Parmelioid lichens form a species-rich group of predominantly foliose and fruticose lichenized fungi encompassing a broad range of morphological and chemical diversity. Using a multilocus approach, we reconstructed a phylogeny including 323 OTUs of parmelioid lichens and employed ancestral character reconstruction methods to understand the phenotypical evolution within this speciose group of lichen-forming fungi. Specifically, we were interested in the evolution of growth form, epicortex structure, and cortical chemistry. Since previous studies have shown that results may differ depending on the reconstruction method used, here we employed both maximum-parsimony and maximum-likelihood approaches to reconstruct ancestral character states. We have also implemented binary and multistate coding of characters and performed parallel analyses with both coding types to assess for potential coding-based biases. We reconstructed the ancestral states for nine well-supported major clades in the parmelioid group, two higher-level sister groups and the ancestral character state for all parmelioid lichens. We found that different methods for coding phenotypical characters and different ancestral character state reconstruction methods mostly resulted in identical reconstructions but yield conflicting inferences of ancestral states, in some cases. However, we found support for the ancestor of parmelioid lichens having been a foliose lichen with a non-pored epicortex and pseudocyphellae. Our data suggest that some traits exhibit patterns of evolution consistent with adaptive radiation. Molecular data have revolutionized our understanding of evolution, especially in groups with relatively simple morphology and high levels of phenotypic homoplasy, such as fungi. The enormous increase in knowledge has led to a revival of fungal systematics. This is especially true in fungal groups, including lichen-forming fungi, for which vegetative characters have traditionally played an important role in classification and inferring relationships. Lichenized fungi usually form persistent thalli to house their photosynthetic partner and produce a wide array of secondary metabolites that play a variety of roles in the maintenance of the symbiotic association -5. TheseAlthough ancestral character state reconstruction approaches provide powerful tools for assessing trait evolution, the methods are not without problems. Ekman and co-workers  pointed Omphalodiella, subcrustose Karoowia, subfruticose Almbornia, umbilicate Xanthomaculina, and even lichenicolous fungal genus Nesolechia, which has been shown to belong to the parmelioid clade [Parmelioid lichens constitute the largest clade within the speciose family Parmeliaceae ,31. Parmid clade ,31,40. Morphological diversity in parmelioid lichens is not limited to different growth forms, but also includes ultrastructural characters and diverse secondary metabolites. Within the group, two major types of thallus perforations can be found that allow gas exchange of the photosynthetic partner. These include pores in the epicortex and cortical structures that reach the algal layer, which are called pseudocyphellae. The epicortex is a polysaccharide layer about 0.6 μm thick, lying over the cortex -44. PoreIn order to understand phenotypical character evolution in parmelioid lichens, we used a previously published molecular phylogeny of parmelioid lichens . SpecifiParmotrema+Xanthoparmelia clade, Cetrelia+Parmotrema+Xanthoparmelia clade, and the entire parmelioid group  the difference was smaller than 2 units of likelihood. However, in order to efficiently process the large number of analyses and results, we decided to use unrestricted models for all analyses.The results of the ancestral character state reconstructions for binary and multistate coding and for the three types of analysis are summarized in Cetrelia+Parmotrema+Xanthoparmelia clade (node 11) in the binary-coded MP and ML, and multistate-coded MP and ML-BMCMC analyses. A foliose growth form received significant support in the binary- and multistate-coded MP, binary- ML, and multistate-coded ML-BMCMC analyses for the ancestral node of the Parmotrema+Xanthoparmelia clade (node 10) but lacked significant support in the binary ML-BMCMC and multistate ML analyses. For the Parmotrema clade (node 1) foliose growth form received strong support in both binary- and multistate-coded MP reconstructions, the binary-coded ML analysis, and multistate-coded of ML-BMCMC analyses. A foliose ancestral state was also inferred in the binary-coded ML-BMCMC and multistate-coded ML reconstructions, although it lacked support (see Xanthoparmelia clade (node 2) was reconstructed as having a foliose growth form in the MP and ML-BMCMC method with significant support using multistate-coding method. However, an ancestral foliose growth form for Xanthoparmelia lacked significant support in the binary-coded MP and ML-BMCMC-based methods, and in ML analyses using both coding methods. A foliose growth form was reconstructed as ancestral for the Cetrelia clade (node 3) in all the analyses using both coding methods. The ancestral growth form for node basal to the Parmelina clade (node 4) was reconstructed as a foliose, with significant result in both coding MP, ML and ML-BMCMC analyses. For node 5 (base of Hypotrachyna clade) all reconstruction methods significantly inferred a foliose growth form as the ancestral state. They also yielded significant support for a foliose growth form at node 6, which included the genus Parmeliopsis. All analyses estimated a foliose growth form as ancestral for the Nipponoparmelia clade (node 7) with significant support. A foliose growth form was also reconstructed for the ancestor of the Parmelia clade (node 8) with significant support by all methods, with the exception of the multistate-coded ML reconstruction, which lacked support. The MP and ML methods using both coding analyses, and multistate-coded ML-BMCMC estimated the ancestral character state for the growth form of the Melanohalea clade (node 9) as being foliose, while the binary-coded ML-BMCMC analysis did not yield significant results. Results of ancestral character state reconstructions of the following four growth forms, foliose, subcrustose, crustose and umbilicate are depicted in port see . The ancResults of the reconstructions of epicortical structure are presented in Cetrelia+Parmotrema+Xanthoparmelia clade (node 11) was inferred as having a non-pored epicortex with significant support in both MP, ML-BMCMC and binary-coded ML reconstructions, but it lacked support in the multistate-coded ML analysis  were less clear (Parmotrema clade (node 1). . . The ML node 1). . Xanthoparmelia clade (node 2) (Cetrelia clade (node 3), a non-pored epicortex and the presence of pseudocyphellae were reconstructed with very significant support in all analyses (Parmelina clade (node 4), a pored epicortex was consistently estimated in all analyses, except binary-coded ML-BMCMC where it lacked support. However, the absence of pseudocyphellae was only reconstructed in the MP and multistate-coded ML-BMCMC analyses (Hypotrachyna (node 5) and Parmelinopsis (node 6), a pored epicortex and the absence of pseudocyphellae were estimated in all analyses (Nipponoparmelia (node 7) and Parmelia (node 8) clades in all analyses , a non-pored epicortex and the presence of pseudocyphellae was reconstructed in ML and binary-coded ML-BMCMC analyses with significant support. However, this reconstruction lacked support in the MP and multistate-coded ML-BMCMC analyses   and 4. Fanalyses , 4. For analyses . For theanalyses , 4. A noanalyses , 4. For analyses , 4.Cetrelia+Parmotrema+Xanthoparmelia clade (node 11) and the Parmotrema+Xanthoparmelia clade (node 10), the multistate-coded ML analysis inferred usnic acid as the ancestral character, while the other analyses did not reveal significant results (Parmotrema clade) the binary-coded ML analysis estimated the presence of usnic acid as ancestral character with significant support (Xanthoparmelia clade (node 2), the multistate-coded ML analysis recovered usnic acid as the ancestral character state with significant support, while the other analyses did not significantly support this ancestral character state (Cetrelia clade (node 3), the Parmelina clade (node 4), and the Hypotrachyna clade (node 5) with significant support (Parmeliopsis (node 6), while the other methods did not support for any character state with statistical significance (Nipponoparmelia (node 7) and Parmelia (node 8) clades with significant support. For the Melanohalea clade (node 9), melanin was reconstructed as ancestral character state in both coding methods in the MP, ML-BMCMC analyses and the binary-coded ML with significant support. In contrast, usnic acid was inferred in the multistate-coded ML analysis with significant support  in order to test whether unrestricted models perform significantly better than restricted models . Using tFor each analyzed node and each character, the ancestral state for this node was successively fixed to each of the possible states (e. g. 0/1 for binary coding) using the 'fossil' command of BayesTraits. Using an unrestricted model of character change, the maximum likelihood for the tree with the ancestral state fixed at this node was estimated and the difference in log likelihood calculated. A difference of 2 log likelihood units was considered significant evidence .In order to include topological uncertainty into the ancestral state reconstruction, we used the “Trace character over trees” method of Mesquite. 1000 trees were randomly sampled from the post-burnin of the 3GENE Bayesian analysis of , and an In contrast to likelihood and Bayesian methods, maximum parsimony does not take into account branch lengths when reconstructing ancestral states. We used Mesquite 2.75  to carryTable S1Results of the different reconstruction methods for parmelioid lichens at selected nodes. Shown are ln likelihood differences , percentage of trees for which the character state has been significantly reconstructed . For ML-BMCMC and MP, values missing to reach a total of 1.00 represent trees for which the node was not present in the set of source trees used for the reconstruction. . All analyses were performed using the binary coding  data set. For MP probabilities only unequivocal reconstructions were counted. For reconstructed nodes see Figure1. (DOCX)Click here for additional data file.Table S2Results of the different reconstruction methods for parmelioid lichens at selected nodes. Shown are ln likelihood differences , percentage of trees for which the character state has been significantly reconstructed . For ML-BMCMC and MP, values missing to reach a total of 1.00 represent trees for which the node was not present in the set of source trees used for the reconstruction. . All analyses were performed with the multistate coding state data set . For MP probabilities only unequivocal reconstructions were counted. For reconstructed nodes see Figure 1.(DOCX)Click here for additional data file.


