It is difficult to detect pancreatic cancer or biliary-tract cancer at an early stage using current diagnostic technology. Utilizing microRNA (miRNA) markers that are stably present in peripheral blood, we aimed to identify pancreatic and biliary-tract cancers in patients. With “3D-Gene”, a highly sensitive microarray, we examined comprehensive miRNA expression profiles in 571 serum samples obtained from healthy patients, patients with pancreatic, biliary-tract, or other digestive cancers, and patients with non-malignant abnormalities in the pancreas or biliary tract. The samples were randomly divided into training and test cohorts, and candidate miRNA markers were independently evaluated. We found 81 miRNAs for pancreatic cancer and 66 miRNAs for biliary-tract cancer that showed statistically different expression compared with healthy controls. Among those markers, 55 miRNAs were common in both the pancreatic and biliary-tract cancer samples. The previously reported miR-125a-3p was one of the common markers; however, it was also expressed in other types of digestive-tract cancers, suggesting that it is not specific to cancer types. In order to discriminate the pancreato-biliary cancers from all other clinical conditions including the healthy controls, non-malignant abnormalities, and other types of cancers, we developed a diagnostic index using expression profiles of the 10 most significant miRNAs. A combination of eight miRNAs  achieved a sensitivity, specificity, accuracy and AUC of 80.3%, 97.6%, 91.6% and 0.953, respectively. In contrast, CA19-9 and CEA gave sensitivities of 65.6% and 40.0%, specificities of 92.9% and 88.6%, and accuracies of 82.1% and 71.8%, respectively, in the same test cohort. This diagnostic index identified 18/21 operable pancreatic cancers and 38/48 operable biliary-tract cancers in the entire cohort. Our results suggest that the assessment of these miRNA markers is clinically valuable to identify patients with pancreato-biliary cancers who could benefit from surgical intervention. Pancreatic cancer is one of the most lethal cancers. Most pancreatic cancers do not accompany any particular clinical symptoms in the early stage, permitting the cancers to progress undetected. In addition, ambiguous radiological images of cancerous lesions and inflammatory conditions in the pancreas prevent pancreatic cancers from being correctly identified. Furthermore, the anatomical location of the pancreas, deep in a retroperitoneal space surrounded by many other organs, hinders the acquisition of a biopsy. All of these factors prevent the early detection of pancreatic cancer. The American Cancer Association estimated that 40,000 people would die of pancreatic cancer in 2014 in the United States [As a diagnostic screening method for pancreatic cancer, ultrasound is one of the most prevalent tests performed. However, this image analysis has its difficulty in differentiating non-malignant tissue from malignant tissue . In addiet al. suggested that serum miR-21 and other miRNAs could predict the aggressiveness of pancreatic cancer [et al. examined a dozen of plasma samples each from patients with and without pancreatic cancer, and concluded that three miRNAs, miR-642b, miR-885-5p, and miR-22, were more than 90% sensitive and specific in the diagnosis of pancreatic cancer [Recently, microRNAs (miRNAs) have been reported as potential biomarkers for various types of cancers. Using plasma samples from 50 cancer patients and ten healthy control subjects, as well as chemo-resistant pancreatic cell lines, Ali c cancer . More reHere, we examined the expression profiles of comprehensive serum miRNAs from the largest cohorts of patients ever attempted: 100 patients with pancreatic cancer, 98 patients with biliary-tract cancer, 150 healthy control patients, 21 patients with non-malignant abnormalities in the pancreas or biliary tract, and 202 patients with other types of cancers. A highly sensitive microarray permitted the simultaneous analysis of more than 2,500 miRNAs that were recently updated in the miRBase (release 20), and serum samples from patients with the various clinical conditions allowed us to evaluate a wide range of diagnostic specificity in the detection of pancreato-biliary cancer.By combining eight miRNA markers, we were able to detect patients with pancreato-biliary cancers among those who were healthy, had non-malignant abnormalities or had other types of cancers, with a diagnostic accuracy of 91.6% and AUC of 0.953.This research on human subjects was approved by the National Cancer Center Hospital East Institutional Review Board (2010-096) and by the Human Tissue Samples Ethics Committee for R&D, Toray Industries Inc. . Written informed consent was obtained from each participant.Blood samples were obtained from a total of 421 patients who were admitted to the Japanese National Cancer Center Hospital East during the years 2010 to 2012. One hundred patients with pancreatic cancer, 98 patients with biliary-tract cancer, 50 patients with colon cancer, 50 patients with stomach cancer, 50 patients with esophageal cancer, 52 patients with liver cancer, and 21 patients with non-malignant pancreatic or biliary-tract diseases were registered in the Biobank and selected for use in this study. For pancreatic and biliary-tract cancers, patients with the following characteristics were excluded: (i) patients with intraductal papillary mucinous neoplasm, (ii) patients simultaneously or previously diagnosed with advanced cancer in another organ, (iii) patients with cancer uncertain to be either pancreatic or biliary, and (iv) patients with special histology other than adenocarcinoma. All the patients were histologically confirmed, and those with cancer were pathologically diagnosed as having adenocarcinoma. All blood samples from cancer patients were taken before any treatment except one pancreatic cancer case with ypStage I which had received therapeutic intervention. The detailed information about these patients is shown in Control blood samples were obtained from healthy individuals recruited from the Japanese affiliate companies of Toray Industries Inc. in 2013. Inclusion criteria for healthy control individuals were age greater than 60 years, no history of any cancer, and no hospitalization during the last 3 months .All peripheral blood samples were processed to serum within a day of acquisition, and serum biomarkers including CA19-9 and CEA were measured before the remaining serum samples were stored at -80°C for miRNA analysis.Formalin Fixed Paraffin Embedded (FFPE) samples of pancreatic tumors and adjacent normal tissues were obtained from 10 randomly chosen patients with pancreatic cancer . In the FFPE sections with the size of 10×10μm, tumor area was marked by ink using the neighboring slides that were H.E. stained. The tumor area and the adjacent normal tissue were then macroscopically dissected.http://www.mirbase.org/).For each clinical group, the miRNA expression analysis was performed simultaneously regardless of the training cohort or the test cohort. Total RNA was extracted from each 300-μL serum sample using “3D-Gene” RNA extraction reagent from a liquid sample kit . Total RNA was also obtained from the FFPE sections using the “Arcturus” “Paradise” Extraction and Isolation kit  in accordance with the manufacturer's instructions. Comprehensive miRNA expression analysis was performed using a “3D-Gene” miRNA Labeling kit and a “3D-Gene” Human miRNA Oligo Chip , which was designed to detect 2,555 miRNAs registered in the miRBase release 20  under the accession number GSE59856.Individual miRNAs were regarded as present if the corresponding microarray signals were more than (the mean + 2x standard deviation) of the negative control signals of which the top and bottom 5% ranked by signal intensity were removed. Once the miRNA was regarded as present, the miRNA signal was subtracted with the mean signal of the negative controls of which the top and bottom 5% ranked by signal intensity were removed. When the signal became a negative value (or was undetected) after the background subtraction, the value was replaced by the number that was the lowest signal intensity on the microarray minus 0.1 on a logerformed . All micIn this study, the miRNA expression profiles of the following five paired-clinical conditions were statistically compared: i) pancreatic cancer versus healthy control, ii) biliary-tract cancer versus healthy control, iii) pancreato-biliary cancer versus non-malignant abnormalities, iv) pancreato-biliary cancer versus other types of cancer, and v) pancreato-biliary cancer versus all other clinical conditions . For each condition, patients with pancreatic and/or biliary-tract cancer were regarded as the cancer group, and the other groups were regarded as the control.6 in more than 50% in either clinical group being compared were selected for further analysis. Using these miRNAs, a Fisher’s linear discriminant analysis was performed, and the resulting diagnostic sensitivity, specificity and accuracy were calculated for each miRNA marker or combination of miRNA markers.After all microarray experiments were performed, the samples of each clinical condition were randomly divided into a training cohort (2/3 of the samples) and a test cohort (1/3 of the samples) by computation. The clinical characteristics of the training cohort and the test cohort was examined and confirmed that there was no significant difference in these two cohorts. The training cohort was used to select significant miRNA markers and to define their discriminant functions, and the test cohort which is independent from the training cohort was used to validate the diagnostic performance of the selected miRNA markers based on the same statistical significance. The analyses of the training cohort and the test cohort were programmed in advance and sequentially performed without overcrossing those cohorts. In the selection process of miRNA markers, any two clinical groups were compared using two-sided Student’s t-test, and a Bonferroni-corrected p-value of <0.01 was regarded as statistically significant. For FFPE analysis, since only eight miRNAs that were preselected was analyzed, Bonferroni correction was not applied to p-value. In order to obtain robust biomarkers, only miRNAs that showed a signal value of 2When expressions of multiple miRNAs were used in the algorithm development, the discriminant functions were created with Fisher’s linear discriminant analysis. The resulting values of the discriminant functions were called as diagnostic indices. Clinical samples that showed the indexed score over 0 were classified to pancreato-biliary cancer, and samples that showed the indexed score under 0 were classified to non-pancreato-biliary cancer . As another statistical tool to analyze