Each year 700,000 infants die due to intrapartum-related complications. Implementation of Helping Babies Breathe (HBB)-a simplified neonatal resuscitation protocol in low-resource clinical settings has shown to reduce intrapartum stillbirths and first-day neonatal mortality. However, there is a lack of evidence on the effect of different HBB implementation strategies to improve and sustain the clinical competency of health workers on bag-and-mask ventilation. This study was conducted to evaluate the impact of multi-faceted implementation strategy for HBB, as a quality improvement cycle (HBB-QIC), on the retention of neonatal resuscitation skills in a tertiary hospital of Nepal.A time-series design was applied. The multi-faceted intervention for HBB-QIC included training, daily bag-and-mask skill checks, preparation for resuscitation before every birth, self-evaluation and peer review on neonatal resuscitation skills, and weekly review meetings. Knowledge and skills were assessed through questionnaires, skill checklists, and Objective Structured Clinical Examinations (OSCE) before implementation of the HBB-QIC, immediately after HBB training, and again at 6 months. Means were compared using paired t-tests, and associations between skill retention and HBB-QIC components were analyzed using logistic regression analysis.One hundred thirty seven health workers were enrolled in the study. Knowledge scores were higher immediately following the HBB training, 16.4 ± 1.4 compared to 12.8 ± 1.6 before (out of 17), and the knowledge was retained 6 months after the training (16.5 ± 1.1). Bag-and-mask skills improved immediately after the training and were retained 6 months after the training. The retention of bag-and-mask skills was associated with daily bag-and-mask skill checks, preparation for resuscitation before every birth, use of a self-evaluation checklist, and attendance at weekly review meetings. The implementation strategies with the highest association to skill retention were daily bag-and-mask skill checks  and use of self-evaluation checklists after every delivery .Health workers who practiced bag-and-mask skills, prepared for resuscitation before every birth, used self-evaluation checklists, and attended weekly review meetings were more likely to retain their neonatal resuscitation skills. Further studies are required to evaluate HBB-QIC in primary care settings, where the number of deliveries is gradually increasing.ISRCTN97846009. Date of Registration- 15 August 2012. Globally, 2.9 million neonates die every year, of which 700,000 die due to intrapartum-related complications . The firNepal has a neonatal mortality rate of 23 per thousand live births and intrapartum-related complications account for 23% of neonatal deaths , 6. In NThere are a number of intervention strategies that have been identified to improve the performance and competencies of health workers for a number of other clinical guidelines . HoweverHelping Babies Breathe® (HBB), developed by the American Academy of Pediatrics, is designed to train birth attendants in developing countries on the essential skills of neonatal resuscitation . It is bThe educational material of HBB consists of a well-tested pictorial representation of the resuscitation protocol, learner workbooks, facilitator flip charts, neonatal simulators and the required equipment – i.e., reusable ventilation bag-and-mask devices and bulb suction devices . An evalAn evaluation study conducted in Ethiopia found that HBB training improved the neonatal resuscitation knowledge of health workers immediately after training and also eliminated the knowledge difference that was present prior to training among health workers . A studyWe therefore conducted this study to evaluate the impact of a multi-faceted implementation strategy for HBB as a Quality Improvement Cycle (HBB-QIC), on change and retention of health workers’ knowledge and skills of neonatal resuscitation.We conducted this study in a tertiary hospital in Kathmandu, Nepal. In 2011, the hospital had around 22,000 deliveries, and an early neonatal mortality of nine per thousand live births . The hosLabor and delivery services are provided in the admission unit, antenatal care unit, labor unit, maternal and newborn service center (MNSC), and the operation theatre. The hospital also has a system that rotates the staff of these units on a periodic basis.This was a sub-set of a larger study conducted to evaluate the change in perinatal mortality before and after the implementation of a simplified neonatal resuscitation protocol, the HBB-QIC, in the hospital . The durAll 137 nurses working in the admission unit, antenatal care unit, labor unit, MNSC, operation theatre and postnatal ward were included in the study. Informed written consent was obtained from each health worker participating in the study.This was a time-series design where a series of periodic measurements were completed to evaluate our multi-faceted implementation strategy for the HBB-QIC, at three different time points Table .Table 1EThe knowledge and skills of health workers were assessed using the standard tools included in the HBB package, which were validated and used in other settings where HBB knowledge and skill evaluations have been conducted . A questInformation on each health worker’s daily routines, including daily bag-and-mask skill checks, preparation for resuscitation before every birth, self-evaluation checklist usage, completion of the peer evaluation process, and attendance at the weekly review meetings was assessed using a direct observation checklist Table .The HBB-QIC was implemented from January until September 2013. The HBB-QIC included multi-faceted implementation strategy including: HBB training, daily bag-and-mask skill checks, preparation for resuscitation before every birth, self-evaluation and peer review on neonatal resuscitation skills and weekly review meetings  suctions™ were provided to each delivery unit. Additionally, HBB mannequins were placed at the entry of each delivery unit for daily skill checks; self-evaluation checklists were also attached to each clinical record form; and HBB schematic posters were placed in front of each resuscitation table for peer review. Weekly review meetings were conducted by each unit in-charge to discuss progress on the implementation of HBB-QIC standards.In order to complete sampling and data collection, an independent surveillance team was formed. The team consisted of eight female surveillance officers with an academic background in nurse-midwifery and sufficient experience in clinical research. A research manager supervised the activities of this surveillance team. The surveillance team was trained to collect information using an observation checklist. Surveillance officers who conducted the HBB trainings collected information from the study participants, including demographic characteristics, academic qualifications and clinical experience.After the surveillance officers collected this information, it was given to the data entry officer for dataset creation and management. The information was reviewed for completeness and then entered into an electronic database that was developed in the Census and Survey Processing System (CS Pro) software . The data was then cleaned and transferred into the Statistical Package for the Social Sciences (SPSS) software (IBM Corporation) for data analysis.The background characteristics of the health workers that were analyzed included age, professional experience, number of deliveries attended per month, number of neonatal resuscitations conducted per month and academic qualifications. The mean ± SD and median (IQR) for age of the health worker were calculated; mean and median years of professional experience in midwifery were calculated; the mean and median number of deliveries and resuscitations attended per month were also calculated. Academic qualifications were categorized into two groups, those who had completed an intermediate level of education in nursing or who were axillary nurse midwives compared to those who had completed a bachelor in nursing or higher.The mean numeric scores obtained on the knowledge questionnaire, preparation for resuscitation before every birth checklist, bag-and-mask skill check, and OSCEs A and B at baseline, immediately after the training, and at 6 months after the training were compared using paired t-tests. The proportion of health workers who scored ≥80% on the bag-and-mask skill checks before the training, immediately after the training, and at 6 months after the training was compared using paired t-tests.The retention of bag-and-mask skills was calculated based on the change in bag-and-mask skill scores immediately after the training compared to 6 months after the training. The health workers who scored the same or better at 6 months after the training, as compared to immediately after training, were categorized as having retained their skills.p < 0.01) between retention of skills and health worker’s completion of HBB-QIC components – i.e., daily bag-and-mask skill checks, preparation for resuscitation before every birth, use of self-evaluation checklists, peer evaluation following each resuscitation occurrence, attendance at weekly meetings – as well as with the completion of at least one neonatal resuscitation per month using the Fischer’s exact test.We then analyzed the association  years. Among the health workers, the mean number of professional years of experience was 10.5 ± 3.3 years and the median was 9 (IQR 6.0–15.0) years. The mean number of deliveries attended by each health worker was 8.0 ± 5.5 and the median was 7.0 (5.0–9.0). The