MPSs) are a group of rare lysosomal storage disorders characterized by the accumulation of glycosaminoglycans in various tissues and organs. Ocular problems that affect the cornea, trabecular meshwork, sclera, retina, and optic nerve are very common in these patients. However, there was limited literature focusing on comprehensive ocular findings in different types of MPS.Mucopolysaccharidoses .We retrospectively reviewed the clinical ophthalmologic features and electrodiagnostic results of 50 Taiwanese patients with a diagnosis of MPS VI, 38% with MPS IV, 29% with MPS I, and 14% with MPS II. Severe corneal opacities existed in 57% of MPS VI patients and 11% of MPS I patients, compared with none for MPS II and MPS IV patients. Among 80 eyes with available data of refraction, 11 eyes (14%) had myopia (≦−0.50 D), 55 eyes (69%) had hyperopia (≧0.50 D), and 55 eyes (69%) had high astigmatism (≧1.50 D). Ocular hypertension was found in 45% (28/62) of eyes. There were 16% (14/90), 11% (10/90), 13% (12/90), 31% (27/86), and 79% (30/38) of MPS eyes with lens opacities, optic disc swelling, optic disc cupped, retinopathy, and visual pathway dysfunction, respectively. Intraocular pressure was positively correlated with the severity of corneal opacity (p < 0.01).Among 44 patients with available data for visual acuity, 15 patients (34%) had a visual acuity of less than 0.5 (6/12) equivalent in their better eye, including 71% of those with MPS patients. Diagnostic problems may arise in these patients with severe corneal opacification, especially for those with MPS VI and MPS I.Ocular complications with significant reduction in visual acuity are common in  Seven distinct types of MPS disorders  with 11 specific lysosomal enzyme deficiencies have been reported. The clinical manifestations of MPS are progressive and chronic with a wide spectrum of clinical severity and prognosis among the different types . We also evaluated the relationship between each ophthalmologic manifestation and different types of MPS.22.1The study protocol was approved by the Ethics Committee of Mackay Memorial Hospital, and written informed consent was provided by a parent of the children and from the patients themselves if they were over 18 years of age.2.2We retrospectively reviewed the medical records, clinical ophthalmologic features and electrodiagnostic results of 50 Taiwanese patients with a diagnosis of MPS  at Mackay Memorial Hospital between January 1996 and December 2017. The diagnosis of the type of MPS was confirmed by specific enzyme activity assays in serum, leukocytes and/or skin fibroblasts, two‐dimensional electrophoresis of urinary GAGs, and/or identification of a pathogenic mutation , lens opacity, and intraocular pressure (IOP) measurement (if possible) were documented. The examinations comprised assessment of best‐corrected visual acuity by Snellen charts, measurement of IOP by noncontact air‐puff tonometry  in the clinic, slit‐lamp examination of the anterior segment , as well as direct and indirect funduscopy. The degree of corneal opacity was subjectively graded by one observer (WCC) as mild (+), moderate (++), or severe (+++). For refractive error examinations by the use of an autorefractor, myopia was defined as sphere power ≦−0.50 D, hyperopia was defined as sphere power ≧0.50 D, and high astigmatism was defined as cylinder power ≧1.50 D , and significance was tested using Fisher's r–z transformations. Two‐tailed p‐values were computed. All statistical analyses were performed using SPSS version 11.5 , and differences with p < 0.05 were considered statistically significant.All results were calculated using descriptive statistics, including numbers and percentages for categorical variables, as well as mean, median, and range  for continuous variables. We compared ocular characteristics among different types of MPS. The relationship between IOP and severity of corneal opacity was determined using Pearson's correlation coefficient  had a visual acuity of less than 0.5 (6/12) equivalent in their better eye, including 71% of patients with MPS VI, 38% with MPS IV, 29% with MPS I, and 14% with MPS II. Among 100 eyes with a medical record of amblyopia evaluation, 22 eyes (22%) had amblyopia, including 71% of cases with MPS VI, 28% with MPS I, 15% with MPS IV, and 6% with MPS II.3.2Among 100 eyes with a medical record of cornea condition, all patients with MPS I and VI and 94% of those with MPS IV had various degrees of corneal opacity, compared with all patients with MPS II having clear corneas. Severe corneal opacities were manifested in 57% of patients with MPS VI and 11% of MPS I patients, compared with none for MPS II and MPS IV patients  had myopia (≦−0.50 D), 55 eyes (69%) had hyperopia (≧0.50 D), as well as 55 eyes (69%) had high astigmatism (≧1.50 D). Twenty‐five percent of MPS IV patients had myopia, followed by 11% of MPS II cases. However, none of the patients with MPS I and MPS VI had myopia. All MPS VI cases had hyperopia, followed by 92% of MPS I patients, 68% of those with MPS II, and 53% of MPS IV patients. Eighty‐three percent of MPS I cases had high astigmatism, followed by 72% of patients with MPS IV, 63% with MPS VI, and 61% with MPS II   was found in 45% (28/62) of eyes, including 64% of MPS VI cases, 55% with MPS II, 33% with MPS I, and 31% with MPS IV. Severe ocular hypertension (IOP > 30 mmHg) was identified in 11% (7/62) of eyes, including 29% with MPS VI and 25% with MPS I, compared with no patients having MPS II and MPS IV  of eyes, including 38% of the patients with MPS I, 18% with MPS II, 6% with MPS IV, and none with MPS VI.3.6Among 90 eyes with available data for optic disc condition, optic disc cupped was identified in 13% (12/90) of the eyes, including 31% with MPS II, 6% with MPS IV, and none of MPS I and MPS VI.3.7Among 86 eyes with available data for retina condition, retinopathy  was found in 31% (27/86) of the eyes, including 50% with MPS II and VI, 43% with MPS I, and 6% with MPS IV  showed VEP delay indicative of visual‐cortical pathway dysfunction, including all MPS VI patients, 80% with MPS I, 67% with MPS II, and 50% with MPS IV.4To the best of our knowledge, this is the first large cohort to describe the ophthalmologic features in Asian patients with MPS. There was limited original literature focusing on comprehensive ocular findings in different types of MPS  is a disorder of sight owing to the eye and brain not working well together leading to decreased visual acuity in an eye that otherwise typically appears normal. Ashworth et al.  describen = 31) had some degree of corneal opacity. Both two MPS II patients had clear cornea. Thirty‐one percent of MPS VI patients (n = 16) had mild corneal opacity, 25% had moderate, and 38% severe. In our study of 100 eyes, all MPS I patients had certain degree of corneal opacity, all MPS II patients had clear cornea, 29% of MPS VI patients had mild corneal opacity, 14% had moderate, and 57% severe. Our results agree with theirs.Pastores et al.  reportedHyperopia may be postulated to be reduced refractive power due to a more rigid and flattened cornea, as well as shortening of the axial length and sclera thickening as a result of GAGs storage in the sclera , which was consistent with the findings by Ashworth et al. , and the number of patients was small. Despite these limitations, our results were consistent with those of other case series in the literature that reported a high prevalence of ocular impairment among MPS patients. Thus, further exploration of the issue in larger cohorts with longer follow‐up periods is warranted.5Ocular complications, including corneal clouding, refractive errors , ocular hypertension, lens opacity, optic disc cupped or swelling, retinal pigment epithelial change, visual‐cortical pathway dysfunction, amblyopia with significant reduction in visual acuity are common in Taiwanese MPS patients. These patients require regular ophthalmologic evaluations for the early detection and management of their ocular complications. MPS VI and MPS I were associated with the most severe types of ophthalmologic manifestations among different types of MPS. IOP was positively correlated with the severity of corneal opacity. Diagnostic problems may arise in patients with severe corneal opacification, especially for those with MPS VI and MPS I. These findings and the follow‐up data can be used to develop quality of care strategies for such patients.The authors declare that they have no competing interests.