biomarkers’ diagnostic performance, Receiver Operating Characteristic (ROC) analysis and its Area Under the Curve (AUC) values were also used.http://www.R-project.org), MASS package version 7.3–30 [All computations were performed using R version 3.0.2 . All other patients with pancreatic cancer who did not undergo surgical resection were classified as cStage III or IV. On the other hand, biliary-tract cancer cases were classified simply by operable and unoperable cases, due to the complex TNM staging system and distinct biological identities across different primary sites  within biliary tract.Using the training cohort, the patients with pancreatic cancer were statistically compared with the healthy control individuals, and 120 miRNAs showed a potential to separate those two clinical groups with Bonferroni-corrected p-value of less than 0.01 by Student’s t-test. Out of the 120 miRNAs selected in the training cohort, 81 miRNAs were statistically validated in the test cohort (p<0.01). Among those, 40 miRNAs were up-regulated and 41 miRNAs were down-regulated in pancreatic cancer. The list of these selected miRNAs, their p-values, the expression levels and the diagnostic performance were provided in The best marker, miR-125a-3p, was able to detect 30 of 33 (90.9%) pancreatic cancers in the test cohort. In contrast, the widely used blood biomarkers for pancreatic cancer, CEA and CA19-9, detected 52 (52.0%) or 77 (77.0%), respectively, out of 100 pancreatic cancers in the entire cohort. Overall, 40 of 81 validated miRNA marker candidates for pancreatic cancer showed sensitivities higher than that of CA19-9 in the test cohort .Similarly, we performed a comparative analysis of the patients with biliary-tract cancer and the healthy control individuals. One hundred twenty-four miRNAs showed statistical significance (p<0.01) in the training cohort, and out of those 124 miRNAs, 66 miRNAs were validated in the test cohort . Among tWe also found that, despite apparent histological differences, a number of miRNAs were selected as markers not only for pancreatic cancer but also for biliary-tract cancer; of the 81 miRNAs selected as marker candidates for pancreatic cancer, 55 (67.9%) were also selected as marker candidates for biliary-tract cancer. In contrast, when miRNAs that could discriminate pancreatic cancer from biliary-tract cancer were sought, none reached statistical significance. The miRNA that showed the smallest Bonferroni-corrected p-value (0.071) in comparison of pancreatic and biliary-tract cancer was miR-1227-5p; however, that miRNA was selected as a marker both for pancreatic cancer (ranked 78) and for biliary-tract cancer (ranked 46) . Those rThis strong overlap in miRNA expression profiles between pancreatic cancer and biliary-tract cancer led us suspect that these miRNA markers were affected by jaundice, a common clinical condition over pancreas and biliary-tract. Therefore, we examined a jaundice marker, blood D-bilirubin, in pancreato-biliary cancer patients. Eighty-one of 100 pancreatic cancer patients and 78 of 98 biliary-tract cancer patients had blood D-bilirubin under the cutoff value of 0.8 mg/dL, and the median value of blood D-bilirubin concentration was 0.2 mg/dL both for pancreatic and biliary tract cancer patients, suggesting that a majority of these patients were non-jaundice. Furthermore, none of those 55 miRNA markers that overlapped in pancreatic cancer and biliary-tract cancer was correlated with D-bilirubin level (the highest Pearson’s correlation was -0.37). These results confirmed that the common miRNAs selected above were not likely a marker for jaundice but a marker for pancreato-biliary cancer.Based on these observations, we therefore decided to pursue the markers that would simultaneously detect both pancreatic and biliary-tract cancers.The ideal pancreato-biliary cancer biomarker should prove its specificity not only against healthy control but also against non-malignant disease conditions as well as other types of cancers. In order to test this thesis, we compared 198 pancreato-biliary cancer patients with 21 patients who had some abnormalities in either the pancreas or the biliary tract, but no trace of malignancy . We assuFurthermore, in order to examine organ-specificity of serum miRNA markers in malignant environment, a total of 202 serum samples obtained from patients with colon, stomach, esophagus, and liver cancers  were anaThe comparative analyses so far suggested that the resulting miRNA markers for pancreato-biliary cancers would largely affected by clinical characteristics of the control population. In order to obtain more robust and universal markers that could identify pancreato-biliary cancer patients from clinically heterogeneous population, the supposed-control: healthy individual, patients with non-malignant abnormalities, and patients with other cancer types, were all combined, and compared with pancrato-biliary cancer patients by discriminant analysis. The analysis identified 143 miRNAs that showed differential expression in the training cohort. Among them, 98 miRNAs were statistically validated in the test cohort. Ninety-one out of those 98 validated miRNAs were already identified in the previous analyses of pancreato-biliary cancer either with healthy control, with non-malignant abnormalities, or with other types of cancer. The 10 miRNAs that showed the smallest Bonferroni-corrected p-values are listed in According to the data above, a single serum miRNA achieved the detection of pancreatic and biliary-tract cancers with >80% accuracy. We aimed to achieve higher discriminant performance by combining multiple significant miRNAs. The 10 miRNAs  listed in We then focused on the algorithm that used the combination of four miRNAs: miR-6075, miR-6799-5p, miR-125a-3p, and miR-6836-3p, which was the combination with the least number of miRNAs that achieved the best accuracy calculated . The numOn the other hand, 5 of 29 pancreatic cancers and 7 of 37 biliary-tract cancers in the test cohort gave false-negative scores by the miRNA index. These false-negative cases did not show any partiality in their clinical characteristics; between the false-negative and the true-positive cases, there was no significant difference in the patients’ age (p = 0.111), serum CA19-9 (p = 0.581) or CEA (p = 0.063) concentrations nor was there a difference in the histological location of the tumor or the sites of distant metastasis. Similarly, 4 of 124 control samples gave false-positive scores by the miRNA index. The false-positive samples varied in their clinical status  and did not show any particular clinical characteristics that might indicate any bias. There was one non-malignant abnormality case that was initially diagnosed as chronic pancreatitis, but after 3-year follow-up, pancreatic cancer was found. This case was designated in the training cohort, and was negative by both CEA (1.2 ng/mL) and CA19-9 (10 U/mL) test, but positive by our diagnostic index.These results suggest that the diagnostic index is able to detect pancreato-biliary cancers at various stages and histological locations, and discriminates not only against healthy individuals but also against individuals with non-malignant abnormalities in pancreato-biliary organs as well as those with other types of cancers.The expression of the miRNAs selected for the diagnostic indices was validated in the FFPE tissue samples obtained from 10 patients with pancreatic cancer. Paired samples of cancerous tissue and the surrounding normal tissue were separately obtained from the same patients by macroscopic-dissection. The expression of those ten miRNAs that were used in the discriminant index was quite strong in the pancreatic cancer tissue ; however, they did not show statistical significance in differentiating the cancerous tissue from the normal surrounding tissue , indicatDespite the desperate need for the early detection of pancreatic cancer, there is no simple, least-invasive screening method. Pancreatic cancer has such a low prevalence in general population that it may be difficult even for accurate diagnostic tests to achieve sufficient positive-predictive values in population-wide screening . Since iThe current blood biomarkers CEA and CA19-9 have been prevalently used for post-operation monitoring, but their poor specificity for non-malignant diseases and other cancers as well as their poor sensitivities to early cancers prevent them from being widely used in screening systems. In this study, CEA and CA19-9 failed to detect not only the early stage of pancreatic and biliary-tract cancers but also progressed cancers. A simple blood screening-test alternative to those protein markers is necessary for pancreato-biliary cancers.Several prior studies have shown the value of serum miRNAs in the detection of several kinds of cancers , 15, 16.In this study, the best single marker achieved an accuracy of 84.7% in detecting pancreato-biliary cancers. By combining multiple significant miRNAs, we had thought to attain higher diagnostic performance as previously demonstrated , 18. UtiWe further validated the detailed result of the discriminant function composed of four miRNAs (the combination with the least number of miRNAs). The detailed validation of the discriminant performance revealed the clinical value of the diagnostic index; the index identified the early phase of pancreato-biliary cancers that were surgically resectable. Because the current best treatment for pancreato-biliary cancers is the resection of tumors while the tumors are still localized in the organs, a diagnostic index capable of detecting small resectable tumors is promising. Furthermore, this index was able to detect any histological type of tumors, some of which  could be difficult to detect by image screening if not well attended. This index which utilizes blood miRNA markers could be used beforehand to provide a rationale to perform more costly and/or invasive screening tests such as a CT scan or endoscopic ultrasound.Although we found that a combination of serum miRNAs could become a specific marker for pancreato-biliary cancers, the biological roles of those serum miRNAs are under discussion. Nine out of the ten miRNAs used for the diagnostic indices had an ID name higher than 1,000, indicating that they were discovered relatively recently. We were therefore not able to find many references about those miRNAs except for miR-125a-3p. Serum miR-125a-3p has been reported to show different expression profiles between patients with pancreatic cancers and healthy individuals . In the One of the mRNAs targeted by miR-125a-3p is MYST/Esa1-associated factor 6 (MEAF6), which encodes a nucleic protein that is associated with transcriptional activators. It is reported that MEAF6 binds with NuF4 complex, which acetylates histone