mean number of resuscitations conducted by each health worker per month was 3.0 ± 1.5 and median was 3.0 (2.0–4.0). More than half, 65%, of health workers had intermediate level nursing qualifications or were axillary nurse-midwives, and the remaining health workers had bachelor degrees in nursing or higher (Table p = 0.6). There was a significant change (<0.001) from baseline in the mean skills scores for preparation for resuscitation before every birth, bag-and-mask skills, OSCE A, and OSCE B after the training, which was retained 6 months after the training (Table There was a significant change (<0.001) in the mean knowledge score after the HBB training, 16.4 ± 1.4 compared to 12.8 ± 1.6, and the knowledge was retained 6 months after the training (16.5 ± 1.1) (p < 0.001); and 99% of the health workers were competent 6 months after the training (p = 0.8)  between retention of bag-and-mask skills and health worker’s completion of daily bag-and-mask skill checks, preparation for resuscitation before every birth, use of self-evaluation checklists and attendance at weekly review meetings . Health workers who prepared for resuscitation before every birth had a two-fold increased likelihood of retaining the skills than those who did not . Health workers who used self-evaluation checklists after each delivery were four times more likely to retain their skills than those who did not . Health workers that attended weekly meetings had a 2.6 times increased likelihood of retaining their skills than those who did not attend   in changing health worker’s behaviors. The strength of our study was that we used a tested tool to evaluate the neonatal resuscitation knowledge and skills of the health worker .Several studies in high-income and low-income countries have shown that resuscitation knowledge and skills improve immediately following training, however, these resuscitation skills tend to deteriorate over a period of time , 20–25. Several systematic reviews have shown that the review of clinical performance among health workers on a periodic basis provides an opportunity to share the challenges faced in the clinical setting and to improve the clinical skills and performance as a team, especially if the baseline performance on the clinical protocol is poor –30. We sIn the context of Nepal, the current number of neonatal deaths due to intrapartum-related complications needs to be reduced by at least three quarters in order to reach the pledged Every Newborn Action Plan target of a neonatal mortality of 10 or less per 1000 live birth by 2035 . FurtherTo our knowledge this is the first study done to evaluate the impact of a multi-faceted implementation strategy of HBB on the retention of neonatal resuscitation skills in low-income settings. We demonstrate that health workers, who conduct bag-and-mask skill checks daily, make preparation for resuscitation at every birth, use self-evaluation checklists after each delivery, and attend weekly review meetings have a higher likelihood for retention of neonatal resuscitation skills, even at 6 months post-training. Since, the HBB-QIC was implemented in a setting with a high number of deliveries, with a large team of nursing staff in each delivery unit, the implementation strategy such as weekly review meetings can be suitable in similar settings, but may not be suitable in primary care settings where there are fewer nursing staff. The other effective implementation strategies of daily bag-and-mask skill checks, preparation for resuscitation at every birth, and use of self-evaluation checklists can be applied in primary care settings to improve retention of resuscitation skills. However, the combination of different strategies needs to be evaluated in primary care settings, the point of access for delivery services among most women in rural areas. The innovative aspect of HBB-QIC is the multi-faceted implementation strategy approach. Future research should focus on further determining and refining those components that improve retention of neonatal resuscitation skills and clinical performance.


The modifying effects of long noncoding RNAs (lncRNAs) in rheumatoid arthritis (RA) recently have drawn much attention; however, the underlying mechanisms remain largely unknown. Herein, we aim to investigate the expression profile of lncRNAs in RA and identify promising targets for RA diagnosis and treatment. Microarray screening and real-time PCR of lncRNAs were performed by use of serum samples from 3 RA patients and 3 healthy controls. Significantly differentially expressed lncRNAs were verified in serum samples from 43 RA patients and 40 healthy controls by real-time PCR. We found that there were 73 up-regulated and 61 down-regulated lncRNAs as well as 128 up-regulated and 37 down-regulated mRNAs in serum samples of RA patients. Validation in RA clinical samples indicated 5 of these lncRNAs were significantly up-regulated including RNA143598, RNA143596, HIX0032090, IGHCgamma1, and XLOC_002730. Significant association was observed between these lncRNAs and the disease course, erythrocyte sedimentation rate (ESR), rheumatoid factor (RF) as well as anti-cyclic citrullinated peptide (anti-CCP) antibody. Additionally, 55 of the differentially expressed mRNAs were associated with 41 lncRNAs and were involved in signaling pathways of toll like receptors (TLRs), nuclear factor-kappa B (NF-κB), and cytokine, especially the IRF3/IRF7 mediated signaling transduction. Our study firstly shows the specific profile of lncRNAs in the serum of RA patients and potential signaling pathways involved in RA pathogenesis, which may provide novel targets for the diagnosis and treatment of patients with RA. Rheumatoid arthritis (RA) is a common autoimmune inflammatory disease, whereas the pathogenesis of it is not fully elucidated. RA is characteristic of systemic and chronic inflammation, abnormal immune response, and irreversible joint destruction , 2. SomeLncRNAs are RNA transcripts with more than 200 nucleotides in length. They are a new class of regulatory RNAs that are not translated into proteins. LncRNAs are transcription products of RNA polymerase II, which are widely expressed in normal tissues of human body and may be aberrantly and specifically expressed in certain pathological tissues. A number of studies have implicated that lncRNAs can be transported and released into the periphery by circulating exosomes, mediating interacts between cells and their microenvironment , 11. DurThe aim of this study is to investigate the underlying effects of lncRNAs in RA by microarray screening and bioinformatics analysis. The findings will provide new insights into the pathogenesis of RA and help to identify prospective targets for RA.The hierarchical clustering analysis, scatter plot and volcano plot all showed that some lncRNAs and mRNAs were differentially expressed in the serum of RA patients compared with healthy controls Figure . A totalMost of the aberrantly expressed lncRNAs were validated in the serum samples from 43 RA patients and 40 healthy controls by real-time PCR. 5 of these lncRNAs were found to be significantly up-regulated in the serum of RA patients when validating by real-time PCR, including RNA143598, RNA143596, HIX0032090, IGHCgamma1, and XLOC_002730 , rheumatoid factor (RF), anti-cyclic citrullinated peptide antibody (anti-CCP Ab) of RA Figure .Figure Computational analysis revealed that 55 mRNAs were associated with 41 differentially expressed lncRNAs Figure . These sLncRNAs, as a class of noncoding RNAs, play critical roles in the regulation of autoimmunity and maintenance of homeostasis , 19. MulRA is one of the most frequent rheumatoid diseases worldwide with kinds of strong autoimmune components, such as autoantigens of citrullinated proteins , 3. To tIn the present study, we tried to find the differentially expressed lncRNAs and mRNAs profiles in the serum of RA patients by microarray screening. 5 significantly differentially expressed lncRNAs were identified in serum samples from patients with RA, including RNA143598, RNA143596, HIX0032090, IGHCgamma1, and XLOC_002730. Besides, significantly positive association was observed between these lncRNAs and the disease course, ESR, RF and anti-CCP antibody of patients with RA. The correlation analysis showed that RNA143598 and HIX0032090 were significantly associated with the disease course and ESR level in patients with RA. In addition, increased levels of RF were observed in RA patients with XLOC_002730 and HIX0032090 up-regulated in the serum among RA patients. Moreover, the expression of RNA143596, RNA143598 and HIX0032090 was also positively related to the level of anti-CCP antibody in the serum. It has been well established that elevated level of RF is highly associated with synovitis, vasculitis and extra articular symptoms, while increased level of anti-CCP antibody is closely related to bone erosion and destruction . AccordiAs shown in the pathway enrichment analysis, TLRs/NF-κB signal pathways are highly enriched in RA associated with the aberrantly expressed mRNAs. Thus, TLRs/NF-κB mediated inflammation may contribute to the development of RA. Currently published studies have suggested a crucial role of TLRs/NF-κB signaling transduction in the development of some autoimmune diseases including RA, Sjogren's syndrome and systemic sclerosis –32, suppIn this study, the cytokine signaling pathway has been significantly enriched by the bioinformatics analysis in the present study, especially the IRF3/IRF7 mediated signaling transduction. IRF-3 and IRF-7 are the vital transcriptional factors for the generation of IL-28A and IL-28B, whereas IRF3 and