Nonadherence to disease-modifying drugs (DMDs) for multiple sclerosis (MS) is associated with poorer clinical outcomes, including higher rates of relapse and disease progression, and higher medical resource use. A systematic review and quantification of adherence and persistence with oral DMDs would help clarify the extent of nonadherence and nonpersistence in patients with MS to help prescribers make informed treatment plans and optimize patient care.The objectives were to: 1) conduct a systematic literature review to assess the availability and variability of oral DMD adherence and/or persistence rates across ‘real-world’ data sources; and 2) conduct meta-analyses of the rates of adherence and persistence for once- and twice-daily oral DMDs in patients with MS using real-world data.A systematic review of studies published between January 2010 and April 2018 in the PubMed database was performed. Only studies assessing once- and twice-daily oral DMDs were available for inclusion in the analysis. Study quality was evaluated using a modified version of the Newcastle-Ottawa Scale, a tool for assessing quality of observational studies. The random effects model evaluated pooled summary estimates of nonadherence.n = 4 studies) was 83.3%  and proportion of days covered  was 76.5% (95% CI 72.0–81.1%). Pooled 1-year MPR ≥80% adherence (n = 6) was 78.5% (95% CI 63.5–88.5%) and PDC ≥80% (n = 5 studies) was 71.8% (95% CI 59.1–81.9%). Pooled 1-year discontinuation (n = 20) was 25.4% (95% CI 21.6–29.7%).From 510 abstracts, 31 studies comprising 16,398 patients with MS treated with daily oral DMDs were included. Overall 1-year mean medication possession ratio (MPR; Approximately one in five patients with MS do not adhere to, and one in four discontinue, daily oral DMDs before 1 year. Opportunities to improve adherence and ultimately patient outcomes, such as patient education, medication support/reminders, simplified dosing regimens, and reducing administration or monitoring requirements, remain. Implementation of efforts to improve adherence are essential to improving care of patients with MS. Multiple sclerosis (MS) is a progressive, inflammatory, autoimmune, neurodegenerative disease of the central nervous system that often begins in early adult life. Guidelines recommend that clinicians should offer disease-modifying drugs (DMDs) to people diagnosed with relapsing forms of MS (RMS) , 2. DMDsMedication adherence and persistence are challenging for patients with MS , 10. NonA systematic review and quantification of the real-world adherence to and persistence with oral DMDs would help clarify the extent of nonadherence and nonpersistence in patients with MS. Kantor et al. 2018 conducted a systematic review and meta-analysis of real-world persistence with fingolimod in patients with relapsing-remitting MS (RRMS) and reported a consensus 1-year persistence rate of 82%  . The curA systematic literature search was performed of all studies published between January 2010 and April 2018 in the PubMed database that evaluated adherence or persistence to oral DMDs. The search strategy used the following terms:  AND multiple sclerosis AND (adherence OR compliance OR persistence OR discontinuation). A priori exclusion criteria were: lack of primary data; lack of primary real-world DMD adherence/persistence data; lack of oral DMD adherence/persistence data; pediatric studies; non-English studies; and abstract-only available. Two reviewers independently reviewed the search results and reference lists of selected articles to identify additional appropriate studies and carried out data extraction. Full search strategy and search results are provided in Additional File Information extracted from the screened articles included the type of study/data source; study population ; treatment arms; duration of follow-up after DMD initiation; sample size; outcomes evaluated (including definition of adherence and method of measurement); clinical results; secondary results; and strengths and limitations of the studies.The quality of selected studies was evaluated using a modified version of the Newcastle-Ottawa Scale (NOS), a tool for assessing the quality of observational studies . The NOSThe selection of endpoints was driven by the availability of data in the published, peer-reviewed literature. Adherence was evaluated using either the medication possession ratio (MPR) or the proportion of days covered (PDC). MPR was calculated as the total number of days of medication supply between the first prescription claim and the last prescription claim issued during the follow-up period divided by the total number of days in the follow-up period. A variable follow-up period was used for the MPR denominator (number of days between the index date and the last prescription dispensed inclusive of supply) . PDC wasI2 statistic, which provide a measure of the presence of heterogeneity and the share of dispersion across studies, respectively [I2 was > 50%, the random-effects model (REM) was used to calculate pooled summary estimates; otherwise, a fixed-effect model was used. The studies were weighted according to the extent of variation among the intervention effects. Egger’s test was used to detect publication bias [p-value of < 0.05 was considered statistically significant. Subgroup analyses conducted included study location  and study design . The impact of any single study on the overall results was assessed using a leave-one-out sensitivity analysis, in which each study was iteratively removed and the findings compared to the overall meta-analysis.In line with published recommendations regarding the use of real-world data in meta-analyses,  statistiectively . If Q waion bias . The ‘meion bias  were useFrom a total of 510 abstracts identified, 31 studies comprising 16,398 patients were included in the systematic review after applying exclusion criteria  were conducted in the US, one-quarter  were from Europe, two (6.5%) were multinational, two (6.5%) were from Canada, and one (3.2%) was from Kuwait (Table n = 20 studies). For 1-year adherence, 4 studies reported the mean MPR, 6 studies reported MPR ≥80%, 4 studies reported the mean PDC, and 5 studies reported PDC ≥80%.Most studies . A lack of significance on the Egger’s test suggested an absence of publication bias.A significant Cochran’s Q statistic and an The overall mean MPR during the 1-year follow-up period for once- and twice-daily oral maintenance DMDs 4 studies) was 83.3% (95% CI 74.5–92.1%)   [Adherence to DMDs is an important aspect of optimizing patient care in MS as greater adherence has been shown to be associated with improvements in relapse outcomes and quality of life, fewer hospitalizations and emergency room visits, decrease in neuropsychological issues, fewer days of work lost, and lower MS-related medical costs , 54–57.  0.0001) . Based o 0.0001) . Persist 0.0001)  decrease 0.0001)  and lowe 0.0001) .An increased understanding of barriers to DMD adherence and persistence and the implementation of efforts to improve adherence and persistence are important. Real-world data on adherence to DMDs in patients with MS can help inform therapeutic decision making . FactorsDepression has also been shown to be associated with decreased DMD adherence and persistence in patients with MS. Gerber, et al. and Munsell, et al. both reported a significant relationship between comorbid depression and nonadherence, , 17 wherThe rates of adherence and discontinuation may differ among oral DMDs due to several potential factors such as the dosing regimen (e.g. once- versus twice-daily), efficacy, tolerability, and adverse events , 35, 36.This study was conducted in line with recommendations available in the literature for the use of real-world evidence in meta-analyses . In our I2 statistics obtained in this study ranged from 93.8 to 99.5%. These values are consistent with those noted in other published meta-analyses of medication adherence across indications [The ications , 70–73.Subgroup analyses demonstrated that there were essentially no differences between US- and ex-US-based studies and support the need for a better understanding of why patients worldwide discontinue oral DMD treatment. The study design subgroup analyses showed overlapping CIs, indicating a lack of significant differences. However, numerical differences suggested that administrative claims data analyses may more fully capture discontinuation than prospective cohort studies and medical chart reviews. With prospective cohort studies and medical chart reviews, bias may arise from patient or investigator report, whereas administrative claims database analyses provide objective billing information for medication dispensed. Also, cohort studies may also inherently encourage patients to remain in the study and continue treatment. Chart reviews may not capture all discontinuations, depending upon the availability and quality of follow-up data. Further subgroup analyses were limited by the small number of real-world/observational studies, suggesting that more real-world research is needed.This is the second published meta-analysis of real-world adherence to or persistence with oral DMDs in patients with MS. Kantor et al. 2018 conducted a systematic review and meta-analysis focusing on the real-world persistence with fingolimod in patients with RRMS . In contThere are limitations to this study. Although nonrandomized cohort studies or observational studies may provide a more ‘real-world’ representation of outcomes, costs, and utilization, differences in baseline characteristics can introduce biases, and the influence of unmeasured factors cannot be ruled out. As described, the substantial proportion of heterogeneity found across the studies is also a limitation. The use of a variable follow-up period for the MPR denominator potentially contributed to an inflated mean MPR . AdditioAdherence to treatment is an important issue for the management of patients with MS. This meta-analysis of real-world studies showed that approximately one in five patients with MS do not adhere to once- or twice-daily oral maintenance DMD treatment regimens, and one in four patients with MS discontinue once- or twice-daily oral maintenance DMDs before 1 year. Wide 95% CIs for the estimates of adherence and discontinuation reflect the heterogeneity in the rates that was observed. Opportunities to address barriers to DMD adherence in patients with MS remain, such as patient education efforts to manage expectations and to emphasize the importance of adherence, implementation of medication support/reminder techniques, simplification of dosing regimens, and reducing the need for specialized administration or monitoring requirements . ImplemeAdditional file 1: Supplementary Methods: Electronic Search Strategy. Details of electronic literature search strategy, exclusion criteria and full list of search results .Additional file 2: Supplementary Figure 1. Leave-one-out sensitivity analysis for the proportion of patients discontinuing a DMD. For studies for which results for treatment naive and treatment-experienced patients were reported separately (combined data were not available), data were combined; for studies reporting data for more than 1 oral DMD (combined data were not reported), data were combined; for studies reporting data for subgroups (combined data were not reported), data were combined. Abbreviations: DMD, disease-modifying drug.Additional file 3: Supplementary Figure 2. Leave-one-out sensitivity analysis for subgroup analyses for the proportion of patients discontinuing a DMD. For studies for which results for treatment naive and treatment-experienced patients were reported separately (combined data were not available), data were combined; for studies reporting data for more than 1 oral DMD (combined data were not reported), data were combined; for studies reporting data for subgroups (combined data were not reported), data were combined. Abbreviations: DMD: disease-modifying drug.