H4 ; and thiIt is hypothesized that cancer cells secrete exosomes that encompass various molecular informants including proteins, DNA, and miRNAs into the blood stream and send them to distant organs in order to cultivate a new environment for future metastasis , 16. AccAnother enigma is the discordance of miRNA expression profiles in the serum and in the tissues. The 10 miRNAs that were significantly expressed in the sera of the patients with pancreato-biliary cancers did not show similar significant increases in the FFPE cancerous tissues compared with the surrounding normal tissues. This observation is again not in accordance with the tumor-secreted exosome theory. We think that the underlying mechanism of circulating miRNAs is more complex than previously speculated. Furthermore, we have preliminary data indicating that expression profiles of those miRNAs that were especially derived from clinical samples such as blood or FFPE tend to fluctuate depending on the assay method  used (data not shown). In short, a number of studies remain to be performed to elucidate the biological significance of the miRNAs that we found to be involved in pancreatic and biliary-tract carcinogenesis. Until such a mechanism is clarified, the serum miRNA markers for pancreato-biliary cancers should not be confused with the tissue miRNA markers.In conclusion, our results provide strong data showing that serum miRNAs, particularly diagnostic index conducted by the combination of several predictive miRNAs, can detect patients with pancreatic and biliary-tract cancers against a background of those who are healthy, have other non-malignant diseases or have other types of cancer. We believe that detecting pancreato-biliary cancers using peripheral blood, which is relatively easy to obtain from most subjects, is clinically useful, particularly as a first screening test. Such a test will provide additional information to those who could be at risk and, if necessary, encourage them to take more costly, sometimes invasive, imaging tests. We hope that such a test will lead to the early detection of these cancers and to improvement in the currently devastating survival rate.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Periodic fever, aphthous stomatitis, pharyngitis and adenitis (PFAPA) syndrome is an inflammatory disorder of childhood classically characterized by recurrent fevers, pharyngitis, stomatitis, cervical adenitis and leukocytosis. Little is known about the true incidence, natural course, pathogenesis, and appropriate therapy in this recently described syndrome. While the mechanism is unclear, previous studies have shown that tonsillectomy can be a therapeutic option with improvement in quality of life in many patients with PFAPA, but long-term clinical follow up is lacking.To evaluate the long-term response of tonsillectomy in patients with PFAPA syndrome treated at a tertiary care center in San Diego, CA.We initiated a prospective cohort study in 2008 to better understand the natural history of PFAPA in children treated at a tertiary care center in San Diego, CA. Any patient aged 1-17 years, seen in the Rady Children's Hospital-San Diego Allergy/Immunology clinics, diagnosed clinically with PFAPA syndrome and undergoing tonsillectomy (with or without adenoidectomy) were eligible for inclusion. Patient data was collected on over 200 children with recurrent fevers including 94 patients with PFAPA under an IRB-approved protocol. Using patient charts and a standardized questionnaire, demographic data, including age, gender, and ethnicity, clinical profiles  and detailed family histories were obtained from patients seen in a children's hospital-based clinic over a 7-year period. A subset of samples (10 PFAPA and 10 control) underwent 16S rDNA profiling.To date, 63 patients with PFAPA and 11 patients with other non-infectious recurrent fevers have undergone tonsillectomy. Forty-four patients with PFAPA syndrome have had complete resolution of symptoms after surgery, with the time to resolution of symptoms post-tonsillectomy approximately 2 months (range 1-11 months). The average length of follow up is 31.6 months (range 1-58 months). In 3 patients, there has been a relapse of symptoms, defined as fevers persisting for more than 6 months, which remain responsive to medical therapy. Post-operatively, tonsils from patients with PFAPA are notably smaller and grossly friable. No granulomas or abscesses were noted on histological examination. While small differences exist in the tonsillar microbiome of PFAPA patients compared to controls, these taxonomic groups were only very low abundance, and were not outside the range of normal flora observed in the Human Microbiome Project.Our cohort of patients demonstrates clinical characteristics consistent with PFAPA. This study demonstrates that tonsillectomy is an effective surgical treatment option for management of children with PFAPA syndrome.


There are an estimated half-million children living with HIV in sub-Saharan Africa. The predominant source of infection is presumed to be perinatal mother-to-child transmission, but general population data about paediatric HIV are sparse. We characterise the epidemiology of HIV in children in sub-Saharan Africa by describing the prevalence, possible source of infection, and effects of paediatric HIV in a southern African population.From 2009 to 2011, we conducted a household-based survey of 3389 children (aged 2–14 years) in Manicaland, eastern Zimbabwe (response rate: 73.5%). Data about socio-demographic correlates of HIV, risk factors for infection, and effects on child health were analysed using multi-variable logistic regression. To assess the plausibility of mother-to-child transmission, child HIV infection was linked to maternal survival and HIV status using data from a 12-year adult HIV cohort.HIV prevalence was  and did not differ significantly by sex, socio-economic status, location, religion, or child age. Infected children were more likely to be underweight  or stunted  but did not report poorer physical or psychological ill-health. Where maternal data were available, reported mothers of 61/62 HIV-positive children were deceased or HIV-positive. Risk factors for other sources of infection were not associated with child HIV infection, including blood transfusion, vaccinations, caring for a sick relative, and sexual abuse. The observed flat age-pattern of HIV prevalence was consistent with UNAIDS estimates which assumes perinatal mother-to-child transmission, although modelled prevalence was higher than observed prevalence. Only 19/73 HIV-positive children (26.0%) were diagnosed, but, of these, 17 were on antiretroviral therapy.Childhood HIV infection likely arises predominantly from mother-to-child transmission and is associated with poorer physical development. Overall antiretroviral therapy uptake was low, with the primary barrier to treatment appearing to be lack of diagnosis. In 2012, it was estimated that over 85% of children who became infected with HIV were living in sub-Saharan Africa (SSA) e.g. perinatally, breastfeeding) the infant becomes vertically infected Most infected children are believed to have acquired HIV perinatally from their HIV-positive mothers. Untreated HIV infection in infants is typically characterised by rapid disease progression and death at a median of two years of age or less, with survival depending at what stage  describe patterns of HIV infection in a representative general population sample of children aged 2–14 years in a large-scale generalised HIV epidemic in rural areas of eastern Zimbabwe; (ii) investigate possible sources of horizontal HIV transmission in childhood; (iii) assess whether the observed age-pattern of HIV-positive children is consistent with that expected from survival of children infected from MTCT  program scale-up); (iv) assess the impact of HIV on children's mental and physical health and nutritional status; and (v) investigate the levels and determinants of antiretroviral treatment (ART) coverage in children.The Manicaland HIV/STD Prevention Project is a population-based, open cohort study in eastern Zimbabwe In the most recent round (2009–2011) of the Manicaland survey, all children (aged 2–14 years) in a randomly selected 1/3 of households were invited to participate in an investigation of HIV prevalence amongst children. Children were interviewed about their welfare, health, and healthcare using a structured questionnaire. Children under seven answered with assistance from their primary caregiver. Questions on HIV testing and knowledge of HIV status were addressed to the child's primary caregiver in the presence of the child if he or she was over the age of seven. Additionally, the questionnaire was administered by a nurse who had HIV Testing and Counselling certificates, which include training in how to respond if a child becomes distressed. More sensitive questions were asked only of older children and were answered without their caregiver being present: children aged 7–14 years were asked questions on sexual abuse and on psychological health. If a child reported abuse then the interviewer notified the supervising nurse who would subsequently investigate in the company of a social worker. The information from the supervisor and social worker was then fed back to the Child Protection committee in the study area. All maternal data (religion and HIV status) were collected in the general (adult) survey and linked to child data based on children reporting who their biological mother was, and confirmed through fertility histories and the household roster. In cases where a link could not be made, or if the child was a maternal orphan, maternal data was coded as missing. Dried blood spot samples were collected and tested for HIV in an offsite laboratory using the COMBAIDS-RS HIV 1+2 Immunodot Assay ; for cases in which the child tested HIV-positive but had an uninfected mother, the HIV test results were confirmed using Vironostika HIV Uni-form II Plus O  ELISA tests. Data used in the manuscript are provided in the supporting information file Ethical approval for the Manicaland HIV/STD Prevention Project was obtained from the Research Council of Zimbabwe (Number 02187), the Biomedical Research and Training Institute Zimbabwe's institutional review board (Number AP6/97), and the Imperial College London Research Ethics Committee (Number ICREC 9_3_13). Written informed consent was obtained prior to survey participation from each child's primary caregiver. In addition, children aged 7–14 years provided verbal or written assent, respectively. Participants and guardians were informed that, at any point, they could refuse to answer a question or decline to continue the interview.In this analysis of children aged 2–14 years old, we tested