High-pressure processing (HPP) is a non-thermal technology that is being increasingly applied in food industries worldwide. It was proposed that this method could be used as an alternative to holder pasteurization  in milk banks but its impact on the immunologic, enzymatic and hormonal components of human milk has not yet been evaluated in detail. The aim of our study was to compare the effects of HPP in variants: (1) 600 MPa, 10 min (2) 100 MPa, 10 min, interval 10 min, 600 MPa, 10 min (3) 200 MPa, 10 min, interval 10 min, 400 MPa, 10 min (4) 200 MPa, 10 min, interval 10 min, 600 MPa, 10 min in temperature range 19–21°C and HoP on the leptin, adiponectin, insulin, hepatocyte growth factor (HGF), lactoferrin and IgG contents in human milk. HoP was done at the Regional Human Milk Bank in Warsaw at the Holy Family Hospital on S90 Eco pasteurizer . Apparatus U4000/65  was used for pascalization. Milk samples were obtained from women during 2–6 weeks of lactation. Post-treatment culture showed no endogenous bacterial contamination in any tested option. Concentrations of selected components were determined using ELISA tests. The level of all analyzed components were significantly decreased by HoP: leptin 77.86%, adiponectin 32.79%, insulin 32.40%, HGF 88.72%, lactoferrin 60.31@.%, IgG 49.04%. All HPP variants caused an increase in leptin concentration, respectively (1) 81.79% (2) 90.01% (3) 86.12% (4) 47.96%. Retention of insulin after HPP was (1) 88.20% (2) 81.98% (3) 94.76% (4) 90.31% HGF (1) 36.15% (2) 38.81% 97.15% (3) 97.15% (4) 43.02%, lactoferrin (1) 55.78% (2) 57.63% (3) 78.77% (4) 64.75%. Moreover, HPP variant as 200 + 400 MPa preserved IgG (82.24%) better than HoP and resulted not statistically significant change of adiponectin level (38.55%) compare to raw milk. Our results showed that HPP leads to preservation of adipokines, growth factor, and lactoferrin, IgG much better or comparable with HoP. Mother's milk is a natural first choice feed for every newborn, whether born in term or prematurely. Access to human milk is critical especially for very preterm babies for their current health condition and later life prognosis , 2. In tHepatocyte Growth Factor (HGF) and multifunctional milk protein as lactoferrin act in synergy to support the function of the immature gastrointestinal tract of newborns  after delivery at term with a surplus of milk during the 2–6 weeks of lactation given informed consent. Samples of ~50 ml of milk were collected at home or in the hospital ward using an electric or manual pump, stored in a refrigerator at temperature 4°C and delivered to human milk bank unit within 24 h while maintaining the refrigeration conditions.The Bioethics Committee of Warsaw Medical University has accepted the information about conducting this non–interventional study (admission number AKBE/59/15).High Pressure Processing (HPP): (1) 600 MPa, (2) 200 + 400 MPa, (3) 100 + 600 MPa, (4) 200 + 600 MPa and holder pasteurization (HoP). The control sample was raw, untreated human milk  after which the fat layer and cells were removed and supernatants were aliquoted into Eppendorf tubes prior to freezing at −21°C. Human milk samples were frozen within 48 h of collection.Staphylococcus aureus (PN-EN ISO 6888-1: 2001 / A1: 2004).Microbiological analysis were performed to verify microbiological purity in the case of selected raw and treated milk samples. The analysis were carried out in three replications to the total number of mesophilic aerobic microorganisms  and the number and Human milk samples were exposed to high pressure treatment at the Institute of High Pressure Physics, Polish Academy of Sciences, using U 4000/65 apparatus (designed and produced by Unipress Equipment). The maximum pressure available in the apparatus was 600 MPa, the treatment chamber had a volume of 0.95 L. The pressure-transmitting fluid used was distilled water and polypropylene glycol (1:1). Manufactory designed working temperature of the apparatus ranges from −10°C to +80°C. In our experiments, the temperature of the tested condition was between 19 and 21°C. A pressure of up to 600 MPa was generated in 15–25 s; the release time was 1–4 s.19 and 21°C. Samples were prepared in 4 variants: (1) 600 MPa (2) 200 MPa, 10 min; interval 10 min; 400 MPa, 10 min (3) 100 MPa, 10 min; interval 10 min; 600 MPa, 10 min (4) 200 MPa, 10 min; interval, 10 min; 600 MPa, 10 min.Holder Pasteurization (HoP) of human milk samples was done at the Regional Human Milk Bank in Warsaw at the Holy Family Hospital on automatic Human Milk Pasteuriser S90 Eco . Samples of 50 ml were treated according to Regional Human Milk Bank standard pasteurization protocol at 62.5°C for 30 min. The correctness of the process was confirmed with the data logging system, by recording temperature of the bottle probe every minute.2 fragment of goat anti-human IgG  was used as a coating agent of the wells of a microtiter plate  to bind IgG from the sample. For testing 100 μl of 100 μl of 100-, 250-, 500-, and 1,000-fold diluted milk and IgG standard preparation from 0.2 to 12.5 ng/100 μl  were taken. The amount of IgG bound was quantified by phosphatase-labeled rabbit anti-human IgG Fcγ fragment specific antibodies .All of the bioactive components were determined by the ELISA method. The assay detecting particular components in the milk was done at least in three times using milk samples proccesing in independent experiments. The concentration of IgG was determined according to a procedure described earlier . BrieflyFor lactoferrin determination monoclonal anti-human lactoferrin antibody  was used as a coating agent of the wells of a microtiter plate  to bind lactoferrin from the sample. For testing 100 μl of 5 000-, 10 000-, 25 000- and 50 000- fold diluted milk and lactoferrin standard preparation from 0.8 to 25 ng/100 μl  were taken. The amount of lactoferrin bound was quantified by phosphatase-labeled rabbit anti-human lactoferrin antibodies .The IgG and lactoferrin tests were assayed with 4-nitrophenyl phosphate  as the enzyme substrate and absorbance was measured in a Stat Fax 2100 Microplate Reader  at 405 nm with 630 nm as the reference filter. All ELISA immunobinding and washing steps were carried out in a TRIS-buffered saline  containing 0.2% Tween 20. All samples were analyzed at four different sample dilutions, each in duplicate.The concentrations of leptin, adiponectin, HGF, and insulin were analyzed with commercial ELISA kits using microplates pre-coated with a monoclonal antibody specifically for test substances. The following tests were used: Human Leptin ; Human HMW Adiponectin/Acrp30 ; Human HGF ; Insulin ELISA . For the study, the option for serum/plasma was chosen as the most adequate for human milk. The method was pre-tested on various samples dilutions as proposed in the protocol. As a result of this validation, the final analyses were performed on undiluted milk. Each sample in ELISA assay was measured in duplicate.The detections of adiponectin, HGF and leptin, insulin were done by microtiter reader  set to 450 nm with 570 nm as the reference filter. For data analysis Gen5 Data Analysis Software was used.p ≤ 0.05) were computed. The actual analysis was based on the overview of overlapping of obtained intervals.Statistical analysis concerns results obtained for six conditions: raw milk, pasteurized milk (HoP), and milk exposed to four variants of high pressure processing (HPP): 600 MPa, 200 MPa +400 MPa, 100 MPa+ 600 MPa, 200 MPa+ 600 MPa. As a calculation tool MS Excel was used. The results of determined parameters for each sample are showed as a percentage of the initial value observed for raw milk as 100%. Next, means, SD and 95% coefficient intervals  in IgG content. HPP variants 600 MPa, 100 MPa +600 MPa, and 200 MPa + 600 MPa also decreased statistically significantly the IgG content, 69.68, 69.16, and 68.46%, respectively. The reduction of IgG in 200 MPa +400 MPa (17.76%) was not statistically significant Table .In the case of lactoferrin HoP caused a statistically significant reduction of this protein (60.31%) in the human milk. HPP variants 600 MPa, 100 MPa +600 MPa, and 200 MPa + 600 MPa also decreased the lactoferrin content statistically significantly, 44.22, 42.37, and 35.25%, respectively. The reduction of lactoferrin in 200 MPa +400 MPa (21.23%) was not statistically significant Table .Leptin level was significantly reduced by HoP (77.86%) in comparison to the raw milk. In the matter of high—pressure, all HPP variants caused an increase in leptin concentration, (1) 600 MPa; 81.71%, (2) 200 MPa +400 MPa; 86.12%, (3) 100 MPa + 600 MPa; 90.01%, (4) 200 MPa +600 MPa; 47.96%, respectively. Indeed, none of the high pressure variants were significantly different from raw milk Figure .HoP caused a statistically significant decrease in adiponectin content (31.19%) but not so serious as HPP variants 600 MPa, 100 MPa +600 MPa, and 200 MPa + 600 MPa which reduced the level of protein almost totally: respectively as 97.99, 89.27, and 95.91%. The reduction of adiponectin at 200 MPa +400 MPa was slighter (61.45%), but also statistically significant Figure .Results obtained for HGF were very similar to adiponectin. HoP caused a statistically significant reduction level of HGF detected in human milk (88.72%). Although the level after HPP treatment by 600 MPa, 100 MPa +600 MPa, and 200 MPa + 600 MPa was decreased not more than 63.85, 61.19, and 56.98%, respectively, it was still significant. Only when it comes to HPP variant 200 MPa +400 MPa was the change in HGF level was almost imperceptible and not statistically significant (2.85%) Figure .The level of insulin was diminished under the influence of HoP by 32.40% in comparison to raw milk. Among of HPP variants only the treatment of human milk by high pressure as 600 MPa caused statistically significant destruction of protein (11.80%). The reduction in insulin content in human milk after others HPP variants was as following: 200 MPa +400 MPa-−5.24%, 100 MPa + 600 MPa-−18.02%, 200 MPa +600 Mpa-−9.69% but it was not statistically significant Figure .p ≤ 0.05.The content of selected bioactive compounds in raw milk was assumed as 100%, additionally the range of obtained results (minimum-maximum) was presented as an error bar. The asterisks indicate a pair of results that differ statistically significantly with Low Temperature Long Time pasteurization, LTLT) was combined to assure microbiological safety and nutritional value  and lactoferrin. Preservation on immunocomponents in donor milk, are well documented in spite of contradictory results –38. NeveIn our study the mean IgG concentration in untreated milk samples was 11.22 ± 8.83 μg/ml (range 5.74–21.40 μg/ml) Table In the case of lactoferrin the mean concentration detected in raw milk samples was 1.63 ± 0.47 mg/ml (range 1.17–2.11 mg/ml) which is similar to those reported in the literature  Table 1. All HPPTaking into account the importance of short and long lasting outcome of donor milk nutritional therapy, we decided to extend the research in to evaluate the possibility to preserve the biologically active peptides found in human milk as key metabolism regulation components.We suspected that HPP variant 200 MPa +400 MPa already tested with success for immunocomponents preservation, will be preferable in comparison to thermal treatment, for hormones such as leptin, adiponectin, insulin, and HGF as well.As was revealed before, HPP was even most effective as holder pasteurization in the elimination of inoculated microbiological flora of human milk . In our The most recent update of Cochrane metaanalysis which evaluated growth and development of preterm born infants fed with formula comparing with donor milk has proven that supplementing mother's milk with pasteurized human milk results in lower rates of weight gain, linear growth and head growth . AlthougSmall for-Gestational Age (ang. SGA) neonates has been observed to be inversely related to rates of intrauterine growth, suggesting a possible role of leptin in promoting fetus growth  which is similar to those reported in the literature  Table 1, Table 2In the case of adiponectin, the mean concentration detected in raw milk samples was 5.30 ± 2.05 ng/ml (range 2.94–3.68 ng/ml) which was comparable to others findings Table . The aveThe mean concentration of HGF in raw milk samples was 1306.15 ± 956.99 mlU/ml (range 413.20–2261 mlU/ml) Table . The HGFInsulin has been detected at very high levels in the human colostrum of healthy mothers (114–306 mU/L) after that decreasing by day 5 postpartum to the physiological levels at the fasting state . InsulinIn our study the mean insulin concentration in raw milk samples was 10.24 ± 4.02 pg/ml (range 6.10–14.67 pg/ml) which is similar to data reported in the literature  Table 1.In conclusion, our experiments showed that the 200+400 MPa variant is the best option of high pressure to preserve several metabolic hormones and immunocomponents of human milk. Our findings with persistent bioactive peptides in pascalized human milk is in line with the earliest reports concerning retention of others hormones after HPP with great significance for the infant's health , 61, 62.We showed, for the first time, preservation of several metabolic hormones in donor milk HPP processing. Growing recent studies in nutritional therapy of preterm's have emphasized the role of our discovery , 64.We believed that preservation of important bioactive peptides as hormones and growth factors in human milk is especially important in the context of delivering donor milk for very preterm infants.Lack of protocol for evaluation of new techniques was a severe obstacle to improving the pasteurization stage of donor milk. In the next step we would like to follow current expert's recommendation for validation of new method of human milk processing and fit designed methodology for this, especially in the matter of microbiology.AW: main contributions to the conception and design of the work, analysis and interpretation of data for the work, drafting the work. ES-D and OB: acquisition, analysis and interpretation of data for the work, revising it critically for important intellectual content. KS and MR: acquiring the main part of the data used for the work. KK statistical analysis and interpretation of data for the work. ER: acquiring part of the data for the work. GO, SR, and MB-K: revising it critically for important intellectual content, providing approval for publication of the content. MO-P: acquiring part of the data for the work, revising it critically for important intellectual content. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.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.


