text
string | predicted_class
string | confidence
float16 |
|---|---|---|
Pericytes (5×104) were cultured with GBM cells at 1:1 proportion in 96-well plates for 24-72 hours. Supernatants were collected, and TGF-β, IL-10, TNF-α and IL-6 levels were measured by sandwich ELISA with specific anti-mouse antibodies following manufacturer’s recommendations (Diaclone and Elabscience, respectively). IL-2 levels from T cell supernatants were measured in a sandwich ELISA following manufacturer’s recommendations (BD Biosciences).
|
study
| 100.0 |
Expression of PDL-1 (R&D Systems), CD86, CD80 (eBioscence) and I-A (MHC/H2 class II histocompatibility molecules; BD Bioscience) were analyzed using specific anti-mouse antibodies. Non-specific fluorescence was measured using specific isotype monoclonal antibodies and GBM cells as negative control. Pericytes death was determined using (LIVE/DEAD Fixable Blue Dead Cell Stain Kit, ThermoFisher). Stained cells were analyzed by flow cytometry using a FACS Canto II Flow cytometer (BD Bioscience) and data were analyzed with Kaluza analysis software (Beckman Coulter, Fullerton, CA).
|
study
| 99.94 |
Pericytes (5×104) were cultured with GBM cells at 1:1 proportion in 6-well plates for 24-72 hours. After, pericytes and GBM cells were isolated by GFP or RFP sorting, respectively or only trypsinized to break cell interactions. GBM cells interacting with pericytes were defined as PCC-GB and pericytes interacting with GBM cells as GBC-PC. Both types of cells were counted using an inverted fluorescence microscope (Nikon, Tokyo, Japan). Cumulative population doubling level was calculated using the formula: “PD=(1Log102)xLog10(NtNo)” where No is the number of viable cells (as determined by trypan blue exclusion) at seeding, whereas Nt is the number of viable cells at harvest.
|
study
| 100.0 |
T cells stimulated for 72 hours in functional pericytes assays were transferred to 96 well plate in order to separate from adherent GBC-PC or control pericytes and GBM cells. BrdU was added for 12 hours. After, incorporation of BrdU was measured by ELISA according to the manufacturer’s instructions (Roche).
|
study
| 99.94 |
Cell pellets from human GBM cells and/or murine pericytes that were co-cultured for 72 hours (5×106 cells), were grafted into C57Black/6 wild type mice brains. Xenografts (10 mice received GBM, 10 mice received GBM+PC, 10 mice received PC and 10 mice received GBM/PC without being co-cultured previously) were performed as described previously in an immunocompetent mouse model . Cell pellets, prepared as hanging drops, were grafted into mice. Xenografts (1 pellet/mouse) were introduced into the right hemisphere through a small craniotomy (2 to 3 mm from the midline, approximately 1 mm behind the bregma) at 2.5 mm depth, using a stereotactic apparatus and a Pasteur pipette hand-pulled to an internal diameter of 0.38 mm. This produced grafts that integrated into the cortex or the hippocampus. After 4 and 11 weeks post-grafting, mice were perfused using 4% paraformaldehyde (5 mice of each experimental grafts at each fixation time). Brains were embedded in 30% sucrose and cut at 40 μm using a cryostat. For Immunohistochemistry methods and the antibody information, please see Supplementary Text.
|
study
| 100.0 |
Despite increasing vaccine coverage level, Indonesia is still in distance with target of Global Vaccine Action Plan (GVAP) (1). In 2012, Indonesia was the third place of countries with most unvaccinated infants with three doses of diphtheria-tetanus-pertussis (DTP3) (2) and the achievement on the National Immunization Program (NIP) was not evenly distributed across provinces-only eight provinces reached the national target of Universal Coverage Level (UCI) (1). Aceh province, the westernmost province of Indonesia, rank in the bottom six of UCI status in 2013 (1) and the lowest district in Aceh only gained 39% of UCI, far from national target of 100% (3). Therefore, understanding of factors that hinder or support the likeliness of vaccination among Acehnese inhabitants is an important sight for vaccination strategy in the future.
|
other
| 99.9 |
The aim of this study was to assess the socio-demographic factors influencing Acehnese inhabitants’ view related to vaccine. A cross-sectional survey was conducted during Nov 2014 to Mar 2015 and Aug to Dec 2015 in 11 regencies of Aceh. A set of validated questionnaires was used to assist the interviews. To elicit the view related to vaccine, two questions regarding the importance of the vaccines to prevent diseases and the safety of the vaccines for children were asked with responses on a Likert-type scale ranging from “1=strongly disagree” to “5=strongly agree”. The view related to vaccine was dichotomized into “good” and “poor” based on an 80% cut-off point. A logistic regression analysis was employed to analysis the data.
|
study
| 100.0 |
We included data of 1059 participants in the final analysis. Approximately, 33% of the participants had a poor view regarding vaccines. Education, occupation, monthly income, and economic status were associated with the participants’ perspective regarding vaccine while age, gender, marital status and type of residency had no association (Table 1).
|
study
| 100.0 |
Low education, working as farmer, low economic status and low monthly income were associated with a poor view related to vaccine. Therefore, these groups should be targeted to increase vaccination coverage in the future. One of the visible efforts is to increase the knowledge and attitudes regarding vaccination and this could be implemented using Puskesmas (community health center)-, hospital-, and Masjid (mosque)-based approaches (4).
|
other
| 99.44 |
In addition, one of the focus groups that should be targeted is that of student/ university students, as their views regarding vaccine have no difference compared to farmers. Therefore, intensive vaccination campaigns focusing on those groups might be required in the future to enhance the correct understanding of vaccination.
|
other
| 99.94 |
Although Japan has rapidly become a super-aged society, with more than one-fourth (26.8%) of the total population aged 65 years or older , a survey of dental diseases conducted in Japan in 2011 reported a decrease in the number of edentulous patients and complete denture wearers . While the results of the next survey will not be reported until 2017, this trend is expected to continue. This decrease has also been observed in other industrialized countries, and now that treatment for edentulous patients requires higher levels of expertise and technical skills than ever before, it is increasingly difficult for general dentists to fabricate complete dentures .
|
other
| 99.9 |
One of the most important steps of denture fabrication is impression taking. Boucher reported that the primary objectives of complete denture impressions were as follows: 1) retention; 2) stability; 3) support; 4) esthetic value; and 5) preservation of the alveolar ridge . In other words, impression taking is an integral component of complete dentures. Therefore, the accuracy of the impression greatly affects the quality of the dentures and the level of patient satisfaction [5, 6]. Although complete dentures have been described as having a fundamental form based on anatomy , a substantial part of the complete denture border is due to its location in the area of the mucobuccal fold. In the case of mandibular dentures, the border must be located beyond the point of muscle attachment, which complicates the border molding procedure for general dentists . Dental compounds are useful for defining this unclear border, but dentists must learn this technique through practical experience, and the opportunities to do so are limited.
|
other
| 98.8 |
The dental curriculum for complete denture prosthodontics at Tokyo Medical and Dental University (TMDU) consists of lectures and practical training in the fourth year and clinical training in the fifth and sixth years. As part of practical training, students practice border molding in the practice room. Previously, students used plaster models to pattern impression material (Impression Compound, Kerr Corp., Orange, CA, USA) in custom impression trays. Students learning border molding should replicate clinical conditions as closely as possible during practical training in order to improve their skills ; however, the operation of the plaster model in the practice room is quite different from that actually experienced in the clinical setting. Therefore, the objective of this study was to gain familiarity with the actual materials used in border molding, not the technique itself.
|
other
| 64.75 |
Silicone models (G10-X1231A, Nissin Dental Products Inc., Kyoto, Japan) have been used in practical training since 2009. Silicone models have elastic qualities, high edges, and simulated tongues, and they are therefore more similar to the oral mucosa than plaster models. In 2010, all dental students were provided with mannequins (Phantom DR-11, J. Morita Mfg. Corp., Kyoto, Japan) in which silicone models could be placed; this allowed better replication of clinical conditions than working with a silicone model alone.
|
other
| 99.9 |
Border molding training for mandibular dentures is part of the practical training carried out in the fourth year for dental students at TMDU. During training, each student prepares a custom impression tray made of autopolymerizing acrylic resin. Through 2008, students first applied petroleum jelly on the surface of plaster models, heated the impression materials onto the border of the trays, placed them briefly in warm water, and then placed the trays on the models. Thereafter, the students used their fingers to apply direct pressure to the compound and mold the model form.
|
other
| 99.9 |
After silicone models were introduced in 2009, students in practical training added the compound to the trays and placed them on the models, similar to the technique used for plaster models. They then applied pressure to the edges of the silicone models. The silicone models have artificial tongues and longer edges, which allow students to apply varying degrees of pressure to bend the model in the manner required.