MicroRNA-21 has been proved to be associated with glioma proliferation and invasion; thus, we sought to clarify the clinical value of miR-21 expression in glioma tissues with WHO grade I to IV.One hundred and fifty-two pairs of human gliomas and non-neoplastic brain tissues were evaluated using real-time PCR. The association of miR-21 expression with clinicopathological factors or the prognosis of glioma patients was also analyzed. In this study, survival analysis was performed using the Kaplan-Meier method and Cox’s proportional hazards model.MiR-21 was more greatly expressed in glioma tissues compared to the corresponding non-neoplastic brain tissues (P < 0.001). This observed high miR-21 expression was significantly associated with high pathological grades and the Karnofsky performance score of glioma patients. In addition, overall patient survival for those with low miR-21 expression was significantly longer than those patients with high miR-21 expression (P < 0.001). Moreover, multivariate Cox regression analysis indicated that miR-21 might be an independent prognostic marker for glioma patient survival.Our data show that miR-21 may be a candidate independent marker for gliomas, especially those with high pathological grades, and this could also be a potential therapeutic target for molecular glioma therapy.http://www.diagnosticpathology.diagnomx.eu/vs/1445749171109834.The virtual slide(s) for this article can be found here:  Glioma, the most common primary malignant brain tumor arising from the brain orspinal cord tissue, has a tendency to invade the surrounding brain tissue. Gliomas represent approximately one-third of all intrinsic neoplasms of the central nervous system in both adults and children. The World Health Organization (WHO) classification divides gliomas into grades I through IV, and these values correspond to increasing levels of malignancy, including well-differentiated low grade, astrocytomas and glioblastoma multiforme . Glioma MicroRNAs (miRNAs) are a class of small, evolutionarily conserved, short non-coding endogenous RNA molecules that regulate gene expression at the post-transcriptional level . PrimaryIn the present study, we focus on miR-21, which has been demonstrated to act as either an oncogenic miRNA or an anti-oncomiR (miRNA that negatively regulates oncogenes) in various human malignancies including glioma -15. HoweThis study was approved by the Research Ethics Committee of Tangdu Hospital of Fourth Military Medical University, P. R. China. Written informed consent for the following pathological analysis or experiments was obtained from all of the patients before his/her surgery. All specimens were handled and made anonymous according to the ethical and legal standards.One hundred and fifty-two pairs of glioma tissues including adjacent non-neoplastic brain tissues were selected, and the pathological information was retrieved from the archives of the Pathology Department of Tangdu Hospital, Fourth Military Medical University, P. R. China, from 2000 and 2009. None of the patients had received chemotherapy or radiotherapy prior to surgery. All the samples were resected from primary surgery, and the tumor tissues and the adjacent non-neoplastic brain tissues were divided by tissue laser microdissection . The speU6 gene was used as an internal control to normalize variances. Relative quantification of target miRNA expression was evaluated using the comparative cycle threshold (CT) method. Each sample was examined in triplicate and the raw data were presented as the relative quantity of target miRNA, normalized with respect to U6.MiR-21 expression in glioma and adjacent nonneoplastic brain tissues was measured by real-time quantitative RT-PCR. Total RNA was isolated from frozen samples using Trizol reagent  according to the manufacturer’s protocol. The TaqMan microRNA assay and TaqMan universal PCR master mix were used to detect the expression of miRNA-21, and the Statistical analysis was performed using the SPSS 13.0 for Windows . The associations between miR-21 expression and clinical characteristics were evaluated by Mann-Whitney U test. Survival curves were estimated by the Kaplan-Meier method, and data were analyzed with the log-rank test. Cox proportional hazards analysis was performed to calculate the hazard ratio (HR) and the 95% confidence interval (CI) to evaluate the association between miR-21 expression and survival. In addition, a multivariate Cox regression was performed to adjust for other covariates. A value of P < 0.05 was considered statistically significant.The characteristics of the 152 glioma patients involved in the study cohort are shown in Table U6 gene, the relative miR-21 expression in glioma samples was 20.99 ± 13.04 (mean ± SD), whereas the relative miR-21 expression detected in adjacent non-neoplastic normal tissues was 0.73 ± 0.05  were still alive, but 116 patients (76.32%) died . Kaplan-Meier survival curves suggested that glioma patients with low miR-21 expression had a significantly longer survival time than those with high miR-21 expression . Accurately predicting postoperative outcomes in glioma patients and choosing the correct adjuvant therapy is a present and persistent problem. At this time, traditional pathologic variables are used to make such predictions. Currently, WHO glioma grades are the gold standard for determining glioma patient prognosis, but this information is less useful for individual patients and often fails to discriminate the biological nature of a large number of gliomas. Therefore patients at the same disease stage often have significant discrepancies in survival times. Until now, there are several studies showed that single molecule could be the relapse and prognosis indicator ,18. ThusTo date, studies have shown that miRNA expression is correlated with clinical and biological features of tumors, including tissue type, differentiation and aggressiveness, which can be a potential biomarker for therapy and prognosis ,12-14. MP value < 0.001, 95% confidence interval is 3.12-7.843). All these data indicate that increased expression of miR-21 is correlated with poor glioma patient outcomes. Thus, miR-21 expression may serve as a potential biomarker for overall survival prediction in this patient population.We investigated miR-21 expression in glioma and determined if this marker could predict disease relapse and patient outcomes. We observed that increased miR-21 expression was evident in human glioma tissues compared with non-neoplastic brain tissues. High miR-21 expression in glioma tissues was significantly correlated with aggressive clinicopathological features as well. Kaplan-Meier analysis revealed that glioma patients with high miR-21 expression had poorer overall survival. Moreover, multivariate analysis revealed that high miR-21 expression was a marker of worse patient outcomes which was independent of known clinical prognostic indicators such as KPS and WHO grades. Gliomas with grade I have different genetic abnormality compared with other grade gliomas. In this study, we divided all the gliomas into two groups: low grade glioma (WHO grade I and II) and high grade glioma (WHO III and IV) and performed multivariate analysis. The result also indicated that miR-21 could be the marker of poor outcomes in these two groups (Relative Risk is 4.947, Our results revealed that miRNA-21 was overexpressed in glioma tumors and that high expression level of miRNA-21 was strongly associated with poor prognosis, but further prospective studies are needed to determine the actual clinical relevance of this observation. Our data suggest that miR-21 may be an appropriate prognostic marker for evaluating advanced tumors with high pathological grades and that this marker may assist with the appropriate choice of adjuvant chemotherapy as well as serve as a potential therapeutic target in molecular therapy for glioma.The authors declare that they have no competing interests.ZZ and JZ designed the study. LW participated in the design and coordination, performed the molecular genetic evaluation, and drafted the manuscript. All the patients were followed up by GL and HQ. And DF performed the statistical analysis, and joined into drafting the manuscript. WL, ZZ and JZ all contributed to improving the draft of the manuscript. All authors have read and approved the final manuscript.


