 2009 to 2013 at Poly Health Center in Gondar town, Northwest Ethiopia. Gondar town is located 747 km (kms) north of Addis Ababa, the capital city of the country. The town has a latitude and longitude of 12°36′N 37°28′E with an elevation of 2133 meters above sea level. Gondar has mid-altitude climate and an average annual maximum and minimum temperatures of 27 °C and 16 °C, respectively. A total population of the town is about 207, 044, of whom 98,120 were men and 108,924 were female and the population of the district is predominately Amhara, Orthodox Christianity being the main religion. The town administration has twenty-three urban and eleven rural kebeles . Intestinal parasites were also reported in all age groups in the area but there is statistically significant difference in the proportion of parasitic infections in different age groups. The highest intestinal parasitic infection was reported in the age group of 20–29 years but the least infection rate was reported in the age group of 40–49 years (26.5% vs 6.4%) (P < 0.001).E. histolytica/dispar being the predominant parasite followed by G. lamblia and A. lumbricoides with a prevalence of 40.7%, 27.5% and 16.2%, respectively. The other identified parasites were H. nana, E. vermicularis, Taenia species, S. mansoni, S. stercularis, T. trichiura and Hookworm , 55/183 (30.1%) and 33/103(32.0%), respectively. The age group < 9 was more affected by G. lamblia, H. nana, E. vermicularis and T. trichiura with the prevalence of 460/1516 (30.3%), 105/228 (46.1%), 41/133 (30.8%) and 16/47 (34.0%), respectively. and 10–19 age group where affected by A. lumbricoide, S. mansoni and Taenia species  and the minimum, 586/1973(29.7%), were reported in 2010 , Mojo Health Center (9.3%), Wonago Health center (16.6%) –23 and o%, Mojo HEntamoeba histolytica/dispar (40.7%) was the most prevalent intestinal parasite but it was lower than the previous study in Wonago Health center (53.8%) in Ethiopia [Ethiopia . HoweverEthiopia  and otheEthiopia , PalajunEthiopia , OsmangaEthiopia  and KeniEthiopia . The preEthiopia , Uludag Ethiopia , 30–32. G. lamblia (27.5%) was the second most prevalent intestinal parasite and A. lumbricoides (16.2%) as the leading helminthic parasite followed by H. nana, Hookworm, E. vermicularis, S. mansoni, with the prevalence of 4.1%, 3.3%, 2.4%, 2.4%, respectively and the least prevalence Taenia species (0.7%) which was differ from the studies done in Ethiopia [In the present study, Ethiopia –23 and oEthiopia –32, 35. The result of our study revealed that a slight fluctuating overall trend of intestinal parasitic infection was observed in the study area. A decreasing number of infections occurred in 2010 compared with reported in 2009. However, there was a gradual increment of a number of cases from 2011 to 2013 with higher intestinal parasite infection being reported in 2013. Similar studies in Ethiopia , PalestiS. mansoni. Males were more affected than females by Hookworm and S. mansoni. This study is consistent with the study done in Uludag University Medical School Hospital and Istanbul in Turkey [Out of the total data review in the last 5 years in the health center, males and females were equally affected for intestinal parasites infection except for Hookworm and n Turkey , 26. Butn Turkey , 23, Tokn Turkey , 29, femEntamoeba histolytica/dispar (28.4%), Hookworm (30.1%), and S. stercularis (32.0%) were higher in the earlier age groups. But the G. lamblia (30.3%), H. nana (46.1%), E. vermicularis (30.8%) and T. trichiura (34.0%) were higher in the later age groups. The 10–19 age groups were also affected by A. lumbricoide, S. mansoni, and Taenia species. The higher overall distribution of E. histolytica/dispar, Hookworm and S. stercularis in adolescence groups in the present study was in agreement with other studies in Ethiopia [Regarding the age groups, 20–29 years were highly affected groups, with a prevalence rate of 26.5%), followed by < 9-year-olds (24.9%). Ethiopia , 22 and .5%, folla/dispar .4%, Hookrichiura .0% were Ethiopia , 33. ThiEntamoeba histolytica/dispar was the predominant parasite. Males were more affected by Hookworm and S. mansoni. In the present study, the parasitic infection was mostly common in the age groups of 20–29 years. This indicates the importance of establishing proper infection and prevention strategies by considering different factors like daily activities that exposed to parasitic infections. There should be also a strong surveillance system in the study area to monitor this high prevalence of parasitic infections in the populations.Intestinal parasites were highly prevalent in the study area and fluctuating trend among the study subject for the past 5 years. E. histolytica and E. dispar due to their morphological similarities. In addition, using the wet mount method only might not be enough to diagnose light parasitic infections as compared to like concentration methods. Hence, the magnitude of parasitic infection in the study area might be underestimated due to the nature of the study.Due to the nature of the study, we were unable to get risk factor information’s like the quality of water supply, environmental sanitation, hygiene practice at both individual and community levels. Hence, we are unable to correlate with the main parasites obtained in the present study. Since the laboratory method employed for the identification of intestinal parasites in the study area was saline wet mount preparation, we are unable to differentiate


Data about heart failure in Egypt is scarce. We aimed to describe the clinical characteristics and diagnostic and treatment options in patients with acute heart failure in the Delta region of Egypt and to explore the gap in the management in comparison to the international guidelines.DELTA-HF is a prospective observational cohort registry for all consecutive patients with acute heart failure (AHF) who were admitted to three tertiary care cardiac centers distributed in the Delta region of Egypt. All patients were recruited in the period from April 2017 to May 2018, during which, data were collected and short-term follow-up was done.A total of 220 patients  was enrolled in our registry. The risk factors for heart failure included rheumatic valvular heart disease (10.9%), smoking (65.3%), hypertension (48.2%), diabetes mellitus (42.7%), and coronary artery disease (28.2%). Left ventricular ejection fraction (LVEF) was less than 40% in 62.6%. Etiologies of heart failure included ischemic heart disease (58.1%), valvular heart disease (16.3%), systemic hypertension (9.1%), and dilated non-ischemic cardiomyopathy (15.5%). Exacerbating factors included infections (28.1%), acute coronary syndromes (25.5%), non-compliance to HF medications (19.6%), and non-compliance to diet (23.2%) in acute decompensated heart failure (ADHF) patients. None of our patients had been offered heart failure device therapy and only 50% were put on beta-blockers upon discharge. In-hospital, 30 days and 90 days all-cause mortality were 18.2%, 20.7%, and 26% respectively.There is a clear gap in the management of patients with acute heart failure in the Delta region of Egypt with confirmed under-utilization of heart failure device therapy and under-prescription of guideline-directed medical therapies particularly beta-blockers. The short-term mortality is high if compared with Western and other local registries. This could be attributed mainly to the low-resource health care system in this region and the lack of formal heart failure management programs. Heart failure (HF) is a global public health problem affecting an estimated 26 million people worldwide . In the DELTA-HF is a prospective cohort registry for all consecutive patients with AHF who were admitted to three tertiary care cardiac centers in the Delta region of Egypt within a period from April 2017 to May 2018, during which, data collection and short-term follow-up were done. Two of these centers have catheterization laboratory and they have primary percutaneous coronary intervention (PCI) programs. Very recently, one of them has arrhythmology department with electrophysiology service but still restricted to pacemaker implantation.We included only patients with AHF whether de novo or acute on top of chronic HF. Patients with AHF were defined as those with progressive symptoms and signs of HF who required treatment with intravenous diuretics, inotropes, or vasodilators. We excluded patients who are less than 18 years of age, refused to give consent, and patients who were managed and discharged from the emergency department without hospital admission. For every patient, a standard case report form (CRF) has been filled