NF-κB are critical transcript factors for the production of the IL-29 . IL-29, In summary, this study, for the first time, shows specific profiles of lncRNAs and mRNA in the serum of RA patients and potential lncRNA-mRNA networks involved in RA. There are a total of 73 up-regulated and 61 down-regulated lncRNAs as well as 128 up-regulated and 37 down-regulated mRNAs in RA. LncRNAs of RNA143598, RNA143596, HIX0032090, IGHCgamma1, and XLOC_002730 are significantly up-regulated in the serum of RA patients. The differentially expressed lncRNAs are closely associated with the inflammatory response and autoimmunity. Findings in this study will support novel promising targets for RA.43 RA patients and 40 healthy controls were recruited from the affiliated hospital of Weifang Medical University between September, 2015 and March, 2016. Controls were from the same hospital for health examination. All fresh blood samples were separately collected and sequentially centrifuged at 2000 rpm for 10 min. 1ml cell-free serum from the supernatant was sucked out and stored at -80°C for further detection. Characteristics of all patients and controls were summarized in Table -ΔΔCTmethod and normalized to housekeeping gene GAPDH. Primer sequences used in validation of lncRNAs in this paper were listed in Table Total RNAs were isolated from serum samples by use of Plasma/Serum RNA Purification Mini Kit  according to the manufacturers’ protocols. Purified total RNAs were quantified using a NanoDrop 1000 . Then, cDNAs were synthesized from 0.5 μg RNAs for further assay based on the instructions of PrimeScript™ RT reagent Kit . Real-time PCR was carried out in triplicate assay in accordance with the specifications of SYBR Green Mastermix kit . A total of 5 ng cDNA template was used for real-time PCR assay. Random primers were used in experiments. The expression of each lncRNA was represented as fold changes using the 2P value for t-test was less than 0.05. Most of the differentially expressed genes were validated by real-time PCR, especially those lncRNAs which co-expressed with mRNAs.Capitalbio Agilent LncRNA + mRNA Human Gene Expression Microarray V4.0 was used to screen differentially expressed lncRNAs and mRNAs in the serum of RA patients . In this study, serum samples from 3 patients and 3 controls were randomly selected for microarray analysis. It was considered to be statistically significant between the two groups when the fold changes for differentially expressed lncRNAs and mRNAs were larger than 2.0 and the After microarray screening for lncRNAs and mRNAs in the serum samples from RA patients and controls, the hierarchy clustering, gene ontology (GO) functional enrichment and pathway enrichment analyses were then performed for further estimation by use of KOBAS (KEGG Orthology Based Annotation System) software. In order to predict the potential regulatory effects of differentially expressed lncRNAs on mRNAs, an lncRNA-mRNA co-expression network was conducted by bioinformatics analysis.P < 0.05 was regarded to be statistically significant. Softwares of SPSS (version 16.0) and Graphpad (version 5.0) were applied for statistical analysis.Data were shown as mean ± SEM. We used independent-Samples T test or One-Way ANOVA for statistical analysis. A two-sided


Carbapenem-resistant Enterobacteriaceae (CRE) pose an urgent public health threat in the United States. An important step in planning and monitoring a national response to CRE is understanding its epidemiology and associated outcomes. We conducted a systematic literature review of studies that investigated incidence and outcomes of CRE infection in the US.We performed searches in MEDLINE via Ovid, CDSR, DARE, CENTRAL, NHS EED, Scopus, and Web of Science for articles published from 1/1/2000 to 2/1/2016 about the incidence and outcomes of CRE at US sites.Five studies evaluated incidence, but many used differing definitions for cases. Across the entire US population, the reported incidence of CRE was 0.3–2.93 infections per 100,000 person-years. Infection rates were highest in long-term acute-care (LTAC) hospitals. There was insufficient data to assess trends in infection rates over time. Four studies evaluated outcomes. Mortality was higher in CRE patients in some but not all studies.While the incidence of CRE infections in the United States remains low on a national level, the incidence is highest in LTACs. Studies assessing outcomes in CRE-infected patients are limited in number, small in size, and have reached conflicting results. Future research should measure a variety of clinical outcomes and adequately adjust for confounders to better assess the full burden of CRE.The online version of this article (10.1186/s13756-018-0346-9) contains supplementary material, which is available to authorized users. The Centers for Disease Control and Prevention (CDC) in the United States has deemed carbapenem-resistant Enterobacteriaceae (CRE) an urgent public health threat that requires immediate and aggressive action . The reaKlebsiella pneumoniae carbapenemase (KPC) was first recognized in North Carolina in 1996 and has since spread around the world [Carbapenem-resistance in Enterobacteriaceae can result from a variety of mechanisms . In the he world . Other eKlebsiella spp. reported at least 1 carbapenem-resistant strain [Escherichia coli was associated with duodenoscopes at an Illinois hospital [CRE infections are typically seen in patients with prior healthcare exposure, and medical devices are a common risk factor for CRE acquisition . Accordit strain . In 2013hospital .Despite ongoing control efforts, CRE has become prevalent in several US regions, including Orange County, California and the Chicago metropolitan area , 9. CRE The CDC has published toolkits on preventing the spread of CRE within and between healthcare facilities , 11. WhiThis systematic literature review was performed according to the MOOSE and PRISMA guidelines , 13. An Studies were included if they (1) were conducted in the United States; (2) evaluated incidence of CRE or outcomes attributed to CRE infections including mortality, length of stay (LOS), discharge to a long-term acute-care (LTAC) hospitals, readmission, recurrence, and costs; 3) included at least 7 study sites (incidence studies only); and (4) used either the CDC’s original or revised definition for CRE  included. In the We excluded studies that (1) were less than 6 months in duration; (2) used International Classification of Diseases Clinical Modification diagnosis codes to define CRE infections; (3) used surveillance cultures from non-sterile body sites to detect CRE; (4) did not have a denominator or control group; (5) took place during an outbreak; (6) were mathematical models; (7) did not contain original data; and (8) were published in a language other than English. We excluded studies evaluating LOS or costs if they did not measure post-infection LOS or costs, or if they did not match infected cases and uninfected controls on time at risk  or did not match on propensity scores.One investigator (MLC) reviewed the titles and abstracts of all articles for inclusion and exclusion criteria. Two of four reviewers  independently abstracted data for each included study. Disagreements between reviewers were resolved by consensus.The reviewers abstracted data on the following items: study design, population and setting, location, definition of CRE infection, incidence data, prevalence data, mortality, LOS, discharge to LTAC, readmission, recurrence, and cost. Additional data were collected regarding tests used to identify CRE infection, identification of different species that were identified as CRE, and definitions of resistance.Risk of bias was assessed using the Newcastle-Ottawa scale for included studies .We screened 18,178 unique studies for eligibility  died compared to 14% of the uninfected patients (log rank p < 0.001). Based on a multivariate Cox proportional hazards analysis, mortality at 1-year was significantly higher in patients who developed CRKP infections compared to uninfected patients . In the second study, patients with and without infections were matched by hospital or facility, unit or clinic, calendar year, and time at risk . This study found higher odds of dying among CRE patients compared with uninfected controls, but the difference was not statistically significant  [Each of the 5 outcomes studies included data from 2009 or earlier Table . Two stuK. pneumoniae while the third study included all types of CSE not producing extended-spectrum beta-lactamases (ESBLs). Two studies used multivariable analysis to adjust for important predictors of mortality; only one of these studies adjusted for severity of illness [Three studies evaluated outcomes in patients with CRE compared to carbapenem-susceptible Enterobacteriaceae (CSE) –22. Two  illness  while th illness . The thiK. pneumoniae, cases with carbapenem-resistance had significantly higher odds of all-cause in-hospital mortality  infections in 2011 across the US was 25 per 100,000 persons, or at least 8 times more common than CRE [C. difficile was approximately 200 infections per 100,000 patient-years, which was at least 65 times more common than CRE [Based on our findings, CRE is still relatively uncommon in the US compared to other antibiotic-resistant pathogens. For example, the estimated incidence of invasive methicillin-resistant than CRE . In the than CRE . It is ithan CRE .Despite its low incidence, CRE remains a public health threat due to its limited treatment options and worse clinical outcomes . Based oK. pneumoniae had 2–3 times the odds of in-hospital death as patients with