P < 0.001) and overall survival (OS) . In the multivariable analysis, microinvasion was an independent prognostic factor for worse CSS  and OS . The 10-year cancer-specific mortality rate was 1.49% in DCIS and 4.08% in DCISM . The 20-year cancer-specific mortality rate was 4.00% in DCIS and 9.65% in DCISM . Deepening understanding of the nature of microinvasive breast cancer will be valuable for clinical treatment recommendations.To investigate the clinicopathological characteristics and survival outcomes of microinvasive breast cancer, we conducted an observational study of female diagnosed with DCIS or DCIS with microinvasion (DCISM) from 1990 to 2012 using the Surveillance, Epidemiology, and End Results (SEER) database. There were 87695 DCIS and 8863 DCISM identified. In DCISM group, patients appeared to be younger and more black patients were identified in comparison with DCIS group. Furthermore, DCISM was associated with more aggressive tumor characteristics like higher rates of oestrogen receptor (ER) and progesterone receptor (PR) negativity, HER2 positivity, and lymph node metastasis. With a median follow-up of 91 months, patients with DCISM had worse cancer-specific survival (CSS) (hazard ratio [HR], 2.475;  Therefore, a population-based study was designed to assess the differences in clinicopathologic characteristics and long-term outcomes between DCIS and DCISM using the National Cancer Institute’s Surveillance, Epidemiology, and End Results (SEER) database.87695 patients with DCIS and 8863 patients with DCISM met the inclusion criteria. Demographic and clinicopathologic characteristics of the study population are summarized in P < 0.001) and more black patients were identified in comparison with DCIS group. In addition, DCISM was associated with more aggressive tumor characteristics like ER negative , PR negative , HER2 positive  and lymph node metastasis . In terms of the treatment, 99.3% of DCISM patients underwent surgery, higher than that (97.6%) of DCIS patients (P < 0.001). A similar trend of radiotherapy was observed between two cohorts that 45.4% of DCISM patients and 45.7% of DCIS patients received radiation (P = 0.631).In DCISM group, more patients appeared to be younger than 40 years old , among which 1230 deaths were related to breast cancer. 699 deaths were observed in the DCISM group (n = 8863), among which 284 deaths were attributable to breast cancer.P < 0.001; log-rank P < 0.001; P < 0.001; P < 0.001). The 20-year CSS rates were 4.00% and 9.65%, respectively .Survival distributions of two groups were demonstrated in P < 0.001) after adjusting for other prognostic factors. Furthermore, younger age (P < 0.001), black race (P < 0.001), higher tumour grade (P ≤ 0.028), lymph node metastasis (P < 0.001), no surgery treatment (P < 0.001) and no radiation (P < 0.001) were also independent variables to predict worse CSS. As shown in P < 0.001) with adjusting for other prognostic factors. In addition, older age (P < 0.001), black race (P < 0.001), lymph node metastasis (P ≤ 0.01), no surgery treatment (P < 0.001) and no radiation (P < 0.001) were also identified as independent prognostic variables for worse OS.Univariate and multivariate Cox proportional hazard regression models were used to calculate hazard ratio and 95% confidence interval and investigate prognostic factors that were associated with CSS and OS. As shown in P < 0.001; CSS, HR, 3.801; 95% CI, 3.245–4.451; P < 0.001).These findings were confirmed even after the weighted Cox proportional hazards regression models with the inverse probability of treatment weighting (IPTW) adjustment. IPTW-adjusted analysis for OS and IPTW-adjusted competing risk analysis for CSS suggested the mortality increased in DCISM , black race , lymph node metastasis , no surgery treatment  and no radiation  were independent prognostic factors for worse CSS. As shown in the P < 0.001), lymph node metastasis, no surgery treatment  and no radiation  were independent prognostic factors for worse OS. For the set of DCISM data, there were small or no number of events in the group of certain variables like HER2 variable (SEER database provided HER2 status after 2010), resulting in calculating extremely small or large HRs in the univariate and multivariate analysis.To further determine the independent prognostic factors for CSS and OS in DCISM patients, both univariate and multivariate analysis were conducted , and in the multivariate analysis, microinvasion is an independent prognostic factor for worse CSS  and OS . Based on the available information, in DCISM group, patients appear to be younger and more black patients are identified in comparison with DCIS group. Furthermore, DCISM is characterized by more aggressive clinicopathologic features. Further multivariate analyses show lymph node metastasis, no surgery treatment and no radiation are independent prognostic factor for worse CSS and OS in DCISM patients.Over the past three decades, more early stage breast carcinoma including ductal carcinoma et al. evaluated the clinical significance of microinvasion in the ever largest series of patients with DCISM (243 cases)P < 104). However, there has also been disagreement on the impact of microinvasion on survival121516P = 0.95), respectively, with a median follow-up of 8.94 years. Wang et al. also reported that the outcomes of patients with microinvasive carcinoma were similar to those with DCIS. The 5-year OS rate for microinvasive carcinoma and DCIS patients was 99.0 and 99.2%, respectivelyOur current analysis showed the 10-year and 20-year breast cancer-specific mortality after a diagnosis of DCIS were 1.49% and 4.00%, respectively, consistent with the results reported in previous studies181913P < 0.001) and OS (P ≤ 0.010). The incidence of pathologically positive axillary lymph node metastases for patients with microinvasive ductal carcinoma of the breast has been reported as 0–20%21222324252627282930313233343536et al. reported the frequency of nodal metastases was 10.5% in the 1229 female patients with microinvasive breast cancer who underwent axillary staging in the SEER database from 1997 through 2003P = 0.004). To some extent, the presence of lymph node metastases results in the poor prognosis of microinvasive breast cancer. These patients should receive more aggressive treatment in the clinical practice.On the basis of our large dataset, 7.6% of DCISM had lymph node metastases. In our multivariate analysis, lymph node metastasis was an independent prognostic factor for worse CSS (P < 0.001), PR negative , and HER2 positive . Margalit et al. reported 39% ER negative and 49% HER2 positive in 83 consecutive patients with microinvasive breast cancer from 1997 to 2005, more frequent than in DCISP < 0.001)P = 0.039)Previous studies have suggested that DCISM may represent a distinct entity with more aggressive pathological features associated with worse survival outcomes1139404142In order to precisely estimate the mortality, it is necessary to conduct a large cohort study with an extended period since death events rarely happen in DCIS or DCISM. Despite the utility of large, high quality cancer data registries such as the SEER database, there are several limitations in our study, including lack of certain characteristics such as surgical margin status, adjuvant endocrine therapy, the process of adjuvant chemotherapy and body mass index. Additionally, key data such as tumor grade and hormone receptor status were unavailable for approximately 30–50% of DCIS or DCISM, and HER2 status were missing in more than 90% patients. Another important limitation is that the SEER registry records multiple primary cancers but not recurrences which may result in inaccurate estimation on disease free survival.In conclusion, the clinicopathological characteristics of breast cancer patients with microinvasion are more aggressive than those of DCIS. Furthermore, microinvasion is an independent prognostic factor for worse CSS and OS. The direction in the future will be to further explore and differentiate those subtypes of microinvasive carcinoma associated with a higher incidence of recurrence or progression to invasive disease in order to tailor treatment strategy accordingly.http://seer.cancer.gov/about/).Data for this study were obtained from the recent SEER 18 registries research database (November 2014 Submission). The SEER18 database contains data from the SEER13 registries  and the registries of greater California, Kentucky, Louisiana, New Jersey, and greater Georgia. SEER database of the National Cancer Institute (NCI) is the largest population-based cancer registry in the United States, which covers approximately 28% of the population (in situ), 8230/2 , 8500/2 , 8501/2 , 8503/2 , 8201/2 (Cribriform carcinoma in situ) and 8507/2 ; and without ductal carcinoma with microinvasion. To identify the eligible DCISM cohort, the inclusion criteria included females aged 20 to 69 years old; the first and only cancer diagnosis with stage T1mic breast cancer between 1990 and 2012; patients with ICD-O-3 codes of 8201/3 (Cribriform carcinoma), 8230/3 (Solid carcinoma), 8500/3 (Infiltrating duct carcinoma), 8501/3 (Comedocarcinoma), 8503/3 , and 8507/3 . Patients for whom DCIS or DCISM was not the first and only cancer diagnosis were excluded from analysis. Patients without microscopic confirmation of the diagnosis and those identified at autopsy or on death certificate only were also excluded from the analyses. Together, 87695 patients with DCIS and 8863 patients with DCISM were eligible for the study.We conducted a retrospective cohort study and SEER*Stat (version 8.2.1) was used to generate a case listing. To identify the eligible DCIS cohort, the inclusion criteria included females aged 20 to 69 years old; the first and only cancer diagnosis with stage Tis breast cancer between 1990 and 2012; patients with the International Classification of Diseases for Oncology Version 3 (ICD-O-3) codes of 8201/2 . Tumor-specific variables included: grade , estrogen receptor (ER) status , progesterone receptor (PR) status , epidermal growth factor receptor 2 (HER2) status , and lymph node metastasis . Treatment-related information included: surgery  and radiation . Survival- specific variables included survival months, vital status recode , cause-specific death, and other cause of death.The distribution of patient and clinicopathologic characteristics between two groups was compared using Pearson’s Chi-square test or Fisher exact test, as appropriate. Cancer-specific survival (CSS) was defined as the time from diagnosis to death from breast cancer. Overall survival (OS) was calculated from the date of diagnosis to the date of death from any cause. Kaplan-Meier estimates and Cox regression analyses of CSS and OS were done. The log-rank test was used to compare the distribution between the DCIS and DCISM groups. Fine and Gray’s competing-risks regression models were conducted to assess cancer-specific survival (CSS). Univariate and multivariate Cox proportional hazard regression models were used to calculate hazard ratio (HR) and 95% confidence interval (CI) and identify factors that are associated with CSS and OS.In order to adjust potential confounders by indication , the weighted Cox proportional hazards regression models with the inverse probability of treatment weighting (IPTW) were conducted. Subgroup analyses were undertaken to investigate the effect of multiple prognostic factors on CSS and OS by use of a Cox regression model. For the subgroup analysis of survival, the HR and 95% CI within each subgroup were summarized and displayed in the forest plot.P < 0.05.All statistical analyses were performed using R statistical package version 3.1.1  or SAS version 9.2 . All analyses were 2-sided and statistical significance was defined as How to cite this article: Wang, W. et al. The Demographic Features, Clinicopathological Characteristics and Cancer-specific Outcomes for Patients with Microinvasive Breast Cancer: A SEER Database Analysis. Sci. Rep.7, 42045; doi: 10.1038/srep42045 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Aspergillus, Cyberlindnera and Candida were predominant at the early stage of manufacturing process (from “primary dark tea” to “fermentation for 3 days”), but after the stage of “fermentation for 3 days” only Aspergillus was still dominated, and maintain a relatively constant until to the end of manufacturing process. The effects of metabolites on the structure of the fungal community were analyzed by redundancy analysis (RDA) and variation partitioning analysis (VPA). The results indicated that GCG , EGCG  and GA as well as the interactions among them were the most probably ones to influence, or be influenced by the fungal communities during the fermentation process of Fu brick tea. This study revealed fungal succession, metabolite