out with an emphasis on clinical and demographic characteristics of included patients, course during hospital stay, and diagnostic and therapeutic options entertained prior to admission, during hospital stay and upon discharge. All CRFs were filled out by a cardiology resident with no interaction with the management plans of the treating physicians. Thirty-day and 90-day follow-up included all-cause mortality and re-admission rates were sought via telephone contact or pre-scheduled hospital visits. An ethical approval letter has been obtained from the ethics committees of the recruiting hospitals. Verbal informed consent has been obtained from each included patient or his substitute decision maker.Continuous variables were summarized using means or medians based on the normality; normally distributed variables were summarized using the mean and standard deviation (SD), while the non-normally distributed variables were summarized using the median. Categorical data were summarized as the frequency and percentages.A total of 220 patients were enrolled in this registry, and 22 patients were lost for follow-up after 1 month from hospital discharge, while 8 patients were lost after 3 months. Overall, patients had a mean age of 58.79 years ± 14.36 and a median age of 61.50. As regard the gender, 65.5% were males. A review of medical history showed that 50.9% were known as heart failure patients before, 48.2% had hypertension, 42.7% had diabetes mellitus, 56.3% had smoking history, 28.2% had coronary artery disease , and 10.9% had rheumatic heart disease . For other comorbidities, 26.4% had atrial fibrillation, 6.4% had cerebro-vascular accidents (CVA) or transient ischemic attacks (TIA), 8.1% had bronchial asthma and COPD, and 10.9% had chronic kidney disease (CKD). None (0%) had a history of implantable cardioverter-defibrillator (ICD), cardiac resynchronization therapy (CRT) implantation, or permanent pacemaker (PPM) implantation. Table Dyspnea was reported by 96.4% as a main complaint, 49.1% were in New York Heart Association Classification (NYHA) class III, 41.5 % were in NYHA class IV, and 9.4% were in NYHA class II. Orthopnea and or paroxysmal nocturnal dyspnea (PND) were reported in 76%, 31.8% had lower limbs swelling, 9.1% had palpitation, 1.8% had syncope and/or pre-syncope, 20% had chest pain, and 9.1% reported other symptoms like fatigue, cough, hemoptysis, and fever. Median systolic blood pressure was 110 mmHg; median diastolic blood pressure was 70 mmHg; median heart rate was 110 beats/min; and median oxygen saturation was 94.0%.Regarding the ECG at presentation, 35.5% had atrial fibrillation, 4.5% had wide complex tachycardia/VF, 17.3% fulfilled acute ST elevation myocardial infarction (STEMI) criteria, and 21.8% had pathological Q waves at presentation. The QRS width mean ± SD was 101.5 ± 20.64. LV EF estimation was calculated exclusively by using the M-mode method. The left ventricular ejection fraction (LVEF) mean ± SD was 38.69 ± 11.94, and the median was 35.0%. The percentage of patients with LVEF of less than 40% was 62.6%. A total of 33.6% of our study population has a known coronary anatomy by coronary angiography either before time of admission or during their hospital admission where 43.2% of them had evidence of multi-vessel disease (MVD), 20% had single-vessel disease, 10% had double vessel disease, and 2.7% had significant left main (LM) disease.Coronary reperfusion has been done for 13.6% of our patients where 36.5% of them had primary PCI, 11.5% had pharmaco-invasive strategy, and 52% had thrombolytic therapy. Intravenous inotropic agents have been prescribed in 34.5% of patients, 41.8% had non-invasive respiratory ventilation (CPAP or BiPAP), 12.7 % had invasive endotracheal intubation and mechanical ventilation, 38.2% had worsening of serum creatinine, 1.8% had renal replacement therapy , 5.5% had blood transfusion, and 7.3% had ventricular tachyarrhythmias (VT and/or VF).Ischemic heart disease has been suggested as the underlying primary etiology in 58.1% of patients, valvular heart disease in 16.3%, dilated non-ischemic cardiomyopathy in 15.5%, and systemic arterial hypertension in 9.1% as shown in Fig. In-hospital mortality rate was estimated to be 18.2%. Mortality rate after 1 month was estimated to be 2.5% and 5.3% after 3 months. Re-admission rate was 10% after 1 month and 18.6% after 3 months follow-up , 3.6% were on dihydropyridine CCB, 24.5% were on statins, 24.5% were on warfarin, 32.7% were on aspirin, 13.6% were on P2Y12 receptor inhibitors, and 6.4% were on trimetazidine. While upon discharge, 50% have been prescribed beta-blockers, 63.3% ACE inhibitors, 13.3% ARBs, 52.2% MRAs, 10% digoxin, 92.2% diuretics, 17.7% ivabradine, 20% nitrates, 14.4% amiodarone, 1.1% non-dihydropyridine CCB, 5.5% dihydropyridine CCB, 56.6% statins, 38.3% warfarin, 48.8% aspirin, 28.8% P2Y12 receptor inhibitors, and 1.1% trimetazidine as shown in Table Heart failure (HF) is a major cause of morbidity and mortality worldwide and has significant negative impacts on quality of life, healthcare costs, and longevity . RegistrThe average age of presentation in our patients was approximately one decade younger relative to patients in the Western countries but was very comparable to the age of patients in registries from the Middle East region , 7, 8. TAtrial fibrillation on ECG was found in approximately one-third of our patients, while its prevalence was lower in ADHERE (20%) , HEARTS LVEF estimation via TTE was done exclusively by M-mode method; however, recent 2016 ESC guidelines have recommended for measurement of LVEF, the modified biplane Simpson’s. This method relies on accurate tracing of endocardial borders . More thNone of our patients has undergone natriuretic peptide (NP) testing, and this was attributed mainly to unavailability of the test, lack of physician orientation of its role in AHF management, and the high costs of the test. Measurement of NPs is recommended in patients presented with acute dyspnea and confusion about its etiology being cardiac versus other non-cardiac and also to assess the prognosis and to guide treatment in both patients with acute and chronic heart failure , 14.We have 38 patients presented with STEMI and 18 patients presented with non-STEMI; however, reperfusion treatments have been offered to only 32 patients which represent slightly more than half of patients who presented with acute coronary syndrome (ACS). Primary PCI strategies are generally underutilized in our Delta hospitals especially in non-STIMI patients, and this is attributed mainly to economic issues. Non-STEMI 2015 ESC guidelines recommend an immediate invasive strategy with intent to perform revascularization, irrespective of ECG or biomarker findings in patients with at least one very high-risk criterion  .None of our patients received device therapy  or even offered these therapies despite we had 40 patients with known LVEF less than 35%, 4 patients with QRS width (ms) ≥ 150, and 8 patients with QRS width (ms) ≥ 130. Underutilization of device therapy is largely related to socioeconomic issues, unavailability of the service and trained teams, and lack of physician orientation. According to 2016 HF ESC guidelines, ICD is recommended to reduce the risk of sudden death and all-cause mortality in patients with symptomatic HF (NYHA classes II–III) and an LVEF ≤ 35% despite ≥ 3 months of optimal medical therapy (OMT), provided they are expected to survive substantially longer than 1 year with good functional status and they have ischemic heart disease “unless they had MI in previous 40 days” and dilated cardiomyopathy. Also, it is recommended as a secondary prevention measure to reduce the risk of sudden death and all-cause mortality in patients who have recovered from a ventricular arrhythmia causing hemodynamic instability, and who are expected to survive for > 1 year with good functional status. Also, CRT is recommended for symptomatic patients with HF in sinus rhythm with a QRS duration ≥ 130 ms and LBBB morphology and with LVEF ≤ 35% should be considered for those in sinus rhythm with a QRS duration ≥ 150 ms and non-LBBB morphology and with LVEF ≤ 35% and may be considered for those in sinus rhythm with a QRS duration ≥ 130 ms and non-LBBB morphology and with LVEF ≤ 35% despite OMT in order to improve symptoms and reduce morbidity and mortality [Before the time of admission, less than one-quarter of our patients were on beta-blockers, less than one-third on angiotensin-converting enzyme inhibitors (ACEIs)/ARBs, and about one-fifth were on MRAs. Upon discharge, the figures improved but still below the ideal with half of them were put on beta-blockers, about 