|
other
| 99.9 |
In 2010, the silicone models were attached to the mannequins, but the technique used to mold the borders remained the same. However, the mannequins had openings for the mouth, a limited handling direction, and an adjustable height and head angle, which allowed students to mold the borders in a standing position, which is more consistent with actual clinical conditions. Students were given guidance by instructors throughout the training, and border molding was considered to be complete when the custom trays were completely filled with material (Fig. 1).Fig. 1Border molding training schema: (a) plaster model, press the compound directly with fingers; (b) silicone model, press the edges of the silicone model; and (c) mannequin with silicone model, use in the same manner as the silicone model
|
other
| 99.9 |
The participants in this study were sixth-year dental students between 2010 and 2012. The practical models were plaster models in 2010, silicone models in 2011, and silicone models mounted in mannequins in 2012. All students were asked to complete an anonymous questionnaire immediately after completion of their clinical training. The questionnaire was composed of 21 items: 10 of these items related to their clinical comprehension of border molding, and the remaining 11 related to evaluations of the overall practical training. All items were rated on a five-point Likert scale (Additional file 1).
|
study
| 100.0 |
This study was carried out with the approval of the Ethics Committee of the Faculty of Dentistry at TMDU (registration number: 915) following their review of the questionnaire; the Committee confirmed that the results could be used without the students’ consent.
|
other
| 99.94 |
Students rated all items related to their comprehension of border molding and their level of satisfaction with the overall practical training on a five-point Likert scale as follows: 5 = “yes, definitely”; 4 = “I think so”; 3 = “no opinion”; 2 = “I don’t think so”; and 1 = “no, definitely not”. The mean scores for each year were then calculated.
|
other
| 89.8 |
In order to reduce the large number of interrelated questions to a smaller number of underlying common factors, exploratory factor analysis was carried out using maximum likelihood estimation with promax rotation (κ = 4) and Kaiser normalization. The number of factors was chosen using the Kaiser-Guttman rule, which states that the eigenvalue should be larger than 1, and the scree plot criteria. Items that scored less than 0.25 in communality and exhibited factor loading greater than 0.35 for more than one item were excluded.
|
study
| 99.94 |
The internal consistency of the subscales was assessed using Cronbach’s alpha coefficient. The defined factors were analyzed for the plaster models, the silicone models alone, and the silicone models with mannequins using the Kruskal-Wallis test and follow-up tests using Bonferroni-corrected Mann-Whitney U tests. The significance level was set at p < 0.05. All statistical analyses were performed using SPSS software (version 8.0; IBM SPSS PASW Statistics Base 17.0; IBM Japan, Tokyo, Japan).
|
study
| 99.94 |
The response rates were 48.3% (28 students) with plaster models, 82.5% (52 students) with silicone models, and 92.3% (60 students) with mannequins (Table 1), and the questionnaire results were shown in Additional file 2.Table 1Number of students and mean scores for the questionnaire itemsResponses/total (response rate)Mean scorePlaster model28/58 (48.3%)3.573Silicone model52/63 (82.5%)3.941Mannequin60/65 (92.3%)4.017
|
study
| 100.0 |
As shown in Table 2, exploratory factor analysis identified three factors. Five questions (“Did you understand the technique for border molding on the labial side?”; “Did you understand the anatomic locations?”; “Are important matters adequately emphasized?”; “Did you understand the technical steps?”; and “Were you motivated for clinical training?”) were excluded from analysis because of insufficient factor loading. The Kaiser-Meyer-Olkin measure was 0.846, and Bartlett’s test of sphericity showed a significant difference between variables (p < 0.001). These results validated the application of exploratory factor analysis. In addition, the validity of the three factors was confirmed by the Kaiser-Guttman rule and the scree plot criteria.Table 2Results of exploratory factor analysis with each item divided into three factorsFactorItems1235.Did you understand the method for border molding at the buccal side?.957.029−.0877.Did you understand the method for border molding at the lingual side?.953−.095.0104.Did you understand the method for border molding at the retromolar pad?.821.077−.0533.Did you understand how to adjust the custom trays?.649.113−.0399.Did you understand the mobility of mucosa?.643−.292.1341.Did you understand how to use the impression material?.588.061−.0808.Did you understand the image of the denture shapes?.449.058.2312.Did you understand how to use the alcohol torch?.446.129.11719.Is practical training well organized?−.113.794−.05213.Did you understand the instructions of the teacher?.083.738−.09421.Do you value practical training overall?.161.645.04620.Is practical training easily comprehensible?−.004.606.10612.Was the level of practical training appropriate for students?−.059.538.14015.Did you find proper literature and references?.061−.081.84014.Did you prepare for or review practical training?−.027.032.78516.Are you satisfied with what you have learned?−.046.168.652Interfactor correlation 1–.339.459 2–.449 3–Percent of variance explained33.30913.6696.813Cronbach’s alpha.893.800.818Mean3.7004.3903.380SD.991.7481.040 SD standard deviation
|
study
| 100.0 |
The first factor, consisting of eight items, showed high factor loading for items specific to the molding technique, such as “Did you understand the method for border molding on the buccal side?”. Therefore, this was termed the “knowledge of border molding” factor. The second factor, consisting of five items, showed high factor loading for items specific to the practical environment, such as “Is practical training well-organized?”. Therefore, this was termed the “contents of practical training” factor. The third factor, consisting of three items, showed high factor loading for items specific to the students’ attitudes, such as “Did you find proper literature or references?”, and was therefore termed the “personal learning attitude” factor.
|
study
| 99.94 |
As shown in Table 2, a weak positive correlation was observed between “knowledge of border molding” and “contents of practical training”. Medium positive correlations were observed between “knowledge of border molding” and “personal learning attitude”, and between “contents of practical training” and “personal learning attitude”. The internal consistencies of the three subscales were good because Cronbach’s alpha coefficients were greater than 0.8.
|
study
| 100.0 |
When comparing the three student groups on the “knowledge of border molding” subscales, the students using silicone models and those using mannequins provided significantly better evaluations than those using plaster models (p < 0.001, both), and on the “contents of practical training” subscales, the students using silicone models and those using mannequins provided significantly better evaluations than those using plaster models (p = 0.046, p < 0.001, respectively). However, no significant differences were observed between those using silicone models and those using mannequins. In addition, no significant differences were found among the three groups on the “personal learning attitude” subscale (Table 3).Table 3Results of multiple comparisons with each factor analyzed for the three model typesPlaster modelSilicone modelMannequinKnowledge of border molding3.18 ± 1.14b 3.84 ± 0.98a 3.83 ± 0.83a Contents of practical training4.19 ± 0.80b 4.38 ± 0.74a 4.50 ± 0.71a Personal learning attitude3.26 ± 1.02a 3.28 ± 1.13a 3.53 ± 0.95a Data are expressed as means ± standard deviation. Different lowercase letters indicate significant differences among three model types considering each line
|
study
| 100.0 |
Although the current method for border molding is very popular, the practical environment varies among countries and dental schools. According to the previous studies in the United Kingdom [3, 10, 11], students have limited opportunities to fabricate complete dentures due to decreasing lecture times and declining numbers of edentulous patients. Instead, they are forced to make copy or repair dentures [12, 13]. Although this is also required knowledge, it is not fundamental knowledge. In the United States, many dental schools and postgraduate curricula continue to use custom tray impressions with silicone after border molding with dental compound [14–16]. While some studies suggest that using dental compounds requires advanced skills, others suggest that adequate training can be achieved through the treatment of several cases [9, 16]. It is true that patients exhibit different denture shapes and muscle activation patterns, and dentists should familiarize themselves with these differences through clinical experience. However, as was mentioned by Swenson , complete dentures do have a basic form, and skills cannot be applied without mastery of the fundamentals. As the number of patients continues to decline, practice with models can provide a proper foundation for students’ clinical skills.
|
review
| 99.06 |
Many studies have investigated the effect of denture impression method. As discussed in a review , a few studies with high-quality evidence indicated that the simplified impression method (single alginate impression) showed better results than the conventional impression method (preliminary and final impression), primarily in cost and time [18–22]. However, the high-quality papers such as those mentioned above came from only four research groups. There are a few who advocate the necessity of the two-step impression method . Whichever method is chosen, additional research is needed. In many countries, dentists take impressions using border molding, which alludes to the fact that border molding is valuable for the treatment of edentulous patients.
|
review
| 99.9 |
This study did have some limitations. The response rate for plaster models was quite low, at 48.3%, and it was likely influenced by the time of collection. In addition, the reliability of the results is low, since the questionnaire was only conducted with students from one dental school. However, the histograms of the three student groups showed the same frequency distribution and had the same median with the highest frequency; therefore, the samples were considered valid for statistical analysis. However, the results of this study should not be over-generalized, since high external validity is not likely with just one study. Therefore, the external validity of this field will increase as many researchers complement each other.