This cohort study evaluates the existence of language and indexing biases among Chinese-sponsored randomized clinical trials on drug interventions. Do language and indexing biases exist among Chinese-sponsored randomized clinical trials on drug interventions?This cohort study included 891 eligible Chinese-sponsored randomized clinical trials identified from English- and Chinese-language clinical trial registries. Among 470 published trials as of August 2019, positive trial findings were more commonly published in English-language journals and indexed in English-language bibliographic databases than negative trial findings.These findings suggest that language and indexing biases may lead to distorted, more positive results of drug interventions when synthesizing evidence. Language and indexing biases may exist among Chinese-sponsored randomized clinical trials (CS-RCTs). Such biases may threaten the validity of systematic reviews.To evaluate the existence of language and indexing biases among CS-RCTs on drug interventions.In this retrospective cohort study, eligible CS-RCTs were retrieved from trial registries, and bibliographic databases were searched to determine their publication status. Eligible CS-RCTs were for drug interventions conducted from January 1, 2008, to December 31, 2014. The search and analysis were conducted from March 1 to August 31, 2019. Primary trial registries were recognized by the World Health Organization and the Drug Clinical Trial Registry Platform sponsored by the China Food and Drug Administration.Individual CS-RCTs with positive vs negative results (positive vs negative CS-RCTs).For assessing language bias, the main outcome was the language of the journal in which CS-RCTs were published (English vs Chinese). For indexing bias, the main outcome was the language of the bibliographic database where the CS-RCTs were indexed (English vs Chinese).The search identified 891 eligible CS-RCTs. Four hundred seventy CS-RCTs were published by August 31, 2019, of which 368 (78.3%) were published in English. Among CS-RCTs registered in the Chinese Clinical Trial Registry (ChiCTR), positive CS-RCTs were 3.92  times more likely to be published in English than negative CS-RCTs; among CS-RCTs in English-language registries, positive CS-RCTs were 3.22  times more likely to be published in English than negative CS-RCTs. These findings suggest the existence of language bias. Among CS-RCTs registered in ChiCTR, positive CS-RCTs were 2.89  times more likely to be indexed in English bibliographic databases than negative CS-RCTs; among CS-RCTs in English-language registries, positive CS-RCTs were 2.19  times more likely to be indexed in English bibliographic databases than negative CS-RCTs. These findings support the existence of indexing bias.This study suggests the existence of language and indexing biases among registered CS-RCTs on drug interventions. These biases may distort evidence synthesis toward more positive results of drug interventions. It is established that RCTs with positive results (hereinafter referred to as positive RCTs) are more likely to be published in English-language journals, a phenomenon termed 1; however, estimates indicate that almost 40% of systematic reviews were reportedly restricted to English-language articles indexed in English bibliographic databases.3 This estimate raises the concern that such reviews may miss negative RCTs that are only published in the non-English literature, leading to biased evidence.4Ideally, this bias would not threaten the validity of systematic reviews because reviewers should comprehensively search for all existing evidence regardless of the language5 Publications of RCTs sponsored by researchers in Mainland China (Chinese-sponsored RCTs [CS-RCTs]) are also split between English- and Chinese-language journals (English CS-RCTs and Chinese CS-RCTs).6 However, limited evidence is available regarding language bias among CS-RCTs. Whether we should include Chinese CS-RCTs indexed in English bibliographic databases to reduce the effect of speculated language bias is unknown.Recently, scientific publications from Mainland China have been surging.7 This challenge implies that systematic reviewers must not only remove language restrictions from searching English bibliographic databases but also actively search Chinese bibliographic databases to capture all Chinese CS-RCTs, a practice seldomly adopted by the systematic review community.8 Bias may exist if Chinese CS-RCTs with positive results are more likely to be indexed in English bibliographic databases than their negative counterparts, which are more commonly seen in Chinese bibliographic databases. We term this potential residual of language bias as indexing bias.A further challenge, and one more difficult to address, is that most Chinese journals have not been indexed in English bibliographic databases owing to large quantity and varying quality.Cochrane Handbook for Systematic Reviews of Interventions only recommends searching Chinese bibliographic databases for systematic reviews on Chinese herbal medicine.1 Whether the recommendation should be extended to drug interventions is unknown. The objective of this study was to evaluate the existence of language and indexing biases among CS-RCTs on drug interventions to inform the potential update of the recommendation.Currently, the STROBE) reporting guideline.9In this retrospective cohort study, we retrieved CS-RCTs from trial registries and searched bibliographic databases to determine their publication status. Two hypotheses were predefined: (1) positive CS-RCTs were more likely to be published in English than negative CS-RCTs (language bias), and (2) positive CS-RCTs were more likely to be indexed in English than Chinese bibliographic databases (indexing bias). Because this study was a literature review based on open-source data and did not include research participants, it was not subject to institutional review board approval. We followed the Strengthening the Reporting of Observational Studies in Epidemiology  sponsored by the China Food and Drug Administration.11 A substance was considered a drug if recognized and regulated by the US Food and Drug Administration and/or the European Medicines Agency.13 We included all CS-RCTs that started January 1, 2008, and were completed by December 31, 2014, to allow a minimum of 4.5 years from trial completion to publication.14 We excluded phase 1 trials  and CS-RCTs missing the study period or RCTs with an unclear study interval  or any unnamed experimental drug, principal investigator, or sponsor in the registries.We retrieved CS-RCTs from all 17 primary registries recognized by the World Health OrganizationThe search and analysis were conducted from March 1 to August 31, 2019. We only included journal articles produced from eligible CS-RCTs. Conference abstracts, research letters, and dissertations were not included. Publications of protocols, subgroup analyses, secondary analyses, and meta-analyses were also excluded.15 we developed search strategies for individual CS-RCTs with informationists from the Welch Medical Library at The Johns Hopkins University (L.R.) and the Institute of Information and Medical Library at Peking Union Medical College (Q.C.)  independently searched bibliographic databases and identified matching PDFs of eligible CS-RCTs. Discrepancies were discussed and resolved by a third author (J.W.). If multiple journal articles existed, we only considered the one with the largest sample size or the earliest one if identical sample size was reported in multiple articles.19 If multiple primary outcomes were reported in a CS-RCT, we selected the first one reported in the result section. If no primary outcome was defined, the selection of the CS-RCT’s primary outcome was based on the following hierarchical order: the first outcome used in the sample size calculation, the first outcome defined in the study objective, or the first outcome reported in the Results section. When the time point was not specified for the CS-RCT’s primary outcome that was measured at multiple time points, we considered the last point in our main analysis and the first point in sensitivity analyses.The exposure was the finding of individual CS-RCTs (positive vs negative) according to the CS-RCT’s primary outcome reported in the journal article.We defined a positive result as favoring the experimental group with statistical significance in superiority trials or showing no difference between treatment groups for equivalence or noninferiority trials. Results that were not statistically significant, significantly favored the control group, or failed to show equivalence or noninferiority were defined as negative.Two main outcomes were defined: the language of the publication (English vs Chinese) and the language of the bibliographic database where the publication was indexed (English vs Chinese). An article published in both Chinese and English was considered published in English; similarly, an article indexed in both English and Chinese bibliographic databases was considered indexed in an English bibliographic database. We assumed all English-language articles were indexed in English bibliographic databases, but Chinese-language articles were possibly indexed in English or Chinese bibliographic databases.21 Industrial funding was considered as long as 1 funder was from industry; prospective registration was considered when registration occurred before the first participant was recruited.22 We included an interaction term in the models to evaluate the heterogeneity of bias across registries. Statistical significance was defined as 2-sided P < .05 for the main effect and P < .10 for interaction, generated by Wald χ2 tests as in the log binomial models. SAS, version 