3 quarters on ACEIs/ARBs, and more than one half on MRAs. There is relatively large gap in prescribing guideline-directed medical therapy (GDMT) particularly beta-blockers (BB) when we compare our data with the regional and international registries which might be explained, at least in part, by limited hospital resources and lack of heart failure programs or protocols in these hospitals.There were some limitations of this registry; most of them related to the limited sample size and limited geographical representation. Some cases were not selected on a consecutive basis due to the lack of hospitals’ database and unavailability of a person responsible for collecting the data during the course of admission of some patients. Small sample is another important limitation which might make this registry less representative for heart failure in Egypt. Absence of hospital database systems for patients’ admission and follow-up made it difficult for us to collect the data, and some cases were missed during follow-up phases. Natriuretic peptide testing was not included in the diagnosis of AHF which was based mainly on clinical examination and TTE findings.There is a clear gap in the management of patients with heart failure in the Delta region of Egypt with confirmed very low use of NPs in the diagnosis and prognostication of heart failure, under-utilization of heart failure device therapy, low rate of coronary intervention, and under-prescription of guideline-directed medical therapies particularly beta-blockers. Limited resources and the lack of heart failure management programs are the main causes of this gap. We are planning to have a second phase of this registry with a bigger sample size and more hospitals representing the Delta region in Egypt.


Based on our recent studies the prevalence of polyclonal infection in tuberculosis clinical specimens is more than 50% in Tehran, Iran. With this background, Spoligotyping was performed on clinical specimens and their respective cultures, and we examined whether mixed infections interfere with the results or not.Based on the Spoligotyping pattern, among the fourteen patients, 57.1% had different genotypes in clinical samples and their respective cultures. These discrepant patterns were suggestive of polyclonal infections in clinical samples with possible overlapping Spoligotype patterns. We propose that in societies with high mixed infections (e.g. Iran), direct Spoligotyping on clinical samples can be controversial. M. tuberculosis culture is also slowly [Tuberculosis (TB) is still a major health problem all over the world. An essential tool in clinical microbiology and molecular epidemiology is genotyping of bacterial pathogens . In manyo slowly . Mycobaco slowly . Also, to slowly . Therefoo slowly . Severalo slowly . In veryo slowly .One of the major issues with TB is mixed  infections, which is a person’s infection with several different genotypes of Mycobacterium . MIRU-VNThe aim of the present study was comparing the direct and indirect Spoligotyping on clinical specimens and their respective cultures, respectively. We also examined whether mixed infections interfere with the results or not.This study was conducted between October 2017 and January 2018. Samples were from patients with definite diagnosis of pulmonary TB. Ethical reviews and informed consent approval were granted by the Ethical Committee of the Pasteur Institute of Iran. The written informed consent was obtained from all patients enrolled in the study. Study samples were included 14 clinical samples (11 smear positive and 3 smear negative). In our study, 12 clinical specimens including sputa and 2 samples were gastric juices. Clinical samples were classified by microscopy according to CDC guidelines .N-acetyl-l-cysteine method and inoculated in Lowenstein–Jensen (LJ) medium.Fourteen clinical specimens were decontaminated by For DNA extraction, the volume of clinical sample was 1.5 mL; after centrifugation, 500 μL of pellet was used. For their respective cultures single colony was selected and diluted in 500 μL TE buffer. After that, genomic DNA was extracted from clinical samples and their cultures with Proba-NK DNA extraction kit  according to the manufacturer’s instructions. The DNA was stored at − 20 °C until used for molecular studies.M. tuberculosis H37Rv and M. bovis BCG (Pasteur strain) were used as control strains. Data analysis was performed by MIRU-VNTR plus [Spoligotyping was performed in duplicate with a commercially available kit  . The PCRNTR plus , 16.Based on the Spoligotyping pattern, among the fourteen patients, 57.1% (8/14) had different genotypes on clinical samples and their respective cultures. Direct and indirect Spoligotyping yielded the patterns in 100% (14/14) of the specimens and their respective cultures. Microscopy was negative in 3 sediments after decontamination. Of these, all of them was successful in direct Spoligotyping. In our study, 42.86% at Spoligotype had similarity between clinical samples and culture. According to SpolDB4 Lineage, among the 14 clinical samples, five (35.7%) belonged to MANU2 genotype which had the highest proportion in the genotype of clinical specimens. In clinical samples, other strains were classified into families included three strains (21.42%) from the H4 family, two (14.29%) from the CAS1-DELHI, one (7.14%) from the T1 family and three  strains were Unique.On the other hand, among their respective cultures, T1 was the largest lineage  and other strains were classified into families included four strains (28.57%) from the CAS1-DELHI family, three (21.4%) from the H4 family, one (7.14%) from the CAS2 family and one (7.14%) strain was Unique. Interestingly, 62.5% (5/8) and 37.5% (3/8) of the samples with different genotypes in clinical samples and their respective cultures showed a genotype of MANU2 and Unique strains in their clinical sample, respectively. Additionally, 50% (4/8) and 37.5% (3/8) of the samples with different genotypes in clinical samples and their respective cultures showed Lineage 4 (SpolDB4 lineage T1) and Lineage 3 (SpolDB4 lineages CAS1-DELHI and CAS2) in their culture, respectively. All the Spoligotype patterns in clinical samples and their respective cultures from the 14 TB patients is summarized in Table Our recent studies have shown that the rate of polyclonal infections in clinical specimens is more than 50 percent in our region –11. WithThe availability of all Spoligotype profiles in our study (100%) is higher than the 98.5% (196/199) found by Sanoussi et al., 90.9% 159/175) by Goyal et al., 49.1% (28/57) by Heyderman et al. on sputa and also higher than Suresh et al. and Zanden et al. on smears , 17–20. 5 by GoyaM.tb cultures in Iran. In none of these studies, the genotype MANU2 has been reported [M.tb isolates, and in none of the strains, genotype MANU2 was observed (Unpublished data). Although two studies have suggested the presence of MANU2 in M.tb cultures in Iranian patients, but the percentage of this genotype in these two articles is very small in compare to our study (4.2 and 7.9% versus 35.7%) [Of the 14 studied patients, 57.1% (8/14) had different genotypes on clinical samples and their respective cultures. These 8 samples with discrepant patterns were suggestive of mixed infections in clinical samples with possible overlapping Spoligotype patterns. Similar to our previous results, approximately 50% of mixed infections were observed in clinical specimens –11. In treported –30. In as 35.7%) , 32.On the other hand, Mozafari et al. worked on 1242 clinical samples in Iranian patients and MANU2 genotypes was detected in 25 cases . This maOur results showed that of 14 TB patients, direct and indirect Spoligotyping were fully successful even for negative smear specimens. These contradict the results of Sanoussi et al. who failed for direct Spoligotyping for their negative smear specimens . AdditioIn the current study, 100% of samples that had a different pattern in Spoligotyping between sputum specimens and their culture, contained inter-lineage discrepancies. This may indicate the presence of completely different strains in samples containing mixed infections. In another study, it was shown that MANU2 contains inter-lineage discrepancy of the Beijing and Euro-American strains (T1 and T2 sublineages) . SimilarAdditionally, 50% and 37.5% of the samples with different genotypes in clinical samples and their respective cultures showed Lineage 4 (SpolDB4 lineage T1) and Lineage 3 (SpolDB4 lineages CAS1-DELHI and CAS2) in their culture, respectively. In this regard, it has previously been established that the lineage 4 has grown faster (in liquid medium) than the lineage 1 and the ancestral lineage . Also, aIn conclusion, direct Spoligotyping is very efficient in regions where mixed infections are low in their clinical specimens. On the other hand, in societies with high mixed infections (e.g. Iran) in clinical specimens, we recommend to rule out mixed infection by the MIRU-VNTR method in the first step and after that direct Spoligotyping can be performed more accurately.The present study must be confirmed by investigation on more clinal samples from TB patients.