for associations of HIV infection with socio-demographic characteristics , community type, and mother's religion) using logistic regression. Socio-economic status was measured using a summed asset-based wealth index developed for the study population in Manicaland To test the hypothesis that HIV infection in children occurs primarily through MTCT, where available, we examined maternal survival/infection status  by child HIV status to establish the plausibility of vertical HIV transmission. The odds ratios of being a maternal orphan and of being a maternal orphan or having an HIV-positive mother amongst infected and uninfected children were evaluated using a one-sided Fisher's exact test. We tested for associations between HIV infection and risk factors for horizontal HIV transmission, which included blood transfusion, vaccination, non-vaccination medical injections, breastfed by a non-biological mother, cared for a sick relative, and sexual abuse.http://apps.unaids.org/spectrum/.To assess whether the observed age-pattern of HIV prevalence in children is consistent with that which would be expected in Zimbabwe if infections were due to mother-to-child transmission, we compared the age-specific HIV prevalence data to national estimates of child HIV prevalence reported by UNAIDS et al., 2010 The impact of HIV on measures of physical and mental health was evaluated using linear (continuous outcomes) or logistic (binary outcomes) regression, adjusting for age. Z-scores for height- and weight-for-age and weight-for-height were calculated using WHO child growth standards Four thousand six hundred and eleven children aged 2–14 years were enumerated and selected for inclusion in the study, of which 3389 (73.5%) completed the survey and gave a dried blood spot for HIV testing. Children who did not complete the survey did not have significantly different age or gender distributions and their household of residence did not have significantly different mean SESs than those who completed the questionnaire . Reasons given for non-response included: away from home for work (7.4%), away from home for school (7.0%), another reason for being away from home (67.1%), whereabouts unknown (1.1%), refused (12.3%), and other (5.0%). Seventy-three  were HIV-positive. Prevalence was 1.6% (11/688), 2.5% (33/1296), and 1.8% (25/1405) among children aged 2–4 years, 5–9 years, and 10–14 years, respectively. Demographic characteristics of children aged 2–14 years by HIV status are presented in All but one HIV-positive child were either maternal orphans or had an HIV-positive surviving mother, consistent with the primary source of childhood infection being MTCT . HIV-posHIV prevalence in children aged 2–14 observed in Manicaland was lower than the national estimates for Zimbabwe as a whole in 2010 from the Spectrum model (3.6%). However, the age-pattern of HIV prevalence amongst children observed in the data was consistent with the model estimates . This suHIV-positive children were significantly more likely to be underweight (low weight-for-age)  and stunted  than HIV-negative children, but were not more likely to be wasted (low weight-for-height)  or to report a recent illness  . HIV stai.e. were on anti-retroviral therapy (ART)). Despite high ART coverage when HIV status is known, overall, less than a quarter  of the HIV-positive children was receiving ART.Of the 73 HIV-positive children, 26.0% (19/73) reported that they knew their HIV status. Of the HIV-negative children, 114/3309 (3.5%) had had an HIV test, significantly less than reported testing prevalence in HIV-positive children (p<0.001). Children of mothers who reported that they knew they were HIV-positive were 5.17 times (95% CI: 2.27–11.76) more likely to have had an HIV test and know the result than children of mothers who self-reported as HIV-negative. Knowledge of HIV status was not associated with psychological wellbeing score for HIV-positive children . All but two of the children (17/19) who were aware of their HIV-positive status reported taking drugs that stop HIV from causing AIDS  of children aged 2–14 years tested positive for HIV, at a time when HIV prevalence was 11% and 17%, respectively, amongst male and female adults (15–54 years) in the same population. This estimate, from a representative general-population sample, is lower than those from a sample of children in 2005 in Chimanimani district in southern Manicaland, where HIV prevalence was 3.2% (41/1290) in children aged 2–14 years Our finding of a relatively even pattern of HIV prevalence by age is consistent with official national estimates derived from the Spectrum model. Survival data for children infected with HIV through MTCT suggest high mortality Our data further confirm the belief that MTCT is the primary mode of HIV transmission in children in eastern Zimbabwe The need to understand HIV infection in children is particularly important given that HIV is increasingly becoming a cause of hospitalisation amongst adolescents in SSA et al. (2010) There are few data from SSA on the psychological manifestations of HIV infection in children, although studies from developed countries report significantly higher incidence of psychiatric admissions for HIV-positive children than HIV-negative children, with knowledge of HIV status increasing the risk of admission Only a quarter of HIV-positive children in our study were aware of their HIV status. Across southern and eastern Africa, ART coverage among children is a major problem and high priority for many governments as it continues to lag behind adult coverage  These findings provide evidence that MTCT is the principal source of HIV infection in children in southern Africa and that current initiatives to increase the availability and effectiveness of PMTCT should result in reductions in HIV prevalence in children over time. Effort should be made to encourage HIV testing in children because, despite low overall ART coverage, children who are aware of their HIV status were highly likely to be on treatment.Dataset S1(CSV)Click here for additional data file.


Furthermore, the erosion resistance of graphene nanosheets-based epoxy resin coating was investigated by electrochemical measurement. The results showed also that the Rrcco (ca. 0.3 mm/year) of graphene nanosheets-based epoxy resin coating was far lower than neat epoxy resin (1.3 mm/year). Thus, this approach provides a novel route for improving erosion resistance and mechanical-thermal stability of polymers coating, which is expected to be used in mechanical-thermal-corrosion coupling environments.The graphene nanosheets-based epoxy resin coating  was prepared by a situ-synthesis method. The effect of polyvinylpyrrolidone/reduced graphene oxide (PVP-rGO) on mechanical and thermal properties of epoxy resin coating was investigated using nanoindentation technique and thermogravimetric analysis, respectively. A significant enhancement ( On the situ-synthesis method. Furthermore, nanoindentation technique was used to measure the mechanical properties of GNS-based epoxy resin coating, in which the Young’s modulus, hardness and plastic index could be obtained. Furthermore, erosion resistance of GNS-based epoxy resin coating was also determined by electrochemical measurements. These results are very important for improving the erosion resistance of polymer coating, which is expected to be used in multi-field coupling environments.In this study, the GNS-based epoxy resin coating was prepared by the −1 were attributed to the location of the carbon-related D peak and the location of the carbon-related graphene peak, indicating the dispersion of the graphene in the epoxy resin matrix . Corrosion current (Icorr) is determined by superimposing the straight line along the linear portion of the cathodic or anodic curve and extrapolating it through Ecorr. Corrosion rate  is calculated from the following equation [EW is the equivalent weight (g), corrI is the corrosion current density (μA/cm2) and ρ is the density (g/cm3).All electrochemical measurements of corrosion potential and corrosion current were performed on a Volta Lab model 21 Potentiostat/Galvanostat in a standard corrosion test cell equipped with a saturated calomel reference electrode (SCE) and a working electrode, and all experimental data were repeated at least three times. The electrolyte was an aqueous solution containing 3.5 wt % sodium chloride. Open circuit potential (OCP) at the equilibrium state of the system was recorded as the corrosion potential [Ecorr (V)equation : (4)Rcorsitu synthesis method, and the graphene modified by PVP could be well dispersed in the epoxy resin matrix and demonstrated good adhesion with epoxy resin matrix. A significant enhancement in the mechanical and thermal properties of epoxy resin coating was obtained with a 0.7 wt % loading of PVP-rGO, such as ca. 213% and 73 °C increase in Young’s modulus and thermal stability, respectively. At the same time, the plasticity index of epoxy resin coating decreased with increases in PVP-rGO content, in which the reduction of this parameter at 0.7 wt % loading was 26.9% compared with that of epoxy resin coating, indicating a significant enhancement in creep properties of GNS-based epoxy resin coating. Furthermore, erosion resistance of GNS-based epoxy resin coating was also investigated by the electrochemical measurements and immersion corrosion experiment in NaCl solution, respectively. It indicated that the GNS-based epoxy resin coating with higher mechanical and thermal properties had better erosion resistance. These results provide a novel route for improving mechanical and thermal properties of inhibited erosion polymer coating, which is expected to be used in thermal-mechanic-corrosion coupled environments.The GNS-based epoxy resin coating was prepared by a