9.4 (SAS Institute Inc) was used for data cleaning and analysis.Bias was estimated by relative risk (RR), including point estimates and 95% CIs. An RR larger than 1.00 indicated that positive CS-RCTs were more likely than negative CS-RCTs to be published in English or indexed in English bibliographic databases. The RRs were estimated using log binomial models with 5 covariates: sample size (<100 vs ≥100), funding source (industry vs nonindustry), study design , number of recruitment centers (single vs multiple), and registration type (prospective vs retrospective).The search through trial registries and bibliographic databases was conducted from March 1 to August 31, 2019. Among the 17 primary registries and DCTRP, 5 were found to include eligible CS-RCTs: the Chinese Clinical Trial Registry (ChiCTR), ClinicalTrials.gov, International Standard Randomized Controlled Trials Number (ISRCTN), the Australia New Zealand Clinical Trials Registry (ANZCTR), and DCTRP. In total, 5084 CS-RCTs were retrieved from these trial registries and screened for eligibility. Eventually, 891 eligible CS-RCTs were identified, 470 (52.8%) of which had been published in 229 English-language journals and 72 Chinese-language journals. The screening results are shown in the Among the 470 journal articles corresponding to 470 CS-RCTs, 368 (78.3%) were published in English and 102 (21.7%) were in Chinese; 432 (91.9%) were confirmed matches to registry records and 38 (8.1%) were probable matches. Thirty of 38 probable matches (79.0%) were published in Chinese. The matching results are shown in eTable 3 in the The distribution of CS-RCTs across bibliographic databases is shown in Most CS-RCTs (306 [65.1%]) were registered in ChiCTR, followed by ClinicalTrials.gov (143 [30.4%]), DCTRP (13 [2.8%]), ISRCTN (4 [0.9%]), and ANZCTR (1 [0.2%]). The CS-RCTs in DCTRP, ISRCTN, and ANZCTR were too few to be analyzed separately. We expected that both biases would be similar across ClinicalTrials.gov, ISRCTN, and ANZCTR, so we combined CS-RCTs from these 3 registries to form an English registry category. On the other hand, DCTRP, which was only available in Chinese and was not a primary registry, was excluded from the inferential analyses. Of the 470 CS-RCTs, 323 (68.7%) were positive, 2 (0.4%) were excluded from inferential analyses because of unknown positivity (no intergroup comparison), 286 (60.9%) recruited at least 100 participants, 322 (68.5%) were conducted at a single center, 377 (80.2%) were supported by nonindustry funding, 315 (67.0%) were retrospectively registered, and 442 (94.0%) were superiority trials.The distribution of covariates, including the sample size, funding source, study design, number of recruitment centers, and registration type, was similar between English and Chinese CS-RCTs , indicating no evidence of heterogeneity of language bias across registries. Other factors associated with increased likelihood of being published in English among CS-RCTs were a sample size of at least 100 , single-center as opposed to multicenter trials , and financial support from a nonindustrial source compared with an industrial source .Four hundred sixty-eight CS-RCTs were included for this analysis. As shown in 2 = 0.22; P = .64), indicating no evidence of heterogeneity of indexing bias across registries. The only other factor associated with an increased likelihood of being indexed in English bibliographic databases among CS-RCTs was sample size of at least 100 .Four hundred sixty-eight CS-RCTs were included for this analysis. As shown in Two sensitivity analyses were conducted. When only confirmed matches (n = 432) were analyzed, the RRs increased regarding language bias  and decreased regarding indexing bias . When the first assessment of the CS-RCTs’ primary outcomes was analyzed (rather than the last assessment at the end of follow-up), the RRs increased regarding both language bias  and indexing bias .Our study supports the existence of language bias and indexing bias among CS-RCTs included in trial registries. As hypothesized, positive CS-RCTs were more likely to be published in English or indexed in English bibliographic databases compared with negative CS-RCTs.24 Positive CS-RCTs are more likely to be submitted to English-language journals because they typically have a higher chance of being accepted; accordingly, English-language journals contain more positive CS-RCTs than their Chinese counterparts.Reputation, job prospects, and academic progress may critically depend on publishing in English-language journals among Chinese researchers.25 With the mean number of RCTs per German-language journal decreasing from a maximum of 11.2 annually from 1970 to 1986 to only 1.7 annually from 2002 to 2004, language bias from German-speaking countries may no longer be a concern.Theoretically, language bias disappears if all clinical trials shift to be published in English. This ideal has been echoed by a trend toward publishing in English in some countries, such as Germany.We did not detect such a trend among CS-RCTs. According to an ongoing study, the number of CS-RCTs published in Chinese may be as many as 44 000 in 2016 as opposed to fewer than 1000 being published in English . The deep gap between the Chinese and English literature has allowed significant space for language bias to develop.27 Because most systematic reviews were constrained to work within English bibliographic databases only, what those studies measured was a fraction of language bias—the difference between English-language and non–English-language trials indexed in English bibliographic databases. The effect of language bias cannot be comprehensively evaluated unless non–English-language trials, especially the ones not indexed in English bibliographic databases, are included and evaluated.Several studies attempted to evaluate the effect of language bias based on non–English-language trials included in systematic reviews.Cochrane Handbook for Systematic Reviews and the United States Institute of Medicine Guidelines for Systematic Reviews have recommended including non–English-language literature indexed in English bibliographic databases.28 Including non–English-language trials indexed in English bibliographic databases may not eliminate the effect of language bias but could reduce it to the scope of indexing bias.As the primary source for systematic reviewers, English bibliographic databases may index some non–English-language literature, but they vary in the amount and scope. The 7 Although we did not simulate actual systematic reviews, it appears plausible that, owing to language and indexing bias, drug interventions might appear more positive than they are when existing evidence is synthesized, for example in systematic reviews.To date, English bibliographic databases do not represent the Chinese-language literature. This is problematic because an ongoing study shows more than 10 000 clinical trials have been published out of China in 2016 . However, Embase only indexes 80 Chinese-language journals.29 Our study tipped the scales in this proposition’s direction: including Chinese-language literature may reduce bias and shrink the CIs of the estimates. Currently, the Cochrane Handbook for Systematic Reviews of Interventions only recommends searching Chinese bibliographic databases for topics related to complementary medicine or Chinese medicine,1 but our results suggest that it might be prudent to expand recommendations to studies on drug interventions as well.The effect of language bias regarding CS-RCTs might be eliminated if reviews comprehensively searched Chinese bibliographic databases, if major English bibliographic databases would index all Chinese-language literature, or if all CS-RCTs would be appropriately registered with results. There are, however, layers of complexity that warrant appreciation. There has been a discussion as to whether scientists should search Chinese bibliographic databases when conducting systematic reviews.31 which some may argue is a reason to not use Chinese CS-RCTs in systematic reviews. However, reporting quality may not completely represent the actual scientific quality.32 One study found no difference between one systematic review mainly using English-language trials and another mainly using Chinese-language trials on the same topic.8 It is the researchers’ decision to include or not include those trials  based on reporting quality, but it might be too simplistic to just ignore them.The reporting quality of Chinese CS-RCTs was low,33 Second, our search strategy relied on the information in trial registries, which may be inaccurate and/or incomplete.35 Third, less than 15% of Chinese-language articles reported registration,6 indicating CS-RCTs in trial registries may not be representative of all CS-RCTs in the period addressed. At this moment, it is unclear how much this study can be generalized to all CS-RCTs. In addition, we studied language bias and indexing bias from the level of the entire RCT community, but we did not assess whether such biases might have effects on the conclusion of individual systematic reviews. Such simulation or empirical studies might further elucidate the extent and direction of systematic error introduced by language and indexing bias.Our study has several limitations. First, we searched 7 prominent, but not all, bibliographic databases.Our study indicates the existence of language bias and indexing bias among CS-RCTs in trial registries. These biases might threaten the validity of evidence synthesis. When synthesizing evidence, drug interventions might appear more favorable than in reality owing to language and indexing bias. Removing language restrictions and actively searching Chinese bibliographic databases may reduce the effect of these 2 biases.