changes and their relationships, provided new insights into the mechanisms for manufacturing process of Fu brick tea.Fu brick tea is a unique post-fermented tea product which is fermented with microorganism during the manufacturing process. Metabolic analysis showed that most metabolites content were decreased during the manufacturing process of Fu brick tea, except GA . Illumina MiSeq sequencing of ITS gene amplicons was applied to analyze the fungal community succession. The genera  These six types of tea have different flavor quality and health benefits because of their characteristic chemical components. The manufacturing process of Fu brick tea involves steaming, piling, pressing, fermentation  and drying. A complex biochemical changes take place during the manufacturing process producing its special flavor and health benefits of Fu brick tea, such as anti-hyperlipidemia, anti-obesity, anti-hyperglycemia, and anti-dysentery activities5.In general, Chinese teas are divided into six categories according to the processing technology and oxidation degree: green tea (no oxidization), white tea (slightly oxidized), yellow tea (lightly oxidized), oolong tea , black tea (fully oxidized), dark tea (post-fermented)Aspergillus, Penicillium, and Eurotium were the dominating fungus during the manufacturing process of Fu brick tea7. However, traditional microbiological methods, such as cell cultures and colony counting, are of limited value for exploring the variation and structure of microbial communities. Moreover, isolation media may be suitable for only some types of microorganisms, as strains of microorganisms cannot be accurately discriminated based on appearance8. Several molecular biology techniques, such as terminal restriction fragment length polymorphism (T-RFLP), polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE), and gene library construction, were used for determining microbial communities and structures9. A recent studies using PCR-DGGE analysis revealed that the microorganisms found in Fu brick tea were from or closely related to the genera Aspergillus, Beauveria, Debaryomyces, Eurotium, Pestalotiopsis, Pichia, Rhizomucor, and Verticillium10, while other studies found that Aspergillus niger, Blastobotrys adeninivorans and Bacillus, Enterobacteriaceae were the major fungal and bacterial communities involved in Pu-erh tea, another kind of post-fermentation tea12. However, these techniques provide limited information on community information, because only a few sequences can be separated and analyzed. Recently, a high-throughput sequencing technique, also called next generation sequencing technology, was used to investigate microbial composition and diversity in various fermented foods, such as doubanjiang-meju, wine grape and Pu-erh tea15. Compared to previous culture-dependent or low resolution molecular method (e.g. PCR-DEGG), this technology produces a very large number of reads in a run of many different samples and provides a powerful tool for profiling microbial structure and diversity at genus or even species levels17.Since the quality of Fu brick tea is closely related to the microbial activities during the manufacturing process, microbial community have been intensive studied in the past few years. Microbial counting and identification revealed that genera The aim of this study was to investigate the fungal community succession, metabolite changes, and assess relationships between fungal community and metabolites during the manufacturing process of Fu brick tea. This knowledge could give new insight into the manufacturing process and provide valuable knowledge to improve the quality of Fu brick tea.The metabolites, including water extract (WE), soluble sugar (SS), flavonoid (FLA), organic acid (OA), tea polyphenol (TP), amino acid (AA), gallic acid (GA), caffeine (CAF), epigallocatechin gallate (EGCG), epicatechin gallate (ECG), gallocatechin gallate (GCG), epigallocatechin (EGC), epicatechin (EC), catechins (C), were analyzed during the manufacturing process of Fu brick tea. As shown in Fig. After quality filtering and chimera removal, a total of 285,184 of valid sequences were generated from the samples, with an average sequence number of 28,518 for each sample (range from 20846 to 37278). At 97% sequence identity, 216 OTUs  were identified , Penicillium (2.49% to 0.01%) were also observed during manufacturing process of Fu brick tea . The result of DCA indicated that the gradient length of the first axis was less than 3SD. Therefore, the linear model with RDA was considered to be the appropriate ordination method for direct gradient analysis. As shown in Fig. Aspergillus was significantly (P < 0.05) correlated with WE, TP, AA, CAF, EGCG, GCG and ECG. Cyberlindnera was significantly (P < 0.05) correlated with WE, TP, CAF, GCG and ECG. Candida was significantly (P < 0.05) correlated with AA, GCG and ECG. Wallemia and Fungi_unclassified were significantly (P < 0.05) correlated with WE, AA, GCG and ECG. Penicillium was significantly (P < 0.05) correlated with WE, TP, CAF, EGC, EGCG, GCG and ECG. Ascomycota_unclassified was significantly (P < 0.05) correlated with TP and CAF. Eucasphaeria was significantly (P < 0.05) correlated with TP and CAF. However, no fungal genera were significantly (P > 0.05) correlated with FLA, OA, EC and C.As shown in Table Fu brick tea is a unique post-fermented tea product which is fermented with microorganism during the manufacturing process. To the best of our knowledge, this is the first report on the fungal community succession and metabolite changes during the manufacturing process of Fu brick tea by high throughput Illumina MiSeq sequencing, spectrophotometry and HPLC. The results, from dramatic changes in fungal community structure and metabolites as well as correlation analysis, indicated strong relationships between metabolites and fungal community during the manufacturing process of Fu brick tea.18. Several enzymes secreted from genus of Aspergillus, such as cellulases, hemicellulases, proteases and α-amylases, could catalyze the major metabolites in Pu-erh tea during fermentation process19. The decrease of SS may be related to microbial growth that SS could be used as the carbon source of microbial growth during the manufacturing process20. Our previous study indicated that the degradation of TP and catechins in the early stage of process might be resulted from moisture and heat reaction, but in the later stage of process was mainly related to the microbial metabolism. And this degradation was good for the formation of Fu brick tea’s taste and its special aroma20. The aldehyde compounds of stale aroma and terpene alcohols of flower aroma which specially increased after the fermentation process were from decarboxylation and oxidative deamination of amino acids21. The increase of GA may be also the result of hydrolysis of galloylated catechins and microbial metabolism, which might be helpful on some bioactivity of Fu brick tea10. Interesting, these decreased components were the major functional components of tea, but Fu brick tea still has a strong health benefits. Mo et al. reported that the activities increased with the course of microbial fermentation process (MFP) of Fu brick tea, which indicated that this process produced new varieties of functional metabolites2. Furthermore, new metabolites such as three triterpenoids22, one norisoprenoid23, seven new B ring fission catechins (flavan-3-ols) derivatives26 were reported from a few phytochemical investigations on Fu brick tea. Especially these B ring fission catechins have not been identified in other tea products, which suggested that the biochemical profiles of Fu brick tea were influenced significantly by MFP.Our metabolic analysis showed that WE, SS, TP and catehins were significantly decreased, but GA was significantly increased during the manufacturing process of Fu brick tea. These changes probably resulted from moisture and heat reaction and microbial metabolismAspergillus, Cyberlindnera and Candida were predominant from S0 to S4, but only the genus of Aspergillus was still dominated in Fu brick tea from S5 and maintained constant until the end of manufacturing process. The genus Aspergillus is a group of filamentous fungi consist of more than 250 species, which is the most economically important of the fungal genera27. Many species of Aspergillus are used in biotechnology for the production of various metabolites, such as antibiotics, organic acids, medicines or enzymes, or as agents in many food fermentations28. The fungal genus Eurotium, which is the teleomorph of Aspergillus, has been proved to be a rich source of novel bioactive metabolites30. Since the recent synonymization of the teleomorph-based genus Eurotium with Aspergillus by the International Commission on Aspergillus , which adopted the newly established principle “one fungus, one name” , species formerly included in the genus Eurotium are displayed with their Aspergillus name27. Many previous reports showed that Aspergillus cristatum (Eurotium cristatum) was the dominant fungus during the manufacturing process of Fu brick tea32, and it is considered to be safe under low- and high-osmolarity conditions by genomic, transcriptomic and HPLC analysis33. Several indole alkaloids and indole diketopiperazine alkaloid were identified from the culture extract of Aspergillus cristatum and these compounds were proved to possess brine shrimp lethality, antibacterial activity against E. coli, radical scavenging activity against DPPH radicals, and exhibited marginal attenuation of 3T3L1 pre-adipocytes36. Both emodin and physcion were also identified in Fu brick tea fermented by Aspergillus cristatum10. Non-mycotoxigenic strains of Aspergillus repens and Aspergillus rubrum are used as starter cultures in the manufacture of the traditional fermented food katsuobushi, from bonito (Katsuwonuspelamis), in Japan37. During katsuobushi fermentation these xerophilic fungi produce effective antioxidants, which participate in the suppression of lipid oxidation such that the fermented katsuobushi is resistant to lipid oxidation and has a long shelf-life38.Fungal community succession showed that the genera Lindnera was established to accommodate some Pichia species in the Pichia anomala clade41. But Lindnera is a later homonym of a valid published plant genus, the genus Cyberlindnera was introduced as a replacement name for Lindnera, with 21 new combinations42. The Cyberlindnera clade contains 23 recognized teleomorphic species and 12 Candida species are also related to this clade43. The yeast Cyberlindner ajadinii, previously referred to as Pichia jadinii, Hansenul ajadinii or Candida guilliermondii, was reported to be the teleomorph of Candida utilis 44, which was used for almost one hundred years for various biotechnological applications45. Because of its capability to efficiently assimilate pentose, including xylose and arabinose, and its robust fermentation characteristics, Cyberlindner ajadinii was exploited to grow large amounts on waste hardwood hydrolysates from the pulp industry. Moreover, some species of Cyberlindnera and Candida, which had a high capability of tannin tolerance, were also isolated from Miang (a fermented food product prepared from tea leaves)46. The genus Candida has become one of the largest in species number, present in almost every environment. Yeasts of this genus are abundantly distributed in nature on land and sea, associated with animals or plants and inanimate objects47. This genus is distributed across the ascomycetous yeast domain, overlapping with other genera, according to phylogenetic analysis using ribosomal genes. Some species of Candida genus were used in food fermentation, such as Candida etchellsii, Candida milleri, Candida rugosa and Candida tropicalis28. Some species of Candida has high phenol degradation ability51, bioconversion of corn fiber, sugarcane bagasse and xylose into the sweetener xylitol53, enhance of clove and smoked favors and biotransformation of maltose into ethanol in soy sauce process54. These properties of yeast Cyberlindnera and Candida genera indicate that their enzyme activities improve the quality of Fu brick tea by degradation of phenol, producing the sweet substance xylitol, and other favors flavour substance. The yeast genera may play an indispensable role in the initial stage of Fu brick tea fermentation.The genus Aspergillus, Cyberlindnera and Candida were dominant at the early stage of manufacturing process, but after the stage of “fermentation for 3 days” only the genus Aspergillus was still dominated and maintain a relatively constant until to the end of manufacturing process of Fu brick tea. In addition, the effects of metabolites on the structure of the fungal community were analyzed by RDA and VPA. The results indicated