The aim of the US dialysis Prospective Payment System bundle, launched in January 2011, was reduction and more accurate prediction of costs of services, whilst maintaining or improving patient care. Dialysis facilities could either adopt the bundle completely (100%) in the first year of launch, or phase-in (25%) over four years. Differences in practice patterns and patient outcomes were hypothesized to occur in facilities that phased-in 25% compared to those that did not.Data are from STEPPS, a study of 51 small dialysis organization facilities designed to describe trends in dialytic treatment before and after bundle implementation. Baseline was defined as October-December 2010; follow-up as January-December 2011. Facility- and patient-level data were collected at enrollment and regularly thereafter. Cox proportional hazards and linear multi-level models were used to estimate the effect of opting-in 25% (vs. 100%) on practice patterns and clinical outcomes.12 facilities (patient n = 346) opted-in 25% and 37 facilities (patient n = 1296) opted-in 100% to the dialysis bundle. At baseline, patients at 25% facilities were primarily covered by Medicare, were more likely to be black, and were receiving higher monthly epoetin alfa (EPO) doses. Throughout 2011, patients in 100% facilities received lower monthly EPO doses, and had lower mean hemoglobin concentrations; hospitalization and mortality rates were numerically lower in 25% facilities but not statistically different.The economic pressure for dialysis providers to work within an expanded composite rate bundle whilst maintaining patient care may be a driver of practice indicator outcomes. Additional investigations are warranted to more precisely estimate clinical outcomes in patients attending facilities enrolling into the bundle 100% relative to the previous fee-for-service framework.The online version of this article (doi:10.1186/s12882-015-0059-8) contains supplementary material, which is available to authorized users. In the United States, over 400,000 individuals have end stage renal disease (ESRD) requiring dialysis treatment to sustain life . PatientIn January 2011 the updated prospective payment system (PPS) for ESRD  was launched, with the objective of reducing costs of services and more accurately predicting expenditures while maintaining or improving patient care . InjectaBecause LDOs have both a larger patient-base over which to distribute costs and are able to negotiate volume rebates, it was postulated that they may have an advantage over the IDOs and SDOs in managing the financial implications of the PPS -8. ShiftWe used data from the Study to Evaluate the Prospective Payment System Impact on Small Dialysis Organizations (STEPPS), a study designed to describe trends in dialytic treatment before and after implementation of the expanded bundle in a representative sample of SDO facilities.STEPPS was a multi-center prospective observational cohort study of patients receiving care in 51 dialysis facilities that were members of SDOs within the United States. An SDO was defined as a dialysis provider that was either a stand-alone facility or part of a chain with 50 or fewer facilities. Facility and patient selection for STEPPS has been described previously . BrieflyData were collected between October 2010 and September 2012. For this analysis, we used data between October 2010 and December 2011. The baseline period was defined as October 1, 2010 to December 31, 2010; follow-up was from January 1, 2011 to December 31, 2011 (to capture the first year of the expanded PPS when facilities phasing-in were enrolled 25%).The outcomes of interest for this study include ESA dosing patterns (type/route), medication use, hemoglobin concentrations and other laboratory parameters, hospitalization events, and mortality. Patient- and facility-level data were obtained using standardized electronic data collection forms. Patient-level demographic, comorbidity, anthropometric, laboratory, and medication data were collected at enrollment and scheduled intervals thereafter. Data on hospitalization events (dates and diagnoses), death, and other censoring events were recorded by the facility throughout follow-up. Although data on red blood cell transfusion were collected, we know these data to be under-reported , and thaBaseline characteristics are described for patients on study between October and December 2010. Study participants were required to be receiving in-center hemodialysis (HD); those on peritoneal dialysis or home hemodialysis were excluded due to small numbers. Dialysis modality was assessed monthly. For patients switching modality, only patient-time on HD was included. For the 3% of patient-months with missing modality information, 17% of this missing data were imputed based on information available in the prior and subsequent months.a priori specified acceptable range were set to missing. Medications such as iron, vitamin D, cinacalcet, and phosphate binders are reported as any use (yes/no) within each quarter. Dosing route was obtained for ESAs [intravenous (IV) vs. subcutaneous (SC)] and for iron and vitamin D ; both are reported as last administered route within the quarter.Erythropoiesis stimulating agent (ESA) use is reported as the total monthly dose administered in the last eligible month of each quarter. For this analysis, only epoetin alfa values are reported as no darbepoetin alfa use was recorded in the 25% facilities. Laboratory values are reported as the last non-missing value from the last eligible month in each quarter; values outside an For each patient, person-time accumulated from January 1, 2011 or date of eligibility to the end of follow-up defined as the first of the following events: death, withdrawal from study, facility transfer, or December 31, 2011. Event rates were estimated as first events  or total events  divided by the person-time at risk. For patients who switched dialysis modality during follow-up, person-time continued to accumulate for 60 days post-switch to avoid under-assessment of events unrelated to modality switch. Person-time was not extended past December 31, 2011.t-test, chi-square, or Wilcoxon, as appropriate, and by calculating the standardized difference which is comparable to the z-score of a standard normal distribution and, unlike the p-value, is unaffected by sample size . Causes of death were well-balanced across facility type, with cardiovascular as primary  followed by unknown cause . Similarly, causes of hospitalization were relatively well-balanced across facility type, although cardiovascular-related reasons were nominally higher in 100% facilities . After adjustment, patients receiving treatment in facilities that opted-in 25% (vs. 100%) experienced numerically fewer deaths [RR (95% CI) = 0.78 ], fewer hospitalizations [RR (95% CI) = 0.81 ], and fewer composite events [RR (95% CI) = 0.80 ], received higher mean monthly EPO doses , achieved higher mean hemoglobin concentrations [11.3  vs. 10.9  g/dL], had a smaller percentage of patients with hemoglobin concentrations <10 g/dL [RR (95% CI) = 0.54 ] and a greater percentage of patients with hemoglobin concentrations ≥12 g/dL [RR (95% CI) = 1.85 ]  vs. those electing to receive reimbursement under the bundled payment system in 25% increments over four years (i.e. control group). Leveraging this “natural experiment,” we evaluated the effect of policy changes on dialysis practice patterns and patient outcomes in a representative sample of SDOs during the first year of the expanded composite rate dialysis bundle .The majority of STEPPS facilities opted fully into the dialysis bundle, with 24% choosing to phase-in 25% over four years. As was anticipated by clinicians, dialysis organizations, and policy analysts -15, we oWhile the majority of patient and facility characteristics were relatively balanced among the 25% and 100% facilities before bundle implementation, the few exceptions are revealing. Notably, 25% facilities were more likely to have greater proportions of patients who were black, who were receiving higher monthly EPO doses, and had Medicare as their primary payer. Dialysis providers understand the cost drivers within their facilities; critical evaluation of their patient and payer case-mix and pre-PPS ESA utilization could have easily played a role during the opt-in decision-making process. After statistical adjustment, we continued to observe higher EPO doses and higher mean hemoglobin levels in 25% facilities. However, upon evaluating hospitalizations, mortality, and their composite, we observed effects in the 25% facilities to be numerically lower and directionally consistent, but statistically imprecise. Our findings are consistent with those reported from the United States Renal Data System (USRDS) .Previously, we found changes in anemia management to be more pronounced among black patients as compared to other racial groups . The DPMet al. report an 11.8% decrease in EPO doses and a concomitant increase in the percentage of patients prescribed IV iron, with no change in mean hemoglobin levels (10.4 g/dL before and after the policy change) [et al. evaluated changes in clinical outcomes before and two years after implementation of capitation and observed only minor differences determined to be independent of case-mix [Outside the US there are has been a limited number of investigations assessing the effects of bundling policies on patient outcomes in the dialysis setting. In 2006, Japan implemented a bundled payment policy to cover outpatient hemodialysis therapy with the stated aim of lowering dialysis payments by 4%. Comparing cross-sections of patients in Japanese DOPPS facilities before and after the implementation of the payment policy, Hasegawa  change) . Portugacase-mix . Notablycase-mix . In contOur study is limited in several ways. Sparse data are available prior to PPS implementation during which we evaluated differences in patient- and facility-level characteristics (limited to Q4 2010). Dialysis providers may have begun altering their practice patterns early in 2010, as noted by the USRDS  and furtIn summary, STEPPS is composed of a representative sample of SDOs that provides valuable insights into the effects of the ESRD PPS on practice patterns and patient outcomes. CMS’s implementation of the ESRD expanded composite rate dialysis bundle is an important policy change with substantial implications, given the interest in containing health care costs for other disease conditions via introduction of future bundled payment policies. Leveraging the 25% vs. 100% analytic framework allows us to evaluate policy changes on patient care in a timeframe during which dialysis providers were subject to similar pressures but with differential time horizons. Herein, we observe pronounced effects on practice indicators which are thought to be economically driven, and which might have been anticipated, given the dialysis providers’ need to maintain patient care within a capitated payment structure without being at an economic disadvantage as a result of continuing to treat dialysis patients. Nonetheless, we note that the decision to choose whether to opt in 100% or phase-in 25% over four years was likely a complex one, given each center’s unique situation regarding costs, economic acumen and nimbleness (requiring a sophisticated understanding of the QIP and resulting effects on future payment years), patient and payer case-mix, and stated mission. Given that these results are from a relatively small subset of the entire population affected by the dialysis bundle, additional investigations are warranted to provide more precise estimates of the clinical outcomes experienced by patients in centers enrolling into the bundle 100% relative to the previous fee-for-service capitation framework.