Colletotrichum, Pestalotiopsis, Phoma, Phomopsis, Sporomiella, among others. Since a small fraction of fungi is able to grow in culture, this study investigated the diversity of fungi associated with leaves of Acanthus ilicifolius var. xiamenensis using both isolation and metabarcoding approaches. A total of 203 isolates were cultured from surface-sterilized leaves, representing 47 different fungal species: 30 species from the winter samples (104 isolates), and 26 species from the summer samples (99 isolates). Ascomycota was dominant in both types of leaf samples, while Basidiomycota was isolated only from the summer samples. Drechslera dematioidea , Colletotrichum sp. 3 (7.69%) and Alternaria sp. (7.69%) were dominant in the winter samples; Fusarium oxysporum (13.13%), Diaporthe endophytica (10.10%) and Colletotrichum sp. 1 (9.09%) in the summer samples. Overall, Corynespora cassiicola (6.90%), F. oxysporum (6.40%) and Guignardia sp. (6.40%) had the highest overall percentage of occurrence. In the metabarcoding analysis, a total of 111 operational taxonomic units (OTUs) were identified from 17 leaf samples: 96 OTUs from the winter and 70 OTUs from the summer samples. Sequences belonging to Ascomycota and Basidiomycota were detected in both samples but the former phylum was dominant over the latter. Based on read abundance, taxa having the highest percentage of occurrence included Alternaria sp. (3.46%), Cladosporium delicatulum (2.56%) and Pyrenochaetopsis leptospora (1.41%) in the winter leaves, and Aureobasidium sp. (10.72%), Cladosporium sp. (7.90%), C. delicatulum (3.45%) and Hortaea werneckii (3.21%) in the summer leaves. These latter four species also had the highest overall percentage of occurrence. Combining the results from both methods, a high diversity of fungi (at least 110 species) was found associated with leaves of A. ilicifolius var. xiamenensis. Many of the fungi identified were plant pathogens and may eventually cause diseases in the host.A high diversity of culturable foliar endophytic fungi is known from various mangrove plants, and the core taxa include species from  Mangroves are tropical intertidal forest communities, situated at coastal areas from low to high salinities . These cSporormiella minima, Guignardia/Phyllosticta spp., Phoma spp., Diaporthe/Phomopsis spp., Cladosporium spp., Acremonium spp. and Collectotrichum spp. Xylaria spp. and Pestalotiopsis spp. were also common  were cut out from each leaf, surface-sterilized by immersing in 70% ethanol for 10 s and 4% sodium hypochlorite solution for 30 s, washed twice in sterile distilled water, and plated on 2% malt extract freshwater agar , supplemented with 0.5 g/L each of streptomycin sulfate  and Penicillin G . The inoculated plates were incubated at 25 °C and checked daily to observe fungal growth from the leaf discs for 1 month. Hyphal tips of different mycelial morphotypes from each plate  were isolated and subcultured onto fresh MEAF. All cultures were kept at National Taiwan Ocean University.2 and 1.25 U of Taq Polymerase . The amplification cycle consisted of an initial denaturation step of 95 °C for 2 min followed by 35 cycles of (a) denaturation (95 °C for 1 min), (b) annealing (54 °C for 1 min) and (c) elongation (72 °C for 1.5 min) and a final 10 min elongation step at 72 °C. The PCR products were analysed by agarose gel electrophoresis and sent to Genomics BioSci & Tech  for sequencing. The sequences returned were checked for ambiguity and the forward/reverse strands were assembled in MEGA7 . Mycelia for each morphotype were ground into fine powder in liquid nitrogen using a mortar and pestle. Genomic DNA was extracted using the DNeasy Plant DNA Extraction Kit  according to the manufacturer’s instructions. ITS was amplified using the primer pairs ITS1 (or ITS5)/ITS4 . PCR reain MEGA7 . The assSeventeen leaves used for the isolation described above were freeze-dried. Total genomic DNA was extracted using QIAGEN DNeasy Plant Mini Kit  according to the manufacturer’s instructions. A nested PCR approach was used to amplify a region of ITS spanning from 18S to 5.8S rDNA. The first set of primers was NSA3 (5′-AAACTCTGTCGTGCTGGGGATA-3′)/NLC2 (5′-GAGCTGCATTCCCAAACAACTC-3′)  and the The raw sequences were filtered with a phred score ≥Q29 . The raw reads were paired into single reads and adaptors, primers and barcode sequences were removed using the QIIME script “split_library.py” . ClusterTotal number of isolates , Richness , Species Richness , Shannon–Wiener Diversity Index, Pielou’s Evenness and Simpson Diversity Indices  were calculated in Microsoft Excel by first computing the variables of the equations and then using the math operators to calculate the different indices.Rarefaction and extrapolation sampling curves were computed and plotted to estimate sample completeness (sample coverage) in R package iNEXT (iNterpolation/EXTrapolation) with the 95% lower and upper confidence limits for the isolation and metabarcoding data . A princAcanthus ilicifolius var. xiamenensis collected in January and July 2014 at Kinmen Township, Taiwan and ITS of the representative isolate for each morphotype was sequenced , Colletotrichum sp. 3 (7.69%) and Alternaria sp. (7.69%); and in the summer samples, Fusarium oxysporum (13.13%), Diaporthe endophytica (10.10%), Colletotrichum sp. 1 (9.09%), Fusarium sp. (8.08%), Corynespora cassiicola (8.08%), Guignardia sp. (7.07%) and Alternaria alternata (7.07%). Overall, C. cassiicola (6.90%), F. oxysporum (6.40%), Guignardia sp. (6.40%), Colletotrichum sp. 1 (5.91%), D. endophytica (5.42%) and D. dematioidea (5.42%) had the highest percentage of occurrence.A total of 104 and 99 isolates were cultured from the winter (January) and summer (July) samples, representing 30 and 26 fungal species, respectively –2. Nine currence  in the wDiversity indices were calculated for the fungal communities in the winter and summer samples . The funSeventeen samples (leaves) were analyzed by the metabarcoding analysis: 10 for the winter and 7 for the summer samples. A total of 773685 reads were obtained after QIIME analysis, including 314692 reads from the winter samples ranging from 2169 to 54606 reads, and 458993 reads from the summer samples ranging from 39151 to 93295 reads . From thAt the class level, 11 different fungal classes were obtained from both the winter and summer samples . Seven cTwenty-two and nineteen different fungal orders were identified in the winter and summer samples, respectively . AgaricaAlternaria sp. (3.46%), Cladosporium delicatulum (2.56%) and Pyrenochaetopsis leptospora (1.41%) in the winter samples, and Aureobasidium sp. (10.72%), Cladosporium sp. (7.90%), C. delicatulum (3.45%) and Hortaea werneckii (3.21%) in the summer samples  than that in the summer samples (5.29) but the Shannon–Wiener Diversity Index was comparable between the two samples, 1.98 and 2.09 respectively . The oveThe fungal community among isolation/metabarcoding and winter/summer samples were analyzed by PCA and the result is shown in A. ilicifolius var. xiamenensis were obtained  was used to identify the cultures. ITS is easily amplifiable by PCR and has the highest probability of successful identification for the broadest range of fungi as compared to other rDNA regions and protein genes  than July 2014  for the summer samples. Generally, higher richness and abundance of endophytic fungi were found in hotter and wetter seasons .A. ilicifolius var. xiamenensis were common between the two sampling times, showing a seasonal variation of fungal diversity using the culture method. However from the metabarcoding analysis, 55 taxa were found to be common between the winter and summer samples , suggesting there was an overall similarity in fungal diversity between the samples. These results show the weakness of using isolation techniques as the sole methods to study diversity of endophytic fungi of mangrove plants , Phoma, Phomopsis  and Sporomiella  was much higher than those found in related studies in this species: 11 species from roots in Udupi, India  were obtained from both isolation and metabarcoding studies suggesting a much higher fungal diversity associated with leaves of  methods  and with methods .Avicennia marina and Rhizophora stylosa and Lecanoromycetes in R. stylosa using 454 pyrosequencing of 18S and ITS rDNA genes  of the mangrove plants NA genes . LecanorA. ilicifolius var. xiamenensis are well known pathogens such as Cladosporium, Colletotrichum, and Fusarium, which might ultimately cause plant diseases. The Botryosphaeriales was reported to potentially cause diseases of mangrove plants  stress when this fungus was added to the growth pots , F. oxysporum (6.40%) and Guignardia sp. (6.40%) had the highest overall percentage of occurrence. In the metabarcoding analysis, Ascomycota was also dominant over the Basidiomycota. Based on reads, Aureobasidium sp. (10.72%), Cladosporium sp. (7.90%), C. delicatulum (3.45%) and H. werneckii (3.21%) had the highest percentage of occurrence. The use of both methods discovered a much higher diversity of endophytic fungi associated with A. ilicifolius var. xiamenensis. The association of these fungi with the plant is not known and future studies should focus on the ecological roles of these fungi. However, a chemical analysis of the spent culture liquid of the fungal isolates in this study suggests that 28 isolates produced antimicrobial substances against some Gram-positive and Gram-negative bacteria and fungi and thus might protect the plant from microbial diseases (In conclusion, this study discovered a high diversity of fungi associated with leaves of diseases .10.7717/peerj.7293/supp-1Supplemental Information 1The fungal isolates were cultured from leaves of Acanthus ilicifolius var. xiamenensis collected at Lieyu Township, Kinmen County, Taiwan on malt extract freshwater agar supplemented with antibiotics.Click here for additional data file.