Tonsillectomy is one of the most commonly performed surgical procedures in children. It is performed for sleep-disordered breathing and infective symptoms. Despite advances in instrumentation and perioperative care, posttonsillectomy haemorrhage (PTH) remains a significant complication, which should be audited regularly. A retrospective case series of all tonsillectomies performed in patients <15 years old in the Auckland region during 2017 was performed. Demographic, clinical, and surgical data were extracted and analysed. Univariate analysis was performed to compare patients with and without PTH.p=0.004) and were less likely to have associated OME (p<0.001). A total of 2177 tonsillectomies were performed during the study period, 64% in a public hospital and 36% in a private hospital. The overall PTH rate was 3.6% (0.23% occurring within the first 24 hours (primary) and 3.4% after 24 hours (secondary)). Mean time to PTH was 6.6 ± 3 days (range: 1-16 days). 90% of PTH occurred within the first 10 days and 99% by 14 days. Return to theatre was required in 28% of these cases, representing 1% of all tonsillectomies. There were no deaths or major complications in this cohort. The only differences observed between patients with PTH and those without were that children with PTH had smaller tonsils ( It is important to report institutional tonsillectomy outcomes and associated complications. These results show that PTH rates in Auckland remain within acceptable limits according to the literature. Tonsillectomy is one of the most commonly performed surgical procedures in the paediatric population worldwide . 530,000Despite significant advances in surgical instrumentation and perioperative care, posttonsillectomy haemorrhage (PTH) remains a significant complication associated with tonsillectomy –6. Clinist of January and the 31st of December 2017.Data were obtained from Auckland District Health Board (ADHB), Counties Manukau District Health Board (CMDHB), and private hospitals in the Auckland region following national ethics committee approval (17/NTA/148). Extraction of all clinical information was performed following the retrospective identification of all patients under the age of 15 years undergoing tonsillectomy between the 1The following demographic data were collected, as previously described by Johnston et al. : age, se Chi-square tests were performed to assess categorical variables and the ANOVA test was conducted to evaluate continuous variables. A two-tailed p-value < 0.05 was regarded as statistically significant. IBM® SPSS® version 24 software was used to perform all statistical analyses.Patient demographics and clinical characteristics were summarised using descriptive statistics. Univariate analysis was used to assess potential factors that were associated with differences in patients with PTH versus those without this postoperative complication.From January 1 to December 31 of 2017, a total of 2177 tonsillectomies were performed on paediatric patients in the Auckland Region. Tonsillectomy alone was performed in 15% (n=326) of cases, whereas all other surgeries included at least one additional procedure including adenoidectomy and inferior turbinate cautery. 64% (n=1395) of tonsillectomies were performed in a publicly funded hospital, with the remainder  being performed in a private hospital.p = 0.33). Mean patient age was 7.5 ± 3.5 years (range 1–15 years). 51% (n=38/78) of PTH patients were male.The rate of PTH in this cohort was 3.6% (n=78). 2.6% of all PTH patients had more than one episode (n=2/78). In the public hospital cohort, PTH occurred in 3.9% (n=54/1395) of patients, compared with 3.1% (n=24/782) in the private hospital group (Mean time to PTH from the day of surgery was 6.6 ± 3 days (range 1 – 16 days). PTH occurring within 24 hours of surgery is regarded as primary PTH and occurred in 0.23% (n=5/2177), accounting for 6% (n=5/78) of all patients with PTH. One of these patients had a secondary PTH on postoperative day 16. Secondary PTH occurred in 3.4% of all tonsillectomies (73/2177); one of these patients had two episodes, on days 4 and 6. 90% of all PTH occurred within the first 10 days (n=72/80) and 99% in the first 14 days (n=79/80). The day of presentation to hospital with PTH in all patients is shown in Indications for tonsillectomy in patients with PTH included sleep-disordered breathing in 54% (n=42/78), recurrent tonsillitis in 24% (n=19/78) and a combination of indications in 6% (n=5/78). In the remaining 16% (n=12/78) of patients this information was unavailable. In regard to surgical technique, cold steel dissection with bipolar diathermy was used in 36% (n=28/78) of cases, bipolar diathermy only in 37% (n=29/78) of cases, monopolar diathermy in 10% (n=8/78) of cases, and coblation in 5% (n=4/78) of cases. In the remaining 12% (n=9/78) of cases information about the surgical technique was not available. Non-steroidal anti-inflammatory drugs (NSAIDs) were prescribed to 82% (n=64/78) of patients with PTH.In regard to the management of patients with PTH, 28% (n=22/80) had a general anaesthetic and arrest of haemorrhage performed in theatre ; the remaining 72% (n=58/80) were managed conservatively. In primary PTH, 40% returned to theatre (n=2/5) while in secondary PTH 27.4% did (n=20/73). There are 80 episodes of PTH included in this analysis given that two patients had a second PTH episode. Both of these patients were managed conservatively. The mean haemoglobin level on admission was 121.4 g/L (range from 64 g/L to 162 g/L). Only one patient required a blood transfusion, as haemoglobin had dropped to 64 g/L. There were no deaths related to PTH in this cohort.A comparison of clinical and demographic factors was undertaken in patients who had PTH versus those who did not. Only the 1395 patients who were operated on in publicly funded hospitals were included in this analysis given the paucity of data available in those patients operated on in the private sector. There were 54 patients who had a PTH and 1341 who did not. Demographic and clinical characteristics are summarised in  p=0.004). There was no significant difference in adenoid grade between groups . No significant differences were noted in the presence of medical comorbidities including asthma, eczema, allergic rhinitis, and gastroesophageal reflux disease (GORD) .Patients in the PTH group had smaller tonsils than those in the non-PTH group . There was no statistical difference in the incidence of a documented history of speech delay between groups .Children with PTH were less likely to have associated otitis media with effusion requiring ventilation tube insertion at the time of surgery . These swab results were positive for GAS in 27.8% of children with PTH and 35.2% with no PTH in the year preceding surgery (p=0.26). There was no difference between groups in regard to medications prescribed at the time of discharge including antibiotics, paracetamol, ibuprofen, and tramadol .A throat swab for Group-A Streptococcus (GAS) was performed in 75.9% of children with PTH and 77.1% with no PTH in the year preceding surgery , finding a correlation with newer surgical instruments (such as diathermy and coblation), and a change in the use of routine drugs (NSAIDs and steroids) , while aIn the majority of our PTH patients, sleep-disordered breathing was the most frequent indication for tonsil surgery, which is consistent with the shift from infectious to obstructive indications observed worldwide , 25. AltRetrospective studies, such as this, rely upon accurate clinical records. Therefore, only PTH severe enough to warrant admission to hospital would be registered and included. It is possible that the rate of PTH may be significantly underreported. In fact, one study showed that up to one-fifth of all PTH episodes could be missed by this approach , especiaIt is important to report institutional tonsillectomy outcomes and associated complications. Our report highlights the current status of paediatric PTH in Auckland over a 1-year period and its demographic characteristics, showing no major changes with previous reports and remaining within acceptable limits according to the literature.