Pancreatic cancer, composed principally of pancreatic adenocarcinoma (PaC), is the fourth leading cause of cancer death in the United States. PaC-associated diabetes may be a marker of early disease. We sought to identify molecules associated with PaC and PaC with diabetes (PaC-DM) using a novel translational bioinformatics approach. We identified fatty acid binding protein-1 (FABP-1) as one of several candidates. The primary aim of this pilot study was to experimentally validate the predicted association between FABP-1 with PaC and PaC with diabetes.We searched public microarray measurements for genes that were specifically highly expressed in PaC. We then filtered for proteins with known involvement in diabetes. Validation of FABP-1 was performed via antibody immunohistochemistry on formalin-fixed paraffin embedded pancreatic tissue microarrays (FFPE TMA). FFPE TMA were constructed using148 cores of pancreatic tissue from 134 patients collected between 1995 and 2002 from patients who underwent pancreatic surgery. Primary analysis was performed on 21 normal and 60 pancreatic adenocarcinoma samples, stratified for diabetes. Clinical data on samples was obtained via retrospective chart review. Serial sections were cut per standard protocol. Antibody staining was graded by an experienced pathologist on a scale of 0-3. Bivariate and multivariate analyses were conducted to assess FABP-1 staining and clinical characteristics.Normal samples were significantly more likely to come from younger patients. PaC samples were significantly more likely to stain for FABP-1, when FABP-1 staining was considered a binary variable. Compared to normals, there was significantly increased staining in diabetic PaC samples (p = 0.004) and there was a trend towards increased staining in the non-diabetic PaC group (p = 0.07). In logistic regression modeling, FABP-1 staining was significantly associated with diagnosis of PaC , though age was a confounder.Compared to normal controls, there was a significant positive association between FABP-1 staining and PaC on FFPE-TMA, strengthened by the presence of diabetes. Further studies with closely phenotyped patient samples are required to understand the true relationship between FABP-1, PaC and PaC-associated diabetes. A translational bioinformatics approach has potential to identify novel disease associations and potential biomarkers in gastroenterology. Pancreatic cancer, composed principally of pancreatic adenocarcinoma (PaC), is the fourth leading cause of cancer death in the United States. At diagnosis, more than 85% of patients with PaC have unresectable disease, with a median survival of 4-6 months ,2.PaC is a diabetogenic state with 45-65% of PaC patients having diabetes mellitus (DM). New-onset DM, occurring within approximately 2 years prior to cancer diagnosis, may be a paraneoplastic effect of the tumor itself secondary to a circulating tumor-associated protein that causes insulin resistance and beta-cell dysfunction . Adult pSeveral strategies have been used to identify pancreatic cancer-associated diabetogenic factors, including microarray work, genotyping, quantitative real time polymerase chain reaction, immunohistochemistry, serum analysis, gel electrophoresis and cell culture, and serum radioimmunoassay. These have yielded putative biomarkers, including connexin-26, insulin gene promoter polymorphisms, glucagon/insulin ratio, S1000-A8 calcium binding protein, islet amyloid polypeptide, and insulin-like growth factor-I -10. NoneWe sought to identify molecules associated with PaC and PaC with diabetes (PaC-DM) using an integrative genomics approach, building from our previous methods in intersecting publicly-available gene expression measurements to find DNA variants associated with disease ,12. We iWe have previously described the creation of a database of gene expression microarray experiments across human diseases, built from publicly-available repositories . SpecifiOur resulting data set incorporates over 200 diseases studied across over 350 GEO datasets comprising nearly ten thousand individual microarrays, studied in over 100 tissues. For every gene in the genome measured across microarray platforms, we calculated a change in rank normalized expression level in that gene from normal samples to disease samples, resulting in a calculated measure ranging from +1.0  to -1.0 .Within this database of disease-related gene expression changes, we searched for genes selectively upregulated in pancreatic tissue from pancreatic adenocarcinoma, but not in pancreatic tissue from samples with acute pancreatitis and type I diabetes mellitus; comparison groups were limited to those conditions with robust available GEO data.Of those genes selectively fitting this profile, the rank normalized expression level for the top ten genes ranged from 0.19-0.12 with FABP-1 measuring 0.14, fifth on the list.http://geneticassociationdb.nih.gov/. Type II DM susceptibility genes were compared given the similar phenotype in pancreatic cancer-associated diabetes of beta-cell dysfunction and insulin resistance [We then compared this list of genes selectively upregulated in pancreatic tissue from pancreatic adenocarcinoma with that of genes known to be associated with type II DM from the NIH Genetic Association Database sistance . Type IIWith a list of genes upregulated in both pancreatic adenocarcinoma and type II DM, we then filtered for a biofluid database containing proteins known to be detectable in the blood or urine, integrating protein lists from the Plasma Proteome Institute, the MAPU Proteome database, and the Urinary Exosome database ,18-20. WFormalin fixed paraffin embedded (FFPE) Tissue Microarrays (TMA) were constructed using 148 cores of pancreatic tissue from 134 patients collected by the Stanford University Department of Pathology between 1995 and 2002 from patients who underwent pancreatic surgery. The FFPE TMA were constructed in 2004 using a manual tissue arrayer  following previously described techniques using 1.0 mm cores . The corFigure Biopsies of normal pancreatic tissue are not routinely obtained. Normal samples on our TMA were derived from histologically normal cores obtained from an organ that often also contained a pathologic pancreatic condition. This is common practice in construction of tissue microarrays -23. In tIRB approval was obtained from the Stanford Research Compliance Office, allowing retrospective collection of patients' age, gender, presence of diabetes, alcohol and tobacco use. Clinical data was collected on a standardized form by one physician-abstractor (RNS). Clinical data was obtained from notes in a given patient's chart.Serial sections of 4 um were cut from the TMA blocks, deparaffinized in xylene, and hydrated in a graded series of alcohol. The slides were pretreated with EDTA pH9 buffers for FABP-1 in a microwave step. Immunohistochemistry (IHC) staining was then performed using the DAKO EnVision + System, Peroxidase (DAB),  for FABP-1 . Antibody staining was graded by a single experienced pathologist per standard protocol on a scale of 0-3  based on the extent of cytoplasmic staining in an individual histologic core . FABP-1 2, Fishers Exact test, Wilcoxon Rank Sum with Bonferroni correction, and non parametric Kruskal-Wallis ANOVA) and multivariate analyses (logistic regression with binary outcome variables) to assess FABP-1 staining and clinical characteristics. Statistical analyses were performed using SAS Enterprise Guide 9.2 .Patient data was reported using bivariate , and PaC with diabetes (PaC-DM) samples. PaC-DM samples represent tissue from patients with pancreatic adenocarcinoma-associated diabetes as well as PaC with coexisting chronic type II diabetes. The information to differentiate between these two groups was not available during chart review. Nonparametric analyses revealed a significant difference in FABP-1 staining (using both binary and FABP-1 score) between samples from normal, PaC no DM, and PaC-DM groups, with significantly increased staining as one moves from left to right in the Table To elucidate relationships between the three groups, individual paired tests were conducted using the Wilcoxon Rank sum test with Bonferroni correction for multiple comparisons. There was significantly increased staining in the PaC-DM samples compared to normals (p = 0.004) and there was a trend towards significantly increased staining in the PaC no DM group versus normal (p = 0.07). There was no statistical difference in FABP-1 staining between PaC no DM and PaC-DM groups (0.09).In logistic regression modeling, FABP-1 staining (binary) was significant associated with diagnosis of PaC with an odds ratio of 8.6 . Age was a confounder in this model. Patient gender did not modify the relationship between FABP-1 staining and diagnosis.Using methods in integrative genomics, we predicted an association between FABP-1 and pancreatic adenocarcinoma and pancreatic adenocarcinoma with diabetes. Compared to normal controls, we found a significant positive association between increased FABP-1 staining and PaC on FFPE-TMA, strengthened by the presence of diabetes.FABP-1 is a cytosolic chaperone protein that is expressed in the liver, kidney, lung, and GI tract . The exaPlausibility of FABP-1 as a biomarker for diabetes comes from animal and human data. FABP-1 null mice demonstrate age dependent weight gain, though may be protected from obesity and hepatic steatosis when fed a saturated fat diet -36. In hTo our knowledge, this is the first study to attempt to validate the FABP-1 association for PaC and PaC with DM. The intent of our study was to examine whether a computational approach could identify proteins associated with a particular disease, as a preliminary step in biomarker discovery. As mentioned, we narrowed our search to proteins present in serum/urine because of the postulated presence of a circulating diabetogenic factor that exerts peripheral effects. Also, a diagnostic test would necessarily be a serum/urine assay.Of note, FABP-1 has been in detected in animal and human serum/urine and commercial ELISA-kits are available for FABP-1 serum/urine detection -47. PossGlobal gene expression analysis in patients with PaC-associated DM is a complimentary and contributory approach to biomarker discovery using integrative bioinformatics. Gene expression analysis could confirm the FABP-1 overexpression we predict. There is no published literature that specifically investigates the gene expression of tumor tissue in PaC-associated DM. Future work will be important to fill that knowledge gap.There are several limitations of our study. When considered a binary variable, FABP-1 staining was defined as a score ≥ 1. A more stringent cutoff for FABP-1 staining would change our reported associated with pancreatic cancer. However, we do feel that in this pilot study, grouping pancreatic cancer groups by the presence or absence of FABP-1 staining was reasonable, given that only one normal sample stained positive for FABP-1 and that the degree of FABP-1 expression is likely to vary between tumors. The extent of FABP-1 staining will be explored in future studies with more patients samples that are better phenotyped. In multivariate analyses, age was noted to be a confounder of FABP-1 staining, though it should again be noted that only one sample in the normal group stained with FABP-1 and this sample was from a patient that was younger than the average age of the staining population in the PaC group. Rather than a true confounder, the association between age and FABP-1 staining may be a result of selection bias from our convenience sample or a spurious association. Larger studies are needed to further explore this relationship.There are limitations of the bioinformatics technique itself, including potential low specificity for a given biomarker, the assumption that differential gene expression is necessarily reflected in plasma or urine, and that we excluded proteins currently not known to be detectable in serum or urine. Biomarker discovery using this methodology is necessarily limited by