Orexin-A (OXA) has been originally isolated from a precursor peptide prepro-orexin from the lateral hypothalamus. The orexin system has been attributed to important functions in sleep, arousal and regulation of energy homeostasis. In addition to its high levels in cerebrospinal fluid, OXA is present in blood. However, reported peptide concentrations in plasma vary significantly depending on the method used. Therefore, a specific and sensitive OXA radioimmunoassay (RIA) with solid phase extraction method was developed to determine whether plasma OXA concentrations is affected by acute feeding and/or wake and sleep in young healthy males. Blood samples were collected for 24 h from nine healthy males  every 2 h starting at 11 a.m. Food was served at 12 p.m, 5:30 p.m, 8 p.m and 8 a.m and the sleep time was between 10 p.m and 7 a.m. Plasma samples were analyzed in addition for cortisol and melatonin levels. Blood pressure was monitored through the experimental period. OXA antibody was raised in rabbits. OXA antiserum had only minor cross-reactivity with prepro-orexin precursor (<0.001%), amino-terminal peptide (<0.001%), carboxy-terminal peptide (0.001%), and orexin-B (0.3%) with high sensitivity (0.15 pg/tube). Plasma OXA levels varied between 0.5 and 16 pg/ml in seven subjects and were undetectable (below 0.5 pg/ml) in two subjects. The OXA concentrations did not correlate to feeding nor wake/sleep, whereas cortisol, melatonin and mean arterial blood pressure presented a clear circadian rhythm in each subject. In conclusion, OXA is present in blood in low amounts and its levels do not follow autonomic nor neuroendocrine circadian rhythms. Thereby, studies examining regulatory mechanisms and influences of OXA from blood samples should interpret results very cautiously. Orexin-A and orexin-B, also called hypocretins, are peptides which were originally isolated from rat hypothalamus and are derived from the precursor peptide prepro-orexin (PPO) . DefectsNarcoleptic patients with cataplexy have low levels of OXA in their cerebrospinal fluid . They alImmunoreactive OXA is present in human plasma, but its source for secretion is still unclear . In the CSF OXA shows a circadian rhythm with peak concentrations late in the night . DespiteNine young healthy male volunteers with no anamnestic narcolepsy  participated in the study. The experiment followed the standards set by the Declaration of Helsinki. The study protocol was approved by the institutional ethics committee . All subjects gave their informed consent. The Municipal Board of ethics committee approved the trial under the registration number 14/2009.No alcohol, smoking or heavy exercise was allowed 24 h before the study. Participants arrived at 9:00 a.m to the Institute. A catheter was inserted into the antecubital vein of the forearm. The testing facility offered computers, television and books/magazines for entertainment. Energy controlled food was available at 12 p.m , 5:30 p.m , 8 p.m , and 8 a.m . Lights were turned off at 10 p.m, and back on at 7 a.m. The volunteers were woken up for each blood sampling and continued sleeping thereafter. Blood samples were drawn and collected in 10 ml EDTA-coated tubes every 2 h starting at 11 a.m. Blood tubes were put immediately on ice, centrifuged at 4°C at 3,000 rpm for 10 min and the plasma was separated into tubes and stored in −70°C until analysis. Blood pressure was monitored throughout the study after each blood draw, except at 1 p.m, with an automatic blood pressure monitor .ad libitum. The animals' pain and suffering was minimized during the experiment and euthanasia. The animal experiment was approved by the University of Oulu Ethical Committee for Animal Experiments and by the Regional Committee of the State Provincial Office. The animal experiment was performed according to the “legislation for the protection of animals used for scientific purposes” (the European Community Council Directive 86/609/EEC).OXA antigen was prepared by coupling 2 mg of synthetic OXA  to 8 mg of bovine thyroglobulin  by 100 mg carbodiimide  at (+4°C for 1 h). Two adult male rabbits weighing 4 kg were immunized with 0.5 mg of the conjugate in Freund's complete adjuvant  and boosters of 0.3 mg were given at 6 and 10 weeks. At 12 weeks the rabbits were bled and the antisera collected. The rabbits had access to their regular rabbit diet and water Antiserum from the rabbit 9/3 bound 40% of the tracer at the dilution of 1:500,000 and showed significant displacement of the tracer at the level of 0.015 pg OXA/tube. Cross-reactivity of OXB  was 0.3% and that of PPO , 1-15 OXA (Orexin A (1-15), Catalog# 003-49, Phoenix Pharmaceuticals) and 16-33 OXA amide (Orexin A (16-33) Amide, Catalog# 003-36, Phoenix Pharmaceuticals) <0.001%.125I to OXA (1 μg) by Chloramine T (10 μg) and purified in a 0.5 × 5 cm Sephadex G25 column eluted by 30% acetic acid followed by RP-HPLC on a Symmetry C18 column . The column was eluted with a 30 min gradient of 20–50% acetonitrile in 0.1% trifluoroacetic acid.The tracer was prepared by radioiodination 0.5 m Ci NaCalibrator solutions, samples and antisera were made up in RIA assay buffer . Duplicate calibrators (OXA 0.075–25 pg/tube), orexin related peptides  and dilutions of evaporated plasma samples were prepared. Tracer containing 5,000 counts per minute and antiserum at dilution 1:500,000 were incubated with calibrators or samples for 24–48 h at 4°C, each in volumes of 100 μl. The immunoglobulins were precipitated by centrifugation with donkey anti-rabbit IgG in 0.5 ml of 80 g/l polyethylene glycol 6,000 containing normal rabbit serum as a carrier.18 cartridges in an automated Gilson Aspec system . After the loading the columns were washed with 1% acetonitrile and OXA was eluted by 2 ml of 80% acetonitrile in. The eluates were evaporated by SpeedVac  and the extracts were reconstituted with 250 μl of the RIA buffer.Human plasma samples (1 ml) were mixed with of 1 mol/l HCl (0.2 ml) containing 16 g/l glycine and submitted to solid phase extraction with Sep-Pak Cn = 8) and for 50 pg 60 and 65% (n = 8). The OXA levels in all the subjects were measured in duplicates in one assay with intra-assay variation of coefficient of 8.2%.Serial dilutions of extracted human plasma samples were parallel with OXA calibrators (data not shown). We determined recoveries by measuring plasma pools to which 25 or 50 pg synthetic OXA was added before solid phase extraction. In two separate experiments the recoveries for 25 pg of OXA were 57 and 62% (Evaporated solid phase extracts of human plasma (2–4 ml) were applied to Sephadex G50 fine column (0.9 × 50 cm) eluted with 30% acetic acid. Fractions of 1 ml were collected, evaporated and assayed for OXA and absorbance at 280 nm. OXA containing fractions were further submitted to RP-HPLC and assayed for OXA. Gel filtration and HPLC runs were also performed in samples in which 50 pg of synthetic OXA was added. Synthetic and plasma OXA eluted at 13–15 ml and was subjected to HPLC after lyophilization.18 Cartridges and further subjected to HPLC. Elution profile was a linear gradient from 0 to 60% of acetonitrile in 0.1% trifluoroacetic acid. A flow-rate of 1 ml/run was used. Only one OXA immunoreactive peak was observed eluting as the synthetic OXA.Four ml of human plasma was extracted with Sep-Pak CPlasma melatonin levels were analyzed using an in-house RIA . SamplesPlasma cortisol was measured using a RIA assay according to manufacturer's instructions . Plasma samples of 20 μl were used per well in duplicates. The intra-assay coefficient of variation was 5.7%.Serial measurements of plasma OXA, cortisol and melatonin concentrations from each subject were analyzed using summary measures . In addiDBP + 1/3 (SBP–DBP), where DBP represents diastolic blood pressure and SBP represents systolic blood pressure.The novel OXA assay used in this study had a high sensitivity of 0.15 pg of peptide/tube. OXA antiserum had minor cross-reactivity with PPO (<0.001%), aminoterminal peptide (<0.001%), and carboxyterminal peptide (0.001%). Antiserum showed 0.3% cross-reactivity with OXB. In RP-HPLC one immunoreactive peak from plasma extracts was observed eluting at 26 min. Synthetic OXA eluted at the same time  between the samples taken at different time points.Diurnal plasma OXA concentrations varied greatly between subjects Figures . Five suPlasma cortisol concentrations varied according to a normal daily cycle with decreasing levels toward the early morning and increasing levels toward the mid-morning Figure . Means fPlasma melatonin concentrations showed a clear circadian variation within all subjects with low concentrations during daytime and high concentrations during nighttime Figure . DaytimeMean arterial pressure showed circadian rhythm with higher pressure during daytime compared to night time Figure .OXA affects wake/sleep cycle and feeding. In the present study we established a sensitive OXA RIA and demonstrated that plasma levels were low but detectable, and did not relate to sleep/wake rhythm or feeding while melatonin and cortisol levels followed their known circadian rhythms.OXA and/or its receptor(s) have been detected in peripheral tissues such as adrenal gland, pancreas, and testis , 28–31. There are different RIAs and ELISAs to measure OXA in human and animal plasma. The measured concentrations, however, vary greatly between the studies depending on study design and methodologies, ranging from lower than 1 pg/ml to about 2,500 pg/ml , 32–37. In CSF, OXA has been shown to follow circadian rhythm with high concentrations late at night , 9. HoweWhether the failure of the orexin system of the narcoleptic patients reflects also changes in plasma OXA levels is currently unknown. Higuchi et al. reported significantly lowered plasma OXA concentrations in narcoleptic patients, while Dalal et al. using an RIA kit from a different vendor did not find any differences between control and patient samples , 33. In Orexins are known for their actions on both short- and long-term feeding. Intra-cerebroventricular injection of OXA induces feeding in rodents –16. SimiWe demonstrated using a sensitive RIA and solid phase extraction of plasma samples that OXA is detectable in low concentrations in most human samples, and not modulated by feeding or sleep/wake cycle in healthy male subjects. OXA is produced throughout the CNS and can cross the blood brain barrier . OXA conKM, JL and K-HH designed the study, acquired the data, took (K-HH) and processed the blood samples, and analyzed the samples. TK and AJA contributed to the development of OXA RIA. OV did the analysis of melatonin and contributed to revision of the manuscript. KM drafted the first version of the manuscript. All authors contributed and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mycobacterium tuberculosis is divided into several lineages, each with a different geographical distribution. M. tuberculosis lineage 1 (L1) is common in the high-burden areas in East Africa and Southeast Asia. Although the founder effect contributes significantly to the phylogeographic profile, co-evolution between the host and M. tuberculosis may also play a role. Here, we reported the genomic analysis of 480 L1 isolates from patients in northern Thailand. The studied bacterial population was genetically diverse, allowing the identification of a total of 18 sublineages distributed into three major clades. The majority of isolates belonged to L1.1 followed by L1.2.1 and L1.2.2. Comparison of the single nucleotide variant (SNV) phylogenetic tree and the clades defined by spoligotyping revealed some monophyletic clades representing EAI2_MNL, EAI2_NTM and EAI6_BGD1 spoligotypes. Our work demonstrates that ambiguity in spoligotype assignment could be partially resolved if the entire DR region is investigated. Using the information to map L1 diversity across Southeast Asia highlighted differences in the dominant strain-types in each individual country, despite extensive interactions between populations over time. This finding supported the hypothesis that there is co-evolution between the bacteria and the host, and have implications for tuberculosis disease control.Tuberculosis presents a global health challenge.  