Enterobacteriaceae (CRE) strains collected from a hospital setting in China, in which consistent emergence of CRE strains were observable during the period of May 2013 to February 2014. Among the 45 CRE isolates tested, 21 (47%) strains were found to harbor the blaNDM-1 element, and the rest of 24 CRE strains were all positive for blaKPC-2. The 21 blaNDM-1—borne strains were found to comprise multiple Enterobacteriaceae species including nine Enterobacter cloacae, three Escherichia coli, three Citrobacter freundii, two Klebsiella pneumoniae, two Klebsiella oxytoca, and two Morganella morganii strains, indicating that cross-species transmission of blaNDM-1 is a common event. Genetic analyses by PFGE and MLST showed that, with the exception of E. coli and E. cloacae, strains belonging to the same species were often genetically unrelated. In addition to blaNDM-1, several CRE strains were also found to harbor the blaKPC-2, blaVIM-1, and blaIMP-4 elements. Conjugations experiments confirmed that the majority of carbapenem resistance determinants were transferable. Taken together, our findings suggest that transmission of mobile resistance elements among members of Enterobacteriaceae and clonal spread of CRE strains may contribute synergistically to a rapid increase in the population of CRE in clinical settings, prompting a need to implement more rigorous infection control measures to arrest such vicious transmission cycle in CRE-prevalent areas.This study investigated the transmission characteristics of carbapenem-resistant  Enterobacteriaceae are common human pathogens which can cause a wide range of community-acquired and nosocomial infections  has posed a huge challenge to clinical infection control. Carbapenem resistance in CRE was mainly mediated by the production of carbapenemases, among which KPC, Metallo-β-lactamases  and OXA-48 type of enzymes were the most common  producing organisms were involved  collected from patients in Second People's Hospital of Jiaxingin Zhejiang Province, China. All isolates were identified using the Vitek 2 system , and confirmed by the MALDI-TOF MS apparatus . These isolates were screened for their ability to produce carbapenemases by a disc diffusion test, in which 10-mg imipenem discs were used   for tigecycline.The MICs of 10 antibiotics, including imipenem, meropenem, ceftazidime, cefotaxime, aztreonam, piperacillin-tazobactam, fosfomycin, amoxicillin-clavulanic acid, amikacin, tigecycline, were determined using the agar dilution method, and the results were analyzed according to the CLSI criteria of 2014 , with a running time of 24 h and pulse times of 3–40 s. Salmonella strain H9812 was used as the control strain. Bands were stained with ethidium bromide (0.5 μg/mL) prior to visualization under UV light. A dendrogram depicting the genetic relatedness of the test strains was generated from the homology matrix with a 0.2% coefficient by the unweighted pair-group method, and by using arithmetic averages (UPGMA), to describe the relationships of the PFGE profiles. Isolates were allocated to the same PFGE group if their dice similarity index was ≥85%.Clonal relationships between blaNDM−1-producing E. coli, K. pneumonia and E. cloacae isolates, which were amplified using primers listed in the online databases . The resultant PCR products were purified and sequenced. Sequence types (STs) were assigned using online database tools.MLST was performed using seven housekeeping genes inblaNDM−1-positive Enterobacteriaceae) and the recipient strains (sodium azide-resistant E. coli J53) were mixed on Luria-Bertani agar at a ratio of 1:1, and the mixtures were incubated for 24 h at 35°C. Transconjugants were selected on LB agar supplemented with sodium azide (100 mg/L) and meropenem (0.3 mg/L). Colonies that grew on the selective medium were picked for identification by the Vitek MS system. Transformants that harbored blaNDM−1 and exhibited resistance to carbapenems and cephalosporins were defined as transconjugants.The conjugation experiment was carried out using the mixed broth method as previously described , who were subjected to various neuronal system surgical procedures, followed by patients from the Respiratory Department (19%) suffering mainly from pulmonary infection, patients from the Pediatric Department and Recovery department (14% each), and one patient each from the Cardio-Thoracic Surgery and Breast Department (Table blaNDM−1 borne CRE strains were isolated from urine (85%), sputum (10%), and breast secretion (5%), respectively.The study period and venue were May 2013 to February 2014 and the Second Hospital of Jiaxing, Zhejian Province, China, respectively. During this period, a total of 45 CRE isolates were recovered from 6598 clinical specimens , among which 21 (47%) were found to harbor the blaNDM−1–borne CRE were recovered have been subjected to different types of antimicrobial treatment including the use of cephalosporins , amikacin, levofloxacin and tigecycline, and the vast majority of patients recovered and were discharged from the hospital. Our record also showed that although blaNDM−1-borne CRE were resistant to all cephalosporins, clinical treatment with cephalosporins for non-blood infections caused by blaNDM−1borne CRE remained effective  and amoxicillin-clavulanic acid  blaNDM−1-positive isolates were found to harbor ESBL genes in various combinations  in Table . PhenotyEnterobacteriaceae have become a worldwide public health concern  being collected during the period of June–November 2013. Our data suggest that a combination of outbreak of CRE infections and sporadic emergence of genetically unrelated resistant organisms contributed to the dramatic increase of CRE infections in this hospital during this period, prompting a need to investigate the molecular basis of these events. It should be noted that the series of CRE infections investigated in this study represents the most serious of its kind in China to date  and K. pneumoniae strains  have been identified worldwide  in this study infers that the size of the existing pool of NDM-1-producing strains has been further expanded.MLST-diverse NDM-1-producing E. coli and K. pneumoniae, MLST type of NDM-1-producing E. cloacae strains in this study included both epidemic ST types and new ST types. A carbapenem-resistant E. cloacae strain belonging to ST89 and producing the OXA-48 carbapenemase was isolated in Poland in 2014 , which generally belong to at least four different Inc groups, including A/C,L/M, FI/FII and an undefined type  and Natural Science Foundation of China (Grant No. 81371871).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.


Basal-like breast cancer (BLBC) is a molecular subtype of breast cancer associated with poor clinical outcome, although some patients with BLBC experience long-term survival. Apart from nodal status, current clinical/histopathological variables show little capacity to identify BLBC patients at either high- or low-risk of disease recurrence. Accordingly, we sought to develop a network based genomic predictor for predicting the outcome of patients with BLBC.We performed network analysis on global gene expression profiling data of BLBCs, and identified BLBC network modules associated with AP-1 transcription, G-protein coupled receptors, and T-, B-, and NK-cells that are significant predictors of BLBC patient survival.In gene expression and tissue microarray (TMA) validation cohorts of 210 and 102 BLBC patients, respectively, the identified network modules were robustly associated with patient outcome. In the gene expression validation cohort, the Kaplan-Meier estimate for 10-year survival in the low-risk group was 90%, whereas in the high-risk group it was a 56%. In the TMA cohort, the Kaplan-Meier estimate for 10-year survival in the low-risk group was 98%, whereas in the high-risk group it was 71%.The capacity to distinguish between patients with BLBC at high- or low-risk of recurrence at the time of diagnosis could permit timely intervention with more aggressive therapeutic regimens in those patients predicted to be high-risk, and to avoid such therapy in low-risk patients. Prognostic stratification of breast cancer patients is traditionally based on a variety of factors such as tumor size, grade, hormone receptor status, HER2 status, lympho-vascular space invasion and lymph node involvement , 2. HoweBasal-like breast cancer (BLBC) was first identified as a subtype of breast cancer in 2000, based on gene expression profiling experiments conducted by Perou and colleagues . SeveralWhereas several genomic based predictors exist to predict breast cancer patient outcome, their prognostic value appears to be mostly derived from their capacity to measure expression of genes associated with proliferation and ER status , 20. BecP < 0.05). Whenever possible, we used disease free survival (DFS) as the clinical endpoint for this analysis, although in some cases distant metastasis free survival was used. The genes represented by the 372 probe sets were then mapped as nodes onto a previously described highly reliable human functional interaction network [We sought to identify gene networks that might be useful to predict outcome in patients with BLBC. Briefly, we compiled gene expression profiles from 5 independent datasets, which represent non-redundant tumor samples, and for which clinical follow-up data was available . Togethe network . Pearson network  . A combination index (BLBC modules), representing the mean of the 7 individual module indices was a stronger predictor of patient outcome than any of the individual modules suggesting that in sum, the modules comprehensively measure the biological programs that drive patient outcome (Table P = 0.000041). We also observed that modules generally did not comprise mixtures of good and poor prognosis genes, but rather were highly enriched for either good or poor prognosis genes. Modules 1, 2, 3 and 6 were enriched in genes whose expression was associated with good outcome, whereas modules 0, 4 and 5, were