quantity, quality, and availability of public microarray datasets for a disease. However, it should be noted that these limitations have not precluded successful discovery of biomarkers for other diseases .Our data collection was retrospective and subject to idiosyncrasies in chart documentation. We could not differentiate pancreatic cancer-associated diabetes from pancreatic cancer coexisting with chronic diabetes. The PaC-DM group presented represents true PaC-associated DM cases as well as those in whom the DM was unrelated to PaC, in unknown proportions. This distinction can be elucidated in future serum FABP-1 studies with more detailed available clinical information.Our sample was a nonrandom small convenience sample, which likely contributed to the lower than published prevalence of diabetes in PaC, the age difference between normal and cancer groups, and the fact that we were not adequately powered to run a matched analysis . HoweverFinally, our normal samples were representative of histologically normal tissue that often did come from organs that had pancreatic pathology. We cannot rule out concerns of tumor microenvironment, though it should be noted that histologic normalcy is the basis for adequate surgical margins during tumor resection, and that this technique is standard practice in construction of microarrays and has yielded reproducible results for other types of cancer/normal tissue ,23. FutuIn conclusion, our study was proof of concept that a translational bioinformatics approach has potential to identify novel disease associations in gastroenterology. Further experimental and clinical research is required to understand the true relationship between FABP-1, pancreatic cancer, and PaC-associated DM.The authors declare that they have no competing interests.RNS, AJB, and PJP conceived of the study and its design and participated in drafting the manuscript. AJB and JTD performed the bioinformatic work. KDM performed the tissue microarray work. RP scored the stained tissue microarrays. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-230X/11/5/prepub


Objective: The presence of appetite hormones, namely glucagon-like peptide-1 (GLP-1), peptide YY (PYY), and leptin in breast milk may be important in infant feeding regulation and infant growth. This study evaluated whether concentrations of GLP-1, PYY, and leptin change across a single feeding (from fore- to hindmilk), and are associated with maternal and infant anthropometrics.Design and Methods: Thirteen postpartum women  provided fore- and hindmilk samples 4-5 weeks after delivery and underwent measurements of body weight and composition by Dual X-ray Absorptiometry. GLP-1, PYY, and leptin concentrations were measured using radioimmunoassay, and milk fat content was determined by creamatocrit.Results: Concentration of GLP-1 and content of milk fat was higher in hindmilk than foremilk (P ≤ 0.05). PYY and leptin concentrations did not change between fore- and hindmilk. Both leptin concentration and milk fat content were correlated with indices of maternal adiposity, including body mass index , and fat mass . Hindmilk GLP-1 was correlated with infant weight gain from birth to 6 months .Conclusion: The presence of appetite hormones in breast milk may be important in infant appetite and growth regulation. In addition to providing optimal nutrition for the infant, breastfeeding offers numerous health benefits that are unmatched by formula. Breastfeeding not only decreases the risk for ear infections, sudden infant death syndrome, lower respiratory tract infections, diarrhea, and vomiting, but also reduces risk for obesity later in life The discovery that two key appetite-regulating hormones, leptin and ghrelin, are present in breast milk—in addition to a variety of bioactive molecules including immunoglobulins, growth factors, and enzymes—provides an attractive explanation for the ability of breast milk to regulate infant body weight. Bouret Leptin, a 146 amino acid peptide hormone produced in white adipose tissue and stomach A hallmark of breast milk is that its composition varies widely and is not static over time The purpose of this pilot study was to determine whether GLP-1 is present in human breast milk and whether the appetite-suppressing hormones, GLP-1, PYY, and leptin, change in concentration from fore- to hindmilk. We hypothesized that breast milk concentrations of GLP-1 and PYY would increase with duration of feeding, sending a signal of increased satiety to the infant. However, as leptin is a long-term regulator of appetite, we hypothesized that concentrations would remain stable from fore- to hindmilk. A second purpose of the study was to examine associations between the concentrations of appetite-suppressing peptides in milk and both maternal adiposity and infant growth. We hypothesized that plasma hormone concentrations influenced by maternal adiposity would be similarly affected in milk. Lastly, if appetite hormones in breast milk play a role in the regulation of infant energy intake, we hypothesized that milk GLP-1, PYY, and leptin would negatively correlate with infant weight gain.Thirteen healthy postpartum mothers and their infants participated in this study from fall 2009 to spring 2011. To qualify for the study, participants had to be first-time mothers exclusively breastfeeding, at least 18 years of age with normal thyroid function (thyroid stimulating hormone [TSH] concentration between 0.47-4.68 μIU/mL) and hemoglobin concentration between 12.0-16.0 g/dL. Women were not eligible if they had given birth to multiples, had major pregnancy complications , reported having any major diseases, had resting blood pressure above 140/90 mm Hg, were taking any medication or herbal supplement that affect metabolism, or had any signs of psychological disorders or substance abuse. Mothers who smoked or had smoked during the previous year were also excluded. The study was approved by the Institutional Review Board of the University of Wyoming. Volunteers were fully informed of possible risks of all procedures before providing written informed consent.Postpartum women visited the laboratory between 29-38 days after delivery to provide fore- and hindmilk samples. Anthropometric measurements of both mothers and infants were collected. Specifically, maternal body weight was obtained in minimal clothing prior to milk collection using a digital scale . Height was measured in bare feet with a stadiometer . Infant body mass was measured using an UltraScale MBSC-55  when clothed in only a dry diaper. Infant length was measured as the parallel distance from the head to the heels as marked on paper. Maternal body composition was measured using Dual X-ray absorptiometry (DXA) . Self-reported infant birth weight/length, pre-pregnancy weight, and weight gain during pregnancy were also collected.Infants were also followed longitudinally for one year after birth. Anthropometric measurements, as previously described, were obtained at 6 and 12 months after delivery. A parent-reported infant weight from a physician visit was accepted in place of weighing for infants whose families were no longer living in the local area or were unable to complete the 6 or 12 month follow-up at the scheduled time. Infant weight gain was calculated by subtracting reported birth weight from the infant weight measured at the milk collection and follow-up visits. Infant weights were calculated as percentiles for weight-for-age and weight-for-length using the sex-based World Health Organization growth charts .Breast milk samples were collected in the morning between 7 and 10 AM, during the mother's usual nursing time. Mothers reported to the laboratory after an overnight fast of at least 10 hours. Milk was collected using a mechanical pump provided by the laboratory  or the mother's own pump. Foremilk (at least 30 mL) was collected before feeding the infant and hindmilk (at least 30 mL) was collected from the same breast after feeding the infant for 6-10 minutes, depending on the mother's perceived milk availability and the infant's feeding rate. Immediately after collection, milk samples were placed on ice and the protease inhibitors aprotinin (37.5 μL/mL)  and dipeptidyl peptidase-4 inhibitor (10 μL/mL) (Millipore) were added to milk samples for analysis of GLP-1 and PYY. Protease inhibitors were not added to samples used for analysis of leptin. Fat content of the milk samples was determined by creamatocrit analysis of whole fore- and hindmilk samples using an Autocrit Ultra 3 , according to the procedures reported by Lucas et al. Leptin was analyzed using a human leptin RIA (Millipore) according to the manufacturer's directions as previously reported t-test. Hormone concentrations in foremilk and hindmilk were compared using the Student's one-sample t-test. In this test, the relative change in concentration was calculated as (Hind-Fore)/Fore and compared to a null value of 0. Hormone and fat contents of fore- and hindmilk were compared to maternal BMI, % body fat, body weight, and fat mass, along with infant weight gain from birth to 12 months using Pearson correlation coefficients. P-values of 0.05 or less were deemed significant. Data are presented as arithmetic means ± SD.Statistical analysis was performed with PASW version 18 . Descriptive data were compared using the Student's two-sample 2 and averaged 26.0 ± 4.2 kg/m2. All infants were born to term with an average weight of 3.3 ± 0.5 kg. A creamocrit analysis was missed on fore- and hindmilk samples of one participant, therefore data regarding fat content of milk are reported on n = 12. Additionally, because of study attrition, follow-up infant anthropometrics were not obtained on three infants at the 6- and 12-month follow-up . Anthropometric data from a physician visit were used for 7 of the 31 infant anthropometric measures.The descriptive characteristics of the mothers and infants are shown in TableDetectable concentrations of GLP-1 and PYY were found in all milk samples Table. Leptin P = 0.051, 95% CI: −0.1-61.3%) from fore- to hindmilk  in hindmilk compared to foremilk (P = 0.205) and PYY (P = 0.909) did not change from fore- and hindmilk , BMI , fat mass , and percentage body fat  . No other correlations were observed.Concentration of average milk leptin was correlated to maternal body weight (= 0.001) . Foremilr = 0.65, P = 0.022; r = 0.67, P = 0.018), BMI , body weight , and percentage body fat  . These correlations did not change after adjustment for pre-pregnancy weight or weight gain during pregnancy. Additionally, the change (fore- to hindmilk) in the percent fat of milk samples was strongly related to BMI  and tended to correlate with percentage body fat . No other correlations were observed.The fat contents of both fore- and hindmilk were correlated with maternal fat mass . However, the concentration of hindmilk GLP-1 at 1 month after delivery was negatively correlated with infant weight gain over the first 6 months  . There was a trend for this relationship  at 12 months after birth. Additionally, the concentration of hindmilk GLP-1 was negatively correlated to the infant weight-for-length percentile at 6 months . No other correlations were observed.Associations between milk GLP-1, PYY, and leptin concentrations or fat content and infant anthropometrics were not observed at 1 month after delivery. Only the percent increase in milk fat content was correlated to infant weight gain at 1 month ( n = 10) . There wIn the current study, all three appetite hormones  were detected in breast milk. Although results for leptin and PYY confirm previous reports, this is, to our knowledge, the first report that GLP-1 is present in human milk. These results also confirm that PYY is present in both fore- and hindmilk. Concentrations of PYY and leptin detected in the current study are similar to those previously reported. The average milk PYY concentration was 39 pg/mL, which is slightly higher than the 23 pg/mL reported by Berseth et al. GLP-1, PYY, and leptin are key anorexigenic hormones that promote satiation and decrease the desire to eat. When infused