Uranotaenia unguiculata mosquitoes in Austria. Phylogenetic analyses suggest that the new strain constitutes a ninth WNV lineage or a sublineage of WNV lineage 4.West Nile virus (WNV) is becoming more widespread and markedly effecting public health. We sequenced the complete polyprotein gene of a divergent WNV strain newly detected in a pool of  West Nile virus (WNV), the most widespread flavivirus, is distributed throughout Africa, Asia, Europe, and Australia, and since 1999, WNV has also been present in the Americas  (GenBank accession nos. FJ154906–49 and FJ159129–31). To determine whether Ur. unguiculata mosquitoes in Austria and the Czech Republic also host WNV, we investigated the mosquitoes collected in 2013 for the presence of WNV, focusing on lineage 4 viruses.WNV is maintained in an enzootic cycle between mosquitoes and wild birds . The relative abundance of Ur. unguiculata mosquitoes among the total collected in Austria was 0.42%. During August 2013, ≈39,000 mosquitoes were trapped at 2 fish ponds (Nesyt and Novy) in Sedlec, Czech Republic, near the border with northeastern Austria. A total of 47 female Ur. unguiculata mosquitoes were grouped into 4 pools . The relative abundance of Ur. unguiculata mosquitoes among the total collected in the Czech Republic was 0.12%.During May–October 2013, ≈11,300 female mosquitoes belonging to 13 species were trapped at 4 sites in Lake Neusiedl-Seewinkel National Park in Burgenland State, Austria. Mosquito species were determined according to morphologic criteria (Uranotaenia unguiculata-Lake Neusiedl-Austria-2013 (WNV-Uu-LN-AT-2013). Primer sequences and amplification protocols are available upon request.The mosquito pools were homogenized in RNase-free water, and RNA was extracted by using the QIAamp Viral RNA Mini Kit . The samples were screened for the presence of flavivirus nucleic acid by reverse transcription PCR, using universal flavivirus primers MAMD . Glycosylation of NS1 at these 3 positions has been implicated in neuroinvasiveness  or can be grouped into lineage 4 as sublineage 4c, with the strains from Russia and Spain as sublineages 4a and 4b, respectively. However, the short sequence available for the strain from Spain does not allow a clear-cut conclusion to be drawn with regard to lineage 4. We suggest that future designation of new WNV lineages should be restricted to viruses for which at least the complete polyprotein gene sequences have been determined. In addition, rules for defining virus lineages should be established by the International Committee on Taxonomy of Viruses.Ur. unguiculata mosquitoes. These mosquitoes are mainly distributed in the southern half of Europe (Ur. unguiculata mosquitoes seem to be an indigenous species, which was first reported in 1970 (Ur. unguiculata mosquitoes have been detected only in Moravia, in the southern part of the country (Ur. unguiculata mosquitoes feeding on mammals, including humans, they feed mainly on amphibians and reptiles (Strain WNV-Uu-LN-AT-2013 has been detected only in The pathogenicity of strain WNV-Uu-LN-AT-2013 in humans and animals has not been elucidated. Genetic data show that the strain carries typical WNV pathogenicity markers and suggest that WNV-Uu-LN-AT-2013 is not restricted to mosquitoes. Additional monitoring studies involving cell culture and animal isolation experiments are necessary to evaluate the pathogenic potential of this virus for humans and animals.


Allogeneic hematopoietic stem cell transplantation (AHSCT) is a curative treatment for a wide variety of hematological diseases. In 30% of the cases, a geno-identical donor is available. Any other situation displays some level of human leukocyte antigen (HLA) incompatibility between donor and recipient. Deleterious effects of anti-HLA immunization have long been recognized in solid organ transplant recipients. More recently, anti-HLA immunization was shown to increase the risk of primary graft failure (PGF), a severe complication of AHSCT that occurs in 3–4% of matched unrelated donor transplantation and up to 15% in cord blood transplantation and T-cell depleted haplo-identical stem cell transplantation. Rates of PGF in patients with DSA were reported to be between 24 and 83% with the highest rates in haplo-identical and cord blood transplantation recipients. This led to the recommendation of anti-HLA antibody screening to detect donor-specific antibodies (DSA) in recipients prior to AHSCT. In this review, we highlight the role of anti-HLA antibodies in AHSCT and the mechanisms that may lead to PGF in patients with DSA, and discuss current issues in the field. Allogeneic hematopoietic stem cell transplantation (AHSCT) is a life-saving procedure for various hematological and immunological conditions. Nowadays ~30% of AHSCT procedures are performed with human leukocyte antigen (HLA)-identical siblings. Any other situation displays some level of HLA incompatibility between donor and recipient, even in 10/10 matched unrelated donors where a mismatch for HLA-DPB1 and/or DRB3, B4, or B5 is present in 80% of the cases. Deleterious effects of anti-HLA immunization have long been recognized in solid organ transplantation (Complement-dependent lymphocytotoxicity (CDC) tests that were used initially have been widely replaced by bead-based assays, such as the single antigen flow bead (SAFB) assays that are more sensitive and resolutive . This te“Natural” anti-HLA antibodies can exist in healthy, non-transplanted, non-transfused, non-parous individuals, at a prevalence estimated to be between <1 and 5% , 7. SomeBesides natural antibodies, main causes of anti-HLA antibody development are pregnancy, blood product transfusion, and previous transplantation. The incidence of anti-HLA immunization through platelet transfusion was strongly decreased by leukocyte depletion of blood products: in a large trial, it plummeted from 45% in controls to 17% in non-parous patients who received leukocyte-depleted platelets . Howeverde novo post-transplantation DSA have been correlated to primary graft failure (PGF). PGF includes graft rejection, defined by the inability to achieve a neutrophil count of 0.5 g/l for three consecutive days at day 28 post transplantation in the absence of donor hematopoiesis. It also includes poor graft function that is a failure to achieve adequate blood counts  for three consecutive days in the presence of complete donor hematopoiesis   .In haplo-identical transplantation, only one gene for each locus is matched between donor and recipient. This procedure is undergoing a rapid development because it offers the possibility of having a donor for AHSCT to nearly every patient. Thus, it was crucial to investigate the impact of DSA in this setting. In a retrospective case-control study, which included 24 patients who received T-depleted haplo-identical transplantation with reduced intensity conditioning (RIC), the incidence of pre-transplant DSA was 21%. Risk of graft rejection was increased from 5% in the non-DSA group to 60% in the group with DSA . More reIn this setting, only HLA-A, -B, and -DRB1 matching is searched for, but not absolutely required. An effect on graft rejection of antibodies against these antigens when mismatched was found in a study on 386 adult patients with MAC conditioning. Importantly, this study was done with single cord blood unit (CBU), which on its own probably partly explains the high rates of graft failure (83% in group with DSA versus 32% in group without DSA) . In a stSeveral studies have shown that higher MFI of DSA were associated with an increased rate of graft failure , 42, 43.Very little is known regarding the role of donor-derived anti-HLA antibodies. Donor-derived antibodies might emerge in recipients of AHSCT because the graft contains memory B cells that can differentiate into plasma cells that could be specific for a given recipient HLA determinant. One study reported the frequent detection of anti-HLA antibodies in patients who underwent AHSCT from anti-HLA antibody-positive donors . One groThe most widely used strategy in case of pre-transplant DSA is to identify and select another donor. Since this is not always possible, several reports mentioned successful strategies to reduce plasma DSA strength prior to transplantation. These reports most often describe single patients or very small series and are possibly biased since failure to reduce DSA might have remained unreported in many cases. The most frequent strategy involved B-lymphocyte depletion with rituximab and plasma exchanges , 39 someThere is no consensus regarding the appropriate titer at which a strategy of antibody reduction should be applied in HSCT, as there is no consensus on the clinically relevant threshold for DSA strength. There is no consensus in organ transplantation either, but recent reports strongly suggest that DSA clinical impact occur earlier as its serum strength increases at time of transplantation , 47 In tIn conclusion, although the mechanisms underlying the association between anti-HLA DSA and graft rejection in AHSCT are unclear, numerous studies report on a deleterious effect of DSA on AHSCT engraftment, without clearly identifying innocuous HLA loci. Further studies are needed, especially regarding the cut-off of MFI values that have clinical consequences on graft survival, and the impact of anti-DP antibodies, since mismatches are most frequent in this locus even for 10/10 matched unrelated donors.SMZ wrote the manuscript. PL, JLT and SCZ commented on the manuscript at all stages of writing and edited it. All authors listed have made substantial, direct, and intellectual contribution to the work and approved it for publication.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.