IL-11) are involved in the enteric nervous system's progress. It was found that IL-11 SNPs (rs8104023 and rs4252546) are associated with HSCR in the Korean population waiting for replication in an independent cohort. This study evaluated the relationship between IL-11 and the susceptibility of patients to HSCR by performing subphenotype interaction examination, HAEC pre-/post-surgical patient-only association analysis, and independence testing.Hirschsprung disease (HSCR) is a hereditary defect, which is characterized by the absence of enteric ganglia and is frequently concurrent with Hirschsprung-associated enterocolitis (HAEC). However, the pathogenesis for HSCR is complicated and remains unclear. Recent studies have shown that pro-inflammatory cytokines such as interleukin-11 (IL-11) and susceptibility to HSCR by replication research, subphenotype association analysis, and independence testing.In this study, a cohort consisting of children from Southern China, comprising 1470 cases and 1473 controls, was chosen to examine the relationship between two polymorphisms (rs8104023 and rs4252546 in IL-11 gene polymorphisms (rs8104023 and rs4252546) are not associated with the risk of HSCR in the Chinese population. The results of both short-segment and long-segment (S-HSCR and L-HSCR) surgery  showed that single nucleotide polymorphisms (SNP) rs8104023 is associated with susceptibility to HAEC.The results showed that This study explored the relationship between genetic polymorphisms and susceptibility to HAEC in HSCR subtypes for the first time. These findings should be replicated in a larger and multicentre study. Hirschsprung disease (HSCR) is a congenital disability characterized by the complete or partial absence of intestinal ganglia . A signiIL-11, participate in neurite growth, and myenteric neurons, and other functions of ENS. For instance, IL-11 plays an important role in the survival, maturation, and myelin formation of oligodendrocytes. A study by Tomuschat et al. reported that the pro-inflammatory cytokines were observed in both the aganglionic and ganglionic and aganglionic regions of the bowels of patients with HSCR [., showing that a number of SNPs on IL-11 such as rs1126760, rs4252546, and rs8104023 have statistically significant associations with HSCR based on 187 cases and 283 controls [IL-11 [IL-11 gene polymorphisms and HSCR susceptibility was investigated by conducting a case–control study of two previously confirmed SNPs (rs8104023 and rs4252546) related to the pathogenesis of HSCR based on an independent Chinese sample (1470 cases and 1473 controls). However, the association of selected SNPs with HSCR failed to be further verified. In addition, rs8104023 was found to be a relevant SNP, whose relationship with HAEC susceptibility was examined.RET proto-oncogene encoding receptor tyrosine kinases can only explain 50% of patients with genetic HSCR and 15–20% with sporadic HSCR . Other mith HSCR , 9. A poP > 0.05). These participants were diagnosed with HSCR as per the histological testing of enteric aganglionosis (using biopsy specimens). Patients with HSCR were divided into three subgroups, including 1033 S-HSCR (70.27%), 294 L-HSCR (20.00%), and 82 TCA (5.58%). In this study, the prevalence of L-HSCR and TCA patients exceeded the general prevalence of HSCR subgroups . Besides, the blood samples of 1473 unaffected controls were obtained from a cohort group. In addition, all the controls were collected without a history of HSCR and neurological disease. Written informed consent was obtained from all the participants' guardians, and the study protocol was approved by the Institutional Review Board of the hospital.DNA samples of 2943 participants (1470 HSCR patients and 1473 controls) were selected from Guangzhou Women and Children's Medical Center  with HSCR [IL-11 and HSCR. Three SNPs were chosen according to 1) 1 SNP exceeded the significance of genome-wide association (P < 1 × 10–8). 2) 2 SNPs showed limited linkage disequilibrium (LD) (r2 < 0.5). The significant signals in potential loci were selected based on the minor allele frequency (< 5%) in the Chinese population. Quality control of SNPs was conducted as follows: (1) 2 SNPs exceeded the filtering criteria with a missing rate of less than 10% (1 SNP was removed). 2) Subjects with a 10% missing rate were excluded. Further analysis of 1469 cases and 1473 controls after the quality control included two SNPs (rs8104023 and rs4252546).A previous GWAS study of the Asian population described the associations of The allelic test was used to compare the risk-allele frequency, and PLINK 1.9 was used to analyze additional tests, including the Cochran-Armitage trend test, genotype test in 3 × 2 contingency tables, dominant and recessive model test, logistic regression, the SNPs were explored for the association with HSCR. Moreover, a comparison of controls and cases with a certain subphenotype was analyzed using subphenotype stratification.This study used HaploView was used to obtain linkage disequilibrium patterns and values . SNPTEST. [IL-11 shows detailed locations of SNPs. Based on gender, HAEC, constipation, and subtypes, stratification analysis was done to assess the association of the two selected polymorphisms with the risk of HSCR. To explore the effect of polymorphisms, the relationship between IL-11 polymorphisms and risks of HSCR subgroups  was also analyzed based on the aganglionic segment range of HSCR. Interestingly, both S-HSCR and L-HSCR subgroups showed a significant increase in signals, which showed a significant association of rs8104023 with the enterocolitis risk no matter before or after the operation. However, IL-11 SNPs  were not significantly associated with HSCR. In the analysis of constipation, no significant association with HSCR, even in the comparison of HSCR subgroups, was observed.The replication study used two SNPs  for the association analysis to identify multiple independent variants related to HSCR. Genotype distribution of all the SNPs failed to violate the Hardy–Weinberg Equilibrium (HWE) controls (P = 0.47). The association of SNPs cannot be replicated, which was inconsistent with the report by Kim et al. . The phyIL-11 SNPs and HAEC was analyzed, and the relationship between the three selected SNPs and pre-/post-operative HAEC was investigated by patient-only analysis (Table The most common and serious complication of HSCR is Hirschsprung-associated enterocolitis (HAEC), which happens either before or after the operation. Therefore, the relationship between two 2 = 0.24). SNPs rs8104023 and rs4252546 showed medium to high LD in Chinese populations (r2 = 0.47).Figure IL-11 gene were unrelated to the risk of HSCR in Chinese populations. Furthermore, IL-11 gene polymorphisms may play a role in HSCR-associated enterocolitis.HSCR results from many genetic factors. Various studies have shown that candidate gene, including SOX10 and Glial cell line-Derived Neurotrophic Factor (GDNF), are important for the diagnosis of HSCR. Several researches have shown the etiology of HSCR. However, the precise mechanism remains unknown. This study showed that two SNPs  in the IL-11 belongs to the IL-6 cytokine family and can stimulate megakaryocytopoiesis, which is essential in pro-inflammatory functionality [IL-11 has been reported to affect the progress of the enteric nervous system [., nine SNPs of the IL11 gene were selected, and several SNPs were identified to have a statistical and significant association with HSCR. The frequencies of rs4252546 and rs8104023 were higher in HSCR cases than those in unaffected subjects [. showed that these two SNPs in the IL11 gene were not significantly related to HSCR of the German population . This study further explored whether the same situation would be observed in a larger Chinese population. In this study, SNP rs4252546 is a putative binding site of NF-1 and may affect gene expression  [IL-11 gene may interact with other genes to exert a limited role in regulating and controlling the aganglionic segment during the ENS development.ionality . IL-11 hs system . The fluIL-11 gene polymorphisms and susceptibility to HSCR in a Southern Chinese population. This study found no association between the 2 SNPs with HSCR. However, small sample size may negatively affect ethnic diversity or sample size, possibly reflected by different genetic backgrounds. The independence test  [., IL-11 restores the ability of longitudinal muscles, thus reversing the inflammatory response and producing active tension in both the jejunum and colon [IL-11 polymorphisms. IL-11 promoter variants may modulate the expression of IL-11 to increase the susceptibility to HEAC, thereby leading to abnormal neuronal maturation and colonic inflammation. However, the functional mechanism of IL-11 and HEAC is unclear, thereby requiring further studies with larger sample sizes, different populations, and functional evaluations to verify these results.Our results showed no significant association with HSCR regarding enterocolitis. Similarly, research by Liao et al. provided evidence that se cases . A studynd colon . This deIL-11 SNPs for fine mapping and replication and then investigates associations. Both IL-11 gene polymorphisms (rs8104023 and rs4252546) may be irrelevant to the risk of HSCR in the Chinese population but play a role in HAEC,which may provide a new way to study HEAC. To address these possibilities, further studies with larger sample sizes, different populations, and functional evaluations should be carried out.Based on other studies, this study uses additional Additional file 1. The subclinical information collected for the subjects in this study.