intravenously, all three hormones decrease energy consumption at subsequent meals In order for appetite hormones in milk to have physiological effects in the infant, the hormones must be able to survive gastric digestion and bind a receptor or be endocytosed. Although there is no direct evidence of these processes, a study in human infants found correlations between milk leptin concentration and subsequent plasma leptin concentration in the infants 60 minutes after nursing If the appetite hormones are biologically active in the infant, the observed increase in the milk GLP-1 concentration across the feeding may serve as a satiety signal to terminate infant feeding, given that GLP-1 decreases food intake and enhances satiety In contrast to GLP-1, the concentration of PYY did not change from fore- to hindmilk. Because milk concentrations of PYY were static, there may be important yet unrecognized biological differences between GLP-1 and PYY and/or differences in their synthesis or secretion by mammary tissue. The change in GLP-1 concentration over the relatively short period of time between fore- and hindmilk suggests it is most likely of breast-tissue origin. However, future studies addressing whether the mammary gland is capable of synthesizing this peptide are needed. As for PYY, the concentration of plasma PYY was roughly four times that found in breast milk, as reported by Berseth and colleagues Supportive of the study hypothesis, the concentration of milk leptin was similar between fore- and hindmilk, providing further evidence that leptin is stable across a single feeding and may not be important for short-term appetite regulation. Similar to leptin's role in adults, concentrations of milk leptin may play a role in long-term energy balance and regulation of infant growth. Milk leptin concentrations are inversely related to infant growth and BMI between 6 and 24 months of age As expected, concentrations of leptin in both fore- and hindmilk were related to multiple markers of maternal adiposity. The correlation between maternal BMI and milk leptin concentration has been previously reported In the current study, maternal BMI was also correlated with the milk fat content of both fore- and hindmilk. This finding could be a result of the higher plasma triglyceride concentrations commonly found in individuals with higher BMI, or higher concentrations of hormones correlated to body adiposity, the most likely candidate being leptin.In addition to leptin and milk fat, maternal adiposity is also correlated with GLP-1 in foremilk, but not hindmilk. A possible explanation for this finding is that leptin increases secretion of GLP-1, but not PYY Although this pilot study provides intriguing results suggesting appetite hormones in breast milk may influence infant feeding, the study was limited by sample size, RIA sensitivity, use of physician-obtained infant anthropometrics in a limited number of cases, and a focus only on satiety hormones. Certainly additional research is needed to confirm the current findings and further explore how synthesis and/or secretion of milk appetite hormones are regulated and determine the origin of these hormones. Although this study evaluated changes in the hormone and fat content of milk at 4-5 weeks postpartum, the importance of appetite regulation and metabolic programming may be greater earlier on in the neonatal period as suggested by animal studies In conclusion, this pilot study has identified a potentially bioactive component in breast milk that changes across a single feeding. The presence of this hormone and its changing concentration suggests that it might regulate infant appetite during individual feedings. Additionally, differences in appetite hormone exposure may create permanent changes in the way the brain reacts to appetite hormones and satiety cues


Default from multidrug-resistant tuberculosis (MDR-TB) treatment remains a major barrier to cure and epidemic control. We sought to identify patient risk factors for default from MDR-TB treatment and high-risk time periods for default in relation to hospitalization and transition to outpatient care. We retrospectively analyzed a cohort of 225 patients who initiated MDR-TB treatment between 2007 through 2010 at a rural TB hospital in the Western Cape Province, South Africa. Fifty percent of patients were cured or completed treatment, 27% defaulted, 14% died, 4% failed treatment, and 5% transferred out. Recent alcohol use was common (63% of patients). In multivariable proportional hazards regression, older age , formal housing (HR=0.38 [0.19-0.78]), and steady employment (HR=0.41 [0.19-0.90]) were associated with decreased risk of default, while recent alcohol use (HR=2.1 [1.1-4.0]), recent drug use (HR=2.0 [1.0-3.6]), and Coloured (mixed ancestry) ethnicity (HR=2.3 [1.1-5.0]) were associated with increased risk of default (P<0.05). Defaults occurred throughout the first 18 months of the two-year treatment course but were especially frequent among alcohol users after discharge from the initial four-to-five-month in-hospital phase of treatment, with the highest default rates occurring among alcohol users within two months of discharge. Default rates during the first two months after discharge were also elevated for patients who received care from mobile clinics. Among patients who were not cured or did not complete MDR-TB treatment, the majority defaulted from treatment. Younger, economically-unstable patients and alcohol and drug users were particularly at risk. For alcohol users as well as mobile-clinic patients, the early outpatient treatment phase is a high-risk period for default that could be targeted in efforts to increase treatment completion rates. Globally and in South Africa, the proportion of multidrug-resistant tuberculosis (MDR-TB) patients who successfully complete treatment remains less than 50% ,2. ComplIdentified risk factors for default from treatment of drug-sensitive (DS) TB include alcoholism and other substance abuse, low socioeconomic indicators, and health systems factors such as inadequate patient education and difficult-to-access care –10. MDR-Mycobacterium tuberculosis, and often after injectable medications are completed, patients are transitioned to the outpatient setting to complete more than a year of additional therapy. The inpatient and ambulatory settings create different sets of obstacles for patient adherence: ambulatory treatment disrupts patients’ lives less than hospitalization, but it requires their active participation and exposes them to competing pressures of family, livelihood, and substance use [Because of the required injections and the high pill burden, MDR-TB treatment is commonly initiated in the inpatient setting. Once patients are stabilized and have sputum that is smear-negative for ance use . Additioance use . In thisThe study protocol was approved by the Health Research Ethics Committee, Stellenbosch University, the Institutional Review Board of Boston University Medical Campus, and the Office of Human Research Administration, Harvard School of Public Health . The approving IRBs specifically waived the requirement of patient consent for information from their clinical records to be placed into a de-identified research database.M. tuberculosis resistant to both isoniazid and rifampin, as determined by culture-based or molecular-based (Genotype MTBDRplus line probe assay) susceptibility testing. We conducted a retrospective cohort study of all adult patients (age > 15 years) who initiated MDR-TB treatment at Brewelskloof Hospital (BKH), the TB referral hospital for the Cape Winelands East and Overberg districts, Western Cape Province, South Africa, between 1 January 2007 and 31 December 2010. BKH serves a largely rural, agricultural population of approximately 600,000 people, and TB is one of the top three causes of death in all the region’s subdistricts, with 6714 cases of TB in 2008 and 74 cases of MDR-TB in 2008-09 [At BKH, standard care for MDR-TB patients included hospitalization for the first four to five months of a 24-month treatment course. While in hospital, patients without contraindication to aminoglycosides received kanamycin injections, in addition to oral therapy of pyrazinamide, ethionamide, ofloxacin, and either ethambutol or terizadone. Patients remained in hospital until their sputum was smear and culture negative for two consecutive months, typically for a minimum of four months, although some early discharges were arranged for personal or disciplinary reasons. Patients then continued oral treatment as outpatients, typically at the health clinics where they were diagnosed. Depending on the distance between patients' homes and clinic, patients either went to clinic daily for directly-observed therapy (DOT), received medications every one to two weeks at clinic with community workers visiting their homes between clinic visits for home-based DOT, or, if they were particularly far from a clinic, received medications monthly from a mobile clinic and were not routinely linked to a community health worker. Care at clinics was carried out by nurses and supervised by BKH doctors who visited monthly. Both hospital and outpatient clinic records for all MDR-TB patients were maintained in a central office at BKH. BKH doctors collected social and economic variables on admission as part of their standard patient-history template. We abstracted hospital and clinic records in a de-identified manner using a standardized form for the following baseline variables: patient age, gender, race, height, and weight; human immunodeficiency virus (HIV) status, CD4 count, and antiretroviral therapy (ART) use; comorbid psychiatric disease and diabetes mellitus; recent alcohol, tobacco, and illicit drug use; marital status; employment status and type of employment; location of residence (in town versus on a farm), type of housing, and presence of electricity, running water, and toilet in the home; prior TB history including outcomes of previous treatment; and patients' current MDR-TB status including sputum collection dates and results, site(s) of disease, the clinic making the MDR-TB diagnosis and referral, dates of hospital admission and discharge, treatment regimens and dates, and final outcome with associated date. Subsequent, post-baseline measurements of these variables were not recorded. Employment was classified as “steady” if the patient was employed in a year-round  position or was a full-time student. Housing was classified as formal  or informal , as the terms are used in South African housing surveys . AlcoholOutcomes were classified according to standard definitions : cure orWe compared baseline patient characteristics between outcome groups, grouping cure with treatment completion and grouping death with failure in order to focus on default . Age, boWe then examined baseline risk factors for time to default in univariable and multivariable Cox proportional hazard regression models. Time was measured from the first day of MDR treatment. Defaults were modeled as events, and other final outcomes were censored when they occurred. We verified proportional hazards assumptions by confirming that interactions between each covariate and time on treatment were not statistically significant, and by visual inspection of graphs of log) versus log(time) stratified by each categorical variable. To generate a multivariable proportional hazards regression model for time to default, we considered all variables that were associated with default at P < 0.2 in univariate analysis, and we retained variables that were associated with default at P < 0.1 when added to the multivariate model. To account for missing data, we performed regression analyses using fivefold multiply-imputed datasets generated by Markov chain Monte Carlo methods , then pooled effect estimates across datasets .To examine how the default rate changed over time and how the relationship between default and risk factors varied during treatment, we calculated default rates for three time periods: during hospitalization, within two months of discharge (“the