that GCG, EGCG and GA as well as the interactions among them were the most probably ones to influence, or be influenced by the fungal communities during the fermentation process of Fu brick tea.In conclusion, predominant fungus and major metabolites were analyzed using Illumina MiSeq sequencing, spectrophotometry and HPLC methods, respectively. The metabolic analysis showed that most metabolites content were decreased during the manufacturing process of Fu brick tea, except GA. Illumina MiSeq sequencing analysis showed that the genera 55. And the manufacturing process of Fu brick tea was descripted as follow: a 100 kg primary dark tea was moistened by steaming then piled up to 1.5 m in height and subjected to temperatures as high as 80 °C overnight. After pile fermentation, the tea materials were mixed with 15 L of tap water, then partitioned and pressed into desired sizes of brick tea. And the brick teas were placed in the fermentation room for 20~22 days. During the fermentation process, the temperature and relative humidity of fermentation room was 30 ± 2 °C and 75 ± 3% from 0 day to 15 day. Then the temperature of fermentation room was increased by 2 °C per day until the end of the manufacturing process during the drying process. Finally, the tea products were packaged and stored. Three independent batches of Fu brick tea were prepared simultaneously by the same manufacturing process. The samples were collected from three independent batches of Fu brick tea at different stage of manufacturing process: primary dark tea (S1), piling-fermentation tea materials (S2), fermenting brick-tea in the fermentation room for 0 days (S3), 3 days (S4), 6 days (S5), 9 days (S6), 12 days (S7), 15 days (S8), 18 days (S9) and newly made tea products (S10) at day 22. The samples were packaged in sterile polyethylene bags, transported to the laboratory and stored at −80 °C until required.Sampling of Fu brick-tea was carried out in a major Fu brick tea production factory, Yiyang Fu Cha Industry Development Co. Ltd. . Primary dark tea, used as raw materials for the process of Fu brick tea, was manufactured from fresh tea leaves as described by DB43/T660-2011  was mixed with sterile water (125 mL), stirred thoroughly, filtered through three layers of coarse sterile gauze to remove large particles, and centrifuged at18,000 g for 10 min at 4 °C. The microbial genomic DNA was extracted by the E.Z.N.A.Soil DNA Kit , and the procedure was according to the manufacturer’s protocols.63. Different barcode sequences were added at the 5′ end of the forward primer for multiplexed pyrosequencing. PCR were carried out in a 20 μL reaction volumes containing 10 ng DNA template, 2 μL dNTPs (2.5 mM), 0.4 μL of each primer (5 μΜ) and0.4 UμL FastPfu Polymerase (Applied Biosystems) in the appropriate 56 FastPfu Buffer (4 μL) and de-ionized ultrapure water (to 20 μL). The protocol was optimized with low cycles for better accuracy and reliability of the subsequent data analysis. The PCR condition were initial denaturation at 95 °C for 2 min, followed by 25 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 sand extension at 72 °C for 45 s, with a final extension phase at 72 °C for 10 min. The PCR products were extracted from 2% agarose gels and purified using the AxyPrep DNA Gel Extraction Kit  according to the protocol and quantified using QuantiFluor™-ST . Samples were then pooled at equal concentrations. Parallel tagged sequencing was performed using Illumina Miseq Sequencing in Majorbio Bio-Pharm Technology Co., Ltd., Shanghai, China.The ITS1 region of fungal was amplified by PCR for MiSeq sequencing. The ITS1 region of fungi was amplified with the forward primer (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and the reverse primer ITS2 (5′-GCTGCGTTCTTCATCGATGC-3′)http://www.usadellab.org/cms/?page=trimmomatic) with the following criteria64: (a) The 250 bp reads were truncated at any site receiving an average quality score less than 20 over a 10 bp sliding window, discarding the truncated reads that were shorter than 50 bp; (b) Exact barcode matching, two nucleotide mismatch in primer matching, reads containing ambiguous characters were removed; (c) Only sequences that overlap longer than 10 bp were assembled according to their overlap region. Reads that could not be assembled were also discarded. These sequences were clustered to OTUs  at 97% sequence identity by using Mothur 65. The OTUs were used for alpha diversity (Shannon and Simpson), richness (ACE and Chao1), Good’s coverage, Rarefaction curve, Shannon-Wiener curve and Venn diagram were performed by Mothur65. In addition, Nonmetric Multidimensional Scaling (NMDS) diagram and rank_abundance curves were generated by using R package vegan . CANOCO 4.5 software was used to conduct multivariate analysis using detrended correspondence analysis (DCA), redundancy analysis (RDA) and variation partitioning analysis (VPA)66. One-way ANOVA test, Pearson’s correlation coefficients and P values were calculated with IBM SPSS 19.0 . All results are presented as the mean value (±SE). Differences between groups were declared significant at P < 0.05.Raw reads were demultiplexed and quality-filtered using Trimmomatic software (version 0.36, Supplementary infromation


Escherichia coli, 10 infecting Pseudomonas aeruginosa, 4 infecting Salmonella enterica, 3 infecting Staphylococcus sciuri, and 6 infecting Enterococcus faecalis). High biological diversity of the collection is indicated by its characteristics, both morphological  and biological , and further supported by hierarchical cluster analysis. By the end of the research no larger collection of phages from a single environmental source investigated by these means had been found. The finding was confirmed by whole genome analysis of 7 selected bacteriophages. Moreover, particular bacteriophages revealed unusual biological features, like the ability to form plaques at low temperature (4 °C), resist high temperature (62 °C or 95 °C) or survive in the presence of an organic solvents  or detergent  making them potentially interesting in the context of biotechnological applications.A large scale analysis presented in this article focuses on biological and physiological variety of bacteriophages. A collection of 83 bacteriophages, isolated from urban sewage and able to propagate in cells of different bacterial hosts, has been obtained (60 infecting  Nevertheless, from the available data one can deduce a tremendous diversity of bacteriophages, which is known only in a small fragment, as indicated in the newest papers focused on this problem68Bacteriophages were discovered about 100 years ago3in silico analyses of nucleotide sequences of their genomes. Such studies provide extremely interesting information about the genetic variability of phage genomes, however, they also indicate that there is a large number of phage genes whose function cannot be predicted due to very low similarity, or lack thereof, to already known genes9A vast majority of current studies on bacteriophage diversity focus on in silico analyses, in our studies, we focus on biodiversity of phages, i.e. investigating the biological and physiological variety of these viruses. Such analyses should indicate the level of biological diversity of bacteriophage properties in the sense of their development characteristics under various conditions. Apart from determining the level of variability of bacteriophages’ features, this could also provide the starting point for further studies on molecular mechanisms of putative phage uncommon traits. Moreover, this kind of research may provide a basis for determining newly discovered features of bacteriophages which can be useful in biotechnological and medical applications. Among them, construction of new vectors and development of bacteriophage therapy appear to be the most likely applicable novel aspects of such studies, as nowadays these fields of bacteriophage research are developing particularly rapidly111213141516171819In our current project, we have applied a different strategy, which is a novel approach considering the previously published works on broad analyses of bacteriophages. While the large-scale genomic projects are based on isolation of large numbers of bacteriophages, sequencing of their genomes and advanced Escherichia coli laboratory strains, 10 phages infecting Pseudomonas aeruginosa clinical strains, 4 phages infecting Salmonella enterica clinical strains, 3 phages infecting Staphylococcus sciuri strain (isolated from sewage), and 6 phages infecting Enterococcus faecalis strain (isolated from urban sewage) . All phages from the collection were then subjected to morphological and physiological studies.For isolation of bacteriophages from urban sewage, two strategies were applied. In one, two multiphage sewage samples were obtained by means of several steps of filtration of raw sewage samples, precipitation of viral-like particles (VLP), and their further purification. One hundred VLPs were selected at random from each of the two samples  and subjected to morphological studies. In the other approach, 10 ml of a raw urban sewage sample was mixed with a culture of a particular bacterial strain, which was the first step to obtain lysates of bacteriophages able to propagate in cells of various hosts. A collection of 83 sewage phages  infecting different bacterial strains has been created . In this sewage) . Colipha results , stronglCaudovirales. In multiphage samples (MPS1 and MPS2), we observed single filaments of morphological features resembling filamentous phages. However, knowing that identification of filamentous phages solely on the basis of microscopic analysis is difficult and prone to errors due to the risk of confusion with other filaments Podoviridae, and the td value was used to classify viruses with longer tails as either Myoviridae (td ≥ 16 nm) or Siphoviridae (td < 16 nm), according to the previously published ruleVirion morphology of all phages isolated from the sewage  was investigated by means of electron microscopic studies. All phages from the collection belonged to the order of tailed phages, Siphoviridae, characterized by long non-contractile tails. Most of them had isometric heads. There were some phages among Sipho- and Myoviridae with elongated heads with hl:hd ratio ≥ 1.3. Most myoviral particles had sheaths with noticeable characteristic criss-cross appearance, while some had visible side fibers. Phages from the family Podoviridae, characterized by short tails, were the least abundant  were calculated. The data is presented in a biplot  and its statistical significance . Similarly, a high correlation was observed between head length and tail diameter . Phages with tails of the largest diameter in the collection – myophages – had the largest capsids at the same time. Over a half of myophages in the collection with the average td = 20 nm had hd ≥ 70 nm, while 87% of podophages and 71% of siphophages with the average td = 11 nm, had hd < 60 nm    . More th = 0.01) . Here, o = 0.01) .Siphoviridae  belonged to oviridae . Interesoviridae .P. aeruginosa strains, isolated from a patient with cystic fibrosis . The phages with larger heads (large fraction of myophages) tend to form smaller plaques  of the phages from the collection and plaque diameter . Some coliphages were able, in addition, to infect clinical E. coli strains , additionally caused the lysis of E. coli strains  showed the ability to lyse their hosts at low temperatures (4–7 °C)  . SimilarBacteriophages were tested for survival at various temperatures, ranging from −20 °C to 95 °C. The vast majority of phages from the collection survived, to some extent, incubations at −20 °C and 40 °C, but they were sensitive to the temperature of 62 °C . In mostMyoviridae and were distinguished by larger heads than the rest of phages from the collection , showed high survivability (70–100%). Twenty-one phages were susceptible to osmotic shock , Fig. 4.llection .The effects of high and low pH on phage virion stability were also studied. Almost all (80 out of 83) phages were stable at pH 10, and the titer of only 3 phages dropped by 80% or more after 1 h of incubation under these conditions . On the The presence of detergents  and organic solvents  caused a significant titer drop of most phages from the collection, though the virions were quite resistant to chloroform. However, a few phages showed high survivability (70–100%) in the presence of SDS or ethanol . One of To assess the level of biodiversity of bacteriophages from the collection, we compared all the phages in terms of morphology and physiology (the response to the studied conditions). We assumed that high biodiversity should allow us to construct a highly divergent dendrogram representing different levels of similarity between phages. However, such a dendrogram would