SPAST gene) and SPG3 (ATL1 gene). In the current research we investigated for the first time the distribution of pathogenic mutations in SPAST and ATL1 genes within a large cohort of Russian HSP patients . We determined the frequencies of genetic abnormalities using Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA), and Next Generation Sequencing (NGS) of targeted gene panels. As a result, SPG4 was diagnosed in 30.3% (37/122) of HSP cases, where the familial cases represented 37.7% (26/69) of SPG4. In total 31 pathogenic and likely pathogenic variants were detected in SPAST, with 14 new mutations. Among all detected SPAST variants, 29% were gross deletions and duplications. The proportion of SPG3 variants in Russian cohort was 8.2% (10/122) that were all familial cases. All 10 detected ATL1 mutations were missense substitutions, most of which were in the mutational hot spots of 4, 7, 8, 12 exons, with 2 novel mutations. This work will be helpful for the populational genetics of HSP understanding.Hereditary spastic paraplegia (HSP) comprises a heterogeneous group of neurodegenerative disorders, it share common symptom - of progressive lower spastic paraparesis. The most common autosomal dominant (AD) forms of HSP are SPG4 ( SPAST gene (protein: Spastin) and demonstrates autosomal dominant (AD) inheritance. It represents 12–25% of all HSP cases (including mutations with non-proven pathogenicity) and 40–60% within familial cases3. Spastin is a microtubule-severing protein that belongs to AAA (ATPase associated with various cellular activities) family of ATPases and regulates the number and mobility of microtubules. SPAST gene contains 17 exons where 723 mutations are mapped been scattered across the coding regions. Most of those mutations identified are missense, however deletions and duplications in SPAST also make a considerable contribution to the HSP pathogenesis4. So far, no mutational hotspot regions for SPAST gene were described and no frequent mutations were found. Therefore, screening of the complete coding sequence of SPAST is necessary for the detection of mutations. But if the new variant located in the region encoding the ATPase domain it is most likely to be pathogenic3.Hereditary spastic paraplegia (HSP) is genetically heterogeneous group of neurodegenerative disorders characterized by progressive pyramidal tract dysfunction due to retrograde degeneration of long axons of the corticospinal tracts. The most prevalent form of HSP is SPG4 (OMIM 182601). SPG4 is associated with mutations in the ATL1 gene (protein: Atlastin). Alastin is a protein implicated in vesicle trafficking and neurite outgrowth. SPG3 is the second most common form of ADHSP and of HSP with other type of inheritance. Although its contribution is much less than that of SPG4, on average it accounts for 2–3% of total HSP cases and 8–10% of family AD HSP cases6. Forty-eight ATL1 mutations were identified so far and they are mainly missense changes. Large rearrangements make however a contribution to SPG3 pathogenesis. Four of 14 gene exons 4 are mutation hotspots in ATL1 whereas no frequent mutations have been described.SPG3  form of HSP is caused by pathogenic variants in the SPAST and ATL1 mutational landscape in a cohort of Russian HSP patients that would help diagnosis and treatment.Many variants can cause different pathogenesis of HSP that requires different treatments. At the same time the mutational spectra of HSP varies strongly on the populational level. Thus the current study is intended to provide for the first time an overview of The current study analyzed DNA of 122 unrelated HSP patients . Cohort included 68 men and 54 women aged between 4 and 68 years. The non-familial cases started to be selected after the next generation sequencing (NGS) panel being implemented.Most patients were diagnosed at the Research and Counseling Department of the Research Centre for Medical Genetics (RCMG) including patients that were referred from the Genetic Counseling Department for the Moscow Region. While the others had been diagnosed by the Genetic Counseling Departments of Voronezh, Yekaterinburg, Khabarovsk and other regions.The study was approved by the local ethics committee of the Federal State Budgetary Institution “Research Centre for Medical Genetics”  and all the patients gave written informed consent. All experiments were performed in accordance with the institutional guidelines.Blood samples were collected, and DNA was extracted at the DNA-diagnostics Laboratory of RCMG. Molecular diagnostics of HSP patients have been performed using NGS, all detected variants were validated by Sanger sequencing. The DNA of HSP patients who were negative for sequencing mutations was analyzed using MLPA to quantify copy numbers.Genomic DNA was extracted from whole venous blood by Wizard® Genomic DNA Purification Kit  following the manufacturer’s protocol.GJC2, AP4B1, AMPD2, IBA57, ALDH18A1, ZFYVE27, NT5C2, ENTPD1, MTPAP, CAPN1, BSCL2, KLC2, KIF5A, C12orf65, MARS, VAMP1, B4GALNT1, SPG20, SACS, ATL1, ZFYVE26, DDHD1, TECPR2, AP4S1, NIPA1, SPG11, SPG21, AP4E1, USP8, SPG7, FA2H, ARL6IP1, KIF1C, AFG3L2, RTN2, PNPLA6, C19orf12, CPT1C, MAG, HSPD1, KIF1A, REEP1, PGAP1, MARS2, SPAST, SLC33A1, TFG, WDR48, CYP2U1, ARSI, ZFR, REEP2, AP5Z1, AP4M1, CYP7B1, KIAA0196, ERLIN2, VPS37A, DDHD2, GBA2, L1CAM, PLP1 and SLC16A2. Next generation sequencing of patient’s DNA was performed by Ion S5 next-generation sequencer  with an Ion AmpliSeq ™ Library Kit 2.0 according to the manufacturer’s protocol. Patient’s DNA samples were prepared using ultra rapid multiplex PCR technology combined with subsequent sequencing (AmpliSeq™).For the current research the Spastic Paraplegia Sequencing Panel of target genes was developed. It comprises the following HSP associated genes; www.gene-talk.de/contact; Gene Talk GmbH, Germany). Sequenced fragments were visualized in Integrative Genomics Viewer (IGV) software .Sequencing data was processed according to the standard bioinformatic algorithm from Thermo Fisher Scientific (Torrent Suite™) and Gene-Talk software (A beta release of the Genome Aggregation Database (gnomAD browser beta) was used to determine the frequencies of new variants.MLPA method was used for the analysis of large deletions and duplications using a SALSA MLPA P-165-С2 HSP kit following the manufacturer’s protocol. MLPA data was analyzed with Coffalayser software (MRC-Holland).SPAST and ATL1 modifications were designated in accordance with HGVS nomenclature (http://www.hgvs.org/mutnomen/) with reference sequences NM_199436.1 and NM_001127713.1, respectively (http://www.Ncbi.nlm.nih.gov/nuccore).Revealed in silico: Mutation Taster (http://www.mutationtaster.org/), UMD-predictor (http://umd-predictor.eu/); SIFT/Provean (http://provean.jcvi.org/index.php); PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/index.shtml); and Human Splicing Finder (http://www.umd.be/HSF/).The following online prediction programs were used to determine pathogenicity 8 were used to define the clinical significance of newly discovered variants.Guidelines for interpretation of NGS dataSPAST mutations within AD HSP forms amounted for 37.7% (26/69) of cases and 20.7% (11/53) were among sporadic cases. A total of 31 pathogenic and likely pathogenic variants were detected in SPAST gene, encompassing 14 novel variants. Whereas 5 newly detected variants were repeated in 2 or more families. Twenty-two pathogenic and likely pathogenic variants in 27 out of total 37 unrelated SPG4 probands were found by sequencing