early outpatient period”), and from two months after discharge until termination of treatment (“the late outpatient period”). We calculated these default rates for all patients and also when stratified by variables which we hypothesized would negatively impact treatment adherence in the outpatient setting: baseline recent alcohol use, whether the MDR-TB diagnosis was made in a stationary or mobile clinic, and whether the patient’s primary address was in town  versus a farm address (indicating a longer distance from routine TB care). We used tests of homogeneity to examine whether the associations between these risk factors and default differed by different treatment time periods .We performed all statistical analyses using SAS version 9.3. We identified 225 MDR-TB patients who started treatment for MDR-TB at BKH between 1 January 2007 and 31 December 2010. MDR-TB was confirmed by culture or molecular resistance testing in 223 patients; two additional patients received empiric treatment based on previous culture history. Thirty-two percent of patients had HIV co-infection . Thirty-Half of patients were either cured  or completed treatment . Sixty-one patients (27%) defaulted, 32 (14%) died during treatment, 11 (5%) failed treatment, and 8 (4%) transferred out. Comparisons of baseline characteristics between outcome groups are shown in With monthly sputum monitoring, 203 patients converted to negative sputum culture on treatment, and the mean time to first culture-negative sputum was 60 ± 47 days. Among the patients who defaulted, the sputum of fifty-four patients (89%) tested negative by culture before default, four patients (7%) remained positive by culture throughout MDR treatment for a median of 5 months (range 1 to 10 months) prior to defaulting, and three patients (5%) defaulted early before a second sputum was collected. Univariate risk factors for default included alcohol, tobacco, and illicit drug use, younger age, unmarried status, lack of steady employment, informal dwelling, rural address, and diagnosis of MDR-TB at a mobile clinic . In multDrug, alcohol, and tobacco use were intercorrelated , but alcohol use and drug use were independently associated with default. Although we found no statistically significant interaction between any covariate and time on treatment, visual inspection of graphs of the log) versus log(time) suggested possible violations of the proportional hazards assumption for alcohol use as well as for MDR-TB diagnosis at a mobile clinic. Patients with outcomes of cure or treatment completion received median 739 (IQR 714-768) total days of treatment and were discharged from the hospital after median 127 (IQR 117-175) days. For patients who defaulted, median time to default was 257 days (interquartile range (IQR) 126-363 days). Of the 61 patients who defaulted, 15 (25%) defaulted during hospitalization–for example, by refusing further treatment or by failing to return from weekend leave. The default rate increased after discharge, and patients defaulted during the early outpatient period at 1.7 times the overall average default rate, although as discussed further below, no increased rate of default after discharge was seen in patients who had denied recent alcohol use . Only onThe relative risk of default associated with alcohol use varied with the hospitalization status (P=0.07 for test for homogeneity). During the in-hospital period, default rates were comparable among those who did and did not report recent alcohol use, but those with recent reported use at baseline defaulted nearly four times as frequently as nonusers during the early outpatient period and continued to default more frequently than nonusers during the late outpatient period . Among pWe also investigated whether patients with a farm address (versus a town address) or those who were diagnosed with MDR-TB at a mobile clinic  experienced higher rates of default after hospital discharge, since these patients may have more difficulty accessing care due to longer distances to health facilities and often do not receive daily DOT. In the early outpatient period, patients who had been diagnosed at mobile clinics defaulted at a seven times greater rate than other patients. This higher rate of default was confined to the early outpatient period (P=0.08 for test of homogeneity). We did not find any association between reporting recent alcohol use and being diagnosed at a mobile clinic (Pearson’s Chi-square P=0.58). Although having a farm address was associated with default in univariable analysis , we did In our rural South African MDR-TB cohort, 27% of patients defaulted from treatment before completing a full 24-month course ‒ twice the 12-15% average default rate in published MDR-TB cohorts worldwide –20. The Alcohol is known to be associated with developing active TB, as well as with poor clinical outcomes including drug resistance. Alcohol is also associated with default from treatment of DS TB, likely because of social destabilization caused by alcoholism, but possibly also in part because alcohol magnifies medication toxicities . The fewThe association we found between informal housing and default agrees with previous identification of substandard housing conditions in Peru  and unstWe did not find associations between default and HIV or prior TB treatment. Although HIV was previously noted as a risk factor for default in South Africa prior to widespread antiretroviral treatment , HIV wasThe elevated default rates we observed in the two months immediately after discharge highlight the transition period between the inpatient and outpatient treatment settings as a high-risk time for MDR-TB treatment adherence. The increased rate of default in that period appears to be driven by individuals with a history of recent alcohol use. Also, although the sample size of patients using mobile clinics was small, having been diagnosed at a mobile clinic was also associated with a greater risk of default in the early outpatient period, and this association did not appear to be explained by alcohol use. South African guidelines recommend but do not require DOT beyond the intensive phase of therapy , and at We note limitations to this study. Drug, tobacco, and alcohol use at intake were available for nearly all patients, but use was not quantified nor re-evaluated later during care. Quantification could help identify a threshold at which alcohol and drug use are problematic for adherence. It is known that drinking in this region tends to involve binging rather than moderate social use, and therefore, reported alcohol use often indicates abuse. In one survey of farm workers in the region, 87% screened positive for problem drinking by the CAGE questionnaire . Women dWe were unable to enroll or quantify those patients diagnosed with MDR-TB who never started treatment. Only 44% of the South African patients who were diagnosed with MDR-TB in 2011 initiated treatment , and theDefault is a major barrier to cure in MDR-TB patients, and in this rural farming population, younger patients, economically-unstable patients, patients receiving care from mobile clinics, and alcohol and drug users are particularly at risk for default. Socioeconomic supports targeting the most at-risk groups may help to increase treatment adherence in the Western Cape and similar areas of high MDR-TB burden. Also, although outpatient treatment may be easier, more affordable, and better for patients in the long run, the transition from in-hospital to community-based treatment is a difficult period in which extra care must be taken to prevent default. Because of alcohol-using patients’ higher default rate during the transition period and in the outpatient setting, MDR-TB programs should assess alcohol use patterns in their patient populations and consider following and supporting alcohol-using patients more intensively.


Clostridium difficile infection.A population-based study in Oxfordshire (UK) hospitals by Sarah Walker and colleagues finds that in an endemic setting with good infection control, ward-based contact cannot account for most new cases of  Clostridium difficile infection (CDI) is a leading cause of antibiotic-associated diarrhoea and is endemic in hospitals, hindering the identification of sources and routes of transmission based on shared time and space alone. This may compromise rational control despite costly prevention strategies. This study aimed to investigate ward-based transmission of C. difficile, by subdividing outbreaks into distinct lineages defined by multi-locus sequence typing (MLST).C. difficile toxin enzyme-immunoassay-positive and culture-positive samples over 2.5 y from a geographically defined population of ∼600,000 persons underwent MLST. Sequence types (STs) were combined with admission and ward movement data from an integrated comprehensive healthcare system incorporating three hospitals  providing all acute care for the defined geographical population. Networks of cases and potential transmission events were constructed for each ST. Potential infection sources for each case and transmission timescales were defined by prior ward-based contact with other cases sharing the same ST. From 1 September 2007 to 31 March 2010, there were means of 102 tests and 9.4 CDIs per 10,000 overnight stays in inpatients, and 238 tests and 15.7 CDIs per month in outpatients/primary care. In total, 1,276 C. difficile isolates of 69 STs were studied. From MLST, no more than 25% of cases could be linked to a potential ward-based inpatient source, ranging from 37% in renal/transplant, 29% in haematology/oncology, and 28% in acute/elderly medicine to 6% in specialist surgery. Most of the putative transmissions identified occurred shortly (≤1 wk) after the onset of symptoms , with few >8 wk . Most incubation periods were ≤4 wk , with few >12 wk . Allowing for persistent ward contamination following ward discharge of a CDI case did not increase the proportion of linked cases after allowing for random meeting of matched controls.All In an endemic setting with well-implemented infection control measures, ward-based contact with symptomatic enzyme-immunoassay-positive patients cannot account for most new CDI cases.Please see later in the article for the Editors' Summary Clostridium difficile, which causes gastrointestinal problems, including diarrhea, leading to severe illness and even death, especially in older patients or patients who are already seriously ill. Between 7% and 26% of elderly adult inpatients in hospitals may be asymptomatic carriers of C. difficile, and the spores that are formed by this organism can live outside of the human body for long periods of time and are notoriously resistant to most routine surface-cleaning methods. Following major hospital-associated outbreaks around the world, C. difficile infection has become a prime target for expensive prevention and infection control strategies.Hospital-acquired infections are common and occur when patients are infected with an organism found in the hospital or health-care environment. Hospital-acquired infections can sometimes cause no symptoms but often lead to illness or even death. A leading hospital-acquired infection is with the anaerobic bacterium C. difficile infection, however, to date, there have not been any robust evaluations of the impact of such strategies in reducing the spread of infection at the individual level. In order to implement improved, cost-effective policies, and to work out how to reduce incidence even further, a better understanding of person-to-person spread is crucial, especially as infection with C. difficile depends on a combination of factors, such as antibiotic exposure and host susceptibility. Therefore, the researchers conducted this study to examine in detail the transmission of C. difficile in hospital wards in order to give more insight and information on the nature of person-to-person spread.Prevention strategies and infection control measures have contributed to reducing the incidence of C. difficile strains from symptomatic patients by identifying C. difficile infection from routine