be relatively simple if biodiversity of the studied phages was low. In the analysis, we considered virion morphology, plaque size, host spectrum, survivability at low and high temperatures, survivability at low and high pH, sensitivity to osmotic shock, sensitivity to a detergent (SDS) and organic solvents  phage, different from other phages from the collection, the actual size of the collection would be 70 rather than 83 phages. This potential reduction, however, would not have a significant influence on phage distribution in families.The results described here indicate high biodiversity of bacteriophages isolated from a single habitat. Previous reports also demonstrated diversity of phages infecting strains of SalmonellaTo investigate genetic variability among phages from the collection, complete genome sequences of bacteriophages vB_Efae230P-4, vB_Pae575P-3, vB_Pae1369P-5, vB_Pae436M-8, vB_SenM-2, vB_SscM-1, vB_SscM-2, have been determined and deposited in GenBank . Maps of these genomes are shown in E. coli isolates Analyses of the sequenced genomes of selected phages indicated that they consist of linear double-stranded DNA of 17,972 bp (vB_Efae230P-4), 72,728 bp (vB_Pae575P-3), 72,508 bp (vB_Pae1369P-5), 66,922 bp (vB_Pae436M-8), 158,986 bp (vB_SenM-2), 139,681 bp (vB_SscM-1), and 139,682 bp (vB_SscM-2). The GC content was determined at 32.76%, 54.72%, 54.72%, 55.76%, 44.71%, 31.83% and 31.82%, respectively. Annotations of the sequenced genomes revealed 25, 92, 91, 91, 210, 203 and 202 coding DNA sequences (CDSs), respectively. Among all identified coding regions, relatively high percentages  of CDSs encoding hypothetical proteins have been determined. One might be surprised that uncharacterized coding sequences dominate in all analysed bacteriophage genomes, especially in the light of 24% of hypothetical proteins encoded in sequenced genomes of Enterococcus phage vB_IME195 (GenBank No. KT932700) whereas two other analysed phages: vB_Pae575P-3, and vB_Pae1369P-5 show 93% sequence similarity with P. aeruginosa phage PA26 (GenBank No. JX194238.1). All of them possess a Podoviridae-like morphology. The genomic sequence of phage vB_Pae436M-8 is similar in 95% to the other P. aeruginosa phage, named LAM2 (GenBank No. FM201282). Curiously, this phage belongs to Myoviridae family of long-tailed phages. Another analysed phage, vB_SenM-2, has also been classified into the Myoviridae family, but presents 94% sequence similarity with the S. enterica phage Det7 (GenBank No. KP797973.1). Two other analysed phages, vB_SscM-1 and vB_SscM-2, which have only 56% sequence similarity with S. aureus phage MCE-2014 (GenBank No. KJ888149.1) and no more closely related phage within NCBI database, have been classified as members of Myoviridae family too.Genomic comparisons of the analysed phages to the most similar and previously sequenced phages from the NCBI database indicated that phage vB_Efae230P-4 presents 74% nucleotide sequence similarity with Enterococcus phage vB_IME195 genome, two CDSs encoding proteins of unknown functions have been identified. The second analysed region within the vB_Efae230P-4 genome (from residue 16480 to16653) encompasses a 174-nt long CDS encoding a hypothetical protein. In a corresponding part of the vB_IME195 genome, there is a 881-nt long fragment with four CDSs predicted to encode uncharacterized proteins. The last analysedregion, extending from residue 17185 to 17972, of the vB_Efae230P-4 genome, shows a low level of sequence identity to vB_IME195 (<50%), however, similarly to the corresponding fragment of the vB_IME195 genome, it also includes two CDSs encoding hypothetical proteins , encode hypothetical proteins, there are also recognizable accessory genes with functions which were determined previously in other biological systems . Curiousproteins .P. aeruginosa phage PA26. An interesting region has also been identified within the genome sequence of phage vB_Pae436M-8 around residue 63100  share only 56% sequence identity. As presented in In summary, our genomic comparisons of newly isolated phages from urban sewage to those published previously, reveal different levels of diversity and spotlight a continuous evolution in a single environmental source. The obtained data highlight the mosaicism of the analysed genomes, resulting from acquisition or loss of genetic material and numerous point mutations. Interestingly, sequencing data indicate that genomes of the analysed bacteriophages are littered with CDS predicted to encode homing endonucleases. They are site-specific DNA endonucleases that function as mobile genetic elements and are known as a significant source of genomic variability. The DNA repair process, catalyzed by these enzymes, results in transmission of the endonuclease gene and flanking segments of DNA between genomesMorphological and physiological analyses of a large collection of bacteriophages from a single environmental source indicated that there is high biodiversity among phages existing in one habitat. Some features of the investigated phages, like the ability to form plaques at low (4 °C) temperature, resistance to high (62 °C or even 95 °C) temperature or survival in the presence of organic solvents  or detergents  make them potentially interesting in the context of biotechnological applications. Ability to propagate on clinical strains of bacteria suggest the potential use of these bacteriophages in phage therapy. Analysis of complete genome sequences of 7 from the newly isolated bacteriophages confirmed a high diversity of these viruses, even though they come from the single environmental source.Pseudomonas aeruginosa, Staphylococcus aureus and Salmonella enterica were received from the National Medicines Institute in Warsaw (Poland), the Laboratory of Molecular Diagnostics of Intercollegiate Faculty of Biotechnology of University of Gdansk and the Medical University of Gdansk, and the National Salmonella Centre at Medical University of Gdansk (Poland), respectively. Enteropathogenic E. coli (EPEC) strains were isolated from stools of patients from the Specialist Hospital of St. Wojciech in Gdansk (Poland). Enterohemorrhagic E. coli (EHEC) strains, as well as laboratory strains of E. coli and P. aeruginosa were from the collection of the Department of Molecular Biology of the University of Gdansk (Poland). Environmental strains came from the collection of the Department of Water and Wastewater Technology of Gdansk University of Technology  and from the collection of the Institute of Oceanology of the Polish Academy of Sciences in Sopot . All bacterial strains employed in this study and detailed information about them are listed in the Raw sewage samples were collected at the Gdansk Wastewater Treatment Plant . The physicochemical parameters were provided by the Laboratory of Gdansk Wastewater Treatment Plant . Clinical strains of E. coli, Pseudomonas, Salmonella and Staphylococcus bacteria were cultivated in Luria-Bertain (LB) liquid medium with aeration achieved by shaking, or plated on solid LB medium supplemented with 1.5% bacteriological agar. For enterococci special Todd Hewitt Broth (THB) and Brain-Heart-Infusion Agar (BHI agar) were preparedFor all phage experiments, 4; pH 7.2) or culture media. Then, 2.5 μl of each dilution of the phage lysate were spotted on the double agar/agarose layer. Plates were incubated at 37 °C for 12–24 h and the phage concentration was quantified on the basis of the numbers of plaques.The phage propagation procedure was performed according to the double overlay plaque assay with modifications4; pH 7.2) containing sodium chloride  and extracted several times with chloroform to remove remnants of PEG and other contaminants. Multiphage lysates were kept at 4 °C for further analysis.For bacteriophage isolation from sewage samples, two methods were employed. The first method was used for isolation of the whole phage population present in the sewage sample. Four liters of raw sewage were subjected to the elution procedure, as described earlierFor the isolation of phages infecting particular bacterial strains one-host enrichment method was used, as described earlierPurification of the virus-like particles (VLPs) was prepared using cesium chloride density gradient centrifugation procedureE. coli O157:H7 ST2–8624) was tested on E. coli MG1655 strain. P. aeruginosa 436/1996 and S. enterica serovar Heidelberg were employed to analyse plaque morphology of Pseudomonas and Salmonella phages, respectively. To determine plaque size, serial ten-fold dilutions of phage stocks were prepared in LB or THB medium. In the next step, 0.2 ml of the host strain culture was mixed with 3 μl of an appropriate dilution of bacteriophage lysate and added to the 4 ml of the top LB or THB agar, supplemented with 0.4% agar/agarose. The mixture was poured onto an LB or BHI plate. The Petri dishes were incubated at 37 °C, and after 20 h, plaque morphology and diameter were assessed.Plaque morphology of all coliphages but one . The host range was created by observing the presence of plaques onto a double layer agar plate, prepared as described in the preceding paragraph. Ten-fold dilutions of phage stocks were performed in LB or THB medium, and spotted on the lawn of a potential host. Plates were incubated at 37 °C, and examined for plaques after 18–24 h.To determine the propagation of bacteriophages at different temperatures, titers of the phage lysates were assayed after storage at 4 °C, 22 °C, 30 °C or 37 °C for 24 h, as described previouslyTo estimate phage stability during thermal inactivation test, four different temperatures were investigated: −20 °C (12 h), 40 °C (40 min), 62 °C (40 min) and 95 °C (5 min). The procedure was conducted as described elsewhereThe effect of an acidic and an alkaline pH on phage particles was studied using LB or THB medium with pH 2, pH 4, pH 10 and pH12, according to a procedure described previously4; pH 7.2) containing sodium chloride  at room temperature for 15 minMeasurement of survival of bacteriophages during osmotic shock conditions were studied by means of incubation of virus particles in TM buffer  and enumerated immediately by the double layer method. The control test was carried out identically without the addition of ionic detergents.To determine antiviral effect of ionic detergents on phage particles three different compounds were tested: anionic sodium dodecyl sulfate (SDS), anionic sodium lauroyl sarcosinate (Sarkosyl) and cationic cetyltrimethylammonium bromide (CTAB), according to the procedures described previously47484; pH 7.2) were prepared and used for plating. Phages incubated in TM buffer under conditions described above, were used as a control.The effect of four different organic solvents: ethanol, acetone, chloroform and dimethyl sulfoxide (DMSO) on phage virions, was studied. The procedures were conducted as described elsewhere5051The viral DNA was liberated from virions using the method described previouslyhttp://blast.ncbi.nlm.nih.gov/Blast.cgi, and Progressive MAUVE, available at: http://darlinglab.org/mauve/mauve.html. Any modifications within sequences were done using Serial Cloner, available at: http://serialbasics.free.fr/Serial_Cloner.html. Automatic annotations were carried out using myRAST softwarehttp://ugene.net/. Sequence analysis and changes to the automatic annotations were done based on the data acquired by protein BLAST program (PSI-BLAST), HMMER software available at: http://www.ebi.ac.uk/Tools/hmmer/, Pfam database (http://pfam.xfam.org/), Phobious webserver (http://phobius.binf.ku.dk/), and TMHMM program available at: http://www.cbs.dtu.dk/services/TMHMM/. Putative coding sequences were added to the annotation list if they contained a plausible ribosome binding site, and both the start and stop codons. The.sqn files, which were sent to GenBank, were created using DNA Master software, available at: http://phagesdb.org/DNAMaster/. Circular comparisons between phage genomes and coordinates were generated using BLAST Ring Image Generator (BRIG) platform available at: https://sourceforge.net/projects/brig/.Phage genomes were sequenced in the Max-Planck Institute  using the pyrosequencing technology and Genome Sequencer FLX with Titanium series reagents. Assembly of the sequences was accomplished using MIRAp value) were calculated for the analysis of the correlations between variables. Correlations, for which p < 0.05 were considered statistically relevant. In addition, hierarchical clustering analysis, using Euclidean distance and complete linkage method, was used for statistical classification of data. All analyses were made employing R for Windows software, version 3.0.1.Principal Component Analysis (PCA) was used for the analysis of morphological and biological data. Spearman’s rank correlation coefficient (ϱ) and its statistical significance .