Most studies of microRNA (miRNA) and disease have examined tissue-specific expression in limited numbers of samples. The presence of circulating miRNAs in plasma samples provides the opportunity to examine prospective associations between miRNA expression and disease in initially healthy individuals. However, little data exist on the reproducibility of miRNAs in stored plasma.We used Real-Time PCR to measure 61 pre-selected microRNA candidates in stored plasma. Coefficients of variation (CVs) were used to assess inter-assay reliability (n = 15) and within-person stability over one year (n = 80). Intraclass correlation coefficients (ICCs) and polychoric correlation coefficients were used to assess within-person stability and delayed processing reproducibility .Of 61 selected miRNAs, 23 were detected in at least 50% of samples and had average CVs below 20% for inter-assay reproducibility and 31 for delayed processing reproducibility. Ten miRNAs were detected in at least 50% of samples, had average CVs below 20% and had ICCs above 0.4 for within-person stability over 1–2 years, six of which satisfied criteria for both interassay reproducibility and short-term within-person stability  and two all three types of reproducibility (miR-27b-3p and -26a-5p). However, many miRNAs with acceptable average CVs had high maximum CVs, most had low expression levels, and several had low ICCs with delayed processing.About a tenth of miRNAs plausibly related to chronic disease were reliably detected in stored samples of healthy adults. Micro RNAs (miRNAs) have recently emerged as key post-transcriptional regulators of gene expression. MiRNAs are ~22bp single-stranded RNA segments that silence gene expression by binding to complementary messenger RNA (mRNA). This binding represses translation and speeds mRNA degradation . Unlike Although the number of promising studies examining miRNAs related to CVD and cancer has grown substantially in recent years, most studies in humans have been cross-sectional and generally limited to 300 or fewer subjects . Large, To evaluate the feasibility of miRNA measurement in large-scale clinical studies, we constructed experiments to examine the inter-assay reproducibility of miRNA in stored plasma, measured by high-throughput quantitative Real-Time PCR (qRT-PCR). Many studies that span a large geographical region require specimen processing delays while samples are shipped to a central laboratory ,5,6. To The Health Professionals Follow-up Study (HPFS) began in 1986 with the recruitment of 51,529 male health professionals age 40 to 75 years, 18,225 of which donated blood samples between 1993 and 1995 . On arriTo assess inter-assay reproducibility, we used two aliquots of EDTA plasma from each of 15 HPFS participants . These pTo assess short-term reproducibility within participants, we used two EDTA plasma samples from 80 HPFS participants donated at two distinct times with a mean of 9.4 (range 6.5 to 12.9) months apart. These plasma samples were mailed overnight to a central laboratory in a styrofoam container with an icepack (at approximately 4°C) and stored in the vapor phase of liquid nitrogen for 13–14 years. We included samples from 40 healthy and 40 less healthy participants , BMI , and/or hypertensive state), hypothesizing that the larger degree of inter-individual variability in health status might lead to greater inter-individual variance and therefore higher intra-class correlation.We selected 61 candidate miRNAs that have been reported to be detectable in circulation and have been cross-sectionally associated with cardiovascular disease ,16,17,18MiRNAs were measured in blinded samples by the High Throughput Gene Expression Core Laboratory at the University of Massachusetts Medical School . All reproducibility and delayed processing samples were measured on one plate in August 2013, and all within-person stability samples were measured on a second plate in January 2014. After thawing, plasma samples were inverted 10 times rather than centrifuged to prevent possible lysing of white blood cells that may be present in plasma and contain their own miRNAs.Total RNA was isolated and purified using the miRCURY RNA Isolation Kit-Biofluids  and manufacturer’s protocol. Isolated RNAs were reverse-transcribed into cDNA using the TaqMan MicroRNA Reverse Transcription Kit  and manufacturer’s protocol. Primers were used for 61 candidate miRNAs that have been cross-sectionally associated with CVD in published studies. All cDNA samples were stored at -80°C until Real-Time PCR analysis.After reverse transcription, a preamplification reaction was performed using the TaqMan PreAmp Master Mix 2X  and the MegaPlex Human PreAmp Primer Pools Set v3.0  using the manufacturers’ protocol. Real-Time PCR reactions (qRT-PCR) were performed using the high-throughput BioMark Real-Time PCR system  and a 96-well plate. Further details have been described previously.We normalized the raw cycle threshold (Ct) values for our main analysis using an adaptation of the global mean normalization method ,21. MethWe imputed a Ct value of 28, the lower limit of detection for this particular assay, for samples with undetectable levels . A single copy of miRNA can be detected in 26–27 Ct in the BioMark System compared to 36–37 Ct in conventional qRT-PCR platforms. We calculated the CV for each miRNA as an average of each participant’s within-subject CV and considered CVs below 20% acceptable . We calcWe also calculated the ICC to compare samples with 0, 24, and 48 hours delay to processing and samples collected one year apart. The ICC is the ratio of between-person variance to the total variance (between- and within-person variance) and takes into account absolute miRNA expression levels as measured by Ct value. We included the first within-person stability measurement (n = 80) in the delayed processing ICC calculation because the between-person variance was close to zero for most miRNAs among the young, healthy male volunteers. The combination of 80 older, less healthy HPFS participants and 12 young, healthy volunteers is more representative of a typical study population and generates more meaningful ICCs.Between- and within-person variances were calculated using a mixed model where the participant was the random variable. We considered an ICC ≥ 0.4 adequate as lower values would greatly reduce our power to detect true and existing associations between miRNAs and disease in epidemiologic studies ,22. FurtThe study protocol was approved by the Institutional Review Board of the Brigham and Women's Hospital and by the Harvard T. H. Chan School of Public Health Human Subjects Committee Review Board and all participants provided written informed consent.Expression levels ranged from 6.6 Ct to 28, where 28 represents no expression or zero copies of miRNA detected . Many miTwenty-four of 61 miRNAs measured for the inter-assay reproducibility experiment had detectable expression levels in at least half of the study samples and 19 were detectable in ≥ 80% of samples . Among tThirty-three of 61 miRNAs measured for the delayed processing experiment had detectable expression levels in at least half of the study samples and 29 were detectable in ≥ 80% of samples . Among tForty-one of 61 miRNAs measured for the within-person stability experiment had detectable expression levels in at least half of the study samples and 33 were detectable in ≥ 80% of samples . Among tOf 61 selected miRNAs, 24 (39%) were detected in at least 50% of inter-assay reproducibility samples, 33 (54%) in at least 50% of delayed processing samples, and 41 (67%) in at least 50% of within-person stability samples. Among those miRNAs detected in at least 50% of samples, 23 (96%) had average CVs below 20% for inter-assay reproducibility, 31 (94%) for delayed processing, and 41 (100%) for within-person stability over one year. Twelve miRNAs had acceptable CVs and were expressed in at least 50% of samples in all three experiments.The 40 miRNAs with low within-person stability ICCs are not necessarily poorly measured, but may change acutely as a result of environmental or phenotypic changes. These rapidly changing miRNAs could be ideal markers of disease presence, severity, and subtype and may thus have substantial clinical relevance. However, for the purpose of risk prediction, these miRNAs are less stable and unlikely to predict risk of experiencing a cardiac event years in the future, especially with a single measurement. Although CVs were below 20% for most miRNAs, many of these miRNAs had high maximum CVs, where at least one individual had low reproducibility. Furthermore, although 31 miRNAs had acceptable CVs for the delayed processing experiment, several had low ICCs, indicating that when possible, temporary storage at 4°C before processing should be avoided.Unlike the CV, the ICC takes within-assay variability into account relative to total variation. Therefore a higher CV may be acceptable if there is large between individual variation and a high ICC, but may not be acceptable if there is small between-individual variation and a low ICC, as in this particular case, because laboratory variability could be larger than between-person differences . Our delUsing standardized values for the short-term reproducibility study, total variation in miRNA expression was reduced and therefore ICCs were lower. However, standardizing with a small set of CVD-related miRNAs may not be an optimal normalization method. Our results suggest that using non-standardized candidates shows useful reproducibility, but further testing with truly global means may be necessary for adequate normalization.It is difficult to calculate reproducibility for several miRNAs that were not detectable in most samples. If some miRNAs are only expressed under certain conditions, such as when disease is present, they may not have performed well in our experiments because the majority of our study participants were healthy. This does not necessarily mean they are poor biomarkers; if these miRNAs were only detectable when preclinical disease is present, they would represent ideal biomarkers for detection of asymptomatic disease. Nonetheless, where miRNAs may be studied specifically within healthy individuals, such as relating them to other biomarkers, our results suggest that a pilot testing phase is highly desirable to minimize futile studies.Our study has several strengths, including the evaluation of inter-assay, delayed processing, and within-person stability over the short term using the gold-standard for gene expression, qRT-PCR. We focused our study on miRNAs that have been previously reported to be associated with varying types of cardiovascular disease and therefore represent interesting targets for future research. We further narrowed our miRNA target selection to candidates with established likelihood of detection in plasma , which are the most likely to be used in future epidemiological studies. Finally, to the best of our knowledge, ours is the first to explore reproducibility of miRNAs in long-term stored plasma samples.Our study is not without limitation. We included healthy, male participants which limited between individual variation and miRNA expression levels in our study participants may not represent levels in less healthy men or in women. Furthermore, CVs may vary when using different laboratories and different study populations, such as those at a higher risk of developing chronic disease. Based on our experience, we would encourage investigators to evaluate laboratory, platform and sample-type specific assay performance prior to proceeding with large ventures.In conclusion, six of 61 miRNAs selected met our criteria for acceptable inter-assay and short-term within-person reproducibility among those miRNAs with high expression levels. These miRNAs may represent targets for the future investigation of the associations between circulating miRNA expression and future CHD risk. Although these six miRNAs had acceptable CVs with delayed processing, ICCs were low for four indicating poor reproducibility without controlled temperature and time during processing. Stored plasma from large cohorts represents an exciting opportunity to further explore the biology of at least some miRNAs in the development of chronic diseases like CVD and cancer, however, many questions remain about the minimum and ideal requirements for such sample processing and storage to conduct valid analyses. To date, miRNA-disease relationships are not always reproducible across studies ,26 or diS1 TableBold indicates acceptable results, italics indicates unacceptable results, and NC = not calculated. *Includes first within-person stability measurement (n = 80 HPFS participants).(DOCX)Click here for additional data file.S2 TableBold indicates acceptable results, italics indicates unacceptable results, and NC = not calculated. *Includes first within-person stability measurement (n = 80 HPFS participants).(DOCX)Click here for additional data file.S3 TableBold indicates acceptable results, italics indicates unacceptable results, and NC = not calculated.(DOCX)Click here for additional data file.