carbapenem-susceptible K. pneumoniae, but a similar difference in mortality was not seen in a study that included all types of Enterobacteriaceae . In contrast, evidence from outside the United States shows increased mortality with CRE. A case-control study from Israel found that, after adjusting for severity of illness, patients with infections from carbapenem-resistant K. pneumoniae had 4 times the risk of death as patients infected with carbapenem-susceptible K. pneumoniae [Surprisingly, however, patients with CRE were not always found to have an increased risk for death compared with controls. Studies that compared CRE to uninfected patients reached different conclusions on mortality, albeit using different definitions of mortality , 23. Coneumoniae .K. pneumoniae bacteremia has an attributable mortality of 50%, or 3 times the risk of death compared with non-bacteremic controls [While it seems intuitive that CRE would be associated with worse outcomes, the lack of consistency across the literature raises several important questions about how CRE cases are defined and how CRE outcomes are measured. First, only 2 studies in this systematic literature review restricted cases to patients with CRE-positive cultures from normally sterile body sites. As a result, there is potential that patients colonized with CRE  were included as “infected” cases. Such a misclassification bias could obscure differences in outcomes. As with any bacteria, the body site of infection is a key determinant of outcomes. A CRE bacteremia, for example, would be expected to have a higher mortality rate than a CRE urinary tract infection. In fact, a case-control study from Israel found that carbapenem-resistant controls . Second,Another potential explanation for the conflicting mortality results is that the current studies were underpowered to find statistically significant results. Each of the 4 studies that evaluated mortality included fewer than 100 CRE-infected patients. In the future, larger well-designed studies should be performed to assess the association between CRE infection and mortality.All the studies included in this systematic literature review evaluated outcomes before the Food and Drug Administration’s (FDA) approval of ceftazidime-avibactam and meropenem-vaborbactam. Now that these new and potentially more efficacious agents are available, a re-evaluation of CRE-related outcomes is warranted.There are several limitations to our meta-analysis. First, definitions of CRE varied across studies. This discrepancy reflects differences in how each study chose to define a CRE case; it also reflects changes in the CDC’s definition for CRE and the CLSI’s breakpoints for carbapenem-resistance. The original CDC definition for CRE did not include ertapenem, which limited its sensitivity for detecting OXA-48-producing CRE. Furthermore, studies that used the pre-2010 CLSI guidelines to define carbapenem-resistance or evaluated just certain species of Enterobacteriaceae may have under-estimated the true incidence of CRE , 31. ThuIn conclusion, while the incidence of CRE infections in the United States remains low on a national level, the incidence is highest in LTACs. Several studies have measured the incidence of CRE, but the percentage of CRE that results from carbapenemase-production needs to be better defined. Studies assessing outcomes in CRE-infected patients have been small and have reached conflicting results. Future research should measure a variety of clinical outcomes, should be adequately powered and should adequately adjust for confounders to better assess the full burden of CRE. Specifically, outcomes of CRE infections treated with recently FDA-approved antibiotics warrant further evaluation.Without adequate studies measuring the burden of CRE infections, proper distribution of resources for research and prevention efforts will be impossible, thereby leaving patients vulnerable to this important emerging pathogen.Additional file 1:Search Methods. (DOCX 32 kb)


To evaluate the cost-effectiveness of human papillomavirus (HPV) DNA testing (versus Papanicolaou (Pap)-based screening) for cervical cancer screening in Nicaragua.A previously developed Monte Carlo simulation model of the natural history of HPV infection and cervical cancer was calibrated to epidemiological data from Nicaragua. Cost data inputs were derived using a micro-costing approach in Carazo, Chontales and Chinandega departments; test performance data were from a demonstration project in Masaya department.Nicaragua’s public health sector facilities.Women aged 30–59 years.Screening strategies included (1) Pap testing every 3 years, with referral to colposcopy for women with an atypical squamous cells of undetermined significance or worse result (‘Pap’); (2) HPV testing every 5 years, with referral to cryotherapy for HPV-positive eligible women (HPV cryotherapy or ‘HPV-Cryo’); (3) HPV testing every 5 years, with referral to triage with visual inspection with acetic acid (VIA) for HPV-positive women (‘HPV-VIA’); and (4) HPV testing every 5 years, with referral to Pap testing for HPV-positive women (‘HPV-Pap’).Reduction in lifetime risk of cancer and incremental cost-effectiveness ratios ).HPV-based screening strategies were more effective than Pap testing. HPV-Cryo was the least costly and most effective strategy, reducing lifetime cancer risk by 29.5% and outperforming HPV-VIA, HPV-Pap and Pap only, which reduced cancer risk by 19.4%, 12.2% and 10.8%, respectively. With an ICER of US$320/YLS, HPV-Cryo every 5 years would be very cost-effective using a threshold based on Nicaragua’s per capita gross domestic product of US$2090. Findings were robust across sensitivity analyses on test performance, coverage, compliance and cost parameters.HPV testing is very cost-effective compared with Pap testing in Nicaragua, due to higher test sensitivity and the relatively lower number of visits required. Increasing compliance with recommended follow-up will further improve the health benefits and value for public health dollars. Strengths of this study include the use of implementation data from the Scale-Up project to estimate the cost-effectiveness of human papillomavirus testing in Nicaragua’s public health system. Findings were robust across extensive sensitivity and scenario analyses.A limitation of this study is that modelled screening algorithms do not entirely reflect the complex downstream management described by in-country guidelines. While the modelled algorithms reflect the prototypical structure of a screening episode and the type of facility at which visits usually take place, these do not capture variation due to geography or health facility capacity.An additional limitation is that while we adopted a micro-costing approach to leverage data from implementation in Nicaragua, individual-level data for each woman were not available; furthermore, we did not have cost data associated with HPV self-collection in community settings, where most self-collection takes place.Cervical cancer is the leading cause of cancer among women in Nicaragua, with an estimated 934 cases and 424 deaths each year.In most high-income countries, routine screening with cervical cytology ) testing has substantially reduced the incidence of cervical cancer.HPV DNA tests are highly sensitive to detect potentially oncogenic HPV infections and present an alternative to Pap-based screening. Because HPV-negative women are at very low risk for developing cervical cancer within the next 10 years,In 2011, the Screening Technologies to Advance Rapid Testing for Cervical Cancer Prevention−Utility and Program Planning (START-UP) project in Nicaragua’s Masaya department demonstrated that screening with careHPV could be effectively implemented in public sector health facilities.To inform decision makers considering the national adoption and scale up of HPV testing within Nicaragua’s public health sector, this study aimed to (1) estimate the economic cost of cervical cancer screening with careHPV testing and (2) project the long-term health and economic impact and value  of careHPV testing in Nicaragua relative to existing Pap-based screening.We used a micro-costing approach to measure and aggregate the cost of all resources used to provide cervical cancer screening at the level of the individual patient within the public health sector in Nicaragua. We considered direct medical costs , direct non-medical costs  and patient time costs . These cost data were input into a previously developed Monte Carlo simulation model (programmed in C++) of the natural history of HPV infection and cervical cancer that was calibrated to epidemiological data from Nicaragua.Model outcomes included the lifetime risk of cervical cancer, total lifetime costs per woman (in 2015 US$) and life expectancy. Incremental cost-effectiveness ratios (ICER) were calculated by dividing the additional cost of a particular strategy by its additional health benefit, compared with the next most costly strategy. Dominated strategies  were eliminated. There is no universal criterion that defines a threshold cost-effectiveness ratio, below which an intervention is considered good value for money; we considered an intervention with an ICER less than Nicaragua’s 2015 per capita gross domestic product (GDP) of US$2090 to be ‘very cost-effective’, and an intervention with an ICER less than three times per capita GDP as ‘cost-effective’.Descriptions of the natural history model of HPV infection and cervical carcinogenesis and model parameterisation process have been previously published,The model was calibrated to epidemiological data on age-specific HPV prevalence and cervical