With tremendous efforts, some new drugs have recently been developed, but there has still been limited success in developing new effective tuberculosis vaccines. Thus a better understanding of the interactions between M. tuberculosis and its host is urgently needed.The burden of tuberculosis is high in developing countries in Asia, with 56% of all global cases found in five countries: India, Indonesia, the Philippines, Pakistan and ChinaM. tuberculosis lineages strongly associate with the geographic location, ethnicity and ages of the hosts2, which could be due to the founder effect or co-adaptation between the bacterial and host population, favouring their stable coexistence4. The latter hypothesis was supported by the sympatric association between bacterial lineages and patients’ origins5 which was weakened in HIV-infected patients6. The global population structure of M. tuberculosis lineage 4 further supported the co-evolution hypothesis7. Moreover, some candidate gene studies revealed an association with diseases caused by only some lineages8. Recently, genome-wide association studies (GWAS) successfully identified the HLA-DRB1*09:01 and CD53 genes associated with tuberculosis when the bacterial genotypes were considered10.13.Other aspects of genotype-phenotype associations have also been studied, particularly for lineages 2 and 4. The association varied between sublineages. The sublineages of the Beijing strains were associated with differential survival rates, virulence and responses to vaccine14 and almost completely congruent with the EAI (East African Indian) strains, defined by spoligotyping. The EAI strains were commonly reported in countries around the Indian Ocean16 and associated with higher ages of patients17. Pulmonary tuberculosis caused by L1 strains was more likely to be sputum negative and had a higher sputum conversion rate at two months after treatment18. L1 was less associated with drug resistance than L220. They were occasionally reported to associate with extrapulmonary tuberculosis21. In general, L1 isolates induced stronger cytokine responses and grew slower in macrophage culture23.Unfortunately, there have been few studies on the phenotypes of lineage 1 (L1). L1, as defined by SNP typing, is congruent with the Indo-oceanic strains, defined by LSP (large sequence polymorphisms)24. It is endemic in 11 of 30 high- burden countries. Some bacterial isolates, such as the “Asian human” type, from India and East Africa had distinct properties25 and were hypothesized to provide an explanation for the lack of efficacy of the BCG vaccine in the monumental clinical trial in Tamil Nadu, Southern India26. The described phenotypes might associate with L1 strains.Studies of L1 are essential for the End TB strategyet al. proposed classification based on a whole-genome single nucleotide variant (SNV) phylogenetic tree and provided a corresponding barcoding scheme27. While there have been many WGS studies of lineage 2 strains, studies of L1 strains are rather limited.Recently, Coll Comparing epidemiological findings between countries requires a reliable and high-resolution classification based on the bacterial genetic relationship. EAI isolates are classified by spoligotyping into “clades”, such as EAI1-EAI8. However, it is not clear whether the “clades” are monophyletic. There are “orphan” spoligotypes that are not yet assigned to any clades while assignment of some spoligotypes may be problematic. Correlation of the SNV phylogenetic tree to spoligotypes would provide better insights into the phylogeography of L1.M. tuberculosis L1 in Chiangrai, the northernmost province of Thailand, were sequenced and revealed significant diversity. Analysis of WGS was done to refine the SNV phylogenetics of L1, verify spoligotype clades and provide mapping between spoligotypes and the SNV phylogenetic tree. This finding provided a basis to map the distribution of predominant sublineages of L1 in various countries in Southeast Asia. Our results suggested an association between predominant sublineages and geography and ethnicity, despite a history of extensive interactions between various ethnic groups, supporting the hypothesis that there is some level of co-adaptation between M. tuberculosis L1 and human hosts.In this study, the genomes of 480 isolates of M. tuberculosis isolates, 480 were initially identified by LSP as belonging to the Indo-Oceanic family and had the 330 SNPs, previously reported to be specific to L127 (Supplementary Table Among 1174 successfully sequenced M. tuberculosis27. Its mean pairwise SNV distance (MPSD) in this study was 645.6 (range 0–965), while that of the globally reported L1 isolates was 7302, indicating high heterogeneity of L1 in Chiangrai. This finding might be related to the knowledge that Chiangrai is an ancient settlement originally with Austroasiatic language-speaking people and subsequently replaced by people who speak the Tai-Kadai language family29. It was historically controlled by several tribal kingdoms that resided now in Thailand, Myanmar and Lao. Chiangrai is home to several hill tribes and recent Chinese immigrants. Its modern roles as a transport hub and a major tourist attraction site lead to rapid urbanization and migration from throughout Thailand and other countries.A total of 41,157 SNVs were identified, with 22,491 SNVs, each present in only a single isolate. The total identified SNVs were more than in the previous reports  and 777777777777771 (SIT523) respectively. Neither pattern belongs to the EAI spoligotypes even though all four had the genetic markers of L1 (TbD1+ and RD239−). Isolates with SIT100 belonging to L1 have been previously reported31.The spoligotypes of 410 (85%) isolates were classified as EAI strains, while those of the other 66 isolates were not previously describedAnalysis of all 68 DVR segments revealed that all EAI isolates and unclassified isolates lacked DVR39-42, DVR44, known characteristics of EAI, and DVR48, which is not used for standard spoligotyping. This finding indicated that all unclassified isolates belonged to the EAI family. Some isolates had all the other 62 DVRs, indicating that the most recent common ancestor of the EAI group (EAI-MRCA) carried the deletion of only DVR39-42, 44 and 48.et al. studied 1601 globally collected WGS deposited in public databases, classifying 121 L1 isolates into five major groups, 1.1.1–1.1.3 and 1.2.1–1.2.227. The phylogenetic trees of the 480 isolates constructed by Bayesian inference  or the fixation indices greater than 0.33  of discrepancy. In seven cases, several DVRs were not identified computationally but they were detected by experimental spoligotyping, shown in Table The SNV phylogeny indicated that EAI2_MNL, EAI2_NTB and EAI6_BGD1 were monophyletic clades, corresponding exactly to L1.2.1.1, L1.2.1.2, and L1.1.3 respectively. Their DR regions were characterized by multiple deletions compared with the EAI_MRCA , typical for EAI5, belonging to 6 sublineages of L1.1.1  has only one more deleted DVR, DVR62, compared with SIT236. In this study, 14 of 20 isolates with SIT48, belonging to L1.2.2, L1.1.1.4 and L1.1.1.9, retained all the other unspoligotyped DVRs. As L1.2.2 separated from the other sublineages early in the evolution of L1 Fig. . Still, There were some sublineages associated with single DVR deletions such as L1.1.2.1-DVR18, L1.1.1.5-DVR18-21, L1.1.1.8-DVR53, and L1.1.1.6-DVR57-60. However, the associations were not completely specific and the single deletion event might allow for homoplasy, as in the case of DVR62 deletion.M. tuberculosis isolates from South and Southeast Asia usually have a single copy or low copy numbers of IS611032. Our study revealed that the isolates could belong to many sublineages except for L1.2.1 and L1.1.3  were usually found between studies performed in the same country, as expected, which might not be true if a large number of samples were intentionally selected from patients with specific forms of the disease, such as extrapulmonary or drug-resistant tuberculosis. There were numerous studies in India, where the percentages of L1 are known to be high in the southern and low in the northern part of the country. Nevertheless, the L1 spoligotype profiles were generally similar throughout the country, excluding eastern India.Correlations between studies in bordering countries were usually intermediate and higher than correlations between countries without common borders. Strong correlations were identified between Pakistan and Indian studies, particularly when homoplastic spoligotypes were excluded, conforming to the close historical relationship between the two countries. There were also strong correlations between the spoligotype profiles in Bangladesh and Myanmar (Supplementary Table Intriguing patterns of spoligotype profile correlations were observed in Southeast Asian countries. Countries in islands of Southeast Asia (ISEA), Indonesia and the Philippines, as well as Singapore and Taiwan are separated by seas but share similar L1 spoligotype profiles comprising predominantly EAI2_MNL (L1.2.1.1). In contrast, there was low similarity of spoligotype profiles between MSEA countries. In all cases, there were some levels of similarity of spoligotype profiles only between countries that shared common borders. The correlation coefficients between spoligotype profiles between countries without common borders were always low  isolates having SIT256, while the others had spoligotypes that could be derived from SIT256 by deletion. Both L1.2.1.2 and L1.1.1.8 had very low intragroup MPSDs of 106.4 and 174.4 and very high fixation indices of 0.61 and 0.63 respectively, indicating a close genetic relationship within each group. Together with the high prevalence, these results suggested high transmission activities of both sublineages.The two most common sublineages (L1.2.1.2 and L1.1.1.8) contributed to a third of L1 isolates in Chiangrai. Of the 94 members of L1.2.1.2, 83 (88%) had SIT89 or EAI2_NTB while the remaining 11 had new spoligotypes that could be derived from SIT89 by additional DVR deletions. The family was initially recognized by ISIn countries bordering Thailand, Cambodia, Malaysia and Myanmar, both SIT89 and SIT256 were reported but at much lower frequencies 39, should also belong to L1.2.1.1.EAI2_MNL or L1.2.1.1 was highly prevalent in the Philippines, accounting for more than 80% of all 40, while Cambodians speak the Khmer branch. The Thai language belongs to the Tai-Kadai family while Burmese belongs to the Tibeto-Burman family. The Malay language belongs to the Malayo-Polynesian branch of the Austronesian family. Each country also had a different predominant spoligotype belonging to different sublineages , while EAI2_MNL accounted for approximately 9%17, which might indicate a specific adaptation. In contrast, L1.2.1.1 was rare in Vietnam. This result is intriguing