Flavivirus) has been reported, representing an emerging threat for both animal and human health. To further study utility of wild ruminant samples in West Nile virus (WNV) surveillance, we assessed spatio–temporal trends and factors associated with WNV and cross-reacting flaviviruses exposure, particularly Usutu virus (USUV) and Meaban virus (MBV), in wild ruminants in Spain. Serum samples from 4693 wild ruminants, including 3073 free-living red deer (Cervus elaphus), 201 fallow deer (Dama dama), 125 mouflon (Ovis aries musimon), 32 roe deer (Capreolus capreolus) and 1262 farmed red deer collected in 2003–2014, were screened for WNV and antigenically-related flavivirus antibodies using a blocking ELISA (bELISA). Positive samples were tested for neutralizing antibodies against WNV, USUV and MBV by virus micro-neutralization tests.During the last decade, the spread of many flaviviruses , the specific south-coastal bioregion (bioregion 5) and presence of wetlands. Red deer had neutralizing antibodies against WNV, USUV and MBV.The results indicate endemic circulation of WNV, USUV and MBV in Spanish red deer, even in areas without known flavivirus outbreaks. WNV antibodies detected in a free-living red deer yearling sampled in 2010, confirmed circulation this year. Co-circulation of WNV and USUV was detected in bioregions 3 and 5, and of WNV and MBV in bioregion 3. Sampling of hunted and farmed wild ruminants, specifically of red deer yearlings, could be a complementary way to national surveillance programs to monitor the activity of emerging flaviviruses. Flaviviridae) in the world has substantially increased over the last decades. During this period, many flavivirus infections have become a major public health concern due to continuous and growing reporting of outbreaks in humans [The distribution of vector-borne flaviviruses (family n humans . FlaviviCamelus dromedarios) [Sus scrofa) and Iberian pigs and red foxes (Vulpes vulpes) [Rupicapra pyrenaica) [Larus michaelis) and ticks (Ornithodoros maritimus) in north-eastern Spain [During the last few years, six flaviviruses, including West Nile virus (WNV), Usutu virus (USUV), tick-borne encephalitis virus (TBEV), Bagaza virus (BAGV), Meaban virus (MBV) and louping-ill virus (LIV), have been detected in Europe , 3. Fiveedarios) , wild bo vulpes) . Clinica vulpes) , 12. USU vulpes) , 13 and  vulpes) , 15. Mor vulpes)  and susprenaica) . An unusrenaica) . FinallyOdocoileus virginianus) from the United States [Rangifer tarandus) in North America [Cervus elaphus), Iberian pigs, red foxes, and others [Wild and domestic artiodactyls can be useful sentinel species for monitoring flavivirus activity –21. Antid States , 23, andd States . In addi America , 26. Serd others , 10. Accd others .As red deer is an important game species in Spain  and is aThe aim of this study was to monitor seroprevalence of WNV and antigenically-related mosquito and tick-borne flaviviruses, particularly USUV and MBV, in wild ruminants in Spain, and, using red deer as the most distributed, abundant and readily available species, to assess the spatial–temporal trends and risk factors associated with the exposure to these flaviviruses in this species.Dama dama), 125 mouflon (Ovis aries musimon) and 32 roe deer (Capreolus capreolus) were collected between 2003 and 2014 from hunting estates across Spain. Red deer were sampled in 130 hunting areas located throughout the five geographical bioregions of Spain previously defined for the national wildlife disease monitoring program [Samples of 4693 wild ruminants, including 3073 free-living and 1262 farmed red deer, 201 fallow deer g for serum separation and serum was stored at −20 ° C until analysis.Blood samples were taken from the thoracic cavity of freshly killed hunted animals or during health inspection in the slaughterhouse. Samples of farmed red deer were obtained by puncture of the jugular vein using a sterile collection system during health inspections. All samples of free-living ruminants were grouped by season and habitat type . Samples were classified by age according to dentition patterns  . The aniAll ruminants were tested for antibodies against an epitope of the WNV pre-membrane-envelope (prM-E) protein shared with other viruses of the Japanese encephalitis serocomplex. A commercially available blocking enzyme-linked immunosorbent assay  was used in accordance with the manufacturer’s recommendations. The bELISA was used as a serological screening tool and bELISA-positive and doubtful sera were confirmed by micro virus neutralization test (VNT) for the detection of specific neutralizing antibodies against WNV  and USUV (It12 strain). Additionally, given the possibility of cross-reaction with antigenically-related flaviviruses not included within the Japanese encephalitis serocomplex such as Meaban virus (MBV), bELISA-positive and doubtful samples were also tested by VNT against this flavivirus . VNTs were performed as previously described . SamplesP-value < 0.20 in the bivariate analysis were included for further analysis. Biologically plausible confounding factors were assessed using Mantel-Haenszel analysis and confounding was considered to be potentially significant if odd ratios (ORs) shifted appreciably. Variables that altered the coefficients for the independent variables of interest by 30 % or more were removed from the model and were classified as confounding factors. Finally, a multiple logistic regression analysis [P < 0.05). The goodness of fit was assessed using the Hosmer–Lemeshow goodness-of-fit test. SPSS 22.0 software  was used for statistical analyses.The prevalence of antibodies against antigenically-related flaviviruses was estimated from the ratio of positives to the total number of samples, with the exact binomial confidence intervals of 95 %. Differences between species were analyzed using a Pearson’s Chi-square test and a Fisher’s exact test (when observations/category were <6). Due to the absence of seropositivity in roe deer and to the limited number of seropositive fallow deer and mouflon, the associated risk factors were only analyzed for red deer, the most widely distributed and abundant wild ruminant species in Spain. A Chi-square or Fisher’s exact test were used to test the relevance of the explanatory variables  to the risk of an red deer being exposed to flaviviruses. Covariates correlated with a analysis  was perf95%: 2.7–3.8 %) wild ruminants were seropostive against antigenically-related flaviviruses using bELISA. A mean seroprevalence of 3.4 ± 0.5 % (148/4335) was obtained in red deer, 1.0 ± 1.4 % (2/201) in fallow deer, and 2.4 ± 2.7 % (3/125) in mouflon. No seropositivity was detected in the 32 roe deer tested. Statistically significant differences in seroprevalence among species were not observed.A total of 153 out of the 4693  from the bivariate analysis (Table P < 0.001). Forty-one out of the 130 (31.5 %) sampling areas presented at least one seropositive animal. The seropositivity by sampling areas was 3.1 % (1/32) in bioregion 1, 42.9 % (3/7) in bioregion 2, 44.3 % (31/70) in bioregion 3, 11.1 % (1/9) in bioregion 4 and 41.7 % (5/12) in bioregion 5. Even though seropositivity was detected in all bioregions, considering farmed and free-living red deer together, a significantly higher seroprevalence was found in bioregion 5  compared to the other bioregions (P < 0.001), and in bioregion 3  compared to bioregion 1  (P < 0.001). Significantly higher seropositivity was also detected in bioregion 5  (P = 0.003), bioregion 4  (P = 0.012) and bioregion 3 (P < 0.001) compared to bioregion 1  when seroprevalence was compared only in free-living red deer. A significantly higher seroprevalence was obtained in summer  as compared to autumn  (P = 0.003) and winter  (P = 0.012). Seroprevalence was significantly higher in farmed 5.9 % (74/1262) than in free-living 2.4 ± 0.5 % (74/3073) red deer (P < 0.001). Seropositivity was confirmed in three out of the five red deer farms tested, with the mean seroprevalence ranging from 0.4 % (1/243) to 9.7 % (71/729). In the red deer farm with the highest seropositivity, a significantly higher mean seroprevalence was found in autumn  compared to spring  and summer , but not in winter . The final multivariate logistic regression model showed that the main risk factors associated with exposure to related flaviviruses in red deer were: year (2011), bioregion (5) and wetland areas (presence)  out of seven variables were selected  could not be analyzed by VNT due to serum cytotoxicity. Of these, 25 (22 from red deer and 3 from mouflon) showed negative results against the three flaviviruses tested using VNT. Specific antibodies against WNV, USUV and MBV were confirmed in 103 , 4  and 2  red deer, respectively (Table Culex family mosquitoes (flavivirus vector) have shown a preference of feeding on deer [Culex species sampled in Canary Islands (Spain) frequently fed on ruminant species [The overall prevalence of antibodies against WNV and antigenically-related flaviviruses detected in wild ruminants (3.3 %) in the present study was consistent with that recently observed in captive zoo artiodactyls in Spain . Similar on deer . Similar species , while a species . Thus, oMultivariate logistic regression identified the year 2011, bioregion 5 and the presence of wetland areas as individual risk factors for antigenically-related flavivirus exposure in red deer. The seroprevalence data obtained indicates widespread but not homogeneous distribution of these flaviviruses in wild ruminants in Spain. Consistently with other studies, seroprevalence of flaviviruses in both farmed and free-living wild ruminants, mainly WNV, was higher in southern Spain, namely in bioregions 3, 4 and 5 , 38, 39.Animals sampled in wetland areas were three times more likely to be exposed to related flaviviruses, which may be due to the larger populations and greater diversity of competent vectors. In addition, in late summer, the number of wetlands and waterholes in southern Spain becomes greatly reduced, aggregating animals in these hotspots , which mSeropositivity of at least one animal in all sample years supports a more-or-less continuous circulation of antigenically-related flaviviruses in the study period. However, a significantly higher seroprevalence was found in 2011, 1 year after the highest number of WNV and Bagaza outbreaks were reported in Spain , 40. In Intrinsic host factors such as age can influence exposure and susceptibility to flavivirus infection. In the present study, seroprevalence increased with age but no significant differences among age classes were observed. The results may be explained by the limited number of samples from yearlings analyzed, due to the fact that yearlings are not usually hunted. A low seroprevalence in yearling red deer from Spain was also previously reported by . MoreoveSignificantly higher seroprevalence to antigenically-related flavivirus was found in farmed red deer as compared to free-living ones, although this variable was not retained in the multivariate analysis. In contrast, free-living red deer sampled in open and fenced sampling areas did not differ in seroprevalence. Our results indicate that samples from farmed red deer, which are accessible during all year, may be a valuable surveillance tool. Variations in the seroprevalence among seasons were also detected in the bivariate analysis, with significantly higher seroprevalence in summer. Moreover, significantly higher mean seroprevalence was found in autumn in farmed red deer sampled in bioregion 5. Most of the WNV outbreaks in horses in Spain have been reported in late summer and autumn , confirmTo the authors’ knowledge, this is the first report on WNV, USUV and MBV antibodies in red deer, which confirms local circulation of these flaviviruses in Spain. Twenty two sera were positive by bELISA but negative to USUV, WNV and MBV in VNT. The results could be due to the circulation of other related flaviviruses . MortaliSpecific antibodies against WNV were confirmed in four out of the five bioregions. The higher WNV seroprevalence in bioregion 3 and 5 is in accordance with WNV distribution reported in horses in Spain . InteresDuring the last decade, USUV has rapidly expanded in different European countries, and has been detected in mosquitoes and vertebrate species . Even thLarus argentatus) gulls from different breeding colonies in France and Spain [Fregata minor) and a sooty tern (Onychoprion fuscatus) in the Western Indian Ocean [Infection with MBV or a Meaban-like virus has been recently detected in yellow-legged and herring (nd Spain , and in an Ocean , althougan Ocean .The results obtained indicate local circulation of antigenically-related flaviviruses in wild ruminants in Spain. The significance of WNV, USUV and MBV infection in this species is unknown at this time and further research on aspects such as the genetic diversity, infection rates and competent vectors of these emerging flaviviruses is needed. Surveillance of hunted wild ruminants, and especially on farmed red deer, may be a useful, cost-effective, rapid and complementary tool to the national surveillance programs to monitor the activity of mosquito and tick-borne flaviviruses in Europe.