|
study
| 100.0 |
The three factors extracted in this study were matched with the factors in the planning phase. Cronbach’s alpha coefficients were greater than 0.8, indicating the high credibility of the results. Moreover, intermediate correlations were observed between two of the three factors, indicating the adequacy of the exploratory factor analysis.
|
study
| 100.0 |
Students’ self-evaluations for “knowledge of border molding” were higher for silicone than for plaster models, and for mannequins than for plaster models. In silicone models, the edges are pressed to form the border indirectly, whereas in mannequins, the structures on the model resembling the lips or buccal mucosa hinder border molding. However, this is not the case with plaster models. Therefore, silicone models and mannequins are more suitable than plaster models for border molding practice. Furthermore, the change from silicone models to mannequins did not affect the students’ self-evaluations, indicating that the limitations created by mannequins did not have a drastic effect on evaluations compared with the change from plaster to silicone models. In addition, the evaluations for the silicone models and mannequins might have been too high to allow any differences to be seen.
|
study
| 99.94 |
The “contents of practical training” was also significantly affected by the change in models, especially from plaster models to mannequins. Many dental schools in the Unites States have used mannequins for more than 10 years, but their specifications and the effects of their use remain unclear . Mannequins have been used to take 73.7% and 57.9% of all primary and final impressions, respectively, in Spain and Portugal . It therefore seems that the use of mannequins has not achieved a high degree of legitimacy. Mannequins are effective for bridging the gap between the laboratory and clinical setting, which, in addition to the learning effects, helps students develop a clearer understanding of actual treatment procedures. The use of different models might contribute to an improvement in the overall evaluation of practical training.
|
other
| 96.1 |
No significant differences were observed in “personal learning attitude” in this study, but mean scores were highest with the mannequin, followed by the silicone and plaster models in descending order. This result indicates the possibility that practice with models had a positive effect. Each student worked with and assessed only one training model, and this should be kept in mind when considering the validity of the results.
|
study
| 100.0 |
The purpose of using silicone models is to help students comprehend the characteristics of modeling compounds and develop skills in border molding. Silicone models are already used in postgraduate training courses and objective structured clinical examinations. In the future, it is expected to expand the use of the silicone models for general improvement in treatment, and border molding training should include movable and immovable mucosa and free lingual movement to make it more consistent with actual clinical conditions. Considering the limited time and number of cases included in the dental curriculum, silicone models are expected to allow an adequate mimicking of clinical conditions and to foster better impression-taking skills.
|
other
| 99.9 |
Additional file 1:The questionnaire in Japanese and English: the questionnaire consisted of 21 items regarding their overall practical training and their clinical comprehension of border molding. (XLS 42 kb) Additional file 2:The questionnaire results: The raw data used in this study. Each subject rated on a five-point Likert scale. (XLSX 22 kb)
|
study
| 90.25 |
Idiopathic normal pressure hydrocephalus (iNPH) is a treatable disorder, presenting with gait and balance difficulties, cognitive impairment and urinary incontinence, and has a prevalence of 1.4–2% in the elderly [1, 2]. INPH is treated by insertion of a CSF-diverting shunt system, which improves more than 80% of the patients on a short-term basis [3–5].
|
review
| 99.8 |
There are several studies on the long-term outcome after shunt surgery for iNPH patients, showing a favorable outcome after 3–7 years in 26–91% of the patients [6–20]. On the basis of five of these studies, published in 2006–2010, a systematic review by Toma et al. concluded that 73% of the patients still benefit from shunt surgery after 3 years or more .
|
review
| 99.9 |
The same review found a revision rate of 13%, subdural hematomas in 4.5% and infections in 3.5% of the patients, and a shunt surgery-related mortality rate of 0.2% . Pujari et al. showed that 74% of the patients improved after shunt revision , but the long-term effects of shunt revision have not been described.
|
review
| 99.9 |
INPH patients have a heavier burden of hypertension [21–25], diabetes mellitus [22–27], hyperlipidemia , obesity , ischemic heart disease [22–24] and arteriosclerotic cerebrovascular disease than control groups. Several findings indicate that risk factors for arteriosclerotic cerebrovascular disease are involved in the pathogenesis of iNPH [21, 28], and it has recently been suggested that iNPH may be a subtype of vascular dementia .
|
review
| 99.56 |
Earlier studies show that a good shunt response is also seen in iNPH patients with extensive presumed ischemic white matter lesions [16, 29], that radiological signs of cerebrovascular disease cannot predict the outcome in iNPH patients , and that the magnitude of improvement in patients with and without vascular comorbidities is the same . However, other studies report contrasting findings, showing less improvement in patients with signs of ischemic cerebrovascular disease [31, 32]. There is little information regarding the effects of vascular risk factors and comorbidities on the long-term outcome in iNPH patients [16, 33].
|
review
| 99.8 |
The Swedish Hydrocephalus Quality Registry (SHQR) is the largest national registry for adult hydrocephalus patients including prospectively collected clinical data. A recent report, based on SHQR data, described the incidence of hydrocephalus surgery in Sweden during 2004–2011 and the 3-month outcome for iNPH patients , but no long-term evaluations have so far been reported.
|
study
| 99.94 |
The 979 patients diagnosed and treated for iNPH during 2004–2011 and registered in the SHQR before September 2014 were included. The patients were registered at five of the six neurosurgical centers in Sweden: the University Hospitals in Umeå (n = 119), Uppsala (n = 313), Linköping (n = 164), Gothenburg (n = 148), and Lund (n = 235). During this period, the sixth neurosurgical center in Sweden did not report to the SHQR.
|
study
| 99.9 |
The treatment was shunt surgery in 974 patients (ventriculo-peritoneal: 953, ventriculo-atrial: 6, not specified: 15 patients), and 5 patients were operated by a third ventriculostomy; 3 of whom were later reoperated with shunt insertion (after 2 weeks, 6 weeks and 8 months, respectively).
|
other
| 99.8 |
Information concerning baseline characteristics, including the presence of hypertension, diabetes mellitus, heart disease and history of stroke (Table 1), the date and type of primary surgery and reoperations, preoperative and 3-month postoperative clinical evaluations, along with long-term evaluations from postal follow-ups, was extracted from the SHQR on the 1st of September 2014.Table 1Baseline characteristics for 979 patients diagnosed and operated on for iNPH during 2004–2011, included in the Swedish Hydrocephalus Quality Registry, SHQR DemographyiNPH patients, n = 979Age (years), median (IQR)74 (68–78)Sex, female, n (%)413 (42)Preoperative modified Rankin Scale (mRS), median (IQR)2 (2–3)Vascular comorbidity n (%)Hypertension438 (49)Diabetes mellitus189 (21)History of stroke119 (14)Heart disease231 (26)Number of vascular comorbidities n (%)0401 (41)1279 (28)2175 (18)365 (6.6)48 (0.8)Not available51 (5.2) INPH idiopathic normal pressure hydrocephalus, IQR interquartile range, mRS modified Rankin Scale
|
study
| 100.0 |
The preoperative and the 3-month postoperative clinical evaluations included the modified Rankin Scale (mRS) , with scores of 0–5 (Table 2).Table 2mRS37 and smRS scales, the latter translated from Swedish. The mRS was used in clinical evaluations pre- and 3 months postoperatively. The smRS was used for patient’s self-assessment of their degree of independent daily function or disabilities in the long-term evaluationsModified Rankin Scale, mRSSelf-assessed modified Rankin Scale, smRSHeadline in the questionnaire: “Disability/need for assistance”0No symptoms at allNo problems1No significant disability despite symptoms: able to carry out all usual duties and activitiesSome problems that do not restrict my lifestyle2Slight disability: unable to carry out all previous activities but able to look after own affairs without assistanceMinor disability, some restrictions to my lifestyle, no need for assistance3Moderate disability: requiring some help, but able to walk without assistanceSome disability, which clearly restricts my lifestyle, need for assistance4Moderately severe disability: unable to walk without assistance, and unable to attend to own bodily needs without assistanceSevere disability, dependent but not in constant need of assistance5Severe disability: bedridden, incontinent, and requiring constant nursing care and attentionVery severe disability, in need of constant care, day and night
|
study
| 99.94 |
mRS37 and smRS scales, the latter translated from Swedish. The mRS was used in clinical evaluations pre- and 3 months postoperatively. The smRS was used for patient’s self-assessment of their degree of independent daily function or disabilities in the long-term evaluations
|
other
| 99.9 |
At the 3-month postoperative visit, the physician assessed if the patients’ general health condition was “better”, “unchanged” or “worse” than before surgery. This variable was included in the SHQR protocol during 2008–2010 and ratings were available for 177 patients.