enriched in genes whose expression was associated with poor outcome.We identified 7 modules that each comprised 8 or more nodes (genes) that displayed an average Pearson correlation of at least 0.25 based on expression. Each module was numbered from 0 – 6 in decreasing module size Figure . Based oP-values ranged from 0.0001 to 0.000009). The module 4 index trended as a predictor of patient outcome, but did not reach statistical significance (Table P = 0.10). As we observed with the training data, the BLBC modules score representing the mean of the 7 individual module indices was a superior predictor of patient outcome than any of the individual modules (Table P = 0.0000021).To assess the robustness of the modules, we compiled a validation cohort that comprised breast tumor sample profiles from additional independent datasets, henceforth referred to as the validation set . TogetheP < 0.0001). Indeed, the Kaplan-Meier estimate for 10-year survival in the low-risk groups was an excellent 90%, whereas in the high-risk group it was a dismal 56%. Hence, we concluded that the network modules were significantly associated with the outcome of patients with BLBC.We also stratified patients comprising the validation set into high- and low-risk groups based on the median module index value and completed survival analysis Figure . In eachP-value scores  of outcome-associated probe sets within the training and validation cohorts. On average, outcome-associated probe sets identified in the training cohort were less accurate in the validation cohort, resulting in a net decrease in average P-value score across all outcome-associated probe sets. However, we did not observe reduced P-value scores between the training and validation cohorts among outcome-associated probe sets that were included within the network modules .We compared the performance of the BLBC modules to multiple other prognostic gene signatures within the BLBC validation set. To this end we calculated and compared de Index , NKI-70 de Index , Oncotyp[® score , CSR/Wou[® score , MS-14 s[® score , Glinsky[® score , Network[® score  and the  [et al.  . The Kaplan-Meier estimate for 10 year survival in the low risk < 0 Allred score group was an excellent 98%, whereas in the > 0 Allred high risk group it was a much worse, 71%  and the BLBC modules IHC score (P = 0.0001) were significantly associated with patient outcome , we also observed enrichment for multiple signaling pathways and biological processes. Given that these modules are associated with poor patient outcome we think it is likely that these biological programs are associated with adverse tumor biology including progression and therapy resistance. Hence, their inhibition might provide therapeutic opportunities in BLBC. Targetable pathways enriched in Module 0 included the AP-1 and ATF-2 transcription factors, and ERBB1 (EGFR) downstream signaling. Whereas EGFR has been investigated as a possible therapeutic target in BLBCs , AP-1 anThere are several limitations of the work described herein. All of our conclusions are based on the analysis of retrospective data, which limits its clinical value. We demonstrated the predictive capacity of the BLBC modules in an independent gene expression cohort, as well as with an exploratory 3-biomarker IHC test in a local archival FFPE cohort. However, a true estimate of the clinical usefulness of the BLBC modules will require additional validation in clinical trial samples, or completion of a prospective clinical trial examining the capacity of the BLBC modules to accurately identify low- and high-risk BLBC patients . In addiAs mentioned above, no robustly validated biomarker test exists to predict BLBC patient outcome. Here we present a network based and genomics driven approach to identify outcome-associated BLBC network modules. Moreover, we also validated the BLBC network modules using an IHC based surrogate assay in an additional series of BLBCs. Given the strong relationship observed between the BLBC modules and patient outcome as well as the widespread availability of IHC, our findings if validated, could be rapidly implemented into the clinic as a means to spare low risk BLBCs patients from aggressive therapy as well as target aggressive therapies to those patients with high risk tumors in a timely fashion.in silico of 5 independent external datasets, obtained using Affymetrix HG-U133A GeneChip arrays, which have been deposited in the Gene Expression Omnibus (GEO); accession numbers GSE1456, GSE2034, GSE3494, GSE6532, and GSE7390 and comprise a total of 1077 samples   and comprise a total of 905 samples with accompanying clinical follow-up data , a procedure that allows one to pre-process microarrays individually or in small batches and to then combine the data into a single dataset for further analysis as previously described . ThereafAll tumors from the independent datasets were classified as basal-like, HER2+, luminal A, luminal B, claudin-low, normal-like or apocrine by assigning them to the standardized centroid of the subtype to which they had the highest Spearman rank correlation , 51-53. BLBC modules identification was implemented using the Cytoscape Reactome FI plug-in . Briefly2 expression) of the poor and good outcome associated genes, respectively, similar to previous reports [We calculated either module indices or signature scores as the difference between the geometric means .Formalin fixed paraffin embedded (FFPE) blocks of triple negative (TN) invasive breast cancer from the pathology archive of Hamilton Health Sciences (HHS) for the years 2005 to 2009 were collected with institutional review board approval. Each tumor was evaluated for the expression of CK5/6 and EGFR by standard immunohistochemical (IHC) techniques. Those tumors identified as TN and positive for either CK5/6 or EGFR were deemed BLBCs as previously described . The patCoxPH package. Kaplan-Meier analysis was completed using GraphPad PrismTM 5; P-values less than 0.05 were taken to indicate statistical significance.Cox-regression analysis was completed in R using the


Results from this study provide some support for an inverse association between serum IGFBP2 levels and risk of AH, particularly in nondiabetic women who are overweight or obese. Further studies are required to confirm these results.Atypical hyperplasia of the breast (AH) is associated with increased risk of subsequent invasive breast cancer, yet little is known about the etiology of AH. Insulin-like growth factor binding protein 2 (IGFBP-2) may contribute to the development of AH due to its proliferative effects on mammary tissue. We conducted a nested case-control study of postmenopausal women enrolled in Women's Health Initiative-Clinical Trial. Cases were 275 women who developed incident AH during follow-up, individually (1 : 1) matched to controls. Levels of IGFBP-2 were determined from fasting serum collected at baseline. Multivariable conditional logistic regression models were used to estimate odds ratios for the association of IGFBP-2 with risk of AH. Serum IGFBP-2 was associated with a nonsignificant decrease in risk for AH, when comparing the highest quartile to lowest quartile . This decrease in risk was most evident when analyses were restricted to nondiabetic, nonusers of hormone therapy (OR = 0.33, 95% CI = 0.13–0.86,  This e cancer , 6. in vitro studies have shown that it has a proliferative effect in mammary and other tumor cell lines [Little is known regarding the etiology of AH and the progression to breast cancer. However, insulin-like growth factors and their binding proteins have been shown to be critical in the development of many epithelial cancers, including breast cancer . Insulinll lines –10 and cll lines . Thus, sll lines .Potentially counterbalancing the proliferative effect of IGFBP-2 is the observation that serum IGFBP-2 levels are inversely associated with obesity . ObesityThese observations suggest a potential dual effect for IGFBP-2 in breast cancer development: IGFBP-2 may reduce the risk of AH via protection against development of insulin resistance and by virtue of its inverse association with obesity, while concurrently increasing risk of AH via its proliferative effects on breast tissue.To our knowledge, no previous studies have investigated the association between circulating IGFBP-2 levels and risk of AH. A greater understanding of the development of AH might enhance our understanding of the origins of breast cancer and help identify women who would benefit from increased surveillance and screening. To this end, we investigated the association of IGFBP-2 with risk of incident AH in a large prospective cohort of postmenopausal women.This investigation was conducted as a case-control study nested within Women's Health Initiative-Clinical Trial (WHI-CT) . BrieflyEvery 6 months, participants in the trial completed medical questionnaires on clinical events including breast procedures. In the Benign Breast Disease Ancillary Study, which was conducted in all WHI clinical centers , 23, womCases in our study were women diagnosed with incident BPBD with AH during follow-up in the WHI-CT. Of the 294 cases of AH ascertained, a total of 275 cases of BPBD with AH were included in the present study, consisting of all cases of atypical hyperplasia that had serum available. Controls were women who did not develop BPBD during the same follow-up period as that for the corresponding cases and who did not have an abnormal mammogram or abnormal clinical breast exam during the same period. Controls were selected from eligible participants using risk-set sampling (with replacement)  and werer = 0.999).Laboratory testing was performed by the Pollak Assay Laboratory at McGill University in Montreal, Quebec, Canada. Serum IGFBP-2 levels were determined by enzyme-linked immunosorbent assay (ALPCO Diagnostics), which has an assay sensitivity of 0.2 ng/mL. All samples but one which did not have sufficient volume were retested as blind duplicates and showed strong correlations  were assessed