Articular cartilage lacks a blood supply and nerves. Hence, articular cartilage regeneration remains a major challenge in orthopedics. Decellularized extracellular matrix- (ECM-) based strategies have recently received particular attention. The structure of native cartilage exhibits complex zonal heterogeneity. Specifically, the development of a tissue-engineered scaffold mimicking the aligned structure of native cartilage would be of great utility in terms of cartilage regeneration. Previously, we fabricated oriented PLGA/ACECM  composite scaffolds. In vitro, we found that the scaffolds not only guided seeded cells to proliferate in an aligned manner but also exhibited high biomechanical strength. To detect whether oriented cartilage regeneration was possible in vivo, we used mesenchymal stem cell (MSC)/scaffold constructs to repair cartilage defects. The results showed that cartilage defects could be completely regenerated. Histologically, these became filled with hyaline cartilage and subchondral bone. Moreover, the aligned structure of cartilage was regenerated and was similar to that of native tissue. In conclusion, the MSC/scaffold constructs enhanced the structure-specific regeneration of hyaline cartilage in a rabbit model and may be a promising treatment strategy for the repair of human cartilage defects. Articular cartilage defects are very common due to the increasing numbers of patients with traumatic injuries and osteoarthritis (OA) . CartilaThe scaffold, the seed cells, and the nature of biochemical or biomechanical stimulation are the three critical elements of tissue engineering and regeneration medicine , 6. NatuBiomimetic structures similar to native tissue are of great utility in scaffold fabrication –19. ArtiTo combine the advantages afforded by synthetic and natural polymeric materials, we previously developed oriented PLGA/ACECM composite scaffolds using an improved, thermally induced phase-separation approach , 23. We Chondrocytes are the (uniquely) functional cells of articular cartilage . HoweverIn the present study, we first isolated autologous bone marrow-derived MSCs and seeded these cells into oriented PLGA/ACECM composite scaffolds. We then implanted the cell-scaffold constructs into cartilage defects created in rabbits . We founv/v) fetal bovine serum  and then plated at a density of 1 × 106 cells/cm2 in a humidified atmosphere under 5% (v/v) CO2 at 37°C. The medium was changed once during the next 3 days until the cells attained 80% confluence. The cells were then deattached using 0.25% (w/v) trypsin solution and expanded in a regular growth medium at an initial concentration of 1 × 104 cells/cm2. The MSCs were not subjected to chondrogenic induction, and passage-3 cells were employed in the following experiments.After the work was approved by the Animal Care and Experimental Committee of the Chinese People's Liberation Army General Hospital, bone marrow-derived MSCs were isolated from anesthetized mature New Zealand white rabbits (weight 2.5–3.0 kg) and cultured as described previously . Brieflyw/v). The same weight of ACECM was added to this solution to form a slurry. The solid concentration was adjusted to 7% (w/w) by the addition of dioxane. The mixed slurry was infused into a 4 mm diameter cylindrical mold, which was then inserted vertically into a metal plate and held at −20°C for 30 min. After becoming completely frozen, the mold was held for a further 2 h at −20°C, transferred to a lyophilizer, and lyophilized for 48 h under vacuum. The scaffolds were removed from the molds, and cross-linking proceeded under ultraviolet light at 258 nm for 4 h. The scaffolds were then immersed in 95% (v/v) alcohol containing 50 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC) and 20 mM N-hydroxysuccinimide  for 24 h at 4°C. Excessive EDAC was washed away repeatedly with phosphate-buffered saline (PBS). The scaffolds were washed in triple-distilled water to remove residual dioxane and then subjected to freeze drying. The scaffolds were finally sterilized by 60Co γ-irradiation (5 mRad) and stored at 4°C prior to use.Previously, we successfully fabricated oriented PLGA/ACECM composite scaffolds , 23; theμL) of the cell suspension (approximately 2 × 106 cells) were added to completely saturate the scaffolds, which were then incubated under 5% (v/v) CO2 in a humidified atmosphere for 4 h at 37°C to allow for cell adherence, with the addition of 20 μL DMEM (with 10% [v/v] FBS) to each scaffold every 30 min. Each well of the 24-well culture plate received an additional 1 mL culture medium, and the culture continued for another 3 days.MSCs were resuspended in culture medium. Oriented PLGA/ACECM composite scaffolds , sterilized by ethylene oxide, were placed into the wells of a 24-well culture plate. Aliquots (50 n = 30). Each rabbit knee joint was opened via a medial parapatellar approach under general anesthesia. Full-thickness cylindrical defects  were created on the patellar grooves of both femora  weighing between 2.5 and 4.0 kg were used in this study , an oriented PLGA/ACECM composite scaffold-alone group (only the scaffold was inserted), and an untreated group. The number of cartilage defects in each group is shown in Rabbits were sacrificed at 12 and 24 weeks postsurgery, and the knee joints were harvested. All evaluations were performed by three independent individuals blinded to the groupings. Repair quality was estimated using the criteria of the International Cartilage Repair Society Macroscopic Evaluation of Cartilage Repair. The scoring system used included rating of the extent of repair, integration with the border zone, and macroscopic appearance  30]..30].Microcomputed tomography  was used to quantitatively and qualitatively assess subchondral bone regeneration at 12 and 24 weeks after surgery. A volume of interest was defined in the subchondral bone region of each defect site. Subchondral bone regeneration was estimated in terms of bone volume per tissue volume (BV/TV) and trabecular thickness (Tb.Th).μm thick slices). Next, the slices were stained with hematoxylin and eosin, toluidine blue, and safranin O. Images from each group were recorded via bright-field microscopy . The thickness of each regenerated cartilage layer (from the cartilage surface to the tidemark) was measured and compared with that of the surrounding native cartilage. The extents of toluidine blue and safranin O staining were assessed by counting the integrated optical density (IOD) units per stained area (μm2) with the aid of ImageJ software . Grading was performed in a blinded manner by three independent individuals. The extent of repair was evaluated based on the International Cartilage Repair Society grading system. Histological grading involved the evaluation of surface regularity, matrix staining, cell distribution, cell viability, and other items  overnight at 4°C. The sections were washed and incubated with biotinylated secondary anti-mouse antibody , followed by development with diaminobenzidine . The sections were counterstained with hematoxylin and examined microscopically . Collagen type II immunohistochemical staining was evaluated by calculating the IOD per stained area (μm2) with the aid of ImageJ software.The expression of collagen type II in regenerated cartilage was examined via immunohistochemical staining at 12 and 24 weeks postimplantation. Collagen type II was stained immunohistologically using a mouse anti-collagen type II polyclonal antibody  according to the manufacturer's instructions. In brief, horizontal and vertical sections were blocked with peroxidase-blocking solution for 10 min, followed by three washes with PBS for 5 min each, and each section was then incubated with 50 E = P(1 − ν2)/2awk  [The mechanical properties of regenerated cartilage were evaluated by indentation testing using a dedicated apparatus , as described previously, at 12 and 24 weeks after surgery. Young's modulus was calculated using the following formula: P values < 0.05 were considered to reflect statistical significance.All data were analyzed with the aid of SPSS software , and the values are expressed as means ± standard deviations. One-way analysis of variance followed by Tukey's post hoc multiple-comparison test was applied to identify significant differences among the groups after assessment of the homogeneities of variance. For 1 week postsurgery, the rabbits reduced their activity levels and appeared to be in a lower mental state than previously; they ate less but exhibited a nearly normal range of joint activity. One rabbit died of a reaction to anesthesia, five died within 1 week due to surgical trauma, three died of diarrhea within 1 month, two died of ear mite infections within 2 months, and one was euthanized because of a joint infection. All rabbits that died before sacrifice were replaced with alternative animals. All remaining rabbits were sacrificed in a timely manner, and the knee joints were obtained.Before harvesting, the knee skin was complete and was healing well; we observed no swelling or fever and no abnormal secretion. The joint fluids were clear. The synoviae lacked thickening and contracture. The regenerated and surrounding cartilage exhibited no obvious exfoliation or hyperplasia, no patellar dislocation, and no obvious joint instability.At 12 weeks postsurgery, the cartilage surface in the untreated group was clearly lower than the native cartilage tissue. Moreover, poor integration with surrounding cartilage was evident, and minimal cartilage-like tissue had regenerated . A majorP < 0.05).At 24 weeks postimplantation, the defective region in the untreated group had increased in size and integration remained poor. Furthermore, the new tissue clearly differed from the native cartilage . In the Macroscopic effects were verified histologically. At 12 weeks postoperation, the defect areas in the untreated group remained concave and no integration with native tissue was evident. No cartilage tissue or subchondral bone regeneration was apparent, except for the development of some fibrotic tissue . In the At 24 weeks postsurgery, integration remained poor in the untreated group and few cellular matrices were stained. Most regenerated regions contained only fibrous tissue. Moreover, no subchondral bone regeneration was apparent; again, only fibrous tissue was evident . ConversP < 0.05; Figures P < 0.05).Toluidine blue, safranin O, and immunohistochemical staining for collagen type II at 12 and 24 weeks postsurgery evidenced chondrogenic differentiation in vivo Figures –7. At 12P < 0.05), but these two groups differed significantly at both timepoints (both P < 0.05). The BV/TV ratios were significantly higher in the cell-loaded scaffold group than in the scaffold-alone and untreated groups at both timepoints  but significant differences between timepoints were evident . The Tb.Th values were significantly higher in the cell-loaded scaffold group than in the scaffold-alone and untreated groups at 12 and 24 weeks postsurgery . Moreover, from 12 to 24 weeks after implantation, cartilage thickness increased in only the cell-loaded scaffold group (P < 0.05) and not in the other two groups (P > 0.05).The thickness of regenerated cartilage was evaluated at 12 and 24 weeks postoperation . It was P > 0.05). In contrast, the compressive moduli in the scaffold-alone and cell-loaded scaffold groups increased from 12 to 24 weeks postsurgery (P < 0.05). Moreover, the compressive modulus was significantly higher in the cell-loaded scaffold group than in the other two