|
study
| 96.44 |
The outcome measures used for the long-term follow-up were a self-assessed mRS (denoted smRS, Table 2), and an enquiry of the present health condition in comparison with before surgery, phrased: “How are you feeling now, compared with your condition before surgery?” with three response alternatives: “better”, “unchanged” or “worse”.
|
study
| 99.94 |
The standardized questionnaires for the long-term follow-up had initially been mailed annually to the patients starting from 2 years after surgery. In 2010, the routine was changed to sending out questionnaires after 2, 5 and 10 years. A cover letter explained the purpose and asked that the questionnaire be completed at home, by the patient, a next-of-kin or caretaker. A reminder was sent to patients who did not reply to the first letter.
|
study
| 97.0 |
Of the 979 patients, 623 (64%) returned 1–6 questionnaires (median 1, IQR 1–2) and the time interval from surgery to returned questionnaires ranged from 1.6 to 10.5 years (median 3, IQR 2.3–4.5). The questionnaire replies were sorted into groups, as shown in Table 3. All responses from the patients are included in the respective groups, except for two patients who replied twice during the same year; only one of these replies from each patient was included.Table 3Replies to questionnaire on long-term evaluation of 979 iNPH patients operated on in Sweden in 2004–2011. Available patients at each year after surgery were defined as living patients with follow-up within each time rangeGroupTime interval (years)Returned questionnaires (n)Proportion of available patients (%)Two years1.6–2.4929733Three years2.5–3.4924729Four years3.5–4.4915023Five years4.5–5.4914432Six years5.5–6.495617Seven years6.5–7.49188Eight years7.5–8.4921Nine years8.5–9.4944Ten years9.5–10.5513Total923Ever replying patients n = 623Proportion64%
|
study
| 100.0 |
Only complications leading to reoperation were counted and grouped into (1) mechanical: most commonly, shunt obstruction or displacement; (2) infections: intraabdominal, skin or CNS infections; (3) subdural hematomas (evacuation of hematoma and/or ligation of shunt), and (4) other or not specified. All reoperations were registered separately even if they referred to the same complication; for instance, management of an intraabdominal infection, first by externalization of the distal catheter and later by removal of the whole shunt, would count as two operations. However, shunt insertions performed after ventriculostomy, reinsertions after earlier shunt removals, and reopening of temporarily ligated shunts were not considered related to new complications and, therefore, not registered in this study.
|
study
| 99.94 |
Only non-parametric tests were used. Proportions were compared using Fisher’s exact test. For comparisons between groups, the Mann–Whitney U test was used, and for within-group comparisons, the Wilcoxon signed rank test was used. To analyze the influence of the vascular comorbidities on long-term outcome, odds ratios with a 95% confidence interval were calculated by logistic regression analysis adjusted for age and sex. Correlations were tested by the Spearman rank correlation. All significance tests were two tailed and the statistical significance was set at the 0.05 level. All analyses were performed with SPSS 24.0 (IBM, Armonk, New York, USA).
|
study
| 100.0 |
The patients were improved on the mRS at the 3-month evaluation (median 2, IQR 2–3 preoperatively vs. median 2, IQR 1–3 postoperatively, p < .001), and remained improved on the smRS after 2 years (median 2, IQR 1–3, p = .001). The smRS at 3, 4, 5 and 6 years were unchanged compared with the mRS preoperatively (median 2–3, p = .22–.86) (Fig. 1).Fig. 1Shift analysis of modified Rankin Scale (mRS) scores (0–5) in 979 iNPH patients: at baseline and 3 months after surgery, and self-reported mRS (smRS) at 2–6 years after surgery. Black numbers within the bars represent the number of patients obtaining each score on the mRS and smRS. White numbers show the proportion of patients with scores between 0 and 2, i.e., able to live a life independent of help from others
|
study
| 99.94 |
Shift analysis of modified Rankin Scale (mRS) scores (0–5) in 979 iNPH patients: at baseline and 3 months after surgery, and self-reported mRS (smRS) at 2–6 years after surgery. Black numbers within the bars represent the number of patients obtaining each score on the mRS and smRS. White numbers show the proportion of patients with scores between 0 and 2, i.e., able to live a life independent of help from others
|
study
| 99.94 |
Around 60% of the patients described their health condition as improved after 2–6 years (Fig. 2).Fig. 2Postoperative health condition in 979 iNPH patients. Percentage of iNPH patients reporting better, unchanged or worse health condition, of whom the following question was asked: “How are you feeling now, compared with your condition before surgery?” after 2–6 years. The 3-month evaluation was carried out in the clinical follow-up setting; results at the 2- to 6-year evaluation come from follow-up questionnaires. The number of available answers at each time point is indicated above the bars
|
study
| 100.0 |
Postoperative health condition in 979 iNPH patients. Percentage of iNPH patients reporting better, unchanged or worse health condition, of whom the following question was asked: “How are you feeling now, compared with your condition before surgery?” after 2–6 years. The 3-month evaluation was carried out in the clinical follow-up setting; results at the 2- to 6-year evaluation come from follow-up questionnaires. The number of available answers at each time point is indicated above the bars
|
study
| 99.94 |
One or more complications were found in 26% of the patients (257 patients), leading to a total of 410 reoperations. Of these, 58% (239 reoperations) took place during the first year after the primary surgery. Of the 257 patients, 61% (157 patients) had only one reoperation, 26% (66) had two and 13% (34) had three or more reoperations (median 1, range 1–5).
|
study
| 99.0 |
In total, 14% (141) of the 979 patients had reoperations due to mechanical complications, 6.4% (63) due to infections, 3.7% (36) due to subdural hematomas and 9.1% (89) due to other or not specified causes. These groups are overlapping, as one patient may have had more than one type of complication at different time points. The patients’ first complications leading to reoperation are shown in Fig. 3.Fig. 3Complications causing first-time reoperations in 979 iNPH patients, visualized year by year after primary surgery. For each year, the percentage of patients who needed reoperation because of complications is represented in the bar chart, in four groups based on the type of complication. The corresponding numbers of patients and the total number of available patients for each year are shown in the table
|
study
| 100.0 |
Complications causing first-time reoperations in 979 iNPH patients, visualized year by year after primary surgery. For each year, the percentage of patients who needed reoperation because of complications is represented in the bar chart, in four groups based on the type of complication. The corresponding numbers of patients and the total number of available patients for each year are shown in the table
|
other
| 99.44 |
There were no differences in long-term outcome for patients who had or had not been subjected to reoperation (Table 4).Table 4Outcome 2–6 years after primary surgery in iNPH patients who underwent reoperation, compared with patients in whom reoperation was not performed. The outcome measures are self-reported mRS (smRS) and “Improved health condition”, signifying the proportions of patients replying “better” to the following question: “How are you feeling now, compared with your condition before surgery?”Reoperated patients, n (% of available questionnaire responses at specified year)smRS, median (IQR) p Improved health condition, n (%) p Re-op.Not re-op.Re-op.Not re-op.2 years53 (18)2 (1–3)2 (1–3).6126 (53.1)147 (63.1).203 years55 (22)3 (1–3)2 (2–3).1529 (55.8)119 (65.7).204 years39 (26)2 (1–3)2 (2–3).2021 (56.8)73 (69.5).165 years42 (29)2 (2–3)2 (1–3).2118 (52.9)68 (68.7).156 years12 (22)3 (1–5)3 (2–3).787 (53.8)22 (55.0)1.0 Re-op. reoperated
|
study
| 100.0 |
Outcome 2–6 years after primary surgery in iNPH patients who underwent reoperation, compared with patients in whom reoperation was not performed. The outcome measures are self-reported mRS (smRS) and “Improved health condition”, signifying the proportions of patients replying “better” to the following question: “How are you feeling now, compared with your condition before surgery?”
|
study
| 81.44 |
Preoperatively, patients with diabetes, stroke or heart disease had higher mRS scores than patients without these factors. Three months postoperatively, patients with stroke or heart disease were still significantly worse (higher mRS), while patients with diabetes were not. Postoperative improvement of the health condition according to physicians’ ratings did not differ between those with or without any of the four comorbidities (Table 5).Table 5mRS scores before and 3 months after surgery, along with physicians’ rating of postoperative relative to preoperative health condition in iNPH patients with comorbidities, compared to those without n (% within available scorings at specified time point)mRS, median (IQR) p Improved health condition, n (%) p WithWithoutWithWithoutPreoperative Hypertension350 (49.7)3 (2–3)2 (2–3).75––– Diabetes147 (21)3 (2–3)2 (2–3).019––– Stroke93 (13.3)3 (2–4)2 (2–3)< .001––– Heart disease171 (24.2)3 (2–3)2 (2–3).002–––3 months Hypertension331 (51.2)2 (1–3)2 (1–3).3666 (70)62 (80).16 Diabetes141 (21.8)2 (2–3)2 (1–3).08228 (68)101 (76).41 Stroke91 (14.2)3 (2–3)2 (1–3)< .00117 (77)111 (74).80 Heart disease161 (24.8)3 (2–3)2 (1–3).01325 (70)103 (75).52 mRS modified Rankin Scale
|
study
| 99.94 |
With regard to the actual smRS ratings, there were no differences between patients with vascular comorbidity and those without, except for patients with a history of stroke in the 3-year group, who had a higher degree of disability (median smRS 3 vs 2, p = .024), i.e., similar to the differences found before surgery and at the 3-month evaluation.