using Spearman correlation coefficients among controls only.2). Adjusting for BMI as a continuous variable did not change the results substantially when compared to the four-category adjustment (data not shown). Additional adjustment for other variables  had no discernible impact on the effect estimates and these variables were not included in the final models. A second model was then run, additionally adjusting for serum levels of insulin, C-reactive protein, and adiponectin, as well as tertiles of estradiol among nonusers of HT only, with HT users as a separate category (as standard estradiol assays cannot accurately measure equine hormones present in most HT preparations). Analyses were also conducted after stratifying separately by hormone use at baseline , BMI based on the median in controls , and age based on the median in controls . These stratified analyses were conducted by introducing interaction terms into multivariate models that also included the main effect variables. We also reran analyses after restricting the study population to those without a history of diabetes (16 cases and 17 controls reported a history of diabetes). For all analyses, tests for trend were performed by assigning median values to each quartile and modeling these categories as a continuous variable. All hypothesis tests were two-sided and all analyses were done using the statistical software Stata S/E 13.0 for Windows .Conditional logistic regression models were used to estimate odds ratios (OR) and 95% confidence intervals (CI) for the association of IGFBP-2 with risk of atypical hyperplasia. Analyses were first conducted with adjustment for age , and BMI  than in those classified as overweight or obese (≥25 kg/m2)  . Further5 kg/m2) . Serum I5 kg/m2) .p = 0.011) that was attenuated with adjustment for insulin, adiponectin, C-reactive protein, and estradiol , and when restricted to those without a history of diabetes .Considering all subjects together, serum IGFBP-2 was associated with a decrease in risk of AH in the age- and BMI-adjusted model only, with those in the highest quartile of serum IGFBP-2 having a 49% decrease in risk compared to those in the lowest quartile  . This asWhen stratified by use of HT, there was a decrease in risk of AH with increasing serum IGFBP-2 that was evident only among nonusers of HT , althougThe association of IGFBP-2 with BPBD also appeared to differ by BMI , althougTo our knowledge, this is the first study to investigate the association between serum levels of IGFBP-2 and the risk of atypical hyperplasia of the breast. Overall, we found a nonsignificant decrease in risk of AH with increasing serum IGFBP-2. The inverse association was of borderline statistical significance in two, prespecified subgroups: nondiabetic, nonusers of HT and women who were overweight or obese.2 showing evidence of decreased risk of breast cancer with increasing IGFBP-2, an association not seen in women under 26 kg/m2 [Although no studies have previously examined the association between IGFBP-2 and risk of BPBD, there is some epidemiological evidence for a role of IGFBP-2 in breast cancer. An early study investigated the association between IGFBP-2 and postmenopausal breast cancer and found a reduction in risk with increasing serum IGFBP-2 , althoug26 kg/m2 , whereas26 kg/m2 . In addi26 kg/m2 .Findings from our study and from these previous studies in breast cancer are in contrast with previous experimental evidence suggesting that IGFBP-2 promotes cell proliferation and tumor growth . This ma in vitro, both via inhibition of IGF-I [With respect to breast cancer and perhaps other obesity-related cancers such as colorectal cancer , the proof IGF-I  and alsoof IGF-I . In addiof IGF-I . There iof IGF-I , 41. Givof IGF-I . Estrogeof IGF-I .Our findings suggest that the effect of IGFBP-2 on risk of AH was modified by both HT use and BMI. The inverse association between serum IGFBP-2 levels and breast cancer risk observed previously seems to be restricted to women who were not using HT and to those women who are overweight or obese. Although suggestive, our study was underpowered to detect significant interactions between these subgroups with respect to risk of AH. A further limitation of this study was that IGFBP-2 was only measured once, at baseline. Repeated measurements over time might have resulted in more accurate estimates of serum levels. Also, the WHI-CT participants were a nonrandom sample of the population, which may limit the generalizability of our findings. However, because the results reported here demonstrate associations between a measured biological marker and risk of AH, it seems plausible that this marker may have similar effects in other populations. There is also the possibility of disease misclassification in this study, which may have biased our results towards the null. Major strengths of this prospective study were the collection of prediagnostic blood samples at baseline and ascertainment of subsequent (postbaseline) development of AH, thereby limiting bias.In conclusion, the results of this study are suggestive of an inverse association between serum IGFBP-2 levels and risk of atypical hyperplasia of the breast. This reduction in risk was most evident among nondiabetic, nonusers of HT at baseline and among obese and overweight women, and the reduction in risk persisted after adjustment for associated serologic factors such as insulin and estradiol. However, these results should be interpreted with caution, as the lack of statistical significance could be due to the relatively small numbers of study subjects available for analysis or could indicate that no true association exists between IGFBP-2 and AH. Nevertheless, given the association between AH and breast cancer, if our finding that serum IGFBP-2 level is associated with reduced risk of AH is confirmed in further studies, this may suggest a role for IGFBP-2 in the early stages of breast carcinogenesis.


Leptospira and compared results to MAT performed on the same specimens.Reference diagnostic tests for leptospirosis include nucleic acid amplification tests, bacterial culture, and microscopic agglutination testing (MAT) of acute and convalescent serum. However, clinical laboratories often do not receive paired specimens. In the current study, we tested serum samples using a highly sensitive real-time nucleic acid amplification test for Leptospira , and in the current study, we demonstrate that this assay amplifies both Leptospira RNA and DNA. Dengue virus RNA was identified in 10 patients, and no cases of malaria were detected. A total of 65 samples (13.6%) were positive for Leptospira: 35 samples (7.3%) in the Lepto-MD assay, 33 samples (6.9%) by MAT, and 3 samples tested positive by both (kappa statistic 0.02). Poor agreement between methods was consistent regardless of the titer used to define positive MAT results or the day of disease at sample collection. Leptospira nucleic acids were detected in the Lepto-MD assay as late as day 22, and cycle threshold values did not differ based on the day of disease. When Lepto-MD assay results were added to the MAT results for all patients in 2008 (n=818), the number of detected leptospirosis cases increased by 30.4%, from 102 (12.5%) to 133 (16.3%).478 serum samples from suspected leptospirosis cases in Rio de Janeiro were tested using a real-time RT-PCR for the diagnosis of leptospirosis, malaria and dengue (the Lepto-MD assay). The Lepto-MD assay detects all species of Leptospira and single-specimen MAT, which may result from the clearance of bacteremia coinciding with the appearance of agglutinating antibodies. A combined testing strategy for acute leptospirosis, including molecular and serologic testing, appears necessary to maximize case detection.This study demonstrates a lack of agreement between nucleic acid detection of  Leptospira species from a normally sterile site; or the detection of DNA from pathogenic Leptospira species by PCR , late acute [6/69 (8.7%)], or convalescent periods . Cycle threshold (CT) values in the Lepto-MD assay also did not differ based on the day of disease at sample collection  vs 34.4 ; p ≤ 0.15). In order to confirm Leptospira results from the Lepto-MD assay, 33/35 (94.3%) positive samples were tested using three other molecular tests  that tested positive for pathogenic species.llection , and meaests see . Two samLeptospira detection using either RT-PCR or MAT despite normal rainfall patterns in Rio de Janeiro during the study period  and extracted nucleic acids from 6 control Leptospira strains. This PCR uses the same Leptospira primers and probes as the Lepto-MD assay at the same final reaction concentrations. Samples were tested side-by-side with the Lepto-MD assay, which is performed using the SuperScript III Platinum One-Step qRT-PCR kit (Life Technologies). CT values in the PCR were, on average, 5.6 cycles later than CT values in the Lepto-MD RT-PCR assay. Results were similar using the TaqMan Universal PCR Master Mix , and the use of three-step versus two-step cycling did not change these findings. To confirm that the improved performance of the Lepto-MD assay was due to the detection of RNA, nucleic acid aliquots from Leptospira controls were treated with RNase A. CT values for treated samples were 10.8 cycles later than untreated controls. RNase A treatment had a larger effect on DENV RNA controls (CT difference 22.7 cycles) and no demonstrable effect on DENV-1 ssDNA or Leptospira plasmid DNA (CT difference 0.1 cycles).An internally-controlled Leptospira outbreaks [This study describes the results of testing 478 serum samples from suspected leptospirosis cases in Rio de Janeiro with the Lepto-MD assay. Thirty-five cases of leptospirosis and 10 dengue cases were identified. It is interesting to note that dengue cases were significantly younger than cases of leptospirosis. This has not been observed during previous studies of concurrent DENV and utbreaks –14. The utbreaks .Leptospira in the Lepto-MD assay demonstrated poor