techniques, namely NGS of targeted panel and Sanger sequencing. These were: 10 missense changes, 3 nonsense mutations, 7 micro-rearrangements and 2 splice site mutations. Most of the variants (19/22) were located in the AAA-domain of SPAST, while 2 were located in the promoter region and 1 in the microtubule interacting and trafficking (MIT) domain. Major limitation of the direct sequencing methods is that large deletions/duplications may not be detected. Therefore, 10 remaining patients out of the total 37 SPG4 patients that fail to reveal mutations by direct sequencing were examined using MPLA assay. As a result, 9 pathogenic variants in 10 unrelated probands, which amounts for 29.0% (9/31) of all pathogenic and likely pathogenic variants, were detected in SPAST.In total 37 SPG4 cases were detected among 122 DNA samples of patients with HSP that represents 30.3% of cohort. Where ATL1 were detected in 10 unrelated probands. All detected mutations were missense substitutions. Large rearrangements were not detected in Russian cohort. The most frequent variants (6/8) in our study were located in the ATL1 gene mutational hotspots in exons 7, 8 and 12. Notably, the variants с.1041G > A (p.Met347Ile) and с.1213G > A  are described here for the first time. Three mutations were found in 2 or more families, whereas the other 5 were distinct in each family.The proportion of SPG3 in Russian patients comprised 8.2% (10/122), where the pathogenic variants were detected only in familial group and amounted to 14.5% (10/69). Seven pathogenic variants of SPAST and ATL1gene mutational landscape is summarized in the Table The data upon in silico prediction programs. At least 3 programs were used to confirm the pathogenic effect of each variant on the gene or gene products. In summary the most of discovered mutations were pathogenic and likely pathogenic with high probability.To determine pathogenicity of the novel variants, we used SPAST range from 14.5% of cases in Spain to 28% of cases in Germany. While ATL1 forms impact range from 1.3% of HSP cases in Germany to 4.6% of HSP cases in Poland14;  and 8.2% (10/122), respectively. In the other studies, however, these forms on average were found to be less frequent. Namely, percentages of pathogenic variants of 12. However, it is significantly lower than the proportion of SPG4 among AD HSP unraveled by researchers from Germany (p = 0.002), e.g. 37.7% versus 61.0% (121/197)14. SPG4 fraction among AD HSP cases in Japan is also relatively high and amounts 55%13. This rates of Japan population however are not significantly higher compare to Russian cohort (p = 0.09). This might be explained by either real lack of differences, or by the smaller sample size that affects statistical significance , which is consistent with the results obtained in Spain, China and Poland6/69, whi12. SPG3 incidence rates however are significantly higher in Russian cohort compare to Chinese data, showing 3.7% (p = 0.028), German data, showing 3.1% (p < 0.0001) and Japanese data, showing 2.0% (p = 0.014)14; , that is compatible to Spanish results showing 11.3% (p = 0.49) and Polish data showing 10.6% (p = 0.4)SPAST and ATL1 may spread through the population and undergo fixation by random genetic drift. This can happen because SPG4 (that has mostly late onset) and SPG3 (which has mostly early onset), exhibit slow progression and a relatively mild symptoms, that don’t interfere with fertility and procreation. Also, particular HSP alleles may accumulate in different populations because of random genetic drift that depends on historical, religious, geographical and other reasons. In this way SPG4 is more common in Asian population (Chinese and Japanese), whereas SPG3, is more common among Europeans. Nevertheless there are exceptions, such as German population that differs from this trend since SPG4 demonstrate higher frequencies, whereas SPG3 is much less common compare to other European countries. In addition, proportions of SPG4 and SPG3 can be influenced by proportions of other forms of HSP in the particular cohorts. Because the research in the field of HSP genetics is very recent the complete mutational spectrum of this disease and its populational genetics in many countries has been described only partially or not at all. Consequently, new forms of HSP in certain populations, may lead to proportional decline of the other forms.Mutations in SPAST gene identified here can be divided into 2 groups: large deletions/duplications detected by MLPA assay  and mutations detected via sequencing methods . The corresponding proportion of large deletions/duplications in SPAST gene in other cohorts was as follows: 37.5% in the Polish cohort12; 2.5% in the Spanish cohort9; 9.0% in The Republic of Bashkortostan cohort15; and 13.5% in the Australian cohort16. The proportion of large deletions/duplications detected in this study and their mutational spectrum, were similar to the results obtained by Polish researchers. This might be explained by the Slavic origin and long-standing historical relationship between these nations. In contrary, the percentage of large deletions/duplications in cohorts of Spain, The Republic of Bashkortostan, and Australia were significantly lower compare to Russian sample.Mutations in SPAST gene. This is consistent with the most available data, except of the results from The Republic of Bashkortostan. Where the c.283delG (p.Ala95Profs*66) variant demonstrated high frequencies in families of Tatar ethnicity17. The c.283delG variant was not detected in our study. The pathogenic variant c.1291C > T (p.Arg431Term) described in 3 unrelated probands of our study was also identified in study from Fonknechten N. et al.18. However, there were no specific symptoms in clinical manifestation of HSP driven by this mutation.The current study did not reveal mutational hotspots or frequent mutations in 20.Among repeated mutations, c.1216A > G , c.1684C > T (p.Arg562Term), and c.1245 + 1G > A variants appeared twice in our cohort. Clinical features of patients carrying these variants were similar to those described in other studies22. In the current studies boundaries of deletion were not identified.Other repeated variant that we found is an exon 1 deletion. It was observed in a couple of other studies with different genetic boundariesATL1 gene. Strikingly, the proportion of large deletions/duplications in the Bashkir study amounted to 1.8% (1/56) that distinguish this population15. Several mutational hotspots in exons 4, 7, 8 and 12, where pathogenic variants acquire more frequently compare to the rest of the coding sequence were described in ATL1. Most pathogenic ATL1mutations detected by our study were also located in the mutational hotspots in exons 7, 8 and 12, however we also found 1 mutation in exon 1023. Two variants c.1243C > T (p.Arg415Trp), and c.757G > A  were reported repeatedly. Clinical profile of the patients carrying these mutations corresponds to pure HSP with age at onset (AAO) less than 10 years old25.Similar to Polish, Hungarian, Spanish and Chinese studies, our study did not unravel large deletions/duplications in SPAST gene mutations identified in our study. This corresponds to the data from other countries and confirms that new mutations occur at a high rate in this gene13. Among 14 newly discovered variants, there were 2 missense variants, 1 non-sense variant, 6 micro-rearrangements and 4 large deletions (Table SPAST gene position c.1107A > G (p.Thr369Thr) that was predicted to be likely pathogenic in silico. Indeed, it