because the Cham people in the historical Champa Kingdom, in what is now southern Vietnam, spoke Austronesian. The kingdom lasted for several centuries and afforded ample interactions between Cham and Kinh, the major ethnic group in Vietnam. The rarity of L1.2.1.1 could be a consequence of the Champa Kingdom acquiring mainly Austronesian languages and culture rather than a large migration of genetic Austronesians42.The most common spoligotype in Vietnam was uniquely SIT139, a rare spoligotype classified as L1.1.1.1 in this study, accounting for 48% of Vietnamese L1 isolates43, which mostly belonged to L1.2.1.1, suggesting high transmissibility. Another study in Vietnam reported the clustering rate among EAI2_MNL (L1.2.1.1), an uncommon sublineage in that region, of only 18.3% while the clustering rate among EAI4_VNM, the predominant clade including SIT139, was 77.4%44. This result suggested that L1.2.1.1 and SIT139 spread differently in both populations, conforming to the co-adaptation hypothesis.Existing studies on the transmissibility of L1 supported the co-evolution hypothesis. A population-based study in Southern Taiwan revealed that the genotypic clustering rates of 60.8% among EAI strainsM. tuberculosis lineages at the population level across several studies. Although many recent studies provided genotypic details of the studied isolates, their phylogenetic relationships were usually not considered in the analysis45. Moreover, many studies used the phenotypes of the other strains in the same studied areas as the controls. The controls, therefore, varied between studies in both proportions of lineages and sublineages. Moreover, in most studies, information regarding host population genetic structures are usually not available.Robust phylogenetics provides a foundation for genotype-phenotype correlation studies. In general, it is difficult to compare phenotypes of In vitro phenotypic studies of M. tuberculosis typically involved a small number of strains and rarely had sublineage information, rendering comparison between studies difficult. A few recent studies, involving L1 isolates with sublineage information, included the autophagy study of EAI2_NTB (L1.2.1.2)46. A number of EAI isolates belonging to SIT1390 (L1.1.3.1)23 or EAI2_MNL (L1.2.1.1)47 were shown to induce high levels of inflammatory cytokines. Unfortunately, there are no known in vitro studies involving L1.1.1 or L1.1.2 isolates that merit further exploration.1, its predominant strain, SIT11 belonging to L1.1.2.2 , the Japan Anti-Tuberculosis Association (JATA), and the Ministry of Public Health, Thailand. The study recruited all newly-diagnosed culture positive tuberculosis patients with all nationality who were also willing to participate in a human genetic study from all pubic hospitals in Chiangrai. During the studied period, there were 15,805 new cases in Public Hospital Tuberculosis Registration System. Bacterial isolates were obtained from 7148 patients, of which both bacterial and host DNA were obtained from 1187 patients. 1174 M. tuberculosis isolates were successfully sequenced and 480 were identified to be Indo-Oceanic strains.The project was approved by the Ethical Committees of Chiangrai Prachanukroh Hospital, Chiangrai and the Thai Ministry of Public Health. Informed consent was obtained from all participants and/or their legal guardians. All methods were performed in accordance with the relevant guidelines and regulations.32. All the processes were performed in Class II biosafety cabinets.Bacterial samples from 1187 patients were successfully regrown in Lowenstein-Jensen medium in an appropriately contained clinical microbiology laboratory in Chiangrai using standard biosafety protocols and equipment. The bacteria were heat-killed and DNA was prepared as previously described51. The primer sequences are shown in Supplementary Table Lineages of the isolates in this study were initially identified by LSP using PCR primers specific to TbD1 , RD239 (for lineage 1), RD105 (for lineage 2), RD750 (for lineage 3) and 7 bp sequence at pks15/1 (for lineage 4)The 487 isolates were identified as Indo-Oceanic strains due the presence of the TbD1 and the absence of RD239.52. Spoligotyping investigates the presence or absence of 43 of the 68 DVRs. The results were coded into a binary pattern of 43 digits, which is typically converted to an octal code by the successive combination of three binary digits from the left, leaving the 15th code as still binary. M. tuberculosis L1 typically has EAI spoligotypes, i.e. the 10–12th octal code of 413. Some octal codes have been designated as spoligotype international type (SIT) and assigned to one of the various clades, such as EAI1, EAI2_MNL, and EAI2_NTB, among others. The SIT and clade assignment was performed by querying SITVITWEB 30. Spoligotypes were also predicted from whole-genome sequences using SpolPred software53.Spoligotyping was performed using a commercial kit  as previously describedM. tuberculosis samples were sequenced on the Illumina HiSeq. 2000 platform at the Wellcome Trust Sanger Institute, UK. The system produced paired-end reads in FastQ file format. We used Trimmomatic version 0.3654 to collect only the paired reads. Five isolates were not further analysed because the numbers of available reads were too small. The raw reads were filtered and discarded if their phred quality scores were lower than 20. The remaining reads were aligned by the BWA program55 and SAMtools56 using M. tuberculosis H37Rv (GenBank NC_000962.3) as the reference genome with alignment score more than 50, SAMtools base quality more than 23 and read depth more than 10. Two samples appeared to contain mixed nucleotide sequences and were also not further studied. The SNVs that were present in any drug-resistance gene, mobile genetic element, phage, PE/PPE region and non-homozygous SNVs were discarded. The remaining SNVs had been converted to an SNV-supermatrix using an in-house Python script before being used in the phylogenetic analysis. The sequencing data for the 480 samples used in this paper were submitted to the European Nucleotide Archive (ENA) of EMBL-EBI mirrored in the Sequence Read Archive (SRA) database. Actual read sequences can be queried and downloaded directly from the SRA database using the accession numbers listed in Supplementary Table SNVs of 480 Indo-Oceanic isolates were used for phylogenetic tree construction, with 24 isolates from the lineage 2–4 as outgroups. We analysed the generated supermatrix with two phylogenetic analytic methods, maximum likelihood (ML) and Bayesian inference (BI).57 via the SeaView program58. The core tree (starting tree) was generated using the BioNJ method. The analysis was run using GTR model, using the nearest neighbor interchange (NNI) method. The bootstrap analysis was calculated for branch support with 1,000 replications of pseudo-data.For ML analysis, we used PhyML54. The parameter “4by4” was used as the nucleotide substitution model. The BI analysis was performed with two simultaneous runs with four n-chain for each run. The analysis was run for approximately 10–20 million generations. The analysis was terminated when the average standard deviation of split frequencies was lower than 0.01. The first 25% of sample trees were discarded as burned-in. All trees were visualized using the FigTree program version 1.4.2. (http://tree.bio.ed.ac.uk/software/figtree/).For the BI methods, we used the MrBayes program57. The analysis was conducted at multiple levels of sublineage classification. The plots between the first three eigenvectors were examined.Principal Component Analysis was performed using Jalview 2.8.256. The fixation index (FST) was calculated based on the following formula (πbetween group − πwithin group)/πbetween group, where πwithin group is the average pairwise SNV distance within a group and πbetween group is the average pairwise SNV distance between all members in the group and all members not in the same group but in the same level of grouping. The statistical tests for the difference between the within-group averages and between-group averages were performed using Wilcoxon rank-sum test at α = 0.05 with Bonferroni correction multiple testing using R statistical package version 3.3.2.Pairwise SNV distances were calculated using MEGA5de novo using velvet software59. Analysis of the DR region in each genome was performed by carrying out a blastn search of the contig files containing the DR region. The identified contigs were mapped to areas around the position of 3119181–3123573 in the genome of H37Rv (GenBank NC_000962.3). The presence or absence of each of the 68 known direct variable region (DVR) sequences was determined and recorded as shown in Supplementary Table The sequence contigs of each isolate were constructed 6110 in the genome of each isolate was identified by ISMapper using the provided IS6110 sequence60 as the query and H37Rv as the reference genome with default parameters.The number of ISThe patient characteristics were described in descriptive statistics (as presented in Table Relevant research articles were identified by PubMed search using the keywords, spoligotype or genotype and tuberculosis and names of the countries in the ASEAN, South Asian and Pacific islands. The full papers were retrieved and examined for the presence of detailed descriptions of spoligotypes. Forty-one articles were identified with 6 having less than 20 EAI isolates in each study. The latter group was excluded. The list of retrieved papers is shown in Supplementary Table Supplementary InformationDataset for supplementary table S1Dataset for supplementary table S5Dataset for supplementary table S10


Docking Plus technique for elbow UCL reconstruction in 324 athletes provided good or excellent Conway score results in 88% of patients. The preponderance of previous studies examining UCL reconstruction outcomes were performed by surgeons at one of only three institutions .This retrospective case series examined 324 athletes who received elbow ulnar collateral ligament (UCL) reconstruction by a single surgeon in a private practice over a 9-year period. The novel Patients undergoing UCL reconstruction from November 2005 to December 2014 were identified and contacted with a mailed survey and phone call. These patients were given a subjective 19 question survey assessing their outcomes from surgery.The participants who responded to our survey were 90% male and 77% baseball players, 73% of which were pitchers. Of the baseball players who responded, 51.9% were in high school at the time of their surgery, 37% college, 6.5% minor leagues, and 2.2% in Major League Baseball. After surgery, 36% of survey responders returned to a higher level of competition than previously. For example, a high school athlete who had UCL reconstruction and went on to pitch in college. Further, 45% returned to the same level, and 7% returned to a lower level. Subjective “satisfaction,” was reported in 92% of responders and 97.2% reported that, “having surgery was a good idea.” Symptom onset in the responding athletes was 58.9% sudden, and 41.1% gradual. Overall, 90.9% of respondents returned to play in less than 1.5 years while 6.3% never were able to return. Re-tear occurred in 2.5% of patients, while 8.8% had subjective nerve dysfunction for at least 3 months following surgery.The Docking Plus technique can produce excellent subjective and objective results in athletes. Further study is warranted to see the effects of this procedure in other settings and determine which method of