agreement with single-specimen MAT. We employed a conservative threshold for calling positive samples by MAT (titer ≥ 1:800), which is consistent with studies by other researchers in Brazil and was selected to maximize specificity [Leptospira in the Lepto-MD assay, 27/33 (81.8%) were confirmed with another molecular test. Although six samples were not confirmed by another molecular test, this is consistent with the improved sensitivity of the Lepto-MD assay relative to these comparators [Leptospira diagnostics employed latent class analysis to estimate the specificity of MAT at 98.8% [Molecular detection of cificity –18. Poorparators ,19. MAT parators . This grat 98.8% . A titerLeptospira detection in the Lepto-MD assay occurred as late as day 22, and samples collected as early as day 1 were positive by MAT. While inaccurate recording of the day of disease at sample collection could affect these numbers, the prolonged detection of Leptospira nucleic acids in serum has been observed by our group as well as others who utilized different molecular assays in varied patient populations [Leptospira can be detected later in the disease course [The classic description of leptospirosis is an acute systemic febrile illness with a biphasic course corresponding to an early leptospiremic phase and a later immune phase. The lack of agreement between molecular testing and single-specimen MAT likely resulted from the clearance of leptospiremia coinciding with the appearance of agglutinating antibodies in serum during the immune phase of illness ,3,22. Thulations ,23,24. Te course .While testing of acute and convalescent serum is recommended for the diagnosis of leptospirosis by MAT, such specimens can be difficult to obtain in a typical laboratory setting. Of the 818 patients who had serum sent to CRNL for testing in 2008, only 69 (8.4%) had multiple specimens collected. This is similar to findings from the Royal Tropical Institute in The Netherlands, where, in a study detailing testing results from 2001 to 2012, only 18.1% of patients had multiple samples available for serology . HoweverLeptospira detection in the Lepto-MD assay, performed as a real-time RT-PCR, compared to an optimized real-time PCR using the same Leptospira primers and probes. The Lepto-MD assay targets the Leptospira 16S rrs gene, and based on our findings using control Leptospira isolates, detection of both RNA and DNA occurs. Further experiments directly comparing PCR and RT-PCR on Leptospira positive clinical specimens with and without RNase A treatment will be required for full characterization. However, the current study lacked sufficient remaining nucleic acids extracted from positive clinical specimens to perform this testing. Of note, nucleic acids from the control Leptospira isolates were extracted using the DNeasy Blood & Tissue Kit (Qiagen). While optimized for DNA extraction, this kit does not specifically remove RNA from the eluted nucleic acids. The increased sensitivity of RT-PCR for the detection of a bacterial 16S target may prove useful in the design of assays for other pathogens that are present at low levels in the blood. Importantly, such assays may not require the use of new or automated extraction protocols.Finally, this study demonstrates the increased sensitivity of Leptospira and single-specimen MAT, regardless of the titer cut-off or day of disease at sample collection. Positive results from these methods demonstrate virtually no overlap, and a combined testing strategy for acute leptospirosis, including a sensitive molecular diagnostic and serologic testing, appears necessary to maximize case detection. Furthermore, we have demonstrated improved sensitivity of Leptospira detection using real-time RT-PCR; similar test designs may prove useful for other systemic bacterial infections that are challenging to diagnose.In conclusion, this study demonstrates a lack of agreement between molecular detection of


Tuberculosis (TB) transmission in prisons poses significant risks to inmates as well as the general population. Currently, there are no data on smear-negative pulmonary TB cases in prisons and by extension no data on the impact such cases have on TB incidence. This study was designed to obtain initial data on the prevalence of smear-negative cases of TB in prisons as well as preliminary risk factor analysis for such TB cases.This cross-sectional survey was conducted in November 2013 at eight main prisons located in the state of Amhara, Ethiopia. Interviews using a structured and pretested questionnaire were done first to identify symptomatic prisoners. Three consecutive sputum samples were collected and examined using acid fast bacilli (AFB) microscopy at the point of care. All smear-negative sputum samples were taken for culture and Xpert testing. Descriptive and multivariate analysis was done using SPSS version 16.Overall the prevalence of smear-negative pulmonary TB cases in the study prisons was 8% (16/200). Using multivariate analysis, a contact history to TB patients in prison, educational level, cough and night sweating were found to be predictors of TB positivity among smear-negative pulmonary TB cases (p≤ 0.05).In the studied prisons, high prevalence of undiagnosed TB cases using AFB microscopy was documented, which is an important public health concern that urgently needs to be addressed. Furthermore, patients with night sweating, non-productive cough, a contact history with TB patients and who are illiterate merit special attention, larger studies are warranted in the future to assess the associations more precisely. Further studies are also needed to examine TB transmission dynamics by patients with smear-negative pulmonary TB in a prison setting. Tuberculosis (TB) is one of the leading causes of death in the world. Globally, around 8.8 million people develop TB and 1.45 million people die every year due to TB, of which 0.35 million deaths are associated with HIV-TB co-infection Prison settings have been often identified as important but neglected reservoirs for TB, including multi-drug resistant (MDR)-TB In Ethiopia, limited work has been done on smear-positive pulmonary TB in prisons http://www.iata.org/ads/issa/htm).This survey was conducted in November 2013 at eight main prisons located in Amhara, Ethiopia. A flow diagram of inclusion and exclusion criteria for study subjects is summarized in On arrival at the mycobacteriology laboratory at the University Hospital Leipzig, all the sputum specimens were treated with the standard N-acetyl-L- cysteine (NALC)-NaOH method In accordance with the NTLCP guidelines Smear-positive pulmonary TB (PTB+) - A patient with at least two initial sputum smear examinations positive for AFB by direct microscopy, or a patient with one initial smear examination positive for AFB by direct microscopy, or a patient with one initial smear examination positive for AFB by direct microscope and radiographic abnormalities consistent with active PTB.Smear-negative pulmonary TB (PTB-) - A patient having symptoms suggestive of TB with at least 3 initial smear examinations that were negative for AFB by direct microscopy, no response to a course of broad spectrum antibiotics, followed by an additional three negative smear examinations by direct microscopy and radiological abnormalities consistent with pulmonary tuberculosis or a patient whose diagnosis is based on a culture-positive result for M. tuberculosis, but has three negative initial smear examinations by direct microscopy.Extra pulmonary tuberculosis (EPTB) - TB in organs other than the lungs, proven by one culture positive specimen from an extraplumonary site or histo-pathological evidence from a biopsy.All data were entered, cleared, and analyzed using the SPSS statistical software package, Version 16 . Descriptive data analysis was used to visualize differences within the data. Frequencies, odds ratio (ORs) and its 95% confidence interval (CI) were calculated. All covariates that were associated and non-associated with the outcome variable in the bivariate analysis were subsequently included in multivariate analysis to determine factors associated with TB positivity. A p value of ≤ 0.05 was used as the cut-off point for statistical significance.The study was reviewed and approved by an Institutional Review Board (IRB) of the University of Bahir Dar and the Amhara regional state prison commission, Bahir Dar, Ethiopia. After the research staff explained about TB transmission, the need for screening, the benefits of receiving treatment and prevention of TB in prison for inmates, written informed consent was obtained from each study subject. Individual records were coded and accessed only by research staff.A total of 200 cases were enrolled in the study, out of which 195 (97.5%) were males and 5 (2.5%) were females. Their ages ranged from 13 to 73 years, with a mean age of 39 years. One hundred fifty (75%) of them were rural residents and 89 (44.5%) were found to be illiterate. Overall the prevalence of culture and/or Xpert test positive smear-negative pulmonary TB cases in the study prisons was 8% (16/200). Of the total culture- and/or Xpert test-positive, smear-negative pulmonary TB cases, culture identified 11 (5.5%) cases of smear-negative pulmonary TB and would still have missed 4 (2%) of the cases. The Xpert test identified 15 (7.5%) cases of TB and missed 1 (0.5%). Out of the total culture and/or Xpert positive cases, five cases were identified from Debiretabor, four cases from Gondar, one case from Woldia and two cases each were identified from Debremarkos, Dessie and Debirebirhan prisons. When the number of registered smear-negative pulmonary TB cases are stratified by prison setting 12 (6%), 17 (8.5%), 23 (11.5%), 24 (12%), 33(16.5%), 35 (17.5%) and 28 (14%) were from Woldia, Bahir Dar, Fenoteselam, Dessie, Deberebirhan, Debiretabor, Debremarkos and Gondar prison, respectively .The most prevalent systemic symptom was cough in 174 (87%) cases, followed by fever in 166 cases (83%), chest pain in 162 (81%) cases and