|
study
| 100.0 |
Looking instead at the differences between the smRS at the different time points and at baseline, the magnitude of improvement did not differ between patients with and without vascular comorbidity, except for the follow-up after 6 years, where patients with hypertension and patients with a history of stroke both had a less favorable development than those without comorbidity (p = .045 for both comparisons).
|
study
| 99.94 |
There were no significant correlations between the number of comorbidities (0–4) and differences between the mRS score at baseline and subsequent smRS scores 2–6 years after surgery, nor with the evaluation of the health condition (“better”, “unchanged” or “worse”) at the same time points (correlation coefficients − .122 to .249).
|
study
| 100.0 |
This study shows lasting improvement on a self-assessed modified Rankin Scale in around 40% of shunted iNPH patients, and lasting subjective improvement in about 60% at follow-up after 2–6 years. The reoperation rate within 10 years was 26%, of which 58% were performed within the first year of surgery. Reoperation did not impede improvement up to 6 years after primary surgery.
|
study
| 99.94 |
Diabetes, heart disease, and a history of stroke were each associated with higher mRS scores at baseline, and the latter also after 3 years. However, neither of these three, nor hypertension, had a negative influence on the degree of postoperative improvement until more than 5 years after surgery.
|
study
| 99.94 |
The reoperation rate of 26% for any cause in our data was rather high. Fourteen percent of the patients had revisions due to mechanical problems. In the systematic review by Toma et al., the revision rate, constituting the need for revision due to shunt failure or “mechanical causes”, was 13% in 30 articles published after 2006 . One explanation for a higher reoperation rate in the current material is the longer follow-up period. Most studies have follow-up periods of up to 1 year, while 42% of the revisions in the present study were performed later. Another explanation could be that patients included in other studies aiming to investigate, e.g., specified measurements may have been carefully selected, whereas the present study includes all operated iNPH patients from the participating centers.
|
study
| 99.8 |
SDH was reported in 4.5% of the patients in the review , which is in the same range as in our study (3.7%). However, our figure shows only those requiring surgical treatment, and the total number of SDH is probably higher. The infection rate of 6.4% is higher than in many other studies (average 3%, but ranging from 0 to 10%) . Similarly, the operation-related mortality of 0.5% is somewhat higher than in contemporary studies. This was calculated in the cited review to be 0.2%, for the years 2006–2010, with a range between 0 and 0.8%, for the 13 studies reporting numbers on shunt surgery-related mortality . Again, the unselected patient material in the SHQR compared with the less extensive single-center studies with stricter inclusion criteria reviewed by Toma et al. may have influenced the results.
|
review
| 99.9 |
One of the important findings in this study is that the complications leading to reoperation had no negative effects on the long-term outcome. This knowledge is essential for clinicians, well aware of the risk of problems with shunt surgery, when advising patients. Of course, complications cause a temporary decline in physical and mental functions but it appears that identification and treatment prevent major decline.
|
study
| 88.25 |
There is increasing evidence that risk factors for cerebrovascular disease contribute to the pathophysiology of iNPH, and a recently published study calculated that multiple vascular risk factors could explain 24% of iNPH cases (population attributable risk, PAR, 24%) . Our study could not show that the specific vascular risk factors of hypertension or diabetes hampered the effects of shunting, either in the short- or long-term perspective, even if patients with diabetes performed worse in the baseline evaluation. This is in line with earlier studies concerning the influence of vascular risk factors on short-term outcome [4, 16, 31], while their influence on the long-term outcome in iNPH patients has not previously been described.
|
study
| 99.94 |
Macro- and microvascular changes, where both hypertension and diabetes are thought to play a role, with stiffening of the arterial walls and development of endothelial dysfunction in small vessels, causing increased permeability , could hypothetically contribute also to the disturbed hydrodynamics in iNPH, with increased CSF pulsations and diminished perfusion in small vessels in the periventricular region [40, 41]. Shunting immediately changes the absorption mechanism and thereby “eliminates” both the vascular and hydrocephalic component of the disturbed CSF dynamics, as well as improves periventricular perfusion . As the effect of the shunt is permanent it may even compensate for the deterioration known to be associated with hypertension and diabetes and thereby explain the lack of negative long-term effects.
|
study
| 99.94 |
Earlier studies showed a less favorable outcome for patients with established cerebrovascular disease (CVD), defined as a history of stroke, infarctions on radiological imaging, or moderate to severe white matter lesions on a CT scan [16, 31]. Boon et al. found 52% of the patients with CVD to be improved, as opposed to 79% of those without , and Spagnoli et al. showed similar results in the long-term perspective (mean 52 months), with 49 vs. 79% improved patients . Our study included only a history of stroke, but the negative impact of CVD, in this narrower sense, regarding the proportion of improved patients in the short or long term could not be confirmed. Patients with a history of stroke performed worse with regard to the smRS score only after 3 years, but the proportion of patients still reporting “better” was not affected at any time point.
|
study
| 99.94 |
The main limitation to this study is the low letter response rates, ranging from 17% of available patients in the 6-year group to 33% in the 2-year group (Table 3). During some periods, letters were not sent from all centers as intended, which explains the lack of data in long-term follow-ups for 258 patients (26%), and why not all patients were followed up at each pre-specified time point.
|
study
| 100.0 |
However, the incomplete mailings were not influenced by patient characteristics. When comparing patients in each of the long-term follow-up groups to those without replies, there were no significant differences regarding the mRS at baseline or age at surgery, and the frequencies of the four reported comorbidities were equal. Furthermore, the prevalence of reoperation before follow-up in the 3-, 4-, 5- and 6-year groups was similar in repliers and non-repliers. Only in the 2-year group, which was closest in time relative to the majority of the reoperations, repliers had undergone fewer reoperations than non-repliers: 18% compared to 27% (p = .004). Altogether, this should indicate that there are no systematic errors in the distribution of follow-up letters, arguing for the representativity and validity of the samples.
|
study
| 100.0 |
Another limitation is the use of subjective outcome measures. The smRS and the patients’ comparisons between their present and their preoperative health condition as measures of outcome are evaluations whose correspondence to objective measures has not been studied. Still, we consider these measures valuable, representing outcome as experienced by the affected patient.
|
other
| 99.8 |
The major strength of the study is that it is a quality registry-based study, which allowed for a very large sample size of 979 patients with data collected prospectively during a long follow-up period of up to 10 years. Patients were diagnosed in five different centers, covering approximately 80% of the referral areas in Sweden , according to standardized and clinically applied routines representing everyday clinical practice. There were no specific exclusion criteria, such as those often applied in other scientific studies, why the study cohort probably reflects the target population of iNPH patients more accurately.
|
study
| 100.0 |
Based on these results, physicians can inform patients about the risk of complications with greater certainty—stating that it is fairly high, but that complications are treatable and that no negative effect on the long-term outcome has been shown. These findings also give support to decisions regarding patients with hypertension, diabetes, heart disease or stroke, as these extensive data did not show a less favorable outcome for these patients, meaning that they should not be excluded from shunt surgery.
|
review
| 99.1 |
The Pacific Coast tick, Dermacentor occidentalis Marx (henceforth D. occidentalis) is the most widely distributed tick in California and is found in chaparral and shrubland areas from northern Baja California to California and Oregon (Furman & Loomis, 1984). D. occidentalis is a three-host, hard-shell tick that feeds on a variety of vertebrates, such as rodents, rabbits, cattle, deer, horses and humans. Surveys of this tick have shown its ability to vector human pathogens such as Francisella tularensis (tularemia), Coxiella burnetii (Q fever), Anaplasma phagocytophilum (human granulocytic anaplasmosis), Ehrlichia chaffeensis (human monocytic ehrlichiosis), Rickettsia rickettsii (Rocky Mountain spotted fever, RMSF) and Rickettsia philipii 364D (hereafter R. philipii) as well as the non-pathogenic spotted fever group Rickettsia, R. rhipicephali (Parker, Brooks & Marsh, 1929; Cox, 1940; Lane et al., 1981; Holden et al., 2003; Wikswo et al., 2008; Shapiro et al., 2010). Rickettsia philipii, was originally described as an unclassified Rickettsia found by Bell in D. occidentalis from California (Philip et al., 1978). It is closely related to Rickettsia rickettsii but can be serologically and genetically distinguished (Philip, Lane & Casper, 1981; Karpathy, Dasch & Eremeeva, 2007). Although discovered in 1966, and long suspected of being able to cause disease, it was only recently confirmed to be associated with eschars and lymphadenopathy in people at the site of a tick bite (Lane et al., 1981; Shapiro et al., 2010; Johnston et al., 2013).