cancer incidence from Nicaragua.10.1136/bmjopen-2016-015048.supp1We considered the following screening strategies for women aged 30 to 59 years : (1) PapScreening and treatment parameters are presented in For all HPV testing strategies, we assumed 20% of women received provider collection of cervical specimens and 80% of women self-collected vaginal specimens, consistent with the proportions in the Scale-Up project to date. We weighted cost and health outcomes for provider and self-collection accordingly when aggregating results for the HPV strategies.All costs were converted to 2015 US$ using GDP deflators and the official exchange rate.Data on programmatic costs are limited, but for HPV strategies, we included the cost of training sessions for health providers offering HPV-based screening, outreach workers, laboratory technicians and providers offering VIA and cryotherapy. While women who self-collected HPV specimens in the Scale-Up project primarily did so in a community setting, micro-costing data were not available for self-collection performed outside of the clinic, so we assumed clinic-based self-collection. However, we conservatively included the cost of training outreach workers to represent a known programmatic cost as self-collection efforts are shifting to community settings.Data on the costs of treating cervical cancer were unavailable for Nicaragua, so we estimated direct medical, direct non-medical and patient and support person time costs using data from El Salvador.Selected cost data are presented in We performed sensitivity analysis to examine the impact of independently varying uncertain parameters, including Pap test performance, triage test performance in HPV-positive women, colposcopy performance, screening coverage, visit compliance, eligibility for cryotherapy following a positive screening and triage test, treatment effectiveness, discount rate and cost data. Ranges selected for sensitivity analysis are displayed in The base-case and sensitivity analyses assumed the availability of all strategies . Additionally, we performed a scenario analysis in which we assumed HPV-Cryo was not available for logistical and programmatic reasons .HPV-based screening strategies were more effective than Pap testing. Among the HPV strategies, HPV-Cryo (every 5 years) was the most effective strategy; under base-case assumptions, it reduced the lifetime risk of cervical cancer by 29.5% on average . HPV-VIA (every 5 years) reduced cancer risk by 19.4% , while HPV-Pap reduced cancer risk by 12.2% . Pap (every 3 years) reduced cancer risk by 10.8% . Under base-case assumptions, HPV-Cryo was both less costly and more effective than all other strategies, thus dominating HPV-VIA, HPV-Pap and Pap alone. With an ICER of US$320 per year of life saved (YLS), HPV-Cryo every 5 years would be considered ‘very cost-effective’ given Nicaragua’s per capita GDP of US$2090. The total discounted lifetime cost per woman and life expectancy associated with each screening strategy is presented in While HPV-Cryo remained the most effective strategy across all sensitivity analyses, the magnitude of reduction in lifetime risk of cancer was dependent on screening coverage of the target population and compliance with recommended follow-up. When coverage was 50% and all other parameters were held constant at base-case values, HPV-Cryo reduced cancer risk by an average of 21.1%; HPV-VIA, HPV-Pap and Pap yielded average cancer risk reductions of 13.9%, 8.7% and 7.7%, respectively. As coverage increased to 80%, all else being equal, HPV-Cryo reduced cancer risk by an average of 33.5%, while HPV-VIA, HPV-Pap and Pap yielded average cancer risk reductions of 22.1%, 14.0% and 12.4%, respectively  Pap test performance (as a primary screening test) improved, (2) VIA and Pap triage test performance improved, (3) colposcopy was assumed to be perfect and (4) the direct medical cost of LEEP was varied from 75% to 125% of the base case. Despite slight fluctuation in the ICER, HPV-Cryo also remained the least costly and most effective strategy as (1) screening coverage varied from 50% to 80%; (2) visit compliance varied from 40% to 85% per visit; (3) the screen-and-treat cryotherapy cure rate was reduced to 75%; (4) eligibility for cryotherapy was reduced to 75% or less; (5) the treatment cure rate following colposcopy was reduced to 85%; (6) women receiving treatment following colposcopy in the Pap and HPV-Pap strategies were assumed to receive cryotherapy instead of the more costly LEEP; (7) the discount rate was varied from 0% to 5%; (8) only payer costs were considered, excluding women’s time and transportation costs; (9) the direct medical cost of HPV self-collection was varied from 75% to 125% of the base case; (10) the direct medical cost of cryotherapy was increased to 170% of the base case; (11) the direct medical cost of colposcopy was reduced to 35% of the base case; (12) programmatic costs associated with HPV-based screening were varied from 50% to 150% of the base case; (13) women’s time and transportation costs were reduced to 50% of the base case; and (14) the costs of cancer treatment ranged from including only direct medical costs to 150% of the base case . Among vWhen only payer costs were considered , the total lifetime cost per woman was lower for all strategies and Pap every 3 years was slightly less costly  than HPV-Pap every 5 years. HPV-Cryo remained the most effective and efficient strategy, with an ICER of US$270 per YLS , a value commonly cited for the cost of Pap in Nicaragua, though the source of this estimate is unknown. However, Pap remained the least effective strategy, and HPV-Cryo had a lower cost-effectiveness ratio, maintaining an ICER of US$320 per YLS  increased from 40% to 85%, the effectiveness or cancer benefit associated with HPV-Cryo rose from 16.2% to 47.9% as more women were linked to treatment; the ICER fell from US$580 per YLS to US$190 per YLS as more cancers were averted. Thus, improved efforts to successfully navigate women to recommended follow-up will enhance screening programme effectiveness and efficiency.While substantially reducing the cost of Pap testing to US$3  made Pap the strategy with the lowest per-woman lifetime costs, the confluence of low test sensitivity and the high number of health facility visits needed to complete screening, diagnostic follow-up and treatment made Pap the least effective strategy since women are lost to follow-up with each additional required visit. Even in this low-cost Pap scenario, HPV-Cryo remained the most- effective and cost-effective strategy.When we assumed a scenario in which HPV-Cryo was not available for logistic and programming reasons, we found that HPV-VIA was usually the least costly and most effective of the remaining strategies. Although the ICER was less attractive than the ICER associated with HPV-Cryo in the main analysis, it was below Nicaragua’s per capita GDP.There are several limitations to this analysis. We did not consider alternative screening intervals or ages for each strategy, but rather restricted the analysis to the ages and intervals currently under consideration by the Nicaraguan Ministry of Health. It is likely that an increased screening interval and fewer lifetime screens will also be cost-effective, although health benefits may be reduced; we demonstrated in a previous analysis that screening once or three times in a woman’s lifetime with careHPV would be very cost-effective in Nicaragua.While we adopted a micro-costing approach to leverage data from the START-UP and Scale-Up projects in Nicaragua, there remain limitations to our cost estimates. First, individual-level data for each woman were not available; thus, our estimates represent average costs in the project populations. Second, we did not have information on the costs associated with HPV self-collection in community settings, where most self-collection takes place. Instead, we assumed all self-collection took place at the clinic. Compared with clinic-based efforts, community-based self-collection may be associated with lower costs for women’s time and travel, and higher direct medical and programmatic costs due to outreach worker involvement in facilitating screening and delivering results. Third, our estimates of programmatic costs were restricted to training sessions, and we did not have information on the costs of social mobilisation and outreach, patient navigation and support or infrastructural improvements that would be required to successfully scale up a screening programme. Fourth, we valued women’s time based on the minimum wage in Nicaragua. This may be a conservative estimate if most women attending screening are formally employed; conversely, it may overestimate the societal value of women’s time spent working in the informal sector or at home. Finally, we extrapolated the cost of cancer treatment using data from El Salvador.As implementation of HPV testing continues, particularly without triage testing, the health system’s capacity to provide cryotherapy will likely need to increase. While the use of triage testing (either with Pap or HPV) reduces the number of cryotherapy procedures performed, we found that the lower sensitivity of triage testing  led to a decline in health benefits as fewer women with persistent HPV infection and precancer received treatment. The cost savings associated with fewer cryotherapy procedures were outweighed by increased costs of additional follow-up and cancer treatment in triage-negative women. A sensitivity analysis on the cost of cryotherapy revealed that HPV-Cryo remained the dominant strategy even when costs increased to 170% of the base case. However, we did not explicitly consider the costs of increasing access to cryotherapy machines or the implications of gas stock-outs, which have been identified as barriers in some low-income and middle-income countries.In summary, using data from the Scale-Up implementation project in Nicaragua, we found that HPV testing followed by cryotherapy for eligible HPV-positive women (a screen-and-treat approach) was a very cost-effective intervention in Nicaragua. As the HPV-Cryo algorithm was not implemented in phase 2 of the Scale-Up project, compliance and cost estimates may need to be further honed to reflect improvements in capacity for cryotherapy if HPV-Cryo is implemented going forward. While it is too early to assume that costs and health impact from phase 2 of implementation are generalisable to other departments in Nicaragua or other settings in Central America, extensive sensitivity analyses indicate the robustness of findings. An HPV-based screening algorithm involving a similar screen-and-treat approach was recently found to be a good value for public health dollars in El Salvador,10.1136/bmjopen-2016-015048.supp2