To investigate whether reactive hyperemia measured by peripheral arterial tonometry correlates with markers of endothelial dysfunction and may be used to identify sepsis in critical illness.A prospective study was performed using a cohort of critically ill patients. Endothelial dysfunction was assessed on admission by quantifying reactive hyperemia-peripheral arterial tonometry and plasma levels of endothelin-1, soluble E-selectin, endocan and syndecan-1. Septic patients were compared to patients without evidence of infection.Fifty-eight septic patients were compared to 28 controls. The natural logarithm of reactive hyperemia-peripheral arterial tonometry was negatively correlated with cardiovascular comorbidities, disease severity and plasma levels of soluble E-selectin (p = 0.024) and syndecan-1 (p < 0.001). The natural logarithm of reactive hyperemia-peripheral arterial tonometry was lower in septic patients than in controls . When adjusted for age, the multivariable model predicted that each 0.1-unit decrease in natural logarithm of reactive hyperemia-peripheral arterial tonometry increased the odds for infection by 14.6%. m.Reactive hyperemia-peripheral arterial tonometry is closely related to soluble E-selectin and syndecan-1, suggesting an association between endothelial activation, glycocalyx degradation and vascular reactivity. Reactive hyperemia-peripheral arterial tonometry appears to be compromised in critically ill patients, especially those with sepsis. Mortality correlates with the severity of sepsis and may exceed 40% when septic shock is present, highlighting the importance of prompt recognition of sepsis and adequate treatment. However, there is no gold standard in the diagnosis of sepsis. Current recommendations suggest using the Sequential Organ Failure Assessment (SOFA) score; however, it does not reflect the full extent of the sepsis mechanisms, and several authors have criticized its lack of specificity along with other limitations.-7Diagnosing sepsis is challenging. It requires the often unclear identification of infection alongside the identification of sepsis-induced organic dysfunction.-14Sepsis is an intricate network of immunologic responses, and many targets have been investigated in the hope of identifying a dynamic biomarker that would help distinguish septic from nonseptic patients. Unfortunately, many of these biomarkers are restricted to research or are expensive or nonspecific.-18 All these changes cause perfusion defects and cell death, triggering organ damage. Reactive hyperemia (RH) is a transient nitric oxide (NO)-dependent vasodilatory response that occurs briefly after a period of flow occlusion, is part of the normal microvascular response to volume changes, and is highly dependent on the endothelial glycocalyx integrity.-23 Peripheral arterial tonometry (PAT) is a noninvasive and indirect method of evaluating the RH response (RH-PAT) with adequate reproducibility and is associated with chronic and acute endothelial dysfunction.,24-27Sepsis-induced acute endothelial dysfunction is a consequence of circulating cytokines and bacterial and viral toxins and has protean manifestations that include microvascular dysfunction, coagulation disorders, increased vascular permeability and loss of vascular tone.,18 Reactive hyperemia-peripheral arterial tonometry is impaired in septic patients compared with healthy controls, but the specificity of this impairment to sepsis compared with other critically ill patients with noninfectious disease remains unknown.,28-30Sepsis-induced damage to the endothelial glycocalyx and NO-depletion may contribute to endothelial dysfunction and microvascular dysfunction.The objective of this study is to evaluate whether RH-PAT correlates with the diagnosis of sepsis and immunologic markers of endothelial activation and whether it can be used to identify sepsis in critically ill patients in clinical practice.Centro Hospitalar de São João, ref. 205/11) and was performed in accordance with the ethical standards of the 1964 Declaration of Helsinki and its later amendments. Written consent was obtained from the patient if awake or from the legal representative.This was a prospective study of a cohort of consecutive patients admitted to the intensive care unit (ICU) of the Infectious Diseases Department in a single tertiary care center. This study was approved by the local ethics committee  with a severity that justifies admittance to the ICU for surveillance or therapy. Exclusion criteria were unavailability to perform RH-PAT in the first 24 hours or clinical and anatomic conditions that interfere with PAT, such as absence of the 2nd or 3rd pair of fingers, severe hypotension , brachial artery stenosis or severe reactive vasospasm in response to recent arterial catheterization, arterial-venous fistulae in any arm, permanent tremor or restlessness and platelets < 20.000/µL or coagulation disorders. The patient was considered infected with a confirmed microbiologic etiology on appropriate diagnostics  or by clinical response to antimicrobial therapy in the absence of microbiological identification where an alternative diagnosis was not likely. Controls were defined when there was no evidence of an infection on admission or no need for antimicrobials and when an alternative diagnosis was made. Patients were further subclassified into septic shock, cardiogenic shock or hypovolemic shock categories according to current guidelines.,31Patients were classified as septic according to the SEPSIS-3 definition. For each participant, the SOFA, Acute Physiology and Chronic Health Evaluation II (APACHEII) score and Simplified Acute Physiology Score II (SAPSII) were quantified.On the first 24 hours of admittance to the ICU, each included participant underwent clinical and demographic data collection, routine blood sampling for biochemical analysis and biomarkers quantification and RH-PAT evaluation. For each patient, plasma was aliquoted and stored at -80ºC for further biomarker quantification. To simplify the multivariable analysis, age was categorized with a cut-off value of 60 years old because this was the most reasonable cut-off after which endothelial dysfunction is independently and significantly affected by age according to studies evaluating the effect of age on endothelial function.Endothelial dysfunction was measured indirectly by quantification of RH-PAT using the Endo-PAT 2000® tonometer . The evaluation was performed after at least 1 hour of hemodynamic stability (defined as absence of vasopressor drugs dose adjustments or need for fluid bolus).Assessments were performed in a controlled environment  with minimal distractions in the dorsal recumbent position. When present, vasopressors doses were stable for ≥ 60 minutes, and only essential routine care was performed during the evaluation. For each assessment, a software profile was introduced, controlling for age, weight, estimated height, heart rate and arterial pressure.nd fingers (or both 3rd fingers in case of finger amputation or severe deformity). Basal pulse wave amplitude (PWA) was registered in both fingers for 5 minutes followed by sphygmomanometer cuff inflation in the 2nd portion of the arm with compression of the brachial artery flow . The PWA was then recorded for another 5 minutes. Compression of arms where vascular catheters were present was avoided whenever possible. The cuff was then deflated, and a new evaluation of the PWA was performed for another 5 minutes. Each evaluation lasted between 15 - 20 minutes. An RH index (RHI), which measures vascular adaptation to a blood flow increase, was calculated automatically using Endo-PAT 2000 software (version 3.1.2) provided by the manufacturer  was selected from a cohort of kidney donors in the Nephrology Department and assessed for RH-PAT.For each admitted patient, we performed a quantification of syndecan-1, endocan, soluble E-selectin (sE-selectin) and endothelin-1 (ET-1) levels in plasma samples using a Human Premixed Multiplex kit , and analysis was performed using Luminex® 200TM xMAPTM Technology (Luminex Corp.).Analytes concentrations were determined by laser monitoring of the spectral properties of the beads, and the amount of associated fluorescence measured was reported as median fluorescence intensity (MFI). The net MFI was calculated from the average of readings (in duplicated) for each standard and sample and subtracting the average of blank MFI. A standard five-parameter logistic (5-PL) curve fit was created for each analyte using Luminex® xPONENT® Software (version 3.1).Descriptive statistics included the mean and standard deviation (SD) for continuous variables and absolute and relative frequencies for categorical variables.At baseline, for the (univariate) identification of variables significantly associated with infection, the chi-squared test was used for categorical variables, and t-test was used for continuous variables. The effects of RHI on infection were investigated using multiple logistic regression models with adjustment for other relevant variables. In accordance with sample size, only variables with a p-value from the univariate analysis less than or equal to 0.1 were considered as potential confounders. The choice of the best model was based on the likelihood ratio test for nested models or the Akaike Information Criterion (AIC). The significance level was set at 0.05.For the models of interest, the area under the Receiver Operating Characteristic (ROC)-curve (AUC) was computed. Cut-off values for RHI were determined by maximizing the sum of sensitivity and specificity. Statistical analyses were performed with R software for statistical computation, version 2.3.3.During a 12-month period, 115 patients were enrolled. Twenty-nine patients were excluded. The main reasons for exclusion were unavailability to perform PAT in the first 24 hours after admission (n = 9), hemodynamic instability leading to death (n = 8), severe thrombocytopenia (n = 3), restlessness (n = 2), arterial-venous fistulae (n = 2), arterial vasospasm (n = 1) and failure to obtain an RHI measurement (n = 4). None of the patients or legal representatives refused to participate. Patient characteristics and comorbidities are shown in Within the septic group, patients were classified according to the new sepsis definition with SOFA scores ranging from 2 to 19 points. A subanalysis of the septic group revealed that 29 (50.0%) patients had septic shock.The control group did not differ significantly from the septic group in terms of characteristics and comorbidities except for age between the two groups, with a higher prevalence of older patients in the control group . There wThe RHI was quantified in the 86 enrolled patients. Due to a skewed empirical distribution, the natural logarithm of RHI (Ln_RHI) was used. Mean Ln-RHI in the study population was 0.58 ± 0.46. Mean Ln_RHI in healthy volunteers was 0.71 ± 0.05, which was significantly increased compared with the study population (p = 0.02).Inflammatory biomarkers  were quantified in samples collected from 68 patients (53 septic and 15 controls). Endothelin-1 values in 6 (8.8%) patients evenly distributed between septic and control patients were below the lower limit of detection. Due to skewed empirical distributions, the natural logarithms of endocan, syndecan-1 and sE-selectin were used.Univariate analysis revealed age > 60 y.o. (p = 0.023), coronary disease (p = 0.043), dyslipidemia (p = 0.015) and solid neoplasia (p < 0.001) were significantly associated with lower Ln_RHI means, higher severity scores, lower serum potassium, hypotension, the presence of shock, higher noradrenalin dose and higher lactate . Ln_synd-1.368*0.1 = 1.146). For any fixed value of RHI, individuals 60 y.o. or older have an 80.4% reduced risk for sepsis than those younger than 60 y.o. The interaction between age group and Ln_RHI was not significant (p = 0.821); thus, the association between Ln_RHI and sepsis does not significantly vary between the two age groups. No other variables (including hypertension and severity markers) significantly improved the model.In the comparison of Ln_RHI between septic and control groups, those with infection had a lower average ; however, the difference was not statistically significant (p = 0.122). To evaluate further interactions between patient characteristics and the presence of sepsis, a multivariable analysis adjusting the effect of Ln_RHI for significant and marginally significant variables from the univariate analysis was performed. The best regression model consisted of Ln_RHI (p = 0.024) and age (p = 0.002) as explanatory variables , sensitivities, specificities and positive (PPV) and negative (NPV) predictive values for the regression models were computed. In Model A, the best Ln_RHI cut-off values were 1.43 (RHI 4.2) for patients younger than 60 y.o. and 0.41 (RHI 1.5) for patients 60 y.o. or older. In Model B, the best Ln_sE-selectin cut-off values were 9.5  and 10.5  for patients younger than 60 y.o. and patients 60 y.o. or older, respectively.