|
review
| 99.44 |
Francisella-like endosymbiotic bacteria (FLEs) have also been detected in Dermacentor occidentalis as well as other tick species (Burgdorfer, Brinton & Hughes, 1973; Noda, Munderloh & Kurtti, 1997; Scoles, 2004; Kugeler et al., 2005). FLEs share 16S rRNA gene homology with Francisella spp., are vertically transmitted, have been observed within tick ovaries and Malpighian tubules, and vary by tick species (Rounds et al., 2012). Although Burgdorfer et al. demonstrated pathogenicity of a Francisella endosymbiont derived from Dermacentor andersoni Stiles ticks (previously categorized as Wolbachia persica, Forsman, Sandström & Sjöstedt, 1994) to guinea pigs and hamsters via injection, most FLEs are not transmitted by tick bites and are considered non-pathogenic (Burgdorfer, Brinton & Hughes, 1973; Niebylski et al., 1997).
|
study
| 99.8 |
Interestingly, the inability of different endosymbiotic Rickettsia species to co-infect the same organ in the same tick, called “interference,” has been demonstrated, although the exact mechanisms are unknown. Early studies seeking to understand the epidemiology of RMSF in the Bitterroot Valley in Montana demonstrated that the non-pathogenic tick endosymbiont Rickettsia peacockii (found on the east side of the valley and originally called the East side agent) colonized the ovaries of D. andersoni ticks and excluded pathogenic Rickettsia rickettsii (more prevalent on the west side of the valley) from infecting the ovaries and being transmitted to eggs (Burgdorfer, Hayes & Mavros, 1981). Similarly, studies of Dermacentor variabilis (Say) infected with R. montanensis or R. rhipicephali demonstrated resistance to transovarial transmission of the reciprocal Rickettsia in challenge experiments (Macaluso et al., 2002). Negative influences between co-infecting species of Rickettsia and other symbionts has been suggested to occur in other vectors such as fleas (Azad & Beard, 1998; Jones et al., 2012). Interference has been postulated to have significant effects in altering the distribution of Rickettsia pathogens in the environment and, consequently, the presence of human disease (Burgdorfer, Hayes & Mavros, 1981).
|
review
| 92.94 |
The use of next generation sequencing has allowed deeper exploration into endosymbionts and complex bacterial communities that colonize different tick species (Nakao et al., 2013), their organs (Budachetri et al., 2014; Qiu et al., 2014), different life stages (Carpi et al., 2011) and different states of nutrition (Menchaca et al., 2013; Zhang et al., 2014). Attention to the microbiome of ticks has been driven, in part, by the fact that ticks can transmit the broadest range of diseases of any arthropod and the recognition that tick co-infections can have dramatic consequences on both the tick host and human patient (Clay & Fuqua, 2010). Microbiome studies using next generation sequencing techniques have demonstrated that each species of tick harbors its own unique bacterial community often dominated by Proteobacteria and one or two endosymbionts (Clay & Fuqua, 2010; Ponnusamy et al., 2014; Hawlena et al., 2012; Van Treuren et al., 2015; Narasimhan & Fikrig, 2015). Given these findings, we hypothesized that next generation sequence analysis of Dermacentor occidentalis ticks microbiomes would reveal patterns of interference or exclusion among pathogenic or non-pathogenic bacteria. We also hypothesized that differences among tick microbiomes would be associated with different geographic locations, and that possible reservoirs of tick pathogens could be found by analyzing ticks for the host origin of prior blood meals or by comparing the tick microbiomes to the skin microbiomes of potential host species. To address these hypotheses, we used culture-independent PCR amplification of the 16S rRNA gene and next-generation sequencing (NGS) to determine whether the microbiomes of SFGR-infected ticks differed from non-SFGR-infected ticks, and if this microbial diversity was consistent with a hypothesis of interference. Our results revealed patterns consistent with partial exclusion between SFGR and FLEs and an association of non-endosymbiotic bacteria with geographic locale. Furthermore, the historical blood meal hosts of the ticks were implicated by the composition of bacterial communities within the ticks and were correlated with SFGR infection. While the precise mechanism of the bacterial interactions (i.e., direct or indirect) need elucidation, our results suggest that carriage of certain pathogenic SFGR in ticks could be modulated by other non-rickettsial endosymbionts, providing a potential non-chemical alternative to SFGR control.
|
study
| 99.94 |
Adult ticks were collected from February to May 2014 from 4 different areas of San Diego County: Escondido Creek, Los Peñasquitos Canyon, Lopez Canyon and Mission Trails Regional Park by dragging a 1 m2 piece of canvas over grass and chaparral and then capturing the ticks with forceps and placing them in individual sterile microfuge tubes. The ticks were transported live back to the Vector Disease and Diagnostic Laboratory at the San Diego County Operations Center where, by visual examination, their species and sex were determined and cataloged before freezing them at −80 °C.
|
study
| 99.7 |
Ticks were processed individually throughout all procedures. The ticks were thawed and washed sequentially in 3% hydrogen peroxide, 100% isopropanol, and sterile distilled water for 1 min in each solution. The final distilled water wash was aspirated from the ticks and then the ticks were sectioned sagittally at midline with a sterile scalpel. Half of the tick was saved at −80 °C; the other half was used for DNA extraction. Briefly, 180 μl of ATL buffer (Qiagen, Valencia, CA, USA) and 20 μl of proteinase K were added to each tick and the ticks lysed overnight at 37 °C in an Eppendorf Thermomixer (Hauppauge, NY) with agitation at 1,400 rpm for 15 s every 15 min, before centrifuging the lysate for 3 min at 18,400× g. The supernatant was transferred into a sterile microfuge tube and DNA extracted using a Qiagen DNeasy Blood and Tissue kit in a Qiacube using the DNeasy Blood and Tissue protocol for Tissue and Rodent Tails (Qiagen, Valencia, CA, USA). Negative extraction controls consisted of sterile water processed via the same washing, chopping and extraction procedure used on the ticks.
|
study
| 99.94 |
The ticks were screened for spotted fever group rickettsia using a Power SYBR Green real-time PCR Mastermix kit (Life Technologies, Carlsbad, CA, USA) and primers for the romp A gene (Eremeeva et al., 2003). Reactions were carried out in a total volume of 20 μL composed of 10 µL Power SYBR Green Mastermix, 0.125 μL each of primers RR190.547F (20 μM) and RR190.701R (20 μM), 7.75 μL of nuclease-free water, and 2 μL of template DNA (Eremeeva et al., 2003; Wikswo et al., 2008). Real-time PCR cycling conditions were: 3 min at 95 °C; 40 cycles of: 20 s at 95 °C, 30 s at 57 °C, 30 s at 65 °C; a holding cycle of 5 min at 72 °C; and a continuous cycle of: 15 s at 95 °C, 1 min at 55 °C, 30 s at 95 °C, 10 s at 55 °C; and a final holding temperature of 4 °C.
|
study
| 99.94 |
DNA from ticks that screened positive for SFGR were subjected to semi-nested PCR amplification of rompA using primers Rr190-70, Rr190-701, and Rr190-602 and the intergenic region (IGR) using primary and nested primers RR0155-rpm B (Eremeeva et al., 2006; Shapiro et al., 2010; Wikswo et al., 2008). Briefly, 20 μL of 2X Taq Master Mix (Qiagen, Valencia, CA), 2 μL of forward primer Rr190-70 (20 mM), 2 μL of reverse primer Rr190-701/Rr190-602 (20 mM), 14 μL of nuclease-free H2O, and 2 μL of DNA was amplified using PCR cycling conditions of 95 °C for 3 min followed by 35 cycles of 95 °C for 20 s, 57 °C for 30 s, and 68 °C for 2 min and then 72 °C for 5 min before holding the products at 4 °C. For the IGR PCR amplification, 20 μL of 2X Taq Master Mix (Qiagen, Valencia, CA, USA), 1 μL of forward primer RR 0155 PF (20 mM), 1 μL of reverse primer 0155 PR (20 mM), 16 μL of nuclease-free H2O, and 2 μL of DNA was amplified using PCR cycling conditions of 95 °C for 5 min followed by 35 cycles of 95 °C for 30 s, 50 °C for 30 s, and 68 °C for 1 min and then 72 °C for 7 min before holding the products at 4 °C.