weight loss in 160 (80%) cases. Among the study participants, 44 (22%) of them had a history of contact with TB patients in prison and 116 (58%) had received family visits during incarceration. The number of inmates per cell varies from 10–587, with the mean number of 117.4. In addition to this high population density, inmates could be held in such cells for as long as 3 years. In the multivariable logistic regression model, smear negative pulmonary TB suspects who had night sweating were more likely to have TB, with an adjusted odds ratio(AOR) of 10.09 . In addition, increased TB positivity was associated with illiterate inmates  and a contact history to TB patients was associated with increased risk to present TB among the prisoners , while decreased TB positivity was associated with patients who had productive cough . Number of inmates per cell, sex, age, fever, weight loss, chest pain, family visit, length of incarceration and presence of symptoms before imprisonment did not have association with the occurrence of TB .Prisons pool and facilitate the spread of smear-positive and -negative pulmonary TB, which is increasingly being reported worldwide Mycobacterium from smear-negative TB cases In this study all AFB microscopy smear-negative pulmonary TB cases were newly diagnosed. Thus, efforts to improve TB case detection in prison should be strengthened. A diagnostic approach using definitive diagnostic methods increased the TB detection rate and is important for diagnosis and treatment of smear-negative TB cases in this difficult setting. The results of the study revealed that the Xpert test has a better performance than culture, because it diagnosed four smear/culture-negative cases and increased the relative proportion of diagnosed smear-negative TB by 2%. However, one smear/Xpert test-negative case was diagnosed positive using culture and increased the prevalence of smear-negative TB by 0.5%, indicating the combined use of different diagnostic tests represented a significant improvement in TB detection rate among smear-negative TB cases in prison settings. Other studies have also shown that culture and Xpert test increased the rate of detection of Frequently occurring clinical features are significant in supporting the diagnosis of smear-negative TB in areas with high TB infection In this study, the disease risk was high for illiterate inmates compared to those inmates with educational status above basic literacy (“above read and write”). One of the main explanations might be the low level of health care seeking behaviors in illiterate patients. Thus, health education for illiterate inmates is important to reduce TB transmission in prisons. A contact history to TB patients in prison was found to be a predictor of TB positivity among smear-negative TB cases, suggesting that transmission may occur in this prison population, although further study may be needed to identify transmission dynamics. Transmission could be facilitated by the nature of the cells shared by the prisoners. Our observation in the study sites showed that the cells are poorly ventilated and dark and that they contained a lot of inmates ranging from 10-587 (the mean number of inmates per cell was 117.4). The high contact rate due to overcrowding combined with poor health care management, transfer of inmates from one prison to another, from room to room and releases of inmates to community before completion of treatment without follow up increases the spread of TB and MDR-TB in prisons and in the general population The strength of our study is that all included cases had culture and Xpert test diagnosed TB, unlike previous reports, where culture and Xpert test were not used In conclusion, a high prevalence of undiagnosed TB cases using AFB microscopy was documented in the studied prisons, which is an important public health concern that urgently needs to be addressed. Furthermore, patients with night sweating, productive cough, a contact history to TB patients and illiterate inmates merit special attention; larger studies are needed in the future to assess the associations more precisely. In-service training of the existing technicians and regular quality control is also warranted. Further studies are required to understand TB transmission dynamics by patients with smear-negative pulmonary TB in a prison setting.


Patients with methamphetamine abuse/dependence often exhibit high levels of impulsivity, which may be associated with the structural abnormalities and functional hypoactivities observed in the frontal cortex of these subjects. Although near-infrared spectroscopy (NIRS) is a simple and non-invasive method for characterizing the clinical features of various psychiatric illnesses, few studies have used NIRS to directly investigate the association between prefrontal cortical activity and inhibitory control in patients with methamphetamine-induced psychosis (MAP). Using a 24-channel NIRS system, we compared hemodynamic responses during the Stroop color-word task in 14 patients with MAP and 21 healthy controls matched for age, sex and premorbid IQ. In addition, we used the Barrett Impulsivity Scale-11 (BIS-11) to assess impulsivity between subject groups. The MAP group exhibited significantly less activation in the anterior and frontopolar prefrontal cortex accompanied by lower Stroop color-word task performance, compared with controls. Moreover, BIS-11 scores were significantly higher in the MAP group, and were negatively correlated with the hemodynamic responses in prefrontal cortex. Our data suggest that reduced hemodynamic responses in the prefrontal cortex might reflect higher levels of impulsivity in patients with MAP, providing new insights into disrupted inhibitory control observed in MAP. Methamphetamine (MA) abuse/dependence is a global public health problem. Approximately 5% of the adult population of the United States has used MA at least once, and there are ~35 million MA users worldwide including Japan , 2. MA aInhibitory control can be defined as the ability to regulate or inhibit prepotent attentional or behavioral actions. A deficit in the ability to inhibit prepotent responses is associated with impulsivity , 13. IndInhibitory control can be clinically assessed using the go/no-go and stop-signal tasks as well as the Stroop color-word task. Indeed, regional brain activities elicited during the Stroop color-word task have been measured using functional magnetic resonance imaging (fMRI) for the assessment of cognitive control –23. PrevIn the present study, we used NIRS to examine prefrontal hemodynamic responses during the Stroop color-word task in patients with MAP. We hypothesized that patients with MAP would show a reduced prefrontal hemodynamic response during the cognitive task. Given that prefrontal cortical activation measured by fMRI was reported to correlate with the Barret Impulsiveness Scale (BIS)-measured impulsivity –38, the Participants were recruited from September, 2014 to August, 2015. We recruited 14 MAP patients  by consecutive sampling, from the outpatient clinic in the Department of Psychiatry at Nara Medical University, Japan . At leasThe study protocol was approved by the appropriate Ethics Committees at Nara Medical University and was in accordance with the Declaration of Helsinki. All study participants or their legal guardians provided written informed consent for their participation prior to the start of the study.Using the Japanese version of the Positive and Negative Symptom Scale (PANSS) , membersThe current BIS-11 contains a 30-item checklist for the measurement of impulsivity . ParticiUsing methods similar to those described previously , we askeBefore starting the task, a well-trained psychologist informed the participants of the following instructions: “This is a test of how quickly you can read the words on the first page, and say the colors of the words on the second page. After you have read the words on the first page for 45 s, we will turn the page. Then you will be required to say the colors of the words on the second page for 45 s. We will repeat this process with the second page three times. Then we will show you the first page again.”We used a design for the Stroop color-word task that has been found to be simpler and easier, as described previously , 48. BriNIRS can detect changes in the concentration of cerebral blood hemoglobins, such as [oxy-Hb] and [deoxy-Hb]. The principle of NIRS is based on the modified Lambert-Beer law, and NIRS monitors the absorption of near-infrared light by [oxy-Hb] and [deoxy-Hb] using two different wavelengths. Increasing [oxy-Hb] and decreasing [deoxy-Hb], as measured by NIRS, have been shown to reflect cortical activation , 52. Rodχ2 test to examine the group difference for categorical variable  In addition, the clinical variables that fit the normal distribution were compared using Student’s t-tests while the clinical variables not normally distributed were analyzed using the Mann-Whitney U test. We performed Student’s t-tests to compare [oxy-Hb] changes between the two groups. For this purpose, we calculated grand average waveforms every 0.1 s in each channel. This analysis enabled a more detailed comparison of [oxy-Hb] changes along the time course of the task. Data analyses were conducted using MATLAB 6.5.2 . We used OT-A4 version 1.63 K  to create the overlap display of the grand average waveforms for both groups in t tests, we corrected for multiple comparisons using the false discovery rate (FDR) . The cot<0.56, p>0.21 for all 3 variables). The results of the psychological examinations are shown in t = −2.13, df = 33, p<0.05,), SCWC-2 , and SCWC-3  scores were significantly lower in the MAP group relative to the controls. Furthermore, patients with MAP exhibited significantly higher scores on the total  and subscale  BIS-11 scores compared with control participants, suggesting that patients with MAP have higher levels of impulsivity.The demographic characteristics of the study participants are presented in ρ = 0.782, p<0.01 