segregates with the disease in a family.We estimated that new pathogenic variants compose 45.2% of all SPAST. In total only 9 variants out of 723 have been mentioned in 3 or more studies, with one mutational case per study.Similar to our observations, The Human Gene Mutation Database (HGMD) describes a small number of repeated mutations in SPAST gene mutational spectrum determined in the current study with the mutational spectrum presented in HGMD, unraveled that the proportion of large insertions in SPAST gene in Russian cohort HSP comprises a group of genetically heterogeneous neurodegenerative diseases that are hard to diagnose in clinics. Such difficulties are caused by the high number of clinical manifestations and clinical complications that can mask main symptoms. Also, differential diagnosis for HSPs is hampered by the existence of many phenocopies within the other non-hereditary neuropathologies. All the above factors emphasize the need of up-to-date molecular diagnostics for HSP.The existence of frequent HSP forms and mutational hotspots in causative genes facilitates the development of a diagnostic algorithms. NGS techniques would perform in the most effective way because mutations can be found in individual genes and/or in many genes simultaneously by sequencing of gene panel, whole-exome or whole-genome. Due to the limitations of direct sequencing methods however large deletions/duplications can’t be detected by NGS. In this respect, it is necessary also to implement MLPA or other assays in the diagnostic algorithm to identify this type of mutations. The current study describes large deletions/duplications along with small DNA alterations in Russian cohort of HSP patients. Altogether that demonstrate necessity of comprehensive molecular examinations for accurate diagnose of HSP in Russian Federation.SPAST gene. To another hand, in ATL1 gene the most of pathogenic and likely pathogenic variants were found in mutational hotspots of the gene . Altogether this re-confirms the global data.In summary, we did not reveal any mutational hotspots or frequent mutations in SPAST gene with 45.2% (14/31) of incidence. 20.0% of detected mutations in ATL1 were novel though the pathogenicity of the novel mutations has still to be confirmed. A comparison between obtained results and published data indicates that HSPs are extremely genetically pleomorphic and the proportions of different HSP forms can vary even among the cohorts of adjacent regions.In our study, we discovered new pathogenic and likely pathogenic variants of It was observed also that the incidence of pathogenic variants was remarkably higher among familial cases compare to sporadic cases (52.2% against 20.7%). However, patients without a family history should not be excluded from extensive genetic testing.


Pyrus bretschneideri) is characterized by being self-incompatible and dependent on cross-pollination to set fruit. Honeybee (Apis mellifera) is considered the most important pollinator of pear. Nevertheless, limited pollen transfer has been cited as the main cause of poor fruit set in many pear orchards. Here, we tested the following hypotheses: (i) colony manipulations increase the pollen collection tendency of honeybees and (ii) the proportion of pollen loads being returned to the hive is from the target plant. The technique reliably and rapidly estimates the pollination of honeybees tested under different colony manipulations: (1) using pollen trapping (PT); (2) PT with sugar syrup feeding (SS) (PTSS); (3) SS alone and (4) control without PT and SS. The results clearly show that the pollen collection of honeybees during the experiment was significantly affected (P < 0.05) by colony manipulations. The mean amount of pollen harvested daily was higher for PTSS (19.4 g) and PT (16.4 g) than for SS (12.85 g) and control (8.7 g) colonies. Therefore, PTSS was the most effective treatment for increasing pear pollen collection; other treatments such as PT and SS could also be useful. This study was important for determining how the behavior of honeybee colonies is shaped through colony manipulation to enhance pollen collection of less preferred pear flowers, which is critical when pollination is required.Pear ( Honeybees are well adapted to collect and transfer pollen and thus are the most commonly used crop pollinators. Unlike other flower-visiting solitary insects, honeybees collect large quantities of pollen and nectar –5. PollePyrus bretschneideri, is widely cultivated in the China due to its great size, good flavor and rich juice [Apis mellifera workers visit pear flowers mainly for pollen for a short duration and often switch to other more attractive plants for nectar when pollination is required [ch juice . Its floch juice –19. Thisch juice ,21. Apisrequired ,20–23. ADifferent colony management techniques were tested to improve honeybee pollination of less attractive crops –27. ThesFeeding honeybee colonies sugar syrup (SS) within hives has also been shown to increase the total amount of pollen collected ,32–34. SThis work is important for testing the use of colony management that enhances pear pollen collection by honeybees as best measures for pear pollination. The objectives were to determine whether PT and SS colonies would enhance their total pollen collection and pear pollen proportion. This study is expected to provide information that will support pollination researchers and pear growers for successful pear pollination management to improve the fruit set.Pear growers that participated in this study were informed about the experiment and provided consent before we started the experiment. Pollen was collected from honeybees with permission from the beekeepers and the land owners.The study was conducted in a potential pear-growing area of Yuncheng  located in Shanxi Province, China. The area is characterized by monoculture commercial pear plantations where no natural habitats exist for wild pollinators to survive. The orchard has 6.6 ha, and pears were planted with a distance of 6 m between rows and 5 m between plants. The cultivar ‘Suli’ is dominantly planted (93%) in the orchard for commercial fruit production; other cultivars, such as Bali, Yali and Hongxiangsu pear varieties, are usually present as pollinizers and all cultivars flowers at the same time.The flower morphology of pear consists of a corolla with five petals, each supported by five sepals, that extends to form a spur where nectar is produced. The flower consists of five longer styles associated with a pair of ovules and 21–23 stamens situated below the styles. This morphology indicates that pollinator-mediated cross-pollination is required for successful fruit set.Taraxacum officinale), a weed grown in the field and field margins, and peach (Prunus mongolica) planted in small patches and/or along borders used as a field demarcation line were grown in the area as competitive flowers that affect the pollination of pear.The blooming time of pear starts in late March and the flowering period ranges from 7 to 10 days but varies with the annual weather conditions. Our field study was conducted from 29 March to 7 April 2016 and from 8 to 17 April 2017 during the peak pear-flowering periods at the study site. Meteorological data such as daily temperature and moisture were recorded. Dandelion , each with two brood frames and two frames with very little pollen and nectar.We brought thirty colonies of Different colony manipulations were tested to determine the response of colonies’ pollen collection behavior. In