Women with signs and symptoms of ischemia, no obstructive coronary artery disease (CAD) and preserved left ventricular ejection fraction (EF) often have diastolic dysfunction and experience elevated rates of major adverse cardiac events (MACE), including heart failure (HF) hospitalization with preserved ejection fraction (HFpEF). We evaluated the predictive value of inflammatory biomarkers for long-term HF hospitalization and all-cause mortality in these women.We performed a cross-sectional analysis to investigate the relationships between inflammatory biomarkers  and median of 6 years follow-up for all-cause mortality and HF hospitalization among women with signs and symptoms of ischemia, non-obstructive CAD and preserved EF. Multivariable Cox regression analysis tested associations between biomarker levels and adverse outcomes.Among 390 women, mean age 56 ± 11 years, median follow up of 6 years, we observed that there is continuous association between IL-6 level and HF hospitalization . In addition, we found significant association between IL-6, SAA levels and all-cause mortality AHR  , respectively.In women with signs and symptoms of ischemia, non-obstructive CAD and preserved EF, elevated IL-6 predicted HF hospitalization and all-cause mortality, while SAA level was only associated with all-cause mortality. These results suggest that inflammation plays a role in the pathogenesis of development of HFpEF, as well all-cause mortality. Signs and symptoms of ischemia in the absence of obstructive coronary artery disease (CAD) is highly prevalent in women and frequently associated with recurrent symptoms, repeated testing and coronary microvascular dysfunction (CMD) . The patWomen with signs and symptoms of ischemia and no obstructive CAD are at elevated risk for major adverse cardiac events (MACE), dominantly heart failure (HF) hospitalization  which weMechanistic pathways and treatment targets for HFpEF remain very poorly understood and evidence-based management strategies are lacking. Given the unexpected predominance of HFpEF hospitalization at 6-year follow-up in WISE , 4, we iThe WISE  design and protocol have been described in detail previously . The stu0C and stored in a central core lab  for subsequent measurement of inflammatory markers. Levels of IL-6 was measured using a commercially available enzyme-linked immunosorbent assay kit . Levels of high-sensitivity CRP (hsCRP) and SAA were measured by a high-sensitivity method on the Hitachi 911 analyzer using reagents from Denka Seiken . All samples were assayed within 5 years of collection at a core laboratory  using previously validated techniques [Plasma sampled at baseline was frozen at -70 chniques .The initial protocol-specified follow-up was conducted by experienced site nurses or physicians through direct, telephone, and/ or mail contact at 6 weeks, 1 year, and annually thereafter using a standardized scripted interview. Women were queried about symptoms, medication use, cardiovascular outcomes, hospitalizations, and diagnostic or revascularization procedures since last contact. For cases cared for at a WISE clinical center, patients’ medical records were also reviewed. The median follow-up time among surviving patients was 6 years. Subsequently, we conducted a National Death Index search for all women who were thought to be alive, and obtained additional death certificates. For this analysis, a major event (MACE) was defined as a first occurrence of all-cause mortality, nonfatal myocardial infarction (MI), nonfatal stroke, or HF-hospitalization.Demographic and patient characteristic variables were summarized using means and standard deviations or percentages where appropriate. Distributions of inflammatory markers are known to be positively skewed, therefore, medians were used to estimate central tendencies and Spearman correlation was used to estimate correlation between markers. For time to event data, the Kaplan-Meier method and log-rank test were used to estimate the incidence rates of all-cause mortality, cardiovascular mortality and HF hospitalization. The hazard ratio for all biomarkers is reported per log change. First, we conducted univariate model to adjust for confounders including age, BMI, history of diabetes, smoking, history of malignancy, history of autoimmune disease and concomitant medications . A multiPertinent baseline characteristics are summarized in Among the 390 women with available follow-up data, 106 (23%) had a MACE over a median of 6 years follow up. These events included 45 deaths (11%), 28 of which (62%) were cardiovascular related causes. There was a total of 14 HF hospitalizations (3.5%), 19 nonfatal MI (4.9%), and 24 nonfatal stroke (6.2%). We observed a significant increase in all-cause mortality associated with IL-6 and SAA, while there was no significant association between hs-CRP and mortality . There wEach individual inflammatory biomarker was then grouped into quartiles and in combination to assess their relative contribution to mortality. When comparing IL-6 and SAA in their highest quartile  to the lowest quartile, there was significant increase in all-cause mortality AHR , respectively . Among tmortality and HF hospitalization during follow up, independent of cardiovascular risk factors and comorbidities, consistent with the epidemic of HFpEF which is now a major contributor to CVD mortality [We observed in this unique population of women with signs and symptoms of ischemia, non-obstructive CAD and preserved EF at enrollment, IL-6 was the strongest predictor of HF hospitalization in these women compared to hs-CRP and SAA level, especially in the highest quartile >4.79 pg/mL). In addition to IL-6 was associated with increase mortality, SAA was linked to high mortality as well which we reported previously [9 pg/mL. ortality , 4. Prevortality . AlthougThe measurement of inflammatory markers for risk stratification of both primary and secondary prevention of cardiovascular disease has been the focus of numerous prior investigations. These efforts have been motivated by observations that inflammation is a key process in the pathophysiology of atherosclerosis, the development of acute cardiovascular syndromes, and heart failure . Not onlA potential mechanism to understand our observed association may be association between elevated levels of inflammatory cytokines and development of HF may include atherosclerosis, endothelial and microvascular dysfunction progression , 16. InfA variety of inflammatory markers have been previously identified as having a potential role in the progression of HF. We found that in this unique population of women with symptomatic ischemia, non-obstructive CAD and preserved EF at baseline, serum IL-6 was associated with an increased risk of HF hospitalization in a continuous fashion without evidence of a threshold. The mechanism is not well understood in our cohort, however elevation of such markers may be a driver or a reaction to the pathophysiological processes of progressive ventricular remodeling. Experimental data have implicated a causative role of IL-6 in cardiomyocyte hypertrophy and apoptosis , 26, advStrengths of our study include a prospective, multicenter design and core laboratory masked assessments of inflammatory markers and coronary angiograms with long subject follow-up of 6 years. This allowed exclusion of obstructive CAD to investigate development of HF hospitalization in a unique population of women with signs and symptoms of ischemia, without obstructive CAD and preserved EF. A potential limitation is the highly selective study population consisting of middle age predominantly Caucasian women with suspected myocardial ischemia referred for specialized centers for clinically indicated coronary angiography. Thus our results may not be generalizable to other populations of women or to men. In addition, there were small number of events during the follow-up period. Furthermore, diagnosis of HF was defined as admission for HF, which was validated in a representative sample from one WISE site using patient hospital records . Thus, dIn women with signs and symptoms of ischemia, non-obstructive CAD and preserved EF, the inflammatory biomarker IL-6 was predictive of HF-hospitalization and all-cause mortality, while SAA predicted all-cause mortality. These results suggest that vascular inflammation may be a mechanistic pathway and treatment target for HFpEF, which is a rising epidemic contributing to CVD morbidity and mortality which predominantly impacts women.