reconstruction or repair is superior. The majority of UCL reconstruction outcomes studies are from just three major institutionsEighty-eight percent of survey respondents who completed their rehab protocols following UCL reconstruction with the Docking Plus technique had a Conway-Jobe score of good or excellentMore studies examining objective outcomes following UCL reconstruction are neededInjury to the elbow ulnar collateral ligament (UCL) and its surgical reconstruction in throwing athletes is well described , 64, 74.The UCL is the primary valgus stabilizer of the elbow. It experiences the most torque in the late cocking and early acceleration phases of the pitching motion kinetic chain , 59, 75.Patients often, though not always, complain of acute or chronic medial elbow pain when throwing, which affects velocity and accuracy. Physical exam generally reveals pain with elbow valgus stress testing and palpation of the UCL . MRI witSince the year 2000, published case series of surgical outcomes for UCL reconstruction have demonstrated rates of return-to-play (RTP) of 74–100% , 69, 72.A variety of surgical techniques have been described, including the Docking Plus technique . Though We identified all UCL reconstructions performed by the senior author (TEK) from November 2005 to December 2014. We did so by searching the senior surgeon’s practice patient database by surgeon and Current Procedural Terminology (CPT) code (24346). CPT code 24346 is defined as, “Reconstruction medial collateral ligament, elbow, with tendon graft (includes harvesting of graft)”. This yielded 655 results of 647 unique patients. These reconstructions were all done with the docking plus technique and utilized the contralateral palmaris longus tendon for the graft when present. If the patient did not have palmaris longus tendons, a gracilis tendon autograft was used.A literature search was conducted to compare our study’s outcomes to previously reported data. We searched PubMed and Google Scholar for UCL reconstruction outcome studies and found 25 such studies published between 1986 and 2014 , 70, 72.For each study, we charted the outcomes and patient and injury characteristics measured. All of the 19 case series measured the competition level of return-to-play with 12 of these studies explicitly using Conway-Jobe or modified Conway-Jobe criteria in their classifications. Several other outcome metrics were used in these case series, including the Andrews-Timmerman elbow score (3 of 19 studies) , 51, 55,A 2-page, 19-question survey was designed with questions about demographics , sports participation , the nature of the injury , subjective outcomes , and objective outcomes .We used contact information  provided by patients at the time of prior clinical encounters to contact the patients. We mailed letters containing a URL link to the survey and a unique, anonymized patient identification number. In addition to standard mail, we contacted patients by telephone to provide them with the survey URL, their identification numbers, and to briefly describe the purpose of the outcomes study. We obtained an advisory opinion which states that our survey posed minimal risk and met criteria for being IRB exempt.The Docking Plus technique for UCL reconstruction Figs.  and 2 waPostoperative rehabilitation utilized standard timelines of immobilization, strengthening, throwing programs, and return-to-play , 42, 68.Of the 647 patients who received a survey, 3 were excluded because they did not undergo UCL reconstruction and 2 because they underwent revision UCL reconstruction. We received 335 responses to the survey, although 9 were found to be duplicate responses, 1 patient failed to respond with either his survey ID number or name, and 1 patient responded with a nonsense ID number. Therefore, we analyzed 324 responses for a 50.1% response rate. These patients had all undergone UCL reconstruction using the “Docking Plus” technique.Survey responders were 90% (290 of 324) male, while 10% (34 of 324) were female.Baseball was the primary sport for 77% (248) of responders. Other primary sports included softball (13), football (14), wrestling (9), volleyball (6), gymnastics (5), golf (5), cheerleading (4), tennis (4), lacrosse (4), rugby (1), and track and field (1).Of the baseball players, 73% were pitchers, 9% catchers, 12% infielders, and 7% outfielders , high school (51.9%), college (37.0%), minor league (6.5%), and major league (2.2%). After surgery, the highest level of performance reached was middle school (1.9%), high school (27.2%), college (54.9%), minor league (14.2%), or major league (1.9%).When asked explicitly about the level of return-to-play postoperative compared with preoperatively, 36% 104) had returned to a higher level of competition for at least one season, 45% (130) had returned to the same level, and 7% (20) had returned to a lower level. Thus, 81% had a Conway-Jobe score of excellent, and 88% had a score of excellent or good. Note that only 88.3% (286) of all respondents had completed their rehabilitation regiment and so were eligible to answer this question  were satisfied with their surgery and 97.2% (315 of 324) thought that it was a good decision to have the surgery Fig. .Fig. 6LeWhile patients described pre-op symptom duration and non-operative management for as little as 0–3 months and as long as greater than 2 years, 43.3% (138) had symptoms for 0–3 months and 68.1% (194) underwent physical therapy for 0–3 months.Symptom onset was sudden in 58.9% (188), but insidious/gradual in 41.1% (131). Of baseball pitchers, the symptom onset was sudden in 50.5% (93), but insidious in 49.5% (91). Then, 7.2% (23) had undergone prior surgery on the same elbow.RTP timing varied from less than 9 months to over 2 years. Further, 90.9% (259) returned in less than 1.5 years, while 2.5% (7) returned in 1.5–2 years, 0.4% (1) returned in over 2 years, and 6.3% (18) did not return to play. Among pitchers, self-reported return-to-play was similar, with less than 9 months , 9–12 months , 1–1.5 years , 1.5–2 years , > 2 years , and never .Complications included UCL re-tear in 2.5% (8) and additional same-side elbow surgery for any reason in 5.6% (18). Then, 8.8% (28) described some element of nerve dysfunction for at least 3 months postoperatively, although the survey did not distinguish between transient or permanent incision site numbness and ulnar nerve dyesthesia.In baseball pitchers, there was self-reported subjective improvement in pitch velocity, control, ERA, and innings pitched as compared with pre-injury. Of the 155 who have by now discontinued playing their sport, only 14.8% (23) state that retirement was caused by elbow disability.There are many UCL reconstruction outcome studies in published literature. However, most are produced by the same few pioneering institutions, such as the Andrews Institute , 66, 69,Despite the increasing incidence of UCL reconstruction surgery Fig. , only seThe Docking Plus technique, a variation on previously described Modified Docking techniques, has been previously described  and testThe goal of this study was to assess the performance of the Docking Plus technique of UCL reconstruction done by a single surgeon outside of the aforementioned pioneering institutions in a high volume patient series. The combined Conway score of good or excellent results of 88% is consistent with the results of similar outcome studies Table .Table 1CSubjective patient satisfaction scores in this study indicate that almost 96% patients think that it was a good decision for them to have surgery. Nearly as many, 92.9%, were satisfied with their surgery. This compares very favorably to patient satisfaction information from a study by Conte et al. in 2015 that found that only 72% of professional baseball players would have the surgery again and 17% would not have the surgery .Our data regarding symptom onset  varies from those results previously published in the literature that are closer to a 1:1 incidence of these two presentations . Though,Time to return-to-play varies based on position and sport. There has been debate about delaying further professional pitchers timing of return-to-play based on some trends in improved pitching in the second year compared with in the first year after return-to-play , 72.The rehabilitation protocol for pitchers dictates a return-to-play at 11–14 months if there are no setbacks. Eighty-one percent of these studies pitchers returned to pitch at 9–18 months postoperative. This is consistent with surgeon outcome studies that have shown a 11–13 month average return-to-pitch timing , 73.Outcome studies that have utilized information from MLB databases have shown a mean return-to-play timing of 16–20 months , 72, 78.The data in this study was self-reported and so is vulnerable to recall bias. Electronic medical records were accessed for the date of surgery and CPT code, but not for details of the patients’ histories and physical exams.Return to sport can be affected by a variety of social and medical factors unrelated to the technical success of the surgical procedure including continued interest in sport, skill level, and commitment to a lengthy rehabilitation process. As much as the senior surgeon’s patient selection includes operating on patients who have the interest and ability to continue competing, these variables may change over time.While a patient response rate of 50.1% of consecutive patients compares favorably with existing literature on UCL surgery outcomes, it is low enough to introduce significant selection bias into the results, especially those regarding patient satisfaction.The Docking Plus technique, performed in a private practice setting outside of the previously mentioned UCL reconstruction pioneering hospitals, produces excellent subjective  and objective  results. Further study is warranted to see if these results can be extrapolated to other surgeons and patient populations.


Since interpersonal violence was recognized as a public health problem in the 1970s, much attention has focused on preventing violence among young persons and intimate partners ; this percentage is expected to reach 28% by 2050 , and data are weighted by the inverse probability of selection to produce national estimates  and were stratified by 10-year age groups . Crude rates per 100,000 population per year were calculated using U.S. Census bridged-race resident population estimates.From 2002 to 2016, an estimated 643,191 nonfatal assault victims were treated in EDs, and 19,059 homicides occurred . CompareNonfatal assault rates among older adults did not change from 2002 to 2008 but increased 53.1% from 2008 to 2016 in the overall sample  Figure . For theFrom 2002 to 2014, the overall estimated homicide rate declined 12.8% (from 2.42 to 2.11)  Figure  and declNonfatal assaults and homicides differed with respect to mechanisms used in assaults. Most (86.6%) nonfatal assaults were related to being intentionally struck or hit . Firearms were the most common weapons used in homicides (42.2%). Other homicide mechanisms included cutting/piercing (14.8%) and suffocation (6.4%).During this study period, the older adult population (persons aged ≥60 years) in the United States grew nearly 3% annually, approximating global projections  with the victim  and 35.4% among women (2007–2016). The estimated homicide rate for men increased 7.1% from 2010 to 2016.Violence against older adults is a growing problem, particularly among men. Emergency departments might be positioned to help prevent violence among this group.