the first manipulation, five colonies were treated with PT alone; in the second manipulation, five colonies were treated with PT with SS (PTSS); in the third manipulation, five colonies were treated with SS alone; and the control colonies were not treated with PT or SS.To minimize the environmental factors, we assigned colonies randomly among treatments and conducting experiments with brief periods of time in the same location. We collected pollen using PT in which honeybees entered the hive through pollen traps whose mesh grid dislodged 70% of the pollen loads from the hind-leg . Pollen The total numbers of pollen foragers were examined to determine if hive manipulations affects in colony level response. Honeybees’ entering the hive with pollen loads were counted from 8:00 to 17:00 within 5 minutes at one-hour intervals using a digital video camera. We also measured the wet weight of pollen collected four times a day from 9:00 to 17: 00 at two-hour intervals and compared these values among the treatments. The pollen weight under the non-PT treatments (SS and control) was estimated from the number of honeybees entering with pollen (two pollen pellets per bee), considering the mass of each pollen load and the 70% trapping efficiency of PT within a given interval.The mass of pollen loads (pellets) was collected to investigate the individual pollen foraging behavior of honeybees. In each interval, bees with pollen were captured, pollens were carefully dislodged from the hind legs, and the weight was recorded and compared across days and times.Pyrus sp.We randomly collected samples of pollen on each experimental day; five grams of pollen samples were taken and sorted by color. Visual identification of pollen color and comparison with reference specimens is the standard approach in pollination ecology . Pollen Statistical analysis was performed using the R-Project software (version 3.3.2). Data were analyzed using ANOVA to investigate the effects. All data were tested for the assumption required by ANOVA, and when the data did not meet the assumptions, the appropriate log transformation was performed. For all data tables, mean values are listed plus or minus with the standard error. Response variables were pooled if necessary, and the mean number of bees foraging pear flowers, mean number of bees entering with pollen, pollen weight and pollen proportion were analyzed. Post hoc analysis was carried out using Tukey’s test for comparison of means at P < 0.05 significance level. Sigma-Plot (version 12.5) was used to sketch graphs.1, 238 = 1.78, P = 0.18). However, pollen collection varied among experimental treatments , days  and times of day .Pollen collection was estimated by quantifying the number of honeybees entering with pollen. The number of honeybees entering with pollen was similar between study years, with no significant variations (FThe mean number of pollen foragers returning to the hive per 5 min was higher for PTSS (41 ± 1.6 bees) and PT (35 ± 1.8 bees) than for SS (27 ± 1.67 bees) and control (17 ± 0.79 bees) colonies. Pollen collection increased with days and was influenced by the times of day. The mean number of pollen foragers was interestingly higher on the second day of pollen collection for all treatments . Pollen 1, 238 = 2.31, P = 0.13), and the average values were 17 ± 1.2 g and 19.1 ± 1.0 g in year 1 and year 2, respectively. ANOVA from year 1 and year 2 pooled together showed that pollen weight was significantly different among experimental treatments , days  and times of day . However, Tukey’s means comparison test showed that there were no significant differences in pollen weight  between PTSS and PT treatments. Colonies under PT, PTSS or SS treatment collected relatively more pollen, with mean weights of 16.4 ± 1.1 g, 19.4 ± 1.2 g and 12.85 ± 1.1 g, respectively, compared to control group (8.7 ± 0.83 g).Pollen weight was inferred from the pollen loads collected through pollen traps. The pollen weight was not significantly varied between years . The pollen weight of the PT and PTSS colonies surprisingly increased on day 2 . On day 3, the pollen weight was relatively stable compared to day 2, with mean pollen weights of 20.44 ± 1.8 g, 20.97 ± 1.2 g, 12.6 ± 1.4 g and 7.88 ± 0.94 g for the PT, PTSS, SS and control colonies, respectively . The pol3,140 = 6.7, P <0.001), and times of day , but not significantly varied across days . The weight of single pollen load was highest for PTSS (10.62 ± 0.27 mg), followed by PT (10.11 ± 0.29 mg), SS (9.05 ± 0.29 mg) and control (8.85 ± 0.41 mg). It decreased with time of day, with mean values of 11.31 ± 0.26 mg, 10.18 ± 0.27 mg, 8.31 ± 0.32 mg and 8.14 ± 0.21 mg at 11:00, 13:00 15: 00 and 17:00, respectively  and peach (P. mongolica) were the most common competitive flowering species. The mean proportion of pollen that honeybees collected from the target crop (pear) by weight was highest (85.7%), followed by peach pollen (8.7%), dandelion pollen (5.1%) and others (0.5%). Moreover, the proportion of pear pollen collected by weight significantly increased  across days and was highest on day 3. The proportion of pear pollen collected by weight was 75.3% on the first day and increased to 88.8% on the second day and 93.2% on the third day. On the other hand, the proportion of peach and dandelion pollen collected by weight decreased as the days advanced  ,33,42. IA. mellifera to pear flowers might be due to the low sucrose content of the flowers’ nectar [Sucrose is an important factor used as a primary signal reward to induce honeybees to collect more pollen . The low’ nectar . Sugar s’ nectar ,32,35.Sugar syrup feeding increased sweet cherry pollen collection by 2.16-fold, field bean pollen collection by 3.27-fold, red clover pollen collection by 5.2-fold and kiwifruit pollen collection by 7.9-fold ,35. In oPollen collection behavior of individual worker bees is crucial for determining the collective behavior of colonies . Pollen T. officinale) and peach (P. mongolica) are found as competitive species grown in pear orchard. Dandelion was sparsely grown in the pear orchard, and the proportion of pollen collected during PT decreased after midday on warm days. On the other hand, honeybees frequently visited peach flowers throughout the day, but the peach plant density in the orchard was too low to significantly compete with pear flowers. Accordingly, the effect of competitive species may be less important as PTSS is continued. Removal of other competitive flowering plant species from pear orchard should significantly enhance the collection of pear pollen and the pollination efficiency of honeybees.Certain plants in the vicinity of a pear orchard may be more attractive to honey bees than pear flower, because they produce more nectar or pollen. As a reIn conclusion, pear flowers have short flowering duration, which requires designed pollination to efficiently utilize honey bees. Honey bee colonies adaptively alter pollen collection behavior responding to colony managements. Given the described behavior of honey bees, that do not prefer for pear flower, the use of pollen traps with sugar syrup feeding increases pear pollen collection and probably its pollination. The techniques can serve as rapid and inexpensive pollination management of honey bee colonies and easily applied by pear growers. Beekeepers might be benefited from renting colonies and pear growers are likely to enhance pear pollination services for optimum pear fruit production.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.