Background: Although the cucumber reference genome and its annotation were published several years ago, the functional annotation of predicted genes, particularly protein-coding genes, still requires further improvement. In general, accurately determining orthologous relationships between genes allows for better and more robust functional assignments of predicted genes. As one of the most reliable strategies, the determination of collinearity information may facilitate reliable orthology inferences among genes from multiple related genomes. Currently, the identification of collinear segments has mainly been based on conservation of gene order and orientation. Over the course of plant genome evolution, various evolutionary events have disrupted or distorted the order of genes along chromosomes, making it difficult to use those genes as genome-wide markers for plant genome comparisons.Results: Using the localized LASTZ/MULTIZ analysis pipeline, we aligned 15 genomes, including cucumber and other related angiosperm plants, and identified a set of genomic segments that are short in length, stable in structure, uniform in distribution and highly conserved across all 15 plants. Compared with protein-coding genes, these conserved segments were more suitable for use as genomic markers for detecting collinear segments among distantly divergent plants. Guided by this set of identified collinear genomic segments, we inferred 94,486 orthologous protein-coding gene pairs (OPPs) between cucumber and 14 other angiosperm species, which were used as proxies for transferring functional terms to cucumber genes from the annotations of the other 14 genomes. In total, 10,885 protein-coding genes were assigned Gene Ontology (GO) terms which was nearly 1,300 more than results collected in Uniprot-proteomic database. Our results showed that annotation accuracy would been improved compared with other existing approaches.Conclusions: In this study, we provided an alternative resource for the functional annotation of predicted cucumber protein-coding genes, which we expect will be beneficial for the cucumber's biological study, accessible from http://cmb.bnu.edu.cn/functional_annotation. Meanwhile, using the cucumber reference genome as a case study, we presented an efficient strategy for transferring gene functional information from previously well-characterized protein-coding genes in model species to newly sequenced or “non-model” plant species. Cucumis sativus L.)  genome sequences were assembled onto chromosomes; (2) Nn-way  collinear segments indicate the group of species consisting of cucumber and the other species that were sequentially incorporated based on the topology of the tree in Figure d  collinear segment represented a pairwise alignment with one of the 14 non-cucumber species indexed by d, where d was incremented with the degree of divergence from cucumber according to the phylogenetic tree  represented, in ascending order, Cucumis melo, Citrullus lanatus, Malus domestica, Glycine max, Populus trichocarpa, Citrus sinensis, Brassica rapa, Arabidopsis thaliana, Arabidopsis lyrata, Vitis vinifera, Solanum tuberosum, Setaria italica, Brachypodium distachyon, and Oryza brachyantha.Two different types of genomic markers, including MAAs identified by multiple alignments and a set of protein-coding gene families constructed by OrthoMCL  were designated as an orthologous protein-coding gene pairs (OPPs) if they met two essential conditions: (i) g1 and g2 shared certain sequence similarity ; (ii) at least 50% of the length of the g1 and g2 sequences were located in the same collinear segment. We also applied two optional conditions: (iii) g1 and g2 were BBH ; (iv) g1 and g2 also met condition (ii) when collinear segments were identified using protein-coding genes as genomic markers.Based on the collinear segments among cucumber and the other angiosperms plants identified by using MAAs as markers, gFor each OPP, we calculated an orthologous pair support score (OPSS) from three parameters as follows: (i) the M-score, indicating the degree of the MAAs-based collinear segment (2~15-way) supporting the orthologous relationship; (ii) the P-score, with weight 0.5, indicating the degree of the protein-based collinear segment (2~15-way) supporting the orthologous relationship; and (iii) the B-score indicating the BBH relationship .To evaluate the quality of the functional annotations from our collinearity-based pipeline, we performed analyses with 6 other commonly-used annotation pipelines, using default parameters, including Blast2GO  and B = , we used three types of similarity indices for comparisons: (i) the Jaccard coefficient  the Jaccard coefficient between GA and GB was used to measure the tendency of pipelines A and B to annotate the same gene set and is a structure-free index, (iii) the semantic similarity of the same gene annotated by GOA and GOB was calculated by Wang's method  to 24.46% (with O. sativa). For coding regions, the alignability ratios were much higher, ranging from 98.13% (with C. melo) to 67.26% (with O. sativa). In addition, similar patterns were observed when calculating the alignability ratios with different genomic features, as illustrated in Figure The summary of the pairwise alignment is presented in Table The 15-way multiple alignments can be viewed as a series of conserved blocks that exist in all 15 species that contain the best match within the cucumber reference. We termed these 15-way blocks conserved across 15 angiosperms genomes as the “multiple alignment anchors” (MAAs), which can potentially be used as genome-wide markers for detecting genomic homology. The rationale for finding MAAs is that due to long enough divergence time any identified regions of sequence similarity are the result of purifying selection rather than neutral carryover. With the cucumber genome as a reference, we assembled the largest comparative genomic dataset in plants to date using whole-genome sequences spanning the breadth of flowering plants. In total, 138,893 MAAs were identified by our local LASTZ/MULTIZ pipeline. The distribution of the MAAs lengths from chromosome 1~7 is shown in Figure P-value < 2.2E-16) higher than the genome-wide average of cucumber  or multiple evidences . Only 10% of OPPs had OPSS values smaller than 3, indicating that these OPPs were uniquely inferred from a 2way-2 collinear segment identified by the MAAs-based method. Interestingly, 31% of OPPs were validated by the BBH strategy, and 25% of OPPs were deduced from more than one level of collinear segments. For example, the orthologous gene pair Csa1P050470.1 from cucumber and NP_199849.2 from A. thaliana was deduced from four types of evidences, including 15-way and 2way-10 collinear segments identified by the MAAs-based method, a 2way-10 collinear segment identified by the protein-coding genes-based method, and the BBH strategy. The distribution of OPPs shown in Figure Each OPPs was further assigned an OPSS that integrated multiple evidences, including the level of collinearity detected with MAAs or protein-coding genes as genomic markers and sequence similarity Table . OrtholoA. thaliana, 4,230 cucumber genes were assigned at least one growth-related Plant Ontology (PO) term  and opp2go (protein coding genes-based), with respect to the following aspects: (i) annotated gene coverage and related numbers of GO terms; (ii) the mean number of GO terms per gene and mean number of genes per GO term; and (iii) similarity comparison among these eight annotation results. All the results produced by the eight pipelines were shown in the Tables Drawing on the experience of Amar  showed the best performance among the eight pipelines for the total GO terms and both the BP and Cellular Component (CC) GO term subsets. However, for the MF subset of GO terms, the ratios of significantly overrepresented terms  among opp2go (MAA-based) and the other methods were all <0.2. We also observed that all eight pipelines showed better performance for the CC subset than BP and MF. One explanation might be that co-expression between genes was evaluated using RNA expression datasets from 10 specific tissues, in which they might contain some degree of tissue-specific and cellular component information. In summary, the annotation quality of opp2go (MAA-based) obtaineds a partial improvement over those of traditional methods, which might be due to its integration of collinearity between related species into the functional annotation process. Our results also demonstrated that inter-species collinearity was a reliable basis for orthologous gene inference.Due to the lack of a high-quality gene functional annotation dataset in cucumber, we validated the annotation results using the gene co-expression patterns from expression data of 11,754 genes in 10 different tissues. The Pearson correlation coefficient was used to measure the co-expression between pairs of genes as detailed in materials and methods. To reduce the GO annotation bias, we removed GO terms associated with more than 300 genes or fewer than 5 genes. Figure cis-regulatory elements for nearby target genes as reviewed previously  that was highly conserved across 15 angiosperm genomes. These MAAs were more numerous, shorter in length, more widely and uniformly distributed than protein-coding genes in cucumber. Using MAAs as genomic markers, we identified multiple levels of collinear segments between cucumber and 14 related species. According to our survey, the mean length of collinear segments decreased dramatically with increasing genetic divergence from cucumber. However, with increasing divergence from cucumber, the number of segments gradually increased in the MAAs-based results, while a nearly opposite trend was observed with protein-coding genes. As a possible explanation, we hypothesized that long collinear segments might have been split into multiple smaller segments due to genomic events that disturbed these plant genomic segments over the course of long-term evolution. In this study, no 9- or 15-way collinear segments were identified by the protein-coding genes-based method, which suggests that using MAAs as genomic markers may be a viable alternative to protein-coding genes for detecting collinear segments among distantly related plants. More importantly, due to