clinical microbiological samples from 1 September 2007 to 31 March 2010. Throughout this period, Oxfordshire hospitals operated a rigorous infection control policy monitored by infection control staff, in which stool samples for C. difficile testing were taken from admitted patients with persistent diarrhea, and from patients with any diarrhea who were 65 years or older. The researchers tested all stool samples for C. difficile toxins by enzyme immunoassay, cultured positive samples, and genotyped C. difficile isolates by using multi-locus sequence typing , and finally, constructed networks of cases and potential transmissions (by tracing contacts for up to 26 weeks) for each sequence type identified.The researchers used a population-based study in Oxfordshire, UK, to investigate hospital ward–based transmission of defined In order to show which ward-based contacts potentially incorporated direct person-to-person spread and indirect transmission via the environment during shared ward exposure, the researchers analysed links (ward contacts) between the first case (the donor) and the second case (the recipient) for all pairs of cases with the same sequence type. The researchers then calculated the minimum infectious period by measuring the time between the first infected stool sample from the donor and ward contact with the recipient, and calculated the incubation period as the time between this ward contact and the first infected stool sample in the recipient. To reduce the possibility of shared ward contacts occurring by chance, the researchers used patients with negative enzyme immunoassay stool samples as controls to estimate how often such ward contacts reflected actual transmission rather than chance.C. difficile, with 4.4%  found positive for C. difficile in enzyme immunoassay and culture. With genotyping, the researchers identified 69 strains (sequence types) of C. difficile. The researchers found that the majority (66%) of cases of C. difficile infection were not linked to known cases, and only 23% had a credible ward-based donor sharing the same sequence type of C. difficile. Furthermore, the researchers found that most probable transmissions occurred less than one week after the onset of symptoms, with a minority (10%) occurring after eight weeks. Most incubation periods were less than four weeks, but a few (13%) were more than 12 weeks. Importantly, even after allowing for the random meeting of matched controls and for persistent ward contamination, the proportion of linked cases did not increase following ward discharge of a C. difficile infection case.Over the study period, almost 30,000 stool samples from almost 15,000 patients were tested for C. difficile infection. Crucially, these findings mean that C. difficile infection might not be effectively controlled by current strategies to prevent person-to-person spread. Although the researchers were able to distinguish different strains of C. difficile, there were insufficient numbers of these different strains to deduce whether the results they obtained might be different if there was a different combination of strain types, that is, if some strains were spreading more in hospitals than others. Finally, in order to determine what other types of control interventions are required to reduce the spread of C. difficile, a better understanding of other routes of transmission and reservoirs of infectivity is needed.These findings show that in an endemic setting with well-implemented infection control measures, ward-based contact with symptomatic, enzyme-immunoassay-positive patients cannot account for most new cases of http://dx.doi.org/10.1371/journal.pmed.1001172.Please access these web sites via the online version of this summary at PLoS MedicinePerspective by Stephan Harbarth and Matthew SamoreThis study is further discussed in a C. difficile infection, as does the UK Health Protection AgencyThe US Centers for Disease Control and Prevention provides information about a guide for preventing hospital-acquired infectionsThe World Health Organization has published  Clostridium difficile is a leading cause of healthcare-associated diarrhoea, which is almost exclusively precipitated by antibiotic exposure that disturbs the normal intestinal flora, allowing C. difficile to flourish C. difficile infection (CDI) has become the focus of multi-faceted and costly prevention strategies Infection with Rigorous implementation of these infection control measures is believed to have reduced disease incidence C. difficile strains from symptomatic cases, the interval between CDI diagnosis and putative onward transmission, incubation periods, and the proportion of cases arising from ward-based transmission from known symptomatic CDI cases, in order to identify and/or better target infection control measures.Here we study endemic CDI in a large geographical region over a 2.5-y period, where a recent genotyping scheme This was a pre-specified analysis within the Infections in Oxfordshire Research Database, an anonymized linked electronic database approved by the Oxford Research Ethics Committee (09/H0606/85) and the National Information Governance Board (5-07(a)/2009).The Oxford Radcliffe Hospitals (ORH) NHS Trust  provides >90% of hospital care, and all acute services, to ∼600,000 people residing in Oxfordshire, United Kingdom. The Trust has two large sites in Oxford—the Churchill  and John Radcliffe —and a smaller district hospital 35 miles north of Oxford (two floors). All ORH hospitals have discrete wards containing 20–30 beds in 4–6 bedded bays with shared bathrooms and only 2–4 side rooms: only a few specific wards (notably elderly medicine) are all single rooms. The ORH microbiology laboratory tests all stool samples from the region, including from other healthcare facilities  and general practitioners (primary care). This population-based study included all CDI cases identified from routine clinical samples at the ORH microbiology laboratory from 1 September 2007 to 31 March 2010.C. difficile testing from any admitted patient with diarrhoea , and for oral vancomycin treatment to be initiated as first-line empiric therapy. Compliance was monitored weekly by infection control staff, with immediate feedback  for C. difficile toxins A and B (Meridian Bioscience). EIA-positive samples were cultured C. difficile isolates genotyped by multi-locus sequence typing (MLST) Throughout this period, the ORH hospitals operated a rigorous infection control policy (Table A in C. difficile sample of each sequence type (ST) from each patient was included in the primary analysis (55/927 [6%] patients had multiple CDI with different STs). A network of ward-based contacts between cases, representing potential transmission events, was created for each ST  EIA-positive sample with this ST prior to the ward contact. To estimate the effect of missing cases, not detected as part of routine testing, the analysis was repeated after randomly removing cases from the complete dataset. Networks were regenerated and the proportion of the remaining cases with a credible ward-based donor calculated. Between 5% and 50% of the original cases were removed, with 1,000 repetitions undertaken for each 5% increment. Extrapolation of the results was used to estimate the possible effect of missing cases.To calculate the proportion of links between cases that might be the result of shared ward contacts occurring by chance rather than actual transmission, ward-based networks were constructed for the same number of patients without CDI for each ST, randomly sampled 1,000 times. The main controls were patients with EIA-negative diarrhoea. The proportion of controls that could be linked by shared time and hospital space was used to adjust the number of linked CDI cases for chance meetings merely due to movements around the hospital, and to therefore provide an estimate of the proportion of cases linked by actual transmission.  were ≤1 wk after the donor was diagnosed , with the majority ≤4 wk , and few >8 wk  . The medResults were similar when (i) restricting links to those where the point of potential transmission was known more precisely (0- to 1-d ward contact durations only), (ii) minimising incubation rather than infectious periods, and (iii) calculating infectious/incubation periods relative to the most recent, rather than the first, donor sample .Investigation of the impact of missing data showed that, under plausible upper limits on the maximum allowable infectious/incubation periods, the proportion of the remaining cases with a credible donor fell from 23% to 22% if 10% of cases were randomly removed, and to 18% if 30% of cases were removed . ExtrapoC. difficile isolates, ∼20% of which were the hypervirulent PCR-ribotype-027/NAP-1/BI/ST-1 strain, was obtained from patients in defined ward locations, in a group of hospitals from a large UK NHS Trust practising rigorous infection control. To our knowledge for the first time for symptomatic C. difficile EIA-positive, culture-positive cases, we have investigated (i) the proportion of cases attributable to credible ward-based transmission from other symptomatic cases, (ii) the time between CDI diagnosis and potential onward transmission (denoted the minimum infectious period) and between putative acquisition and CDI diagnosis (incubation period) based on these putative transmissions, and (iii) the possible impact on transmission of ward contamination persisting following discharge of CDI cases. Crucially, our findings are based on data collected over 2.5 y, and are thus less susceptible to biases inherent during CDI outbreaks. Our CDI rates are all within the 3.8–9.5/10,000 overnight stays typically observed in endemic settings Our uniquely large dataset of genotyped C. difficile shedding is most prominent during episodes of diarrhoea Plausible ward-based transmissions were most commonly observed during the week following the first EIA-positive sample. This supports the current recommendations for immediate isolation of symptomatic patients, and is consistent with evidence showing that Our data suggest that incubation periods most commonly last a few days to 4 wk, but extending these up to 12 wk can provide plausible ward-based transmission links. Previous studies have been inconclusive. Cohort studies taking serial samples from inpatients (likely receiving antibiotics) suggest a median 2–3 d from first positive test to symptoms C difficile, in an intensive care unit setting, investigators found that only 15% of infections could be attributed to ward-based patient-to-patient spread based on molecular typing In our study no more than ∼25% of patients with detected symptomatic CDI could plausibly have acquired the infection from other patients with EIA-positive symptomatic CDI via ward contact. Chance meetings between patients, without transmission occurring, could mean that this 25% is in itself an overestimate. Similarly, in a study of ten nosocomial pathogens, not including C. difficile, large numbers of completely untested inpatient or community symptomatic patients are unlikely. Our 6.2% C. difficile EIA positivity rate is similar to the average 6.45% for 170 English hospitals in 2008 C. difficile colonies from single faecal samples Since (i) 800–1,000 EIA tests were performed each month, (ii) there was an ORH policy of universal testing of nosocomial diarrhoea , and changes during the study (through 31 March 2010). Table B: Network characteristics. Figure A: C. difficile tests and EIA-positive tests over the study period. Using 28-d de-duplication . Figure B: Distribution of infectious and incubation periods for transmissions within 69 sequence types and 705 test CDI cases. All potential transmission links where the direction of transmission is known  are plotted assuming maximum allowable infection and incubation periods of 26 wk and no ward contamination persisting after donor discharge (see Results for distributions from the most plausible links). Times are plotted rounded up to the nearest week, e.g., intervals of 0–6 d are plotted as 1 wk.(DOC)Click here for additional data file.