Understanding contributors to mortality during the initial phase of tuberculosis (TB) treatment in patients co-infected with HIV would guide targeted interventions to improve survival. The aim of this study was to ascertain the incidence of death during the initial 2 months (new cases) and 3 months (retreatment cases) of TB treatment and to assess correlates of mortality in HIV co-infected patients.OR) with 95% confidence interval. A p value < 0.05 was considered statistically significant.We conducted a hospital-based retrospective cohort study from January 2006 to December 2013 at Yaoundé Central Hospital, Cameroon. We reviewed medical records to identify co-infected TB/HIV inpatients aged 15 years and older who died during TB treatment. Death was defined as any death occurring during TB treatment, as per World Health Organization recommendations. We collected socio-demographic, clinical and laboratory data. We conducted multivariable logistic binary regression analysis to identify factors associated with death during the intensive phase of TB treatment. Magnitudes of associations were expressed by adjusted odds ratio , having extra-pulmonary TB , and one year increase in duration of known HIV infection  were independently associated with death during the intensive phase of TB treatment.The 99 patients enrolled had a mean age of 39.5 (standard deviation 10.9) years and 53% were male. Patients were followed for 276.3 person-months of observation (PMO). Forty nine patients were died during intensive phase of TB treatment. Death incidence during the intensive phase of TB treatment was 32.2 per 100 PMO. Having a non-AIDS comorbidity (aMortality incidence during intensive phase of TB treatment was high among TB/HIV co-infected patients during TB treatment; and strongly associated with extra pulmonary TB suggesting advanced stage of immunosuppression and non-AIDS comorbidities. Early HIV diagnosis and care and good management of non-comorbidities can reduce this incidence. Mycobacterium tuberculosis bacteremia [Tuberculosis (TB) remains the most common serious opportunistic infection in people infected with human immunodeficiency virus (HIV) in sub-Saharan Africa and is the leading cause of death –5. In Cacteremia . Also incteremia . A more cteremia  determincteremia , we repoOur hospital-based retrospective cohort study took place at the Infectious Diseases Unit (IDU) of the tertiary-care Yaoundé Central Hospital (YCH), in Cameroon. Inpatients aged 15 years and older diagnosed with TB from January 1, 2006 to June 30, 2013 who were co-infected with HIV and who died during their TB treatment were included. We excluded all outpatients as more than 70% of those initiating TB therapy as outpatients had incomplete data. We also excluded patients who died before initiation of TB treatment or on the date TB treatment began, cases with missing information on follow-up and missing documentation of outcomes in the IDU/TB registers, and patients with incomplete file information (at least 30% of variables). We reviewed a total of 681 TB yellow cards [For new and retreatment cases, we considered the intensive phase as the first two and the initial three months of anti-tuberculous therapy respectively , 10. FivA death for the purposes of this study was defined as a death from any cause during treatment for tuberculosis as per the World Health Organization’s recommendations . All of Data was extracted, coded, entered, and analyzed using the Statistical Package for Social Science (SPSS) version 21.0 for Windows . Continuous variables were expressed as the mean with standard deviation (SD) or median with interquartile range (IQR). Categorical variables were expressed as numbers with percentages (%). Death incidence ratio was analyzed using Windows Program for Epidemiologists version 11.25 and reported with Fisher’s exact 95% confidence interval (95%CI). All follow-ups were censored at three months and two months for retreatment and new cases respectively. This is because the intensive phase of TB treatment is defined as the first three months of therapy for retreatment of TB and the initial two months for new TB cases .Multiple imputation was used to handle missing data, creating a new data set, which was the average of twenty data sets of imputed values imputation , 20. AllOR) were reported with a 95%CI.Firstly, we conducted univariate binary logistic regression analysis. After this, we performed multivariable  binary logistic regression analysis to identify independent predictors of death during intensive phase of TB treatment. We also performed these univariate and multivariable analyses with categorization of all continuous data . A p value < 0.05 was considered statistically significant. Odds ratios , the median CD4 lymphocyte count was 42 cell/mm3 (IQR 15–101), and the median hemoglobin level was 8 g/dl (IQR 6–9).Patients were followed for 276.3 person-months of observation with median duration of 8.1 days (IQR 3.0–24.9) and mean of 20.4 days (SD 27.9). Among the 99 patients who died during TB treatment, 89 died during the intensive phase of TB treatment. Death incidence during the intensive phase of TB treatment was 32.2 per 100 persons-months of observation. OR 1.84, 95%CI 1.11–3.05, p = 0.018) and the presence of any non-AIDS comorbidity . Independent factors associated with death during intensive phase of TB treatment were having non-AIDS comorbidity , having extra-pulmonary TB  compared to localized pulmonary TB, and one year increase in duration of known HIV infection . These results are reported in In our univariate analysis, two factors associated with death during the intensive phase of TB treatment were extra-pulmonary TB at presentation  as compared to localized pulmonary TB and having non-AIDS comorbidity , was associated with death during intensive of TB treatment. Duration of known HIV infected was not associated with early death when is categorized. These results were reported in After categorization of all continuous data, in multivariable analysis, having extra-pulmonary TB  of death during intensive phase of TB treatment among patients co-infected with TB and HIV. This finding is consistent with other studies reporting high rates of early death in TB/HIV co-infected people during ongoing TB treatment –14. As wOur study also reveals that a diagnosis of extra-pulmonary TB at presentation is a risk factor for death during the intensive phase. Due to the difficultly in diagnosing extra-pulmonary TB in our resource-limited clinical setting, often compounded by patients’ limited financial resources that would allow for timely targeted diagnostic testing, such a diagnosis tended to be made late, at a more advanced stage of disease. Also, extra-pulmonary TB is a marker of more advanced host immunosuppression, as compared to pulmonary TB that remains confined to the respiratory system. The same pathophysiologic process related to increasing immunosuppression is invoked as IRIS, which favors death –27, occuThe presence of a non-AIDS medical comorbidity was associated with death during the intensive phase of TB treatment in our study. This is a novel finding. We have not found any published study implicating this clinical factor in mortality during intensive of TB treatment. One likely explanation is that comorbidities further compromise the health of TB/HIV co-infected patients, or their response to the anti-tuberculous medication, or both, thus contributing to premature death. The care of HIV-infected patients with tuberculosis and non-AIDS related comorbidities should therefore be carefully initiated as soon as possible, with close attention to identification and treatment of non-AIDS illnesses. The plan of care especially during the intensive phase of anti-tuberculous medication prescription, should also focus on and address the comorbidity. Identification of patients with this risk factor, and its optimal management could ameliorate the overall survival of TB/HIV co-infected patients undergoing TB treatment. Another likely contributor to the increased mortality in patients with a non-AIDS comorbidity is that the polypharmacy of multiple drugs , along with the required multidrug antituberculous regimen could result in less adherence to treatment –34, to iWe also found as a factor associated with early death, increasing duration to be known as HIV-infected. This result was found in another study ; and sev3; the 2012 revision instead advised prescribing ART once the CD4 cell count reached 350 cells/mm3. This likely lead to more of our patients being started on ART later in the study period; tuberculosis guidelines however, remained unchanged for the entire period of our study. Some data for body weight, hemoglobin level, white blood cell count, and CD4 count were missing. However, multiple imputations have been used to handle the missing data. Whatever the underlying cause, the missing data underscores the challenges of conducting “real-life” clinical research in resource-limited settings, like Cameroon and other sub-Saharan countries. Our results may, therefore, not be generalizable to all patients with TB/HIV, and particularly to those receiving their treatment on an ambulatory basis. After starting treatment in hospital, patients continued their TB as outpatients; it is thus possible that we underestimated the actual overall death rate as some patients were lost to follow-up. This study thus provides at least a conservative assessment of mortality while providing knowledge of risks and preliminary information to guide future research.Our study has some limitations to consider. It captured patients undergoing treatment for tuberculosis over a consecutive 90-month period, from January 1, 2006 through June 30, 2013. The Cameroonian Ministry of Public Health issued new guidelines for prescribing ART in 2012. Prior to 2012, it was recommended that ART therapy in the setting of HIV be started when the CD4 cell count dropped to less than 200 cells/mmIn our setting, among patients co-infected with TB and HIV undergoing TB treatment, the incidence of death during intensive phase of TB treatment was high. The independent factors associated with mortality during intensive of TB treatment were easily identifiable clinical ones, having extra-pulmonary TB and having non-AIDS comorbidity. Timely consideration and identification of extra-pulmonary TB can help to reduce early death by allowing for prompt initiation of TB treatment at a relatively earlier stage of immunosuppression. Treatment of non-AIDS comorbidities and optimal control of chronic ones, along with reduction in the amount of medications prescribed in order to include only essential pills are straightforward measures that may help to reduce early death. Further studies focused on mortality during initial TB treatment are needed and should include investigation into the causes of death in order to better determine if early deaths are due to inadequate TB control, or to other processes including, but not limited to drug toxicities, IRIS, or worsening of comorbid illnesses.S1 Data(XLSX)Click here for additional data file.