and ρ = 0.648 p<0.05), while none of SCWC scores were significantly correlated with age . In the control group, the SCWC-2 and SCWC-3 scores were positively correlated with JART-IQ , whereas none of SCWC scores were significantly correlated with age . The total BIS11 score was negatively correlated with the SCWC-1 score in MAP group only .Because SCWC scores on the Stroop-word color task varied across the patients with MAP and the controls, we calculated Spearman’s rank correlations between SCWC scores, age, JART-IQ score, and BIS-11 scores for all participants . In the U = 87.50, p<0.05) compared with controls. Similarity, we observed a higher mean percentage of errors on the second presentation  in patients with MAP relative to controls.To examine whether disrupted inhibitory control in individuals with MAP was associated with the frequency of incorrect responses given during trials, we assessed the mean percentage of errors made during the incongruent Stroop color-word task . We founp<0.01). Indeed, these channels were nearly identical with the FPPFC and APFC (BA 10). Finally, to examine if the hemodynamic changes in these locations were associated with impulsivity, we conducted Spearman’s rank correlation analyses between the hemodynamic changes and impulsivity scores from the total BIS-11 score. We found that the total BIS-11 scores were negatively correlated with the hemodynamic changes in channel 19 , channel 21 , and channel 23  in all participants , but not in controls , when considered separately. However, the correlations between them in channel 21 and 23 failed to reach the significant level within patient with MAP or controls, probably due to the smaller range of values within each subject group.The grand average waveforms of [oxy-Hb] changes during the Stroop color-word task in the two participant groups are illustrated in icipants . Of the To the best of our knowledge, this is the first NIRS study to compare the prefrontal hemodynamic response during the Stroop color-word task in individuals with MAP and controls. We found that the MAP group showed higher levels of impulsivity, as measured by the total and subscale scores of BIS-11, compared with controls. Additionally, those in the MAP group had lower SCWC scores and a higher mean percentage of errors on the Stroop color-word task, which is at least in part due to deficits in inhibitory control. Finally, our NIRS measurement revealed that, compared with controls, those in the MAP group showed significantly smaller [oxy-Hb] changes in channels 19, 21, and 23 in the PFC, during the Stroop color-word task. These data suggest that patients with MAP have significantly lower [oxy-Hb] changes patterns of blood oxygenation in the FPPFC and APFC (BA 10) compared with controls, which appears to be associated with higher impulsivity in these subjects.Neuropsychological studies have reported that active MA dependent individuals exhibit impaired memory, abnormal manipulation of information, and atypical response inhibition with respect to habitual prepotent responses , 58. As Several researchers have used neuroimaging techniques, including NIRS, to investigate the characteristics of MAP. Consistent with the present study, Okada et al. used NIRS to show that patients with MAP exhibited significantly smaller changes in [oxy-Hb] at the FPPFC during stop-signal tasks which is another task for the assessment of inhibitory control. These individuals also had higher impulsivity compared with controls and schizophrenia patients when assessed by PANSS excitement score . These fp>0.05). Nonetheless, investigating those who are “drug-naïve” or “drug-free” should be considered. Third, our sample size was relatively small. Taken together, future studies using larger samples are required to determine whether reduced hemodynamic change is a conserved feature in individuals with MAP, and to examine whether such changes measured by NIRS are dose-dependent in patients with MAP.The present study has several potential limitations. First, the spatial resolution for the detection of hemodynamic responses from the scalp surface using NIRS is lower than that of fMRI, single-photon emission computed tomography (SPECT), or positron emission tomography (PET). However, the spatial resolution may be within an acceptable range because previous NIRS studies have also found clear distinctions in hemodynamic responses between diagnostic groups , 48, 76.We have shown that patients with MAP performed poorly on the Stroop color-word task, with an associated reduction in prefrontal hemodynamic responses, compared with controls. Additionally, we found that the degree of hemodynamic activity observed during the Stroop color-word task was negatively correlated with impulsivity. Especially given the quick speed of non-invasive NIRS (about 5 min) compared with other functional imaging methods, the present study suggests that NIRS is easily applicable for identifying the reduced PFC activity associated with impaired inhibitory controls commonly observed in individuals with MAP.


Background. Whole lung irradiation (WLI) is a standard treatment component for children with metastatic Ewing Sarcoma (ES), but data on WLI for adults are sparse. Design. An email survey was sent to expert sarcoma-dedicated oncologists worldwide: An adult with excellent performance status presents with primary ES in the leg and multiple pulmonary metastases. The patient achieves complete radiographic response after chemotherapy and resection of the primary. Would you give bilateral WLI to (1) this adult patient?, (2) this patient if 20 years old (yo)?, (3) this patient if 45 yo?, or (4) this patient if 60 yo? Results. 38 experts responded, including 24 adult, 1 adolescent young adult, and 13 pediatric oncologists. 63%, 63%, 62%, and 50% of respondents offered WLI to the adult, 20-year-old, 45-year-old, and 60-year-old, respectively. Pediatric oncologists more likely endorsed WLI across all ages including the adult (P = 0.01), 20-year-old (P = 0.005), 45-year-old (P = 0.01), and 60-year-old (P = 0.08). There were no significant differences between medical and radiation oncologists or between European/Australian and American providers. Conclusions. Almost two-thirds of experts surveyed supported WLI for adults with metastatic ES up to age 45 and half supported WLI for a 60-year-old. Continued collaboration across adult and pediatric oncology is needed to define evidence-based strategies across the age spectrum. Ewing Sarcoma (ES) is a mesenchymal malignancy of unclear histogenetic derivation characterized by distinct chromosomal translocations at the EWSR1 gene , some paHowever, given the limited efficacy and toxicity data pertaining to WLI for adults, the use of WLI in adult ES patients is appropriately more controversial. Like other sarcomas that impact both adult and pediatric patients, ES presents distinct challenges with respect to forming consensus on best practice treatment strategies across the age spectrum. Clinical studies are ongoing, such as the Euro-Ewing-Intergroup EE99 (COG-AEWSO331) trial, which randomizes patients with pulmonary metastatic disease alone to standard consolidation chemotherapy with WLI or to high dose chemotherapy plus peripheral blood stem cell (PBSC) rescue in patients up to 50 years old. In the absence of robust data regarding efficacy and toxicity, there is no clear consensus on the use of WLI for adult ES patients with PM. In this context, we conducted a survey of expert sarcoma-dedicated oncologists to ascertain practice patterns regarding WLI for adults with ES and PM. An adult with excellent performance status presents with a primary ES in the leg and multiple PM. The patient achieves a complete radiographic response after chemotherapy and resection of the primary. Participants were asked: Would you give bilateral WLI to (1) this adult patient?, (2) this patient if 20 years old?, (3) this patient if 45 years old?, or (4) this patient if 60 years old?  The survey was emailed to 54 expert sarcoma-dedicated medical and radiation oncologists from adult and pediatric practices located in the US, Europe, and Australia. Experts were identified on the basis of their active involvement in sarcoma clinical trial groups such as EORTC, Children's Oncology Group (COG), NCI Canada Clinical Trials Group, Sarcoma Alliance for Research through Collaboration (SARC), and NRG Oncology. This study was approved by the Dana-Farber/Harvard Cancer Center Institutional Review Board.We developed a brief survey, in which we described the following clinical scenario:Basic demographic information about survey participants was collected including practice discipline, country of practice, adult versus pediatric specialty, and institution. Responses among participant groups were compared using Fisher's Exact Test.Thirty-eight of the 54 (70%) emailed participants responded to our survey, including 26 medical and 12 radiation oncologists; 24 adult, 1 adolescent and young adult (AYA), and 13 pediatric oncologists; and 1 from Australia, 14 from Europe, and 23 from the US. Overall, 63% of participants opted to offer WLI to the adult patient, 63% to the 20-year-old, 62% to the 45-year-old, and 50% to the 60-year-old .P = 0.01), for the 20-year-old , and for the 45-year-old ; the trend was similar for the 60-year-old, but the results were not statistically different . No statistically significant differences were seen between responses for medical and radiation oncologists or between oncologists in Europe or Australia versus in the US. Among all categories, WLI was recommended the least for the 60-year-old, but still approximately 50% of the time.Despite a relatively poor prognosis among patients with metastatic ES, some of these patients are potentially curable, and as such, aggressive multimodal therapy is a standard approach for metastatic ES. Since the 1970s, WLI has been a component of this aggressive therapy  althoughTo illustrate practice patterns and to gather expert opinions on this topic, we