To evaluate four different commercially available assays for anti-double stranded DNA (dsDNA) detection and compare them with the in-house radioimmunoassay according to Farr (FARR-RIA) in order to select the optimal primary method for use in combination with FARR-RIA.Crithidia luciliae immunoflourescence tests (CLIFT), and FARR-RIA. The specificities and sensitivities of the tests were calculated and compared.Sera from 583 consecutive patients sent to our laboratory for routine diagnosis, 156 selected patients with autoimmune diseases (76 systemic lupus erythematosus [SLE] patients and 80 patients with other autoimmune diseases), and 150 blood donors were tested for anti-dsDNA antibodies with two enzyme-linked immunoassays (ELISA), two FARR-RIA and CLIFT 2 showed the highest specificity for SLE (100%), with CLIFT 2 showing higher sensitivity (33% vs 47%). Both ELISAs showed higher sensitivities (>53%) than FARR-RIA but lower specificities (<93%), whereas CLIFT 1 showed the lowest overall agreement with FARR-RIA.CLIFT 2 was selected as the primary test for use in combination with FARR-RIA. The use of CLIFT 2 reduced the number of sera that needed to be tested by FARR-RIA, the time needed to report the results, and environmental toxicity, cancerogenicity, and radioactivity. Anti-double stranded (dsDNA) antibodies were discovered in 1957 and since then have been well recognized as diagnostic markers of systemic lupus erythematosus (SLE). They are excellent indicators of SLE disease activity  and theiCrithidia luciliae immunofluorescence assay (CLIFT), and radioimmunoassay methods developed according to Farr technique (FARR-RIA) , ARR-RIA) . DiffereARR-RIA) ,10. An iARR-RIA) . The metARR-RIA)  and it eThere are certain considerations regarding the detection of anti-dsDNA antibodies that need to be specified. For the diagnosis of SLE, it is crucial that the anti-dsDNA assay is highly specific for dsDNA, especially since elevated levels of anti-dsDNA antibodies can also be detected in other autoimmune diseases, as well as in blood donors, very much depending on the detection method used -17. FARRThe aim of our study was to evaluate four different commercially available assays for anti-dsDNA detection and compare them to the in-house FARR-RIA assay in sera from 583 consecutively collected individuals, 156 individuals with autoimmune diseases, and 150 blood donors. The overall intent was to substantially shorten the time of reporting results, lower toxicity, and estimate the overall laboratory costs for anti-dsDNA testing.This cross-sectional study analyzed 156 sera of Slovene patients with systemic autoimmune diseases. Seventy-six patients  with SLE fulfilled the criteria established by American Rheumatism Association  and reviTM dsDNA Crithidia luciliae  (CLIFT 1) and Fluorescent nDNA Test system  (CLIFT 2) and two enzyme immunoassays – DiastatTM  (ELISA 1) and Quanta LiteTM dsDNA (INOVA Dignostics) (ELISA 2). All kits were used according to the manufacturer’s instructions. All CLIFT preparations were examined by three biochemical analysts in order to obtain a consensus result. The analysts were blinded to the results of other tests or other clinical information.Patient sera were prospectively analyzed with five different anti-dsDNA assays. Four of them were commercially available kits. We used two indirect immunofluorescent tests – NOVA Lite14C dsDNA extracted from E coli  for 1 hour at 37°C. Samples were stored overnight at 4°C, and the following day saturated ammonium sulfate was added to precipitate proteins (1:1) and incubated for one hour at 4°C. Following a 15-minute-centrifugation at 1800 × g, the supernatants (S) and pellets (P) were divided into separate glass bottles for scintillation counting. Bray scintillation solution was added, and the amount of radiation (cpm counts) was measured in each flask. The ratio (P-S/P+S) above 0.35 was determined as a positive result.The in-house FARR-RIA method used in the Immunology Laboratory since 1976 follows the first published protocol  with somThe international reference standard WO/80 was no longer available from the World Health Organization and therefore it was not included in the study.P value of <0.05 was considered statistically significant.Statistical analysis was performed using the SPSS 15.0 program . Correlations of variables were determined by the Spearman rank correlation, and kappa values for agreement were computed. Normality of distribution was evaluated with Kolmogorov-Smirnov test, normal probability plots, and curve fittings. Since data were not normally distributed, differences between the means were analyzed by the Mann-Whitney test. The relative risks were approximated by odds ratios with its 95% confidence interval. The receiver operating characteristic curves (ROC) were constructed, and sensitivity, specificity, and positive and negative predictive values were calculated. A P < 0.001). In ELISA 1, the 99th percentile estimated on our population was on the border between equivocal and positive samples as determined by the manufacturer and in ELISA 2 it was lower than the manufacturer’s, however their calculations included more blood donors.The main characteristics of the five anti-dsDNA assays are presented in The cut-off value for FARR-RIA anti-dsDNA antibody positivity has been previously determined to be 0.33 . Two parWe used 583 fresh sera from persons who underwent anti-dsDNA testing in our Immunology Laboratory. Their diagnoses/clinical signs were unknown to us at the time of the measurements. We determined the levels of anti-dsDNA with four different assays and compared the results with those obtained by FARR-RIA. CLIFT 1 detected the greatest number of positive sera, while FARR-RIA detected the lowest number of positive sera .There was an overall agreement between CLIFT 2 and ELISA 2 with FARR-RIA of above 90%, and a low agreement between CLIFT 1 and FARR-RIA (73%) .Rho = 0.491 – ELISA 1, Rho = 0.418 – ELISA 2; P < 0.001)  .FARR-RIA and CLIFT 2 showed the highest specificity for SLE (100%) . No falsWe evaluated the diagnostic applicability of all assays with ROC curve and estimated the area under the curve . The greCLIFT 2 and FARR-RIA detected only SLE patients as positive for anti-dsDNA. All the other kits also detected patients with pAPS, rheumatoid arthritis, and Sjoegren syndrome  .Four tests and FARR-RIA (100%) were compared according to the total costs, time needed for reporting the results, environmental toxicity, and cancerogenous characteristics. FARR-RIA was most cancerogenous and toxic to the environment, had the longest time needed for reporting the results, and was most costly. It also requires special safety measures, training of laboratory personnel, and management of workplace and radioactive waste. CLIFT and ELISA tests showed substantial improvement in all of these categories. Specifically, cost benefits of CLIFT tests and ELISA were respectively 50% and 25% higher than those of FARR-RIA. Also, their time needed for reporting the results after having received the samples was respectively one fifth and one half shorter than that required by FARR-RIA .We determined that CLIFT 2 was the most suitable method for anti-dsDNA detection in combination with FARR-RIA. Both CLIFT tests showed higher sensitivity than FARR-RIA and CLIFT 2 showed a higher specificity and a much higher overall agreement with FARR-RIA without losing any truly positive samples. However, CLIFT as a qualitative assay cannot substitute FARR-RIA, but can reduce its use by approximately 80%, since the results of 583 consecutive sera showed that only every 10th to 15th patient was tested positive for anti-dsDNA by CLIFT and needed quantitative confirmation by FARR-RIA.For both ELISAs, as qualitative assays, we determined the reference range and cut-off values in our population of blood donors. Their diagnostic specificities were lower than those of FARR-RIA. Our calculations of 99th percentile set the cut-off values lower than the manufacturers’ and therefore reduced the specificity even further. Including either ELISA in our routine practice would increase the number of false-positive results, which would need to be further tested with a more specific assay. In this way, our aim of reducing the number of tests performed with FARR-RIA, while not missing any truly positive samples, would not be achieved. So, ELISAs are not used in our laboratory to detect anti-dsDNA antibodies.Recently, a similar study  comparedHaving compared CLIFT, ELISA, and RIA, and considered both sensitivity and specificity, we conclude that greater sensitivity does not guarantee a larger number of truly positive samples. The major problem of anti-dsDNA ELISAs represents nonspecific binding to the, plastic surface. However, their availability, ease of use, and quantitative output has kept ELISAs as methods of choice in many laboratories . An alteThere were also reports on a novel surface plasmon resonance biosensor chip analytic method  and an iOur study confirmed that we can decrease the use of FARR-RIA with the introduction of CLIFT 2 assay and obtain the highest possible specificity and sensitivity. Since our original intent was not to choose a screening test, but rather to perform a lower number of FARR-RIA tests, CLIFT 2 proved to be the optimal. Thus, considerably fewer sera need to be tested by FARR-RIA following the use of CLIFT 2 in order to quantify values and perform disease activity monitoring. In this way, we also decreased the time needed for reporting negative results, the amount of toxic, cancerogenous, and radioactive chemicals in the environment, and overall laboratory costs for anti-dsDNA testing. On the basis of all this, we recommend the use of CLIFT 2 followed by FARR-RIA on positive samples, as well as finding a nonradioactive alternative to FARR-RIA.