|
study
| 100.0 |
Amplification products were visualized in a 1% agarose gel stained with ethidium bromide on a UV illuminator and subsequently purified using the PureLink PCR Purification Kit, following the manufacturer’s protocol (Life Technologies, Carlsbad, CA, USA). Products were sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit and purified using the BigDye XTerminator Purification Kit following the manufacturer’s protocols on an AB 3500xL Genetic Analyzer (Applied Biosystems, Grand Island, NY, USA). Due to highly conserved 16S rRNA gene sequences between Francisellaceae, DNA extracts of the ticks were also tested specifically for the presence of Francisella tularensis using a multi-target real-time PCR test employing primers ISFtu 2, iglC and tul4 that are specific for F. tularensis as described in Kugeler et al. (2005) and Versage et al. (2003). All reactions were performed in a final volume of 20 μl and contained LightCycler FastStart DNA Master HybProbe mix (Roche, Mannheim, Germany) at a 1× final concentration, 500 nM forward and reverse primers, 100 nM probes, and 1.25 U of uracil-DNA glycosylase per reaction. For the iglC and tul4 the final MgCl2 concentration was 4 mM, and for the ISFtu2 assay, the final concentration was 5 mM. Real-time PCR cycling conditions were: 50 °C for 2 min; 95 °C for 10 min; 45 cycles of: 95 °C for 10 s, 60 °C for 30 s; and 45 °C for 5 min.
|
study
| 100.0 |
PCR amplification of the cytochrome b gene was used to query the DNA from the ticks for determining the hosts of their prior blood meals using the primers UNFOR403 and UNREV1025 (Kent & Norris, 2005; Lah et al., 2015). PCR reactions were conducted using 2X Taq PCR Master Mix (Qiagen, Valencia, CA) with primer concentrations at 0.2 μM, 8 μL of template per reaction and a total reaction volume of 40 μL. PCR cycling conditions were: denaturation at 94 °C for 3 min followed by 35 cycles of 94 °C for 1 min, 52 °C for 1 min, and 72 °C for 1 min; then final extension at 72 °C for 7 min before holding the PCR products at 4 °C.
|
study
| 99.94 |
For the bacterial community analysis, a segment of the conserved bacterial 16S rRNA gene was amplified from the individual tick DNA extractions using universal primers 515F and 806R that flank the V4 region (Caporaso et al., 2012). The 806R primers also contained a unique 12-nucleotide Golay “barcode” for each sample that allowed us to pool the PCR products from all the samples into one Illumina MiSeq sequencing run but then to identify sequences derived from each individual tick. PCR reactions were conducted in a total volume of 40 μL using Taq98® Hot Start 2X Master Mix (Lucigen, Middleton, WI, USA) with primer concentrations at 0.2 μM. PCR cycling conditions were: denaturation at 98 °C for 2 min followed by 35 cycles of 98 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min; then final extension at 72 °C for 10 min before holding the PCR products at 4 °C. The PCR products were visualized under UV light on 1% agarose gels stained with ethidium bromide before being normalized and sequenced on an Illumina MiSeq instrument by The Scripps Research Institute DNA Array Core Facility using their standard protocols (TSRI, San Diego, CA, USA).
|
study
| 100.0 |
The sequence data was analyzed using the QIIME (Quantitative Insights Into Microbial Ecology) version 1.8.0 software program (Caporaso et al., 2010b). Raw sequence data was demultiplexed into samples by barcode and filtered by mean quality score below 25, homopolymers greater than 6, uncorrected barcodes, barcodes not found in the mapping file, chimeric sequences and mismatched primers. Sequences were grouped into operational taxonomic units (OTUs) at the 97% sequence similarity level using UCLUST (Edgar, 2010) and a consensus taxonomic classification was assigned to each representative OTU using the UCLUST classifier with a Greengenes 13_8 reference database (DeSantis et al., 2006) in which at least 90% of the sequences within the OTU matched the consensus taxonomic classification 16S rRNA gene. Sequences were aligned using PyNAST (Caporaso et al., 2010a) against the Greengenes 13_8 reference core set and a phylogenetic tree of the OTUs inferred using FastTree (Price, Dehal & Arkin, 2010). In order to remove spurious OTU’s and samples with low numbers of sequences, OTU’s that occurred only once in the data and samples with less than 150 OTUs were removed. Rickettsia, Francisella and other selected taxonomic sequence identifications were crosschecked against the NCBI nucleotide database using BLASTn. Sequence, OTU table and map files can be downloaded from Figshare: 10.6084/m9.figshare.2056275, 10.6084/m9.figshare.2068644, and 10.6084/m9.figshare.2056272, respectively.
|
study
| 100.0 |
The OTU dataset was rarefied to an even sampling depth of 150 and weighted and unweighted UniFrac distance measures between all pairs of microbial communities were calculated and visualized by principal coordinate analyses (PCoA) (Lozupone & Knight, 2005). Rarefying at 1,500 even sampling depth resulted in similar results. Several analyses were performed to determine possible factors related to microbiome differences observed within the ticks and if interference between bacteria was observed. To determine if microbial profiles were consistent with the hypothesis of interference between bacteria, the Pearson product-moment correlation coefficient (PPMC) was calculated using R to determine if a statistically significant relationship existed between the number of sequences of Rickettsia and Francisella found in the various locations. Faith’s phylogenetic diversity measure (PD) was used to compare the alpha diversity between male and female ticks. Unlike other ecological diversity metrics (Shannon, chao1) that rely on species counts, Faith’s PD is calculated based on phylogenetic tree branch lengths which captures an additional aspect of diversity, namely evolutionary diversity. To elucidate which bacteria in the microbiome were associated with the presence of SFGR (as identified by rompA and IGR sequences), Random Forest supervised learning was performed in QIIME using 1,000 trees and 10 times cross validation. The ratio of Rickettsia to Francisella was classified as high (ratio > 2), even (0.5–2) or low (<0.5) in each tick. Correlations between non-Rickettsia, non-Francisella genera in the tick microbiomes and the Rickettsia to Francisella ratios (high, even, or low) was determined via a Kruskal–Wallis H test. Associations between microbiome phylogenetic distances with physical separation of the sampling locations was investigated by comparing microbiome UniFrac measures to the physical distances between sampling locations using the Isolation by Distance (IBD) web service http://ibdws.sdsu.edu/ĩbdws/distances.html (Jensen, Bohonak & Kelley, 2005). IBD tests the linear relationship between geographic distance and genetic distance of a population or, in our case, geographic distance and the microbial community phylogenetic distance. It uses a pairwise Mantel test to assess the relationship between location and UniFrac phylogenetic distances. To determine which of the abundant genera were responsible for differences in UniFrac measures between locations, OTUs that occurred in less than 10% of the samples were removed and the null hypothesis that abundances of OTUs were the same for all locations was tested using a Kruskal–Wallis H test in QIIME. A Procrustes least squares orthogonal mapping analysis was performed in QIIME to determine if the beta diversity of Rickettsia and Francisella populations was similar to non-Rickettsia non-Francisella populations with respect to location (Gower, 1975). Procrustes analysis is a statistical scaling method that transforms multidimensional shape data, in this case, beta diversity matrices, into maximal superimposition (least squared distances) to determine the concordance between the matrices. Furthermore, Analysis of Similarity (ANOSIM), which compares the ranked Bray–Curtis similarity between and within groups, was used to determine whether microbial population beta diversity between locations differed significantly. We also compared inter-and intra-subject microbial community variability using PERMANOVA (Anderson, 2001), a non-parametric multivariate analysis of variance test that employs a permutation procedure to test the null hypothesis that there is no difference between and within subjects. We used Bray–Curtis distance and 999 permutations in R studio (Version 0.99.893, vegan package).
|
study
| 100.0 |
SourceTracker was used to compare the tick microbial profiles to microbiome datasets of dog, fish, iguana, human, pigeon, rat, and soil. SourceTracker is a tool that uses Bayesian methods to predict the source(s) of microbial communities in a set of samples (sink) (Knights et al., 2011). (The inclusion of human microbiome information, especially skin, also allowed us to test for investigator-introduced contamination since skin bacteria is the most common source of laboratory and indoor contamination.) To test for sources of the tick microbiomes (sink), microbial source tracking was performed on the merged sink and source OTU file. A logistic regression was perform using the general linear model function in R, was used to determine if Rickettsia presence or absence (binary outcome) in the tick was associated with a particular host source. SourceTracker was also used as a quality control measure to identify possible sample contamination. SourceTracker version 1.0 was implemented in QIIME (version 1.9.1) with default settings. As source datasets, we used publicly available sequence data in QIITA (https://qiita.ucsd.edu/) that included 16S rRNA data from a wide range of samples such as canine skin, mouth, and feces (Study ID 1684), human skin, mouth and stool (Study ID 1684), soil (Study ID 1684, 10363), fish, frog, iguana, pigeon, and rat skin (Study ID 1748) and negative water controls (Study ID 10363) as sources. All source and sink samples were sequenced using Illumina and the same 16S rRNA V4 region primers.