-37 The RHI was negatively associated with sepsis. Thus, the lower RHI, the higher the risk of the patient being septic. This association was independent of disease severity, comorbidities and other factors known to influence the RHI. This finding suggests that although disease severity and comorbidities are the main factors influencing RHI, the presence of infection (irrespective of the state of sepsis or septic shock) seems to decrease the RHI to a greater extent than that in other critical illnesses.The present study aimed to clarify the role of RH-PAT in sepsis by comparing a group of septic patients in the ICU with a group of critically ill patients without evidence of infection. After adjusting for variables known to influence RHI, we found a significant difference influenced by age between patients with and without sepsis.Age highly influenced the predictive values of RHI. For the same RHI, the odds for infection were significantly lower in older patients. Reactive hyperemia index cut-off values also varied widely between the two age groups. Despite good threshold sensitivity (86%), the lack of specificity makes these values clinically unreliable. Endothelial frailty may be a justification for this difference as older patients may already have a dysfunctional endothelium with a lower basal RHI due to age and comorbidities. Therefore, in older patients, the presence of infection may be sufficient to decrease endothelial function, and a lower RHI threshold may be reasonable to aid in the diagnosis of infection. In contrast, younger patients may have a protective functional endothelium; thus, a severe infection with a deep systemic inflammatory response would be required for an effect as intense as that seen in older patients.,39 There are a few reports of increased serum syndecan-1 levels in septic patients.-43 Glycocalyx integrity is essential for the shear stress sensing mechanism that leads to the NO-dependent vasodilatory response observed in reactive hyperemia, and the absence of syndecan-1 significantly decreases endothelial nitric oxide synthetase (eNOS) and vascular reactivity.,45 As syndecan-1 levels increase in serum due to sepsis-induced cleavage, the reactive hyperemia response is hypothesized to be impaired, and RHI would decrease. E-selectin is a glycan binding protein that is typically absent in the endothelial wall and is upregulated and mobilized by vascular endothelial cells to the cell surface facing the blood vessel lumen in response to inflammatory mediators . Its main function is to initiate neutrophil extravasation into tissues in acute and chronic inflammatory diseases.,47 Soluble E-selectin becomes available in plasma upon apoptosis of activated endothelial cell, correlating with the degree of endothelial activation, and higher plasma levels are associated with sepsis and sepsis severity.,48,49 Together, the independent association of RH-PAT with syndecan-1 and sE-selectin levels in our results suggests that glycocalyx integrity and endothelial activation may have a role in vascular reactivity, more specifically with reactive hyperemia.Syndecan-1 is a transmembrane heparan sulfate proteoglycan, and syndecan-1 serum levels increase in the setting of glycocalyx degradation.-53 As pneumonia was underrepresented in our study population and endocan is mainly produced by the kidney and lung in the presence of inflammatory mediators, our results may have been underpowered, and such an association was not identified. Endothelin-1 (ET-1) is an endothelial derived factor that regulates vascular tone and remodeling, induces adhesion molecules in the endothelial wall and adherence of polymorphonuclear cells, and leads to oxidative stress with endothelial damage.,55 Although ET-1 levels are usually increased in cardiovascular diseases and sepsis, we did not find such an association or any association with RH-PAT. Possible explanations include that ET-1 levels may not differ in a population with high comorbidities and critical disease even in the presence of infection. Alternatively, as plasma ET-1 levels are 100-fold lower that on a the vascular wall, plasma quantifications may not be sensitive enough.Despite the promising results of syndecan-1 and sE-selectin, endocan, a proteoglycan secreted by the vascular endothelium in septic patients, was not associated with RH-PAT or sepsis.Our data confirmed our hypothesis that when measured by PAT, sepsis is associated with an impaired reactive hyperemia response to a greater degree than with other nonseptic conditions. This finding has not been previously reported using this method. These results also clarified the association between markers of endothelial activation and glycocalyx degradation and the reactive hyperemia response, which has not been previously explored in other studies. However, although a definite trend is evident, our model using RHI has only moderate sensitivity and specificity, which may be explained by the fact that RHI is affected by numerous variables as evidenced in our initial analysis.Additional work will be required to fully understand the role of RH-PAT in sepsis as there are some limitations in our study. Given the lack of a gold standard for the diagnosis of sepsis, the definition used in our study may have failed to correctly classify patients. A larger sample size would be required to attenuate the effect of variables influencing RH-PAT, but the costs associated with each PAT measurement make this difficult in research and in clinical practice. A multicentered study including other ICUs is necessary to decrease potential selection bias. Although this ICU is part of the infectious diseases department, it is considered a general ICU and receives adult patients with any type of critical condition. Early mortality in the ICU is high, and RH-PAT requires hemodynamic stability to be achieved. Unfortunately, a substantial number of patients died before recruitment, which could have skewed the results. The test was also performed after the start of antimicrobials and inotropes, which directly impact arterial tone and potentially altered the response to the test.-59 Furthermore, sepsis is currently defined as organic dysfunction in the presence of infection, and the latter is responsible for the inflammatory cascade that leads to organic malfunction in some patients, distinguishing the normal inflammatory response to infection from sepsis. If RH-PAT is proven to be correlated with organic dysfunction, it may become a useful method to distinguish patients with simple infections from patients with microvascular dysfunction and progression to sepsis.Nonetheless, although RH-PAT does not at present seem to be of clinical value for distinguishing septic patients from other patients admitted to the ICU, there was a strong association with every measured marker of disease severity, suggesting that RH-PAT may be useful for accessing prognosis. However, except for septic and cardiogenic shocks, where RH-PAT has been proven to be impaired, studies correlating RH-PAT and other conditions in the ICU are lacking.As RH-PAT is noninvasive and has almost no contraindications or complications, it is a desirable method for evaluating reactive hyperemia. However, future studies should focus on the prognostic value of the test.Reactive hyperemia-peripheral arterial tonometry seems to be impaired in septic patients compared with patients with noninfectious critical illness. However, reactive hyperemia-peripheral arterial tonometry is highly influenced by age and lacks specificity, which may render it clinically unreliable to distinguish septic patients from nonseptic patients. Soluble E-selectin and syndecan-1 were independently associated with reactive hyperemia-peripheral arterial tonometry, which may suggest common pathological mechanisms. Our results revealed a strong association between reactive hyperemia index and disease severity, and further studies should clarify whether reactive hyperemia-peripheral arterial tonometry can be used as a prognostic indicator in patients admitted to the intensive care unit.


Saccharum spp.), the weed Columbus grass  and cultivated sorghum (S. bicolor). All three hosts are also colonized by the sugarcane aphid (Melanaphis sacchari), the main vector of SCYLV worldwide. To understand the high incidence of SCYLV observed in sugarcane commercial fields and in germplasm collections, we investigated the transmission efficiency of SCYLV from sugarcane and Columbus grass to sugarcane using the sugarcane aphid and a spider mite (Oligonychus grypus) that also tested positive for SCYLV in Florida. Healthy and SCYLV-infected leaf pieces of sugarcane and Columbus grass carrying viruliferous aphids or spider mites were transferred to virus-free plants of the yellow leaf susceptible sugarcane cultivar CP96-1252. Three- and 6-months post inoculation, the 108 aphid-inoculated plants of Columbus grass and the 90 mite-inoculated plants of sugarcane tested negative for SCYLV by tissue blot immunoassay (TBIA) or reverse transcription polymerase chain reaction (RT-PCR). Similar results were obtained for 162 aphid-inoculated plants of sugarcane, except for two plants that tested positive for SCYLV by TBIA and RT-PCR. In two field experiments planted with SCYLV-free and virus-infected sugarcane (cultivar CP96-1252), only 18–28% of healthy plants became infected during a 24- to 28-month period. SCYLV prevalence in these field experiments did not differ between aphicide treated and untreated plots. Incidence of M. sacchari haplotypes in the Everglades agricultural area also indicated that the predominant haplotype that is currently colonizing sugarcane was not a vector of SCYLV in Florida. Lack of virus transmission by the spider mite suggested that this arthropod only acquired the virus when feeding on infected plants but was unable to transmit SCYLV. The current vector of SCYLV in Florida remains to be identified.Sugarcane yellow leaf virus (SCYLV), the causal agent of yellow leaf disease, naturally infects at least three plant species in Florida: sugarcane ( Saccharum, along with its related genera Erianthus, Miscanthus, and Narenga are all members of the Poaceae grass family. High sucrose content, useful by-products and ethanol, a bioenergy source, are all reasons why sugarcane (Saccharum interspecific hybrids) is an important economical asset to many tropical and sub-tropical areas around the world, including Florida. However, cultivating this crop holds its challenges. One of them is to control the numerous pathogens  that infect and cause diseases in sugarcane, subsequently resulting in yield losses [The genus d losses . Among td losses , 3, 4.SCYLV is the causal agent of yellow leaf, a disease that was originally called yellow leaf syndrome of sugarcane or YLS. The disease was first reported in Hawaii in 1989 but there is evidence that the disease occurred in other locations much earlier , 5, 6. DYellow leaf can cause up to 25% yield losses in commercial fields, and a negative effect of SCYLV on sugarcane growth has been reported worldwide , 13, 14.Worldwide isolates of SCYLV were recently distributed into two major phylogenetic clades and seven genotypes  . AlthougLuteoviridae family and cannot be transmitted mechanically. This virus is spread by infected stalk cuttings and by at least four aphid species in a