surveyed a global sample of oncologists with dedicated expertise in sarcoma management on the use of WLI in adults with ES. Almost two-thirds of sarcoma experts in our survey supported WLI for adults with ES and PM up to age 45, and half supported it up to age 60. Although there were no significant differences in recommendations between medical and radiation oncologists, or by practice location, we found that pediatric oncologists were the most likely to recommend WLI for adults at nearly double the rate across all age groups.In the pediatric literature, there is substantial evidence to support WLI among ES patients with localized disease  as well  per se has not been described for any age group, but there is a preponderance of evidence associating RT with the heart with subsequent adverse cardiac events for both pediatric and adult cohorts [Broad recommendations for the use of WLI in all age ranges of patients with PM are further complicated by the unique side effect profile and comorbidities relevant to each age group. Toxicity concerns in the pediatric population center on the risks of late pulmonary fibrosis, impaired pulmonary function, chest wall hypoplasia/deformity, and secondary malignancy . The lat cohorts –20. For  cohorts .P = 0.05) predicted for inferior overall survival, whereas a history of smoking predicted for poorer event-free survival (P = 0.04) and showed a trend for inferior overall survival (P = 0.06). Smokers and former smokers also appeared to have higher rates of acute toxicity (73% versus 36%), but this did not reach significance (P = 0.11). No significant differences in outcomes were seen according to response of PM to chemotherapy. This study is an important step towards understanding outcomes of WLI in adult patients with ES but is limited by the relatively young age of patients included and likely selection bias acknowledged by the authors related to the retrospective design, high rate of patients with isolated pulmonary metastases (65%), and high proportion of adults who did not receive WLI at their institution (35%).To help fill the data void relating to WLI for adults, investigators from Memorial Sloan Kettering Cancer Center studied a series of 26 adult patients with ES and PM who were treated with WLI . In thisIn our survey, we identified significant differences in recommendations for WLI between pediatric and adults oncologists. These divergent practice patterns illustrate not only the challenges in the interpretation of limited data sets in rare tumors but also some of the inherent biases that may arise through a practice devoted to either adult or pediatric patients. Practice variation across adult and pediatric providers has been reported in other soft tissue cancers as well. For example, a survey of adult and pediatric oncologists on the treatment of adult medulloblastoma patients similarly demonstrated substantial variation in practice, particularly with the choice of chemotherapeutic agents, likely reflecting the limited data available relevant to older adult population . LikewisTogether, these data call for renewed and continued collaboration across the artifactual divides of adult and pediatric oncology to define evidence-based treatment strategies and appropriate prospective trials across the age spectrum. A vibrant form of this collaboration is seen in the emergence of interest in adolescent and young adult (AYA) oncology as a resource-dedicated discipline dedicated to bridging service and survival gaps in the young adult population and in cancers affecting many age groups which span the traditional “lines” demarcating pediatric and adult oncology –26. EffoThe ongoing Euro-Ewing-Intergroup EE99 (COG-AEWSO331) trial comparing standard consolidation chemotherapy with WLI to high dose chemotherapy plus peripheral blood stem cell (PBSC) rescue in patients up to 50 years old with EW and PM is one example of such interdisciplinary collaboration, which promises to help elucidate the role of WLI across a broad range of ages. As we await those results, our survey suggests that the majority of expert oncologists would offer WLI for most adults with ES and PM in complete response, particularly for younger patients up to the age of 45. While it may be reasonable to treat younger adults with WLI, outside of a clinical trial, we would recommend caution in older adults with comorbidities due to limited safety data in this patient population. For clinical scenarios of localized disease, PM that do not completely respond to induction therapy, or PM in combination with extrapulmonary disease, we agree with others that the potential benefits of WLI are limited and we would not endorse the use of WLI in these scenarios.In this survey of worldwide expert sarcoma-oncologists, approximately two-thirds of respondents supported WLI for adult patients with metastatic ES up to 45 years old, and half supported WLI for patients 60 years old. Pediatric oncologists were most likely to recommend WLI at nearly double the rate across all age groups. These data call for continued collaboration across adult and pediatric oncology to define evidence-based treatment strategies and appropriate prospective trials across the age spectrum. As we await the results of the ongoing Euro-Ewing-Intergroup EE99 study investigating the role of WLI, these data suggest that the majority of expert oncologists would offer WLI for most adults with ES and PM, particularly for younger patients up to the age of 45.


Nearly one-in-ten Sri Lankans are employed abroad as International migrant workers (IMW). Very little is known about the mental health of adult members in families left-behind. This study aimed to explore the impact of economic migration on mental health  of left-behind families in Sri Lanka.A cross-sectional survey using multistage sampling was conducted in six districts (representing 62% of outbound IMW population) of Sri Lanka. Spouses and non-spouse caregivers  from families of economic migrants were recruited. Adult mental health was measured using the Patient Health Questionnaire. Demographic, socio-economic, migration-specific and health utilization information were gathered.A total of 410 IMW families were recruited (response rate: 95.1%). Both spouse and a non-spouse caregiver were recruited for 55 families with a total of 277 spouses and 188 caregivers included. Poor general health, current diagnosed illness and healthcare visit frequency was higher in the non-spouse caregiver group. Overall prevalence of common mental disorder  was 20.7% (95%CI 16.9-24.3) with 14.4% (95%CI 10.3-18.6) among spouses and 29.8% (95%CI 23.2-36.4) among non-spouse caregivers. Prevalence of depression  and somatoform disorder 11.7% (95%CI 7.0-16.3) was higher in non-spouse caregiver group. When adjusted for age and gender, non-returning IMW in family, primary education and low in-bound remittance frequency was associated with CMD for spouses while no education, poor general health and increased healthcare visits was significantly associated in the non-spouse caregiver group.To our knowledge, this is one of the first studies to explore specific mental health outcomes among adult left-behind family members of IMW through standardized diagnostic instruments in Sri Lanka and in South Asian region. Negative impact of economic migration is highlighted by the considerably high prevalence of CMD among adults in left-behind families. A policy framework that enables health protection whilst promoting migration for development remains a key challenge for labour-sending nations. Migration for economic reasons has become an important geo-political phenomenon of the modern era ,2. The gDespite the monetary benefits to migrants, their families and to the country, evidence is emerging about numerous unfavorable effects of economic migration, including adverse health outcomes for IMWs and their left-behind families ,7. AlthoMigration & Health in 2011 prompted public health attention and called for an evidence-based research agenda on health of migrants  in an Indonesian population 12 in an .Most IMWs seek overseas employment with the hope of obtaining higher incomes to alleviate poverty and develop their household capital. However, at the onset of the migratory process they may incur debt. Household remittance studies have revealed the pre-migration pathways result in significant financial costs (including hidden costs due to agent exploitation) to most economic migrants and their families, especially those within low-skilled worker categories . Our finRecently, the Sri Lankan government has been promoting a more ‘skilled’ international migrant workforce. However, as yet, the vast majority of Sri Lankans entering into international labour markets are over-represented in low-skilled employment categories such as labourers and domestic maids ,5. Our sCurrently, there are no existing mechanisms to monitor the health of left-behind families of IMWs, either through public health and education systems or labour migration industry in Sri Lanka. There are no specific provisions offering mental health services to affected adult members of left-behind families. We advocate a multi-sectoral approach for monitoring the health of IMW left-behind families to be adopted at district and national levels with involvement of all relevant stake holders such as the Ministry of Health, SLBFE, provincial ministries of health, social services and public health agencies. The government and multiple stakeholders can also play a role in providing educational sessions on potential mental health issues for IMW families during SLFBE’s mandatory pre-departure training programs. We also advocate that the national migration health policy to be linked with relevant sections of national mental health policy, to enable seamless provision of mental health care for left-behind families of IMWs.As the study cannot determine causality due to the cross sectional nature, prospective cohort and longitudinal studies are needed to reveal true impact of migration on physical wellbeing and mental health outcomes, and whether the workers and their families left-behind truly recover from the migration experience. The ethnic profile of the study sample closely matched national population ratios from the 2001 national population census, with 73% of migrant families being of Sinhalese ethnicity . As mentAs labour migration flow increase in a rapidly developing post-conflict Sri Lanka ,5, the i