the co-localization of genes and their respective regulatory elements, the collinear segments may be used to detect potential With these identified collinear segments, we inferred putative OPPs between cucumber and each of the other 14 species. Each of these orthologs to cucumber genes was then used as a proxy to transfer annotation information to its corresponding cucumber gene if its biological function was known. Although the annotation coverage was comparable with traditional methods, our functional annotation strategy greatly reduced the annotation redundancy and obtained a partial improvement of annotation accuracy evaluated by gene co-expression profiles.However, the criteria for MAA inclusion should be refined by additional steps, including phylogenetic and evolutionary model analyses, such as BinCons (Margulies et al., http://cmb.bnu.edu.cn/functional_annotation. In addition, our study offers a pipeline for identifying collinear segments across multiple related plant genomes based on conserved DNA segments, which can potentially be used as genomic markers instead of protein-coding genes.In summary, using the cucumber as a case study, we provided a potential alternative resource for the functional annotation of cucumber protein-coding genes that was an alternative strategy for transferring functional information from previously well-characterized protein-coding genes in model species to genes in “non-model” plant species guided by genomic collinearity, accessible from EP and KL: contributed the central idea; HS and EP: designed the study; HS: analyzed most of the data and wrote the initial draft of the paper; JH: contributed to refining the ideas. All authors discussed the results and revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Xpert MTB/RIF is recommended by the World Health Organization (WHO) as the initial tuberculosis (TB) diagnostic test in individuals suspected of HIV-associated TB. We sought to evaluate field implementation of Xpert among a cohort of HIV/TB co-infected individuals, including availability, utilization and outcomes.Observational cohort study (patient-level data) and cross-sectional study (site-level Xpert availability data).Data were collected at 30 participating International epidemiologic Databases to Evaluate AIDS (IeDEA) sites in 18 countries from January 2012-January 2016. All patients were HIV-infected and diagnosed with TB, either bacteriologically or clinically, and followed until a determination of TB treatment outcome. We used multivariable modified Poisson regression to estimate adjusted relative risk (RR) and 95% confidence intervals for unfavorable TB treatment outcomes.Most sites (63%) had access to Xpert, either in the clinic (13%), in the same facility (20%) or offsite (30%). Among 2722 HIV/TB patients included, median age was 35.4 years and 41% were female; BMI and CD4 count were low. Overall, most patients (76%) received at least one TB test; 45% were positive. Only 4% of all patients were tested using Xpert: 64% were Xpert-positive, 13% showed rifampicin (RIF) resistance and 30% were extrapulmonary (EPTB) or both pulmonary-EPTB. Treatment outcomes were mostly favorable (77%) and we found little association between Xpert use and an unfavorable TB treatment outcome .In this cohort, Xpert utilization was low even though the majority of sites had access to the test. Our findings show the need for expanded implementation and further research exploring barriers to use in low-resource settings. The World Health Organization (WHO) estimates that effective diagnosis and treatment of tuberculosis (TB) saved approximately 49 million lives between 2000 and 2015. TB remaiMycobacterium tuberculosis and rifampicin resistance–a marker for multi-drug resistance) and CD4 count (117 cells/μl [IQR: 41–253]) were low. ART data were not available for (25%); of those with non-missing data, 1354 (66%) were not on ART at the time of TB diagnosis. Over half (56%) of the patients included in the study were from East Africa, with the rest of the patients split between the other study regions.Characteristics of the 2722 patients at the time of TB diagnosis are shown in TB testing utilization and outcomes are shown in Among all 2722 TB patients, 4% received Xpert testing overall . There wIn this large, international study of Xpert MTB/RIF access and utilization, we found that the majority of sites had access to Xpert, but only 4% of TB/HIV co-infected patients in this cohort were tested using Xpert. Eighteen countries participated in this survey and country-specific policy was evolving rapidly during the study period. Additionally, access to Xpert was increasing quickly due to contributions from international non-governmental organizations and public/private partnerships. Therefore, it is difficult to summarize the specific Xpert policy at each site during data collection. However, all data were collected after the WHO guidelines recommending Xpert as the first diagnostic method for suspected HIV/TB co-infected patients. Still, apart from national policy, our results showed a lack of use of Xpert despite availability of the device at the sites. Over half (54%) of the patients with no TB test were treated at facilities with Xpert available on-site. For another 34%, Xpert was available offsite. Thus, for most patients, Xpert was available but it was not used in their case, nor were they tested for TB using any other diagnostic method.This low utilization is concerning given that this study occurred well over one year after the WHO guidelines recommendation that Xpert be used for TB diagnosis among all TB/HIV co-infected persons. Local study site investigators have hypothesized that there are several possible explanations for the low utilization of Xpert, including cartridge stock-outs, interruptions in electricity, and unreliable transportation of specimens at sites with off-site Xpert testing. Additionally, local study site investigators reported anecdotally that provider education on the WHO guidelines and importance of Xpert utilization may be lacking. WHO recommends that sites routinely monitor the number of cartridges used, shelf-life of cartridges, and estimated delivery time in order to improve the supply-chain management and avoid cartridge stock-outs. High cosDespite the low utilization, we did not find that lack of Xpert testing was associated with an unfavorable TB treatment outcome. Overall, 70% of the patients in our study had a favorable outcome. While all patients were diagnosed as co-infected with HIV/TB and initiated TB treatment, nearly one-quarter of patients (24%) had no record of any TB test, showing a heavy reliance upon empiric TB treatment. Other studies in low-resource settings have shown a similar reliance upon empiric treatment –30 and hThis study emphasizes that operational research must address global implementation challenges of Xpert in order to maximize the potential benefits of this new technology. In contrast to two recent multi-country Xpert studies, our study did not provide Xpert machines to sites  or a funWhile Xpert was used only among a small proportion of cases, 64% were Xpert-positive. A Cochrane review reported a pooled sensitivity of Xpert as 79% (95% CI 70–86%) among people living with HIV. However, this pooled sensitivity differed depending on smear status with 61% (95% CI 40–81%) among smear-negative persons compared to 97% (95% CI 90–99%) among smear-positive persons. In our sOur prevalence of rifampicin resistance, a marker of MDR-TB, was 13%. In 2015, the WHO estimated that 3.9% (95% CI 2.7–5.1%) of new cases and 21% (95% CI 15–28%) of previously treated cases had MDR- or rifampicin resistant-TB  these fiAmong 30% of the TB cases diagnosed with Xpert, the site of disease was extrapulmonary or both pulmonary and EPTB. Thus our findings demonstrate the utility of Xpert for testing non-sputum samples and again suggest that Xpert is being utilized for testing of non-sputum samples as recommended by the WHO in a recent update on Xpert policy.We acknowledge several limitations related to this study. First, due to resource constraints, data for all HIV/TB cases could not be collected and the patient population included varied by site. This introduces the possibility of selection bias, although if sites were unable to enter data for all TB cases they were asked to select cases at random. Second, the retrospective nature of our data collection may also result in selection bias or information bias. Related to retrospective data, a possible lag in case identification and/or data entry may have contributed to fewer cases included in 2014. Third, it is possible that Xpert use has increased since our site-level data were collected in 2012, but our results represent a time period well over a year after the WHO guidelines announcement. Additionally, it is unlikely that sites lost access to Xpert over the study period and an increase in access to Xpert at the site-level would further strengthen our conclusion that Xpert utilization was low despite access to the technology. Fourth, data for the timing of TB testing to TB treatment initiation were not available and we were, therefore, unable to measure and control for the use of empiric anti-TB therapy. Similarly, data for the timing of TB treatment outcomes were not available so that information from patients who were lost or transferred could be included in a censored time-to-event analysis.In conclusion, our data suggest that in the low- and middle-income countries participating in this study, implementation of Xpert is low. In order to fulfill the promise of Xpert as a ground-breaking TB diagnostic, we must ensure that its implementation meets the WHO guidelines. Our findings show that there is a long way yet to go in achieving widespread clinic-level access to Xpert and patient-level implementation. Future study should focus on the barriers to implementation of Xpert use in resource-limited settings.S1 AppendixThe combined relative risks (RRs) and 95% CIs were computed based on the results of the region-specific RRs using the meta- analysis approach of DerSimonian and Laird, a random effects method which makes no assumption regarding homogeneity across sites.(PDF)Click here for additional data file.S2 Appendix(PDF)Click here for additional data file.