persistent, circulative, and non-propagative manner: Melanaphis sacchari, Ceratovacuna lanigera, Rhopalosiphum maidis and R. rufiabdominalis [M. sacchari, commonly known as the sugarcane aphid, is the most efficient vector of SCYLV worldwide and the most widespread in the Western hemisphere [M. sacchari to transmit SCYLV from infected to healthy sugarcane [M. sacchari in March-April in the EAA . These observations could explain the high incidence of SCYLV in commercial fields and germplasm collections in Florida [Sorghum species but also found on sugarcane, haplotype H3 that mainly colonizes sugarcane but also found on sorghum and Johnsongrass , and haplotype H6 that infests Johnsongrass and sugarcane [SCYLV is a member of the ominalis , 18, 19.misphere . Despiteugarcane , 22, 23.ugarcane  were supugarcane , supportugarcane .Acari order within the Arachnida class of arthropods. Among them, only members of the Eriophyidae and Tenuipalpidae families are known to transmit virus species of the Fimoviridae, Potyviridae, Secoviridae, Alpha- and Betaflexiviridae families [Oligonychus stickneyi has been present and occasionally a pest of sugarcane in Florida since the 1970s [Oligonychus grypus, was found more recently on greenhouse plants in Canal Point and Belle Glade, FL [Luteoviridae viruses are only known to be vectored by aphids [Mites belong to the families . The spihe 1970s . Anotherlade, FL . These slade, FL . Recently aphids .Hordeum vulgare) in Tunisia [Sorghum almum and S. bicolor in Florida [Zea mays) which is not commercially grown in Florida. On the other hand, S. almum, also known as Columbus grass, is a robust, short-lived perennial grass that can be found worldwide between 25°N and 30°S latitudes and starting at sea level up to a 700 m altitude [M. sacchari and SCYLV, it is a potential reservoir for virus transmission to sugarcane.Although SCYLV was successfully transmitted to several grass hosts under controlled conditions, sugarcane had been considered the only natural host of this virus for more than two decades , 31, 32. Tunisia . More re Florida , 35. Thealtitude . Columbualtitude , 38. In M. sacchari) and a potential vector (O. grypus), 2) the efficacy of virus transmission from a “reservoir” plant (Columbus grass) to sugarcane using sugarcane aphids, 3) the SCYLV transmission capacity of field collected versus greenhouse raised sugarcane aphids, 4) the efficacy of an aphicide to limit the spread of SCYLV to healthy sugarcane under field conditions, and 5) the haplotypes of M. sacchari occurring on sugarcane and sorghum in Florida.The objectives of this study were to determine: 1) the efficacy of transmission of SCYLV from sugarcane to sugarcane using a known vector  or directly from tissue culture . These healthy plants were used for either rearing aphids or inoculation experiments. SCYLV-infected plants of Columbus grass were collected in the field at Belle Glade and transferred to pots in a greenhouse. Single-bud cuttings from SCYLV-infected stalks of sugarcane cultivars CL88-4730 (infected with genotype BRA) and CP88-1762 (infected with genotype CUB) were planted separately in pots in a greenhouse. Plants obtained from these cuttings were used for rearing viruliferous aphids. None of the plants infected by SCYLV showed symptoms of yellow leaf.Virus-free plants of Columbus grass were grown in pots from seeds collected from field plants at Belle Glade, FL, and kept in a screened enclosure until aphid transfer. Virus-free sugarcane plants were obtained from disease-free single-bud cuttings from KleentekM. sacchari was collected in summer 2016 from a single sugarcane plant of cultivar CP96-1252 in the field at Belle Glade. Aphids were transferred on healthy leaf pieces (approx. 14 cm long) of the same cultivar in moist Ziploc bags at room temperature (~25°C). Aphids were moved in the laboratory to fresh virus-free sugarcane leaves every 3–4 days for 6 months to obtain a SCYLV-free colony of M. sacchari. Aphids were then moved onto healthy plants of Columbus grass in a growth chamber at 23°C and healthy plants of sugarcane cultivar CP96-1252 growing in an insect proof cage  in the greenhouse. Aphid colonies were kept on healthy Columbus grass and sugarcane plants until the beginning of the spring 2017. Aphids reared on Columbus grass were then moved onto caged SCYLV-infected plants of Columbus grass and aphids reared on sugarcane were moved onto caged SCYLV-infected sugarcane plants in the greenhouse to build up colonies of viruliferous M. sacchari.A colony of M. sacchari, leaves carrying aphids were regularly collected from April 14 to June 22, 2018, cut in 3–5 cm sections, and immediately used in transmission assays as described below.A field of sugarcane cultivar CP96-1252 with an incidence of SCYLV >90% was identified at Belle Glade in 2018. Plants of this field did not show symptoms of yellow leaf nor of any other known viral disease occurring in Florida such as mosaic, mild mosaic or leaf fleck. When the sugarcane field was colonized with SCYLV-free plants of sugarcane cultivar CP04-1935 and both SCYLV-infected plants of sugarcane cultivars CL88-4730 and CP88-1762 in the greenhouse became infested with spider mites in early spring 2018. Leaves carrying mites were collected from these plants, cut in 12- to 15-cm long sections, and immediately used for transmission assays as described below.Stalks of Columbus grass were cut transversally, and sections of the upper, middle, and lower part of each stalk were used to make imprints on nitrocellulose membranes. Similarly, duplicated imprints of collected sugarcane midrib cross-sections of leaves were made onto a nitrocellulose membrane. The membranes were then processed using SCYLV antibodies as described by Schenck et al.  and modiTotal RNA was extracted from sugarcane and Columbus grass leaves with the Qiagen RNeasy Plant mini kit , and from mites (~100 pooled-mite samples) and aphids (10 pooled-aphid samples) with the Qiagen RNeasy mini kit , following the manufacturer’s protocol. Arthropods were pooled before RNA extraction and molecular diagnosis in order to test a large number of individuals for presence or absence of the virus. Two experimental replicates (pooled samples) were used for aphids reared on virus-free Columbus grass, for aphids reared on virus-free sugarcane, and for spider mites collected from virus-free sugarcane. When arthropods were sampled from virus-infected materials, the number of experimental replicates was as follows: two for aphids reared on Columbus grass, four for spider mites collected from sugarcane, five for field-collected aphids from sugarcane, and 10 for aphids reared on sugarcane. Sample quality and presence or absence of SCYLV were determined by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) using Qiagen’s OneStep RT-PCR kit and ScYLVf1/ScYLVr1 primers as described by Girard et al. . SCYLV aTo determine the genotype of SCYLV in infected plants, RNA samples that tested positive with diagnostic primers ScYLVf1/ScYLVr1 were then subjected to OneStep RT-PCR using genotype specific primers sets BRA-PER-F/BRA-PER-R and CUB-F/CUB-R as described by Abu Ahmad et al. . The expFor all assays, 1- to 2-month-old tissue culture (virus-free) plantlets of cultivar CP96-1252 were transplanted into trays and moved into insect-proof cages in the greenhouse for inoculation with viruliferous aphids or mites . InoculaThree plants of Columbus grass that tested positive for SCYLV by RT-PCR were transplanted into separate pots and moved to insect-proof cages. Colonies of aphids raised on healthy Columbus grass were then transferred to each infected Columbus grass plant for the aphids to acquire the virus on the SCYLV inoculum source. Aphid transfer on a single plant occurred on 24 April 2017, 15 May 2017, and 5 June 2017 for transmission experiments 1, 2, and 3, respectively, as described below. Reared aphids were allowed to probe and propagate on source plants for at least three weeks before transmission experiments were carried out. Twenty to 30 cm long pieces of SCYLV-free or infected leaves or stalks of Columbus grass were transferred onto virus-free 2-month-old plantlets of sugarcane cultivar CP96-1252 . Each leM. sacchari raised on healthy CP96-1252 were transferred to genotype specific infected sugarcane plants (CL88-4730 for genotype BRA and CP88-1762 for genotype CUB) to build populations of viruliferous aphids. Sugarcane leaf fragments carrying 3–50 aphids reared on SCYLV free or infected plants were deposited on 2-month-old virus-free plantlets of sugarcane cultivar CP96-1252  inoculated with aphids collected from SCYLV-infected sugarcane in the field on 14 April 2018. This experiment was repeated on 16 April, 21 April, 27 April, 30 April, 15 May, 29 May, and 22 June 2018 using 24, 19, 17, 10, 8, 14, and 7 plantlets, respectively. The transmission experiment performed on 29 May 2018 also included 21 control plantlets that were not aphid-inoculated. The experiments were carried out and SCYLV incidence was determined by TBIA as described above (Transmission assays). SCYLV incidence was expressed as the ratio of TBIA positive plants over the total number of inoculated plants. Total RNA was extracted from either TBIA positive samples or randomly selected TVD leaves and processed by RT-PCR. The results were expressed as the ratio of positive samples over the total number of samples tested.Twelve to 15 cm leaf pieces from SCYLV-free (CP04-1935) or infected (CL88-4730 and CP88-1762) sugarcane plants harboring 100–1000 spider mites were transferred onto healthy trays of CP96-1252 plantlets maintained in insect-proof cages . Caged pM. sacchari in GenBank using blast search.Aphids were collected from locations that were either public areas (no permit needed) or private sugarcane farms collaborating with the authors on numerous research and extension projects. Farm managers had given permission to collect aphids on their properties. A total of 140 aphids were collected in 2018–2019 from sugarcane  in Belle Glade, Okeelanta and Clewiston, FL; from sorghum  in Belle Glade; and from Columbus grass  at 10 locations in the EAA . Not morSCYLV-free  and SCYLV-infected stalk cuttings of cultivar CP96-1252 were used to set up two trials on commercial sugarcane farms . One trial was established at Okeelanta on organic (muck) soil on 8 December 2016 and the other one was planted at 20 Mile Bend on mineral (sand) soil on 18 December 2016. At both experimental sites, a randomized block design with six blocks (replications) was used to compare plots planted with healthy (with or without insecticide applications) or virus-infected seed cane. The three treatments were randomized to plots that were 13.1 m long and six rows wide (1.5-m row spacing). All plots were surrounded on each side by a 4.6-m alley. Consecutive blocks were separated by same size blocks made of plots of cultivar CP01-1372 at Okeelanta and cultivar CPCL97-2730 at 20 Mile Bend. Each experimental field was 217 m long and 44.2 m wide.2 tanks. The insecticide was applied bi-weekly starting two to three months after planting or harvest. At Okeelanta, 12 applications were done in the plant cane crop and eight in the first ratoon crop. At 20 Mile Bend, nine applications were performed in the plant cane crop and seven in the first ratoon crop.Flupyradifurone  was applied at 87.7 g of active