groups at both timepoints (P < 0.05).The biomechanical properties of the regenerated cartilage were evaluated by calculating Young's modulus at 12 and 24 weeks postoperation . The comArticular cartilage defects of the knee joint are very common in the general and athletic populations . CartilaIn the cell-loaded scaffold group, the cartilage defect was repaired almost completely via formation of new cartilage that was similar in appearance to adjacent normal cartilage. Histologically, the regenerated cartilage integrated well with adjacent tissue. Toluidine blue, safranin O, and immunohistochemical staining further confirmed that the cell-loaded scaffold group contained more GAG-expressing and collagen type II-positive cells at 12 and 24 weeks postsurgery. Notably, the newly formed cartilage exhibited the characteristic lacuna-like structure, providing indirect evidence that the cell-loaded scaffold facilitated chondrogenic differentiation in vivo. In addition, most cells in the deeper cartilage defect regions were organized in columns. The cartilage thickness and subchondral bone formation in the cell-loaded scaffold group were similar to those of native tissue. Moreover, from a functional perspective, the regenerated cartilage was biomechanically competent.Repair was not as good in the scaffold-alone group as in the MSC-loaded scaffold group. Some repaired cartilage was evident in the scaffold-alone group at 12 weeks after surgery; however, the newly formed tissue stained only slightly for GAG and type II collagen. Moreover, the cells in this group were arranged randomly. Some studies have shown that ECM degradation products enhance the recruitment and proliferation of multipotential progenitor cells . We specStem-cell-loaded scaffold-based strategies are more effective than scaffold-alone strategies , as theyOverall, the results showed that autologous MSCs loaded into an oriented PLGA/ACECM composite scaffold enhanced structure-specific regeneration of hyaline cartilage and subchondral bone in a rabbit model. This approach may become a promising treatment strategy for human cartilage defect repair.


Helicobacter pylori (HP) infection is associated with reduced lung function and systemic inflammation in chronic obstructive pulmonary disease (COPD) patients. Azithromycin (AZ) is active against HP and reduces the risk of COPD exacerbation. We determined whether HP infection status modifies the effects of AZ in COPD patients.Plasma samples from 1018 subjects with COPD who participated in the Macrolide Azithromycin (MACRO) in COPD Study were used to determine the HP infection status at baseline and 12 months of follow-up using a serologic assay. Based on HP infection status and randomization to either AZ or placebo (PL), the subjects were divided into 4 groups: HP+/AZ, HP-/AZ, HP+/PL, and HP-/PL. Time to first exacerbation was compared across the 4 groups using Kaplan-Meier survival analysis and a Cox proportional hazards model. The rates of exacerbation were compared using both the Kruskal-Wallis test and negative binomial analysis. Blood biomarkers at enrolment and at follow-up visits 3, 12, and 13 (1 month after treatment was stopped) months were measured.p < 0.001). The median time to first exacerbation was significantly different across the four groups (p = 0.001) with the longest time in the HP+/AZ group  followed by the HP-/AZ group . Hazard ratio (HR) for exacerbations was lowest in the HP+/AZ group after adjustment for age, sex, smoking status, ethnicity, history of peptic ulcer, dyspnea, previous hospital admission, GOLD grade of severity, and forced vital capacity . Circulating levels of soluble tumor necrosis factor receptor-75 were reduced only in the HP+/AZ group after 3 months of AZ treatment ; levels returned to baseline after discontinuing AZ.One hundred eighty one (17.8%) patients were seropositive to HP. Non-Caucasian participants were nearly three times more likely to be HP seropositive than Caucasian participants  contains supplementary material, which is available to authorized users. Helicobacter pylori (HP) infection in patients with COPD [Persistent systemic inflammation affects ~15 to 20% of patients with chronic obstructive pulmonary disease (COPD), which in turn is associated with an increased risk of exacerbations and mortality , 4. Previth COPD . HP infeith COPD –9.in vitro and in vivo [Interestingly, azithromycin (AZ), which is being increasingly used to prevent exacerbations in COPD has bactericidal activity against HP both  in vivo , 11. In  in vivo , 13 and  in vivo . However in vivo , we deteDetails of the MACRO Study design and results have been published previously . The stuFor the current study, we used blood samples that were available from 1018 subjects to determine the prevalence of HP seropositivity (HP+ or HP-). Plasma samples, collected at enrollment and at the 12 month visit, were used to measure concentrations of immunoglobulin G (IgG) to HP cytotoxin-associated gene A (CagA) antigen using a commercially available ELISA kit  according to the manufacturer’s protocol. Samples with values greater than 18 DU/mL were regarded as positive for HP infection (HP+). Values lower than 18 DU/mL were considered HP negative (HP-) . Using thttps://www.r-project.org).Exacerbation-free survival was assessed using Kaplan-Meier curves stratified by HP serostatus and AZ use, and the groups were compared by a log-rank test. Bootstrap methods were used to generate the confidence intervals (CIs) for median time to the first exacerbation . Pair-wip < 0.001), even though the history of peptic ulcer was similar between the two groups . After excluding 15 patients without follow-up visits, the baseline characteristics of the remaining 1003 patients according to HP status are summarized in Table Plasma samples from 1018 subjects at enrollment were analyzed to determine the prevalence of HP seropositivity; 181 patients (17.8%) had positive titres. HP seroprevalence was substantially lower among Caucasian than non-Caucasian subjects ; the HP+/PL group (p = 0.006) and the HP-/PL group (p = 0.001); and between the HP-/AZ and the HP-/PL groups (p = 0.02). A Cox proportional hazards regression was performed to investigate the relationship between the 4 groups in regards to the time to first exacerbation. Hazard ratio (HR) for the time to first exacerbation was the lowest in the HP+/AZ group after adjusting for age, sex, smoking status, ethnicity, history of peptic ulcer, dyspnea, previous hospital admission for COPD during past year, GOLD grade of severity of airflow limitation, and FVC . The rates of exacerbation per patient-year were 1.21, 1.54, 1.73, and 1.85, respectively , whereas the HP-/AZ group had a 14% lower exacerbation compared to the HP-/PL group (p = 0.08).The median time to first exacerbation was the longest in the HP+/AZ group , followed by the HP-/AZ group , the HP+/PL group  and the HP-/PL group  with a significant difference across these 4 groups by a log-rank test  to 16.6% (107/643) after 1 year.i.e. seronegative at the baseline plasma measurement), 21 (8 from the HP-/AZ group and 13 from the HP-/PL group) became seropositive, resulting in an annual seroconversion rate of 4.0% .Of the 530 COPD patients at risk of acquiring HP infection .The seroreversion rate was not statistically different between AZ and PL use group  changes in the log-transformed CRP or sTNFR75 levels within subjects from baseline to 3, 12, and 13 months using paired t-tests  and in the AZ use group  at baseline and 859 (85.6%), 719 (71.7%), and 697 (69.5%) subjects at 3, 12, and 13 months of follow-up, respectively. We analyzed the mean (Helicobacter pylori (HP). The most important findings were that approximately 18% of patients with COPD in the MACRO Study were seropositive for HP and that these individuals experienced the largest reduction in the risk for exacerbation from chronic prophylactic AZ therapy. AZ therapy in these patients was associated with a significant decline in plasma sTNFR75 levels, but not with seroreversion rate as compared to PL use group. Together, these data suggest that AZ therapy alone probably does not eradicate HP infection but may have a significant immunomodulatory role that mitigates the risk of exacerbations in HP seropositive individuals.The present study is the first to evaluate the effect of azithromycin on COPD exacerbations according to serostatus for post hoc analysis of the MACRO data, Han and colleagues showed that the benefits of AZ were greatest in ex-smokers, older patients, and subjects with mild COPD. However, in their study, reductions in exacerbations were still seen in younger patients and those with higher GOLD grades of airflow limitation, particularly for exacerbations that required treatment with antibiotics and steroids [p = 0.04). Interestingly, using the same cohort, Woodruff et al. found that a decline in sTNFR75 concentrations over 3 months identified COPD patients who benefited from AZ therapy [N = 76; −0.87 ± 0.31 μg/L; p = 0.002), followed by ex-smokers , suggesting that potential mechanisms for the reduction in CRP levels include an anti-inflammatory effect independent of the presence of HP infection. Previously it was shown that there was an increase in CRP level in mild to moderate COPD patients who were stable and HP positive [In contrast to sTNFR75, CRP concentration at 3 months was significantly reduced in the AZ group  [Seropositivity to HP, and annual rates of seroconversion and reversal, is not well-known in patients with COPD. Our previous study using serum samples from the lung health study (LHS) and ELISA plates coated with CagA protein demonstrated a cross-sectional prevalence of HP seropositivity of 17.6% . Despiteulation) –27. A prulation) , possiblulation) , 27, 29.ulation) . Since tin vitro and in vivo activity against HP, we did not find a significant difference in the rate of seroreversion between the PL and AZ groups. We also used ≥50% decline in titres at 12 months of follow-up compared to baseline levels as indirect evidence for HP eradication [p = 0.006; Additional file Importantly, in the HP-/AZ group, there was no significant increase in HP antibody titres over the 12 month period; by contrast, there was a significant increase in HP antibody titres in the HP-/PL group, suggesting that AZ use prevented significant increases in HP antibody levels in HP- patients. Moreover, although AZ has some dication , 17 sincdication , 31. IntThere were several limitations that should be noted in the current study. First, we do not know how many patients with an HP infection developed resistance to macrolides after 1 year of AZ exposure. HP readily becomes resistant to macrolides when given alone, and short-term AZ monotherapy is not considered sufficient for HP eradication –35. MoreHP infection status was associated with differential effects of AZ in COPD patients with frequent exacerbations, highlighting the importance of evaluating HP status in these group. Chronic AZ monotherapy suppressed HP antibody levels after 1 year of treatment, but did not significantly modify the rate of seroreversion in the HP+ group. The HP+/AZ group exhibited a decline in sTNFR75 levels at 3 months. Patients who were HP seropositive experienced a significantly greater protection against exacerbation from chronic AZ therapy than those who were HP seronegative. A larger prospective study is needed to definitively prove the benefit of eradication of HP with triple therapy in seropositive HP patients with COPD, who frequently exacerbate.