Methods: Vaccine uptake in 2016/17 and five previous seasons were measured using a sentinel general practitioners cohort in England; the test-negative case-control design was used to estimate pooled aVE by subtype and age group against laboratory-confirmed influenza in primary care from 2010–2017. Results: Vaccine uptake was 64% in 65–69-year-olds, 74% in 70–74-year-olds and 80% in those aged 75 and older. Overall aVE was 32.5% (95% CI: 11.6 to 48.5); aVE by sub-type was 60.8% (95% CI: 33.9 to 76.7) and 50.0% (95% CI: 21.6 to 68.1) against influenza A(H1N1)pdm09 and influenza B, respectively, but only 5.6% (95% CI: - 39.2 to 35.9) against A(H3N2). Against all laboratory-confirmed influenza aVE was 45.2% (95% CI: 25.1 to 60.0) in 65–74 year olds; - 26.2% (95% CI: - 149.3 to 36.0) in 75–84 year olds and - 3.2% (95% CI: - 237.8 to 68.5) in those aged 85 years and older. Influenza-attributable mortality was highest in seasons dominated by A(H3N2). Conclusions: Vaccine uptake with non-adjuvanted, normal-dose vaccines remained high, with evidence of effectiveness against influenza A(H1N1)pdm09 and B, though poor against A(H3N2), particularly in those aged 75 years and older. Forthcoming availability of newly licensed vaccines with wider use of antivirals can potentially further improve prevention and control of influenza in this group.In 2016/17, seasonal influenza vaccine was less effective in those aged 65 years and older in the United Kingdom. We describe the uptake, influenza-associated mortality and adjusted vaccine effectiveness (aVE) in this age group over influenza seasons 2010/11–2016/17.  The United Kingdom (UK), like many other countries in Europe, North America and Australasia, has a longstanding inactivated influenza vaccine programme including for all those over 65 years of age. The UK universal influenza vaccine programme for those aged 65 years and older was first started in 2000/01  informedFollowing this change in vaccine policy, influenza vaccine uptake for those aged 65 years and older has increased, reaching a high point in excess of the World Health Organization (WHO) and European Council recommendation of 75% in 2005/06 in England. Although there has been a gradual decline in uptake since that time, coverage was still 70.5% in 2016/17, ranging from 66.6% to 74.5% in other UK countries [The aim of this study was to further describe the recent uptake of influenza vaccine in those aged 65 years and older (including prior vaccine history) to measure influenza-associated mortality in this age group and to estimate the effectiveness of influenza vaccine over the period 2010–2017. The results will help inform optimal approaches to further mitigate the impact of influenza in this enlarging group.The previously described Royal College of General Practitioners (RCGP) Research Surveillance Centre (RSC) VE cohort was used to measure vaccine uptake and obtain prior vaccine history . The RCGUsing the weekly number of all-cause death registrations for those aged 65 years and older in England, available from the Office for National Statistics (ONS) for the period September 2011–September 2017, a previously described multiplicative Poisson regression model (the FluMoMo model) was used to estimate the number of deaths associated with influenza . In brieThe FluMoMo model was run separately for each age group (65–74 years and those aged 75 years and older) and by sub-type, (influenza A and B).The Test Negative Case (TNCC) control design has been used to measure vaccine effectiveness against laboratory-confirmed influenza infection that has resulted in acute influenza-like illness consultation in primary care. Five sentinel primary care surveillance schemes across the UK provided swabbing data . Details of these swabbing schemes have been published previously . The stuLaboratory confirmation was undertaken using RT-PCR assays for circulating influenza A, influenza B and other respiratory viruses at the national laboratory ,13. SampCases and controls were defined as vaccinated if the date of vaccination that season was 14 or more days before onset of ILI. Cases and controls with less than 14 days between vaccination and onset of illness were excluded, as their immune status was unclear. If the date of vaccination was missing it was assigned 15 October, which was the median of all known vaccination dates. If the date of onset of symptoms was missing, then the individual was excluded. Respiratory samples with more than 7 days delay between onset of illness and sample collection were excluded, as the sensitivity of the PCR test decreases over longer intervals between onset and sampling.Average VE over the entire seven-season period (2010/11–2016/17) was estimated as 1-(OR) using multivariable logistic regression models with influenza A(H3N2), A(H1N1)pdm09 or influenza B PCR results as outcomes and seasonal vaccination status as the linear predictor. Age , sex, season, clinical risk group, surveillance scheme  and date of sample collection (month) were investigated as potential confounding variables.To investigate whether the VE changed in relation to time since vaccination, influenza VE was stratified by time since vaccination . To investigate whether the VE changed with age, an interaction term between vaccination and age (as a continuous variable) was included in the model. To test the significance of changes in VE with the time since vaccination, multivariable logistic regression was performed with time between vaccination and onset of symptoms (days) included as a continuous variable. As testing for evidence of waning immunity was one of the study objectives, multiple testing adjustments were not made.All statistical analyses were carried out in Stata version 13 .During influenza season 2016/17, among 65–69-year-olds in England, influenza vaccine uptake was 64%  compared with 74%  in 70–74 year olds and 80%  for those aged between 75–84 years or aged 85 years and older . The majority  of people vaccinated in 2016/17 had received one or more doses of IIV in the previous 5 years, with most of those aged over 70 years having received all five doses of IIV that they were eligible for over the previous five seasons .During influenza seasons 2011/12–2016/17 in England, influenza-associated mortality estimates were higher in both the 65–74-year-olds and those aged 75 years and older during influenza A(H3N2) compared with A(H1N1)pdm09 dominated seasons . Over fiA total of 3,679 swabs were taken in those over 65 years and older over influenza seasons 2010/11–2016/17, with 2,096 swabs used in the final analysis: of these 1,702 were controls; 101 were flu B, 57 were A(H1N1)pdm09, 215 were A(H3N2) and 21 A non-subtyped. The reasons samples were discarded are outlined in When estimating VE, key confounders were adjusted for in a multivariable logistic regression model, all of which (except age and sex) were associated with a positive swab . Season The crude and adjusted VE (aVE) estimates in those aged 65 years and older by season are shown in Over the seven-season period, for any influenza (A and B), the crude seasonal average VE in those aged 65 years and older was - 12.0% (95% CI: -42.1 to 11.8), which increased to a seasonal average of 32.5% (95% CI: 11.6 to 48.5), when fully adjusted over the entire study period . By sub-We found that the aVE for all influenza types in those aged 65 years and older reduced by 6.1% for each year older (95% CI: - 10.5 to - 1.9: p = 0.004). For A(H3N2) the aVE reduced by 6.6% (95% CI: - 12.8 to - 0.7: p = 0.03); for A(H1N1)pdm09, by 1.9% (95% CI: - 10.4 to 5.9: p = 0.64) and for influenza B, by 5.5% (95% CI: - 14.1 to 2.5: p = 0.19) for each year older.For all laboratory-confirmed influenza cases the aVE by time since vaccination, within 3 months of symptom onset, was 44.7% (95% CI: 21.2 to 61.2) compared with 14.5% (95% CI: - 18.2 to 38.2) for more than 3 months between onset and vaccination. For A(H3N2), the aVE was 13.6% (95% CI: - 41.4 to 47.2) if onset was within 3 months of vaccination compared with - 7.1% (95% CI: - 69.7 to 32.5) for more than 3 months since vaccination. For A(H1N1)pdm09, aVE was 66.7% (95% CI: 30.2 to 84.1) compared with 59.1% (95% CI: 23.2 to 78.2) and finally for influenza B, aVE was 71.5% (95% CI: 43.0 to 85.8) compared to 19.1% (95% CI: - 40.2 to 53.3).We found that despite achieving very high influenza vaccine uptake in those aged 65 years and older, the average annual age group-specific influenza-associated mortality risk in England was highest in those aged 75 years and older over a six-season period and during seasons dominated by circulation of influenza A(H3N2). Over the seven seasons (2010/11–2016/17), the pooled aVE against all-laboratory confirmed influenza diagnosed in primary care across the UK was moderate in those 65 years and older. There was, however, no evidence of statistically significant VE in those aged 75 years and older nor specifically against A(H3N2).We found evidence of high vaccine uptake, with increasing uptake with age in excess of 80% in those aged 75 years and older, which is comparable to other European countries . EnglandWe demonstrate that over the 7 year study period, there was overall evidence of moderate aVE in those aged 65 years and older, though this obscures some important reductions by season, influenza subtype and age group. Immunosenescence is a key contributory factor with overall aVE decreasing with age. Age-related decline in humoral immune response following vaccination is a well-recognised phenomenon ,17. IrreWhat potential interventions do we have to improve vaccine performance and potentially reduce the remaining disease burden in this age group? Improving protection through the induction of higher levels of antibody and/or broader repertoires of antibody responses are ways of improving vaccine-induced immunity. Until recently, only normal-dose, non-adjuvanted influenza vaccines were licensed for use in those 65 years and older in the UK, but newer influenza vaccines are becoming increasingly available. MF59 adjuvanted vaccines provide a future alternative option—with effectiveness data from North America and Europe -30, suggThe UK is also in the process of rolling out a universal childhood influenza vaccine programme, targeting children 2-10 years of age, with the intention of providing both direct protection to vaccinated children but also, by reducing their risk of infection and thus transmission, provide indirect protection to vulnerable at-risk groups including those that are 65 years and older. Modelling work by Hodgson et al has demonstrated the projected impact of a paediatric programme on older age group and predicts that the direct and indirect benefits of vaccinating children against influenza should be realised in future years once programme rollout is achieved . Early iIt is important to highlight that prompt use of influenza antivirals in vulnerable populations for treatment and prophylaxis purposes is also important to mitigate the impact of severe influenza; particularly during A(H3N2) dominated seasons,. These medicines are, however, currently underutilised in primary care in the UK .One of the key advantages of the TNCC approach is that the design allows control for key biases e.g. the propensity to consult, but confounders still need to be adjusted for. Pooling vaccine effectiveness estimates over a number of seasons has allowed for the further exploration of influenza sub-type and age-specific effects. However, the available numbers of samples limit the precision of some of the stratified estimates e.g. by age group and the ability to assess waning by type/sub-type. Some of the sub-set analyses should thus be interpreted with caution. Other published work has suggested that the TNCC design should control for a possible confounding effect due to frailty in older subjects . NeverthDespite high vaccine uptake with non-adjuvanted, normal-dose inactivated vaccine in older people in the UK, influenza remains an important cause of influenza-associated mortality, particularly due to influenza A(H3N2) and in those aged 75 years and older. The forthcoming policy of administering adjuvanted vaccines to older people in the UK will provide the opportunity to improve the prevention and control of influenza in these older age groups, with guidance for their use particularly in those aged 65 years and older recently published . Further