ingredient per hectare to prevent aphid colonization in plots planted with healthy seed cane and treated with insecticide. A two-row boom backpack sprayer with 6 nozzles spaced 50.8 cm apart was used and calibrated to deliver 93.5 l/ha at 241.3 kPa of spray solution using a pressurized system with portable COThe top visible dewlap leaf of 10 randomly selected sugarcane stalks per plot was collected at each of six different sampling dates after planting and over a 24- (20 Mile Bend) to 28-month (Okeelanta) period. Presence of SCYLV was tested by tissue blot immunoassay (TBIA) as described by Schenck et al.  and modiData from the Okeelanta and 20 Mile Bend experiments were analyzed separately. For each experiment, SCYLV prevalence as affected by treatment and sampling date was analyzed using a linear mixed model . TreatmSCYLV was not detected by TBIA or RT-PCR in any of the source plants used to rear healthy aphids or in healthy plants harboring mites. These plants included three plants of Columbus grass and two plants of sugarcane (cultivar CP96-1252) that were used for transmission assays with virus-free aphids and one sugarcane plant (cultivar CP04-1935) used for transmission assays with virus-free mites. SCYLV was detected by TBIA or RT-PCR in all the source plants used to rear viruliferous aphids for transmission assays: 3 plants of Columbus grass and 25 plants of sugarcane . The virus was also found in two sugarcane infected plants (cultivar CL88-4730) harboring mites.Pooled aphid and mite samples collected from virus-free source plants all tested negative by RT-PCR: two aphid samples from Columbus grass, two aphid samples from sugarcane, and two mite samples from sugarcane. Aphids reared in the greenhouse tested positive for SCYLV when collected from virus-infected sugarcane (10 samples) and negative when collected from virus-infected Columbus grass (2 samples). Five aphid samples collected from virus-infected sugarcane plants in the field all tested positive for SCYLV by RT-PCR. Four mite samples collected from virus-infected plants in the greenhouse also tested positive for SCYLV by RT-PCR.Three separate SCYLV transmission experiments from Columbus grass to sugarcane were performed using aphids reared on healthy and infected plants. Three and six months after each inoculation, all plants (three leaves per plant) were tested for infection by SCYLV using TBIA. At both time points, the virus was not found in any of the 66 plants inoculated with aphids from virus-free Columbus grass plants or in the 18 non-inoculated plants (negative control plants). SCYLV was not detected either in a total of 108 plants inoculated with aphids reared on virus-infected plants. Fourteen leaf samples randomly collected across the three experiments also tested negative for the virus by RT-PCR: 8 plants inoculated with aphids raised on SCYLV-infected Columbus grass, 4 plants inoculated with aphids raised on SCYLV-free Columbus grass, and 2 plants not inoculated with aphids.Two separate SCYLV transmission experiments from sugarcane to sugarcane were performed using aphids reared on plants infected by two different genotypes of the virus. Inoculated plants were tested for virus infection by TBIA three and six months after each inoculation, using three leaves per plant. Three months after inoculation, SCYLV was not found in the 23 plants  inoculated with aphids from sugarcane plants infected by SCYLV genotype CUB. Six months after inoculation, two of these 23 plants tested positive for SCYLV by TBIA and RT-PCR. These two plants were both from the set of 12 plants inoculated on 2 August 2017. The 26 plants inoculated with aphids collected from sugarcane infected by genotype BRA tested negative by TBIA at both time points. Eight leaf samples collected across the two experiments and that tested negative by TBIA also tested negative for the virus by RT-PCR.Eight separate transmission experiments from sugarcane to sugarcane were performed with viruliferous aphids collected in the field and transferred immediately on healthy sugarcane plants in the greenhouse. Three and six months after inoculation, the 113 plants inoculated with viruliferous aphids and 21 negative control plants all tested negative for SCYLV by TBIA. Sixteen randomly selected samples from inoculated plants and two from control plants also tested negative for the virus by RT-PCR.Two separate SCYLV transmission experiments from sugarcane to sugarcane were performed with viruliferous mites. Inoculated plants (three leaves per plant) were tested by TBIA three and six months after inoculation, and results were identical at both time points: SCYLV was not detected in any of the 90 plants inoculated with mites collected from plants infected with either genotype BRA (45 plants) or CUB (45 plants). The 20 plants inoculated with mites from virus-free sugarcane leaves and the eight non-inoculated control plants also tested negative for SCYLV by TBIA. The virus was not detected either by RT-PCR in 12 leaf samples collected randomly across the two experiments.M. sacchari (All 61 aphids collected from Columbus grass and sorghum belonged to haplotype one (H1) of sacchari . Haplotysacchari . Four apFrom December 2016 to April 2019, SCYLV prevalence in infected plots of cultivar CP92-1252 planted on organic soil at Okeelanta varied from 55% to 77% . PrevaleM. sacchari while ensuring the presence of SCYLV in source plants (Columbus grass and sugarcane) and aphids (from sugarcane). Several studies are available in the literature reporting from 29–100% transmission efficiency of SCYLV using this aphid species . This is the first report of failure of transmission of SCYLV using  species . In succVirus transmission can be affected by several factors such as plant age, the number of aphids used for transmission, the feeding time and the inoculation access period. In our study, we used young tissue cultured plantlets similar to those reported for successful transmission of SCYLV by Chinnaraja and Viswanathan . In prelM. sacchari was an efficient insect vector of SCYLV [M. sacchari occurred on sorghum in the USA [M. sacchari [M. sacchari in the USA. In Louisiana, aphids collected on sugarcane and Johnsongrass in 2007 all belonged to lineage MLL-D or haplotype H3 whereas haplotypes H1 and H3 were found on sugarcane in 2013–2017 [Although this is the first SCYLV transmission study done in Florida, these findings were unexpected because aphids originating from Florida  were used to demonstrate that of SCYLV . On the of SCYLV , 35. Fur the USA . These o the USA . This dosacchari . Invasio013–2017 . In a su013–2017 .M. sacchari present in Florida before the 2013 aphid outbreaks on sorghum are unknown. Aphids collected in this study in 2018 and 2019 belonged to three haplotypes . Only haplotype H1 aphids were found on sorghum species  whereas aphids of haplotype H1, H2, and H3 were all three collected from sugarcane. Nevertheless, H1 aphids were rarely found on sugarcane (only one of 79 samples) and most aphids (>88%) belonged to haplotype H3 which was used in our transmission assays and failed to spread SCYLV from infected to healthy plants. The third haplotype (H2) was found at low frequency (10%) and its presence in Florida is reported here for the first time. Interestingly, haplotype H2 is the only one so far reported in Guadeloupe (French West Indies) where SCYLV has been efficiently transmitted from infected to healthy sugarcane plants under controlled conditions [M. sacchari are able to transmit SCYLV and the capacity of H1 and H2 aphids to transmit SCYLV to sugarcane in Florida has yet to be explored.The haplotypes of nditions , 25. TheM. sacchari only recently, and this is the first reported attempt to use Columbus grass in virus transmission studies. In contrast to aphids collected from infected sugarcane, SCYLV was not found in aphids reared on infected Columbus grass plants. SCYLV may have not been found in aphids raised on Columbus grass because the virus titer was low in this plant species or because the aphid haplotype that was used for these transmission experiments originated from sugarcane and was not adapted for transmission to sorghum species. Previous studies showed that the virus can be detected in M. sacchari (most likely haplotype H1) collected from SCYLV-infected plants of S. bicolor in Florida [Columbus grass has been identified as a secondary host of SCYLV and  Florida .Buchnera aphidicola, a symbiotic bacterium of aphid vectors, is involved in Luteoviridae virion transmission [Buchnera-regulated plant defense against aphids or the synthesis by Buchnera of a chaperonin that facilitates the virus movement in the aphid hemolymph [M. sacchari may occur in Florida and the lack of Buchnera symbiont may result in incapacity to acquire/transmit SCYLV. Furthermore, insects (and aphids in particular), can also induce a range of plant responses associated with pathogen resistance instead of insect resistance [Cucumis melo), the Vat gene, reduces both aphid feeding and virus transmission [Inefficient spread of SCYLV by haplotype H3 from sugarcane to sugarcane could be caused by several factors such as mutations in the virus affecting its transmission efficiency or by a modification of aphid transmission capacity. smission . Luteoviemolymph , 47, 48.sistance . A locussmission , 51. On smission , 53, 54.smission .Although source plants used in our study for virus transmission did not show any virus disease symptoms, it cannot be excluded that these plants were infected by a cryptic virus or another microorganism that interacted with SCYLV, thus preventing virus transmission. Further investigations need to be undertaken to test this hypothesis.M. sacchari was currently not, if at all, efficiently spreading SCYLV in Florida sugarcane fields. Consequently, other vectors might be involved in infection of sugarcane by this virus.In two separate field experiments conducted in this study, sugarcane that was healthy at planting became infected by SCYLV. However, prevalence of the virus was low (maximum 28%) during two crop seasons (plant cane and first ratoon crop) although healthy plots of cultivar CP96-1252 were planted in proximity to infected plots. Fluctuations of SCYLV prevalence during these two crop seasons, especially the apparent disappearance of the virus at some time points could be ascribed to sample size  or to titer variations at different growth stage of the sugarcane plant (low and undetectable virus concentrations). The latter is often associated with luteovirids and virus detection that was impeded using serological methods such as TBIA , 56, 57.M. sacchari.Spider mites have been occasional pests of sugarcane in Florida. Unlike luteoviral aphid vectors that use their long stylets to probe and directly feed from the phloem, these mites use their sharp stylets to penetrate and remove the cell contents from plants. Although SCYLV was systematically detected in spider mite samples collected from virus-infected sugarcane, it was never found in inoculated plants, thus suggesting that this arthropod only acquired the virus while feeding on infected plants but was unable to transmit SCYLV. Future research needs to focus on other possible arthropod vectors of the virus, as well as transmission efficiency of all known haplotypes of S1 Table(XLSX)Click here for additional data file.