Despite advances in asthma treatment, severe asthma (SA) still results in high morbidity and use of health resources. Our hypothesis was that SA patients would achieve adequate control with a systematic protocol, including oral corticosteroids, budesonide/formoterol maintenance and reliever therapy and a multidisciplinary approach to improve adherence.Non-controlled (NC) SA patients were enrolled to receive 2 weeks of oral corticosteroids and 12 weeks of formoterol + budesonide. Assessments included asthma control questionnaire (ACQ), asthma control test (ACT), daily symptom diary, lung function and health-related quality of life (HRQoL) questionnaires.p = 0.03). Both groups had low HRQoL at baseline with improvement after intervention.Of 51 patients, 13 (25.5%) achieved control. NC patients had higher utilization of health resources and higher exacerbation rates. Both controlled (C) and NC patients had significantly reduced ACQ scores after oral corticosteroid treatment. After 12 weeks, C patients continued improving. NC patients did not have significant changes. A similar pattern was found regarding lung function, use of rescue medication, and days free of symptoms. After 2 weeks of oral corticosteroids, an increase occurred in those who achieved the ACQ cut off; however, 53.8% of C patients had an ACQ < 1.57 versus 21.1% of NC patients .Retrospectively registered at ClinicalTrial.gov on 22 February 2010 ( There is still debate if severe asthma (SA) patients may acquire symptoms control with available treatment. Our findings support that the application of a systematic, low-cost, easy to conduct protocol can prevent up to 25% of uncontrolled SA patients from up titrating their treatments to new and complex therapies.The knowledge of the pathogenesis, pathophysiology and treatment of asthma has made great progress in recent decades. However, several surveys have shown that a significant part of patients do not achieve adequate control of the disease despite proper management according to guidelines , 2. FactThese factors have a particularly significant impact in severe asthma (SA), in which this small proportion of patients is subject to high morbidity and disproportionate use of health resources , 5. In tSeveral validated tools are available to evaluate asthma control, such as diaries for symptoms, clinical questionnaires, and inflammatory/functional measurements , 8. EachWe have previously published the characteristics of a group of patients who did not reach full clinical control and maintained persistent airflow limitations, despite regular monitoring and treatment for at least 4 years - Brazilian Severe Asthma São Paulo (BRASASP) cohort . Here, wThis was a prospective study including a group of SA patients already followed in a specialised SA centre (BRASASP cohort) at least for 1 year. Patients were between 18 and 65 years old, had confirmed SA treated for at least 1 year, documented airway reversibility, presence of at least one asthma exacerbation in the previous year, were non-smokers or former smokers of ≤ 30 pack-years, and receiving high-dose IC plus LABA. If the patient was a smoker, asthma symptoms must have been present before the onset of smoking, and the patient could not smoke more than 10 cigarettes/day. The local Institutional Review Board approved this study (CAPPesq 757/05). All enrolled patients signed an informed consent. The project was retrospectively registered at ClinicalTrial.gov on 22 February 2010 (NCT 01089322).The study design is shown in Fig. At baseline, all patients self-reported their past medical history and had their medical records evaluated. Comorbidities were evaluated prior to the protocol intervention as the standard clinical practice of our institution and aimed to confirm or exclude known diseases related to non-controlled asthma such as vocal cord dysfunction, gastroesophageal reflux disease, chronic rhinosinusitis, and other pulmonary diseases. Whenever present, these comorbidities were treated according to the same attending physician.Exacerbation was characterized based on the expert panel of 2009 . PatientWe used ACQ 7 score to measure our primary objective of asthma control achievement at W12. Patients who maintained ACQ scores >1.57 were classified as the non-controlled group (NC) .Secondary clinical outcomes included ACQ 7 score at W2, the Asthma Control Test (ACT) , lung fu1 and asthma duration) to try do identify predictors of asthma control. The statistical package Sigma Stat version 3.5, Sigma Plot version 10, and PASW Statistics (SPSS) version 18 were used for statistical analysis. Statistical significance was considered p < 0.05.All continuous variables were evaluated for normality using Kolmogorov-Smirnoff or Shapiro-Wilk tests and are shown as mean ± standard deviation or median (IQ25% – 75%). ANOVA repeated measures were used to compare variables at three moments  with Bonferroni’s correction. Unpaired t (independent groups) was used to analyse the measures between groups. Wilcoxon Signed-Rank and Mann–Whitney tests were used for non-parametric variable analysis. Categorical variables are presented as numbers and percentages and were analysed with the chi-square test. Correlations were performed using Pearson or Spearmen tests whenever appropriate. Multivariable logistic regression analysis was performed (C versus NC) with pre-defined relevant clinical variables  did not reach the controlled criteria of our primary objective (ACQ < 1.57), thus were classified as the NC group, and 13 (25.5%) achieved control and were classified as the controlled group (C). Table p = 0.05). Regarding hospitalization in the last year, no difference existed between groups (NC 37% versus C 23%). Nearly 40% of patients of both groups had undergone previous orotracheal intubation. Regarding comorbidities, there was no difference between self-reported diseases at baseline.NC patients had higher previous health care system utilization. Of NC patients, 71% had more than five hospitalizations compared with 38% in C patients (p = 0.03). Despite not achieving asthma control, the majority of patients presented a clinically significant improvement in their symptoms at the end of the study, represented by a decrease of at least 0.5 in ACQ (C = 86.7% versus NC = 66.7% - p = 0.13). We were unable to identify predictors of asthma control in multivariable analysis.ACQ analysis showed that both C and NC had a significant reduction in ACQ score after oral corticosteroid treatment W2). After 12 weeks of follow-up, C continued to improve, while NC did not . A significant correlation was noted between ACQ and ACT scores in NC patients at the end of the protocol , but not in C patients.ACT values were significantly higher in C compared to NC after the course of oral corticosteroids W2) and at the end of the protocol (W12)  patients experienced at least one exacerbation. No difference existed between C and NC groups  in NC compared with C (0.46 ± 0.5).p =0.058) compared with NC, indicating clinical improvement in HRQL. In the SF-36 questionnaire, the two groups reported lower HRQoL at baseline. As observed in the SGRQ questionnaire, C had greater improvement in SF-36 measurements than NC had at the end of protocol.Both groups had low HRQoL at baseline involving all SGRG domains. At the end of the intervention, the C had significantly better HRQoL in all SGRQ domains Table . The C gr = 0.681; p <0001) and all independent domains . SF-36 correlated with ACQ score in all domains, except pain.The ACQ score showed a good correlation with HRQoL baseline and ending questionnaires, mainly with SGRQ. Total SGRQ score was positively correlated to ACQ  would lead to disease control.All patients were uncontrolled despite regular use of inhaled corticosteroids associated with LABA, including the run-in period. During the 12-week study, we applied a maintenance and reliever strategy with formoterol and budesonide proven to be effective in patients with moderate to severe asthma, especially in symptom scores and number of exacerbations . In the 1 measurement) and a 1-week recall time. It assesses the adequacy of asthma control and the change in asthma control over time. A change in score of > 0.5 is considered to be clinically important and an ACQ score of 1.57 discriminates between controlled and non-controlled asthma [This strategy was clearly effective in a subset of patients. The ACQ incorporates seven items  and ACT (3 points) after the use of oral corticosteroids, as well as significant improvement in FEV1. On the other hand, virtually no patients were daily free of symptoms during the period. In the following 10 weeks, despite a higher dose of inhaled corticosteroids compared to the control group (shown by the number of rescue doses of formoterol/budesonide), there was a trend to a clinical and spirometric worsening. Similar response to a course of systemic corticosteroids have been previously reported. ten Brinke et al. demonstrated a reduction in rescue medication and an increase in FEV1 2 weeks after intramuscular triamcinolone in patients with severe asthma who were using inhaled corticosteroids or chronic oral prednisone . In a reWe identified a similar profile of patients characterized by predominantly female gender, long term disease, and overweight and atopic patients compared to other large cohorts of SA such as SARP and ENFUMOSA , 24. We There are some limitations in our study. The relatively low number of patients in the C group limits the statistical power to some correlations, although the data were consistent with those previously published , 18, 25.Our study has important clinical implications. Patients classified as GINA steps 4 and 5 followed at our asthma clinic for at least 4 years, with free access to anti-inflammatory therapies, educational programs and proper evaluation of factors associated with non-control composed our population –31. GiveOur findings support that in patients with severe uncontrolled asthma in daily practice an automatic step up in treatment may lead to unnecessary increase in expenses and risks related to new treatments, since the control can be achieved with a clinical targeted treatment approach. Nevertheless, despite rigorous and optimized follow-up treatment, the majority of patients with SA did not achieve adequate symptom control, and had high exacerbation rates and impaired QoL. These findings indicate the necessity to redefine the goals and monitoring tools related to SA. The application of a systematic, low-cost, easy to conduct protocol can prevent up to 25% of SA patients from up titrating their treatments to new and complex therapies, thus reducing costs (possibly) and morbidity (certainly).