|
study
| 100.0 |
Four hundred seventy-four adult D. occidentalis ticks were collected. No immature ticks were caught. Although no ticks were positive for Francisella tularensis, 39 ticks (8.2%) were positive for R. rhipicephali and 12 (2.3%) were positive for R. philipii 364D as identified by sequencing of the rompA gene and IGR. No significant difference in infection rate between male and female ticks by R. rhipicephali and R. philipii was observed (Fisher’s exact test; P = 0.47). From this group, 114 ticks were selected for Illumina sequencing. Amplification and gel electrophoresis of the V4 segment of the 16S rRNA gene produced visible PCR products of the expected 300 bp size from all ticks, while negative PCR and DNA extraction controls yielded no visible bands and were not sequenced. After quality filtering, 102 ticks remained: 44 positive for SFGR (as identified by rompA and IGR sequencing) and 59 negative for SFGR (forty-five male and fifty-seven female) from the four locations (Table 1); the total number of sequences was 6,799,927 with sample depths ranging from 2013 to 250403 reads (Table S1). Clustering sequences at the 97% level of similarity and discarding OTUs that occurred only once yielded 105,174 different OTUs and 535 different taxa including one unassigned taxon. Rickettsia and Francisella genera were the most prevalent genera present in the ticks, representing 46.8% and 41.4% of all genera, respectively. The next most frequently occurring genera were Sphingomonas (3%), Methylobacterium (1%) and Hymenobacter (0.4%) (Fig. 1).
|
study
| 100.0 |
One Rickettsia sp. (OTU 83718) accounted for 89% of all Rickettsia OTUs and matched 99–100% to R. rhipicephali (GenBank accession numbers CP013133.1, NR_074473.1, CP003342.1, NR_025921.1, and U11019.1). It also had 99% identity to other Rickettsia; however, E values were 100× higher to these other Rickettsia sequences. The second most abundant OTU (553807) accounted for 0.7% of all Rickettsia OTUs and was 99% identical to several different R. rickettsii strains including R. philipii str. 364D (GenBank: NR_074470.1) as well as other strains of R. rickettsii (including GenBank accession numbers CP006010.1, NR_102941.1, and CP003311.1). All other Rickettsia OTUs comprised less than 0.09% of total Rickettsia OTUs. OTU 840032 comprised 87.4% of all Francisella OTUs and matched 100% with Francisella-like endosymbiont (FLE) of D. occidentalis (GenBank accession numbers AY805304.1, and AY375402.1). The next closest matches were Francisella endosymbionts of other tick species D. albipictus, D. andersoni and D. variabilis (GenBank accession numbers GU968868.1, FJ468434.1, and AY805307.1, respectively). The next most abundant Francisella OTU (399541) (GenBank acc. KU355875.1, this paper) accounted for 3.1% of all Francisella OTUs and matched 97% with gene sequences of endosymbionts previously determined from a spectrum of Dermacentor species including Dermacentor occidentalis (AY375403.1), D. albipictus (GU968868.1), D. variabilis (AY805307.1), D. nitens (AY375401.1) and D. andersoni (AY375398.1). All other Francisella OTUs accounted for less than 0.4% of the total Francisella OTUs.
|
study
| 100.0 |
Female ticks had significantly less microbial diversity (alpha diversity) than male ticks as measured by Faith’s Phylogenetic Diversity which measures diversity based on phylogenetic tree lengths (Faith’s PD, two sample t-test; t = 3.63, P < 0.01; Fig. 2). Although there was no significant difference between the mean number of Rickettsia and Francisella sequences in male versus female ticks (Student’s t test P = 0.36, 0.06, respectively), Rickettsia and Francisella endosymbionts comprised a greater percentage of the microbiome of female ticks than male ticks 74.9% and 60.1%, respectively (Student’s t test P = 0.02; Fig. 3).
|
study
| 100.0 |
Escondido Canyon had lower average alpha diversity than Lopez and Peñasquitos canyons, P =0.05 (Fig. 4). Beta diversities of unweighted and weighted tick microbiomes had small but statistically significant associations with location as measured by analysis of similarity (ANOSIM) of UniFrac distances and visualized on Principal coordinate analysis (PCoA) (Figs. 5A and 5B). When only Rickettsia and Francisella were assessed for association with location, ANOSIM results were not statistically significant (ANOSIM, unweighted UniFrac; R = − 0.06, P = 0.92; ANOSIM, weighted UniFrac R = 0.02, P = 0.13). However, in order to determine if location influenced the non-dominant species separately from the dominant Rickettsia and Francisella endosymbionts, Rickettsia and Francisella were removed from the data and the analysis was repeated. After Rickettsia and Francisella were removed, the remaining microbiome association with location was low but statistically significant (ANOSIM, unweighted UniFrac; R = 0.20, P < 0.01; ANOSIM, weighted UniFrac; R = 0.28, P < 0.01). Procrustes analysis also demonstrated that the beta diversity of microbiomes in which Rickettsia and Francisella were removed had a different association with location than Rickettsia and Francisella endosymbionts (error, M2 = 0.91, P < 0.01). Isolation by distance (IBD) analysis using unweighted UniFrac distances that incorporated all members of the microbiome revealed little geographic IBD (Mantel test, unweighted UniFrac, R = 0.09, P < 0.01). However, a pattern of IBD was significant after excluding Rickettsia and Francisella (Mantel test, unweighted UniFrac, R = 0.14, P < 0.01). (Unlike weighted UniFrac, unweighted UniFrac distances only incorporate the presence or absence of microbial taxa and to not take into account the abundance of the particular taxa. This allowed us to focus on the taxonomic differences among locations rather than the abundance differences of particular taxa. In a separate analysis, we found significant differences in the relative abundances of three bacterial genera, Nevskia, Curtobacterium and Sphingomonas, between locations (Kruskal–Wallis H = 25.7, 24.2, 22.9; Bonferroni corrected P < 0.01, respectively). Ticks in Peñasquitos and Lopez Canyons had higher abundances of Nevskia than ticks collected in Mission Trails. Peñasquitos and Lopez Canyons ticks had higher relative abundances of Curtobacterium and Sphingomonas than ticks in Escondido Creek and Mission Trails (Table 2).
|
study
| 99.94 |
Stars indicate statistically significant differences between samples; Faith’s PD, two sample t-test, Escondido Creek versus Lopez Canyon; t = − 3.28, P = 0.02; Escondido Creek versus PQ, t = − 3.31; P = 0.04; other comparisons were not statistically significant. PQ = Peñasquitos Canyon.
|
study
| 99.94 |
Bonferroni correction (Bonferroni correction is used to reduce the chances of obtaining false-positive results (type I errors) when multiple pair wise tests are performed on a single set of data because the probability of identifying at least one significant result due to chance increases as more hypotheses are tested).
|
other
| 99.44 |
Rickettsia and Francisella were negatively correlated in the ticks (Pearson’s product moment correlation; R = − 0.44, P < 0.01; Fig. 6). In order to assess whether the tick microbiomes were predictive of infection with spotted fever group Rickettsia (as determined by real-time PCR of the rompA gene and IGR sequences), a Random Forests supervised learning analysis using 1,000 trees and 10× cross validation was performed on the OTU dataset minus Rickettsiaceae and Rickettsia OTUs. The ratio of baseline error to the estimated generalization error was 8.8 (i.e., more than 8 times greater than random chance). The most predictive OTU was the FLE OTU 840032 and it accounted for 13% of the model. OTUs 866436 and 639277 each accounted for 3% of the model and the closest database matches to it were the Firmicutes Geobacillus and Aeribacillus (Geobacillus), respectively (Minana-Galbis et al., 2010). Non-Rickettsia, non-Francisella bacteria associated with Francisella to Rickettsia > 2 (range 2.4–119.0) were Planococcaceae and Geobacillus (Kruskal–Wallis test; H = 23.8, 14.2, Bonferroni P < 0.001 and P = 0.011, respectively). In addition, PERMANOVA with Bray-Curtis distances was used to test for the impact of tick sex, collection site and endosymbiotic infection status on the composition of microbial communities between samples (Anderson, 2001). These environmental differences showed a significant influence on the tick associated microbial communities. SFG (P = 0.001), tick sex (P = 0.021), location (P = 0.001), and Rickettsia to Francisella ratio (P = 0.008) all had significant effects on the bacterial communities.
|
study
| 100.0 |
Subsets and Splits
No community queries yet
The top public SQL queries from the community will appear here once available.