Real-time, single-cell multiplex immunophenotyping of circulating tumor cells (CTCs) is hypothesized to inform diagnosis of tissue of origin in patients with carcinoma of unknown primary (CUP). In 20 to 50% of CUP patients, the primary site remains unidentified, presenting a challenge for clinicians in diagnosis and treatment. We developed a post-CellSearch CTC assay using multiplexed Q-dot or DyLight conjugated antibodies with the goal of detecting multiple markers in single cells within a CTC population. We adapted our approach to size-based CTC enrichment protocols for capturing CTCs and subsequent immunofluorescence (IF) using a minimal set of markers to predict the primary sites for common metastatic tumors. The carcinomas are characterized with cytokeratin 7 (CK7), cytokeratin 20 (CK20), thyroid transcription factor 1 (TTF-1), estrogen receptor (ER) or prostate-specific antigen (PSA. IF has been optimized in cultured tumor cells with individual antibodies, then with conjugated antibodies to form a multiplex antibody set. With IF, we evaluated antibodies specific to these 5 markers in lung, breast, colorectal, and prostate cancer cell lines and blood from metastatic prostate and breast cancer patients. This advanced technology provides a noninvasive, diagnostic blood test as an adjunct to routine tissue biopsy. Its further implementation requires prospective clinical testing. Metastasis is responsible for the majority of cancer-related deaths . A limitCarcinoma of unknown primary (CUP) accounts for only 3-5% of metastatic tumors and presents a challenge for clinicians in diagnosis and therapy . CUP patQuantum dots (Q-dots) are nanocrystal fluorophores with long-term photostability and improved brightness, which allow simultaneous excitation of multiple fluorescence colors , 12. DyLIsolation of circulating tumor cells involves both selection and enrichment. The cell surface marker most commonly utilized for isolation of CTCs is the epithelial cell adhesion marker (EpCAM). The Janssen (previously Veridex) CellSearch system with the EpCAM kit uses EpCAM-antibody bound ferrofluid to capture EpCAM positive cells with the use of a magnet. The cells are permeabilized and subjected to further positive selection by immunofluorescence with DAPI, antibody staining with Cytokeratins 8, 18 and 19 followed by negative selection with CD45 antibody staining -15.The Janssen CellSearch is utilized in this study to diagnose carcinoma based on the presence of CTCs as described above and subsequent enumeration. CTCs are also enriched and captured based on their size utilizing either the flexible micro spring array (FMSA) device , 17 or tCytokeratin 7 (CK7), cytokeratin 20 (CK20), thyroid transcription factor 1 (TTF-1), estrogen receptor (ER) and prostate-specific antigen (PSA) are markers commonly used by pathologists in determining the site of origin from tissue biopsies , 18-24. We have developed an immunofluorescence (IF) protocol with a minimal set of markers to predict the primary sites for common metastatic tumors from lung, colon, breast or prostate cancer Figure , and thiThe minimal set of markers we have chosen are commonly used by pathologists in identifying the primary site from tissue biopsies . The antFigure CEA is a secreted cell-surface glycoprotein, which is not included in our algorithm due to its poor tissue specificity. Using an unconjugated primary antibody and fluorescence-labeled secondary antibody, CEA expression was found to be positive in HT29 and weakly positive in HCT 116 colon cancer cells. Specifically, HCT 116 cells have lower levels of CEA and in some cells the expression levels were barely detectable. When the CEA antibody was conjugated to Q-Dot 655, these differences were more subtle, which is attributed to non-specific binding . To incrTo further confirm this, we recruited a patient with ER-negative breast cancer based on prior pathology who had bone metastasis and positive CTCs (2 cells) by CellSearch analysis. Two additional tubes ∼7.5 mL each) of blood were enriched for CTCs with two FMSA devices. The results from one of the tubes showed at least 2 CK7-positive, Hoechst-positive, CK20-negative, TTF1-negative cells in the blood consistent with her breast cancer diagnosis  of blood was evaluated with antibodies to TTF-1, CK7, PSA (and Hoechst) whereas the second tube was evaluated for PSA, CK7, CK20 (and Hoechst) as shown in Figure CTCs were captured from a second breast cancer patient with ER-positive, progesterone receptor (PR)-positive, human-epidermal growth factor receptor 2 (HER2)-negative breast cancer. This patient had bone metastasis and was treated with an aromatase (an enzyme in estrogen synthesis pathway)-inhibitor plus a mammalian target of rapamycin (mTOR) inhibitor. She had 38 CTCs by the clinical CellSearch test. She had blood drawn for this study 37 days later (referred to as the first IF/on-study blood draw) with 167 CTCs by CellSearch Figure  and subsOur study established a new algorithm-based protocol, which enabled determination of the tissue of origin in patients with breast and prostate cancers, two of the most common metastatic cancers. Our proof-of-principle study showed that applying our algorithm in multiplex IF analysis of individual CTCs successfully identified the primary tumor in metastatic breast and prostate cancer patients. Future studies with more patients of known metastatic carcinomas  as well as CUP patients will pave the way for clinical application of this diagnostic blood test.After evaluating different commercially available antibodies to CK7, CK20, TTF-1, ER and PSA, we have identified antibodies, which work well in the appropriate positive and negative cancer cell types. After conjugating various primary antibodies to Q-Dots, and reducing non-specific binding, we determined that DyLight conjugated antibodies allow better distinction between positive and negative expression of the marker than Q-Dots. DyLights 488 and 594 were successfully applied; However, since DAPI or Hoechst occupies another excitation/emission cube this ruled out the use of DyLight 3 The use of Alexa Fluor 680-labeled secondary antibody was much more effective than DyLight 650. Since we had no success with DyLights in the near IR range, this ruled out the simultaneous use of a fourth marker/fluorophore pair. Our primary antibody/fluorescent antibody pairs provided a feasible test to allow for interpretable results relevant to the goal of the study and the eventual clinical application. Finally we developed a protocol, which utilizes a combination of DyLight conjugated primary antibodies and primary /secondary antibody pairs for IF evaluation of two 7.5 mL tubes of blood from each patient. PSA , 31 exprER-negative breast carcinoma poses a particular challenge for accurate diagnosis. Thus our algorithm focused the diagnosis in ER-negative breast cancer or previously ER-positive breast cancer with loss of ER in their CTCs. The CK7-positive, TTF-1-negative, CK20-negative, Hoechst-positive signature observed in our ER-negative patient Figure  is consiThe presence of ER-positive, Hoechst-positive, CK7-negative cells in the blood of the second breast cancer patient could be interpreted as CK7 loss in CTCs. Only a subset of cytokeratins are down regulated during epithelial to mesenchymal transition (EMT). There is a dearth of information on the frequency of CK7 loss in breast cancer CTCs, where antibody cocktails targeting several cytokeratins are often utilized , 33. AltOur prostate cancer patient demonstrated a CK7-positive, TTF-1-negative, PSA-positive profile in Hoechst-positive CTCs which can only be diagnosed as prostate cancer. Moreover, PSA marker expression has been shown to be stable after metastasis in prostate cancer patients and no metastatic tumors of non-prostatic origin express PSA in this study . CK7-posA third tube of blood from each patient is used for FDA-cleared CellSearch analysis of the CTCs using the epithelial kit to enumerate the CTCs and diagnose carcinoma. Although our initial approach was to further immunophenotype the same cells captured by the CellSearch using 4 of the 5 markers, it was determined that both the FMSA and CellSieve enable superior IF analysis of cells captured on the device. It is important to note that we obtained comparable or greater numbers of CTCs using these size-based approaches as with FDA-cleared CellSearch. A broader limitation of the utilization of EpCAM for CTC selection is that many of the CTCs may not express EpCAM. Cells undergoing EMT and poorly differentiated stem cell-like tumor cells are likely EpCAM negative. Our study identified metastasis of the four most common carcinomas  from CTCs, which typically express high levels of EpCAM . NeverthCUP can have early  and unusOur future studies involve implementation of an approved clinical study, which is divided into two phases/stages. In the first stage/phase of the CUP-CTC study, we will recover CTCs in 20 patients with metastatic cancers of known primary , and then use our novel assay to obtain informative marker expression information to aid in the diagnostic process. The primary site of origin as determined from the novel CTC-CUP assay will be verified using classical immunofluorescence and common IHC markers as is routinely performed in the course of clinical care of patients. In second stage/phase of the CTC-CUP study, we plan to enroll 20 patients being evaluated for suspected CUP.The results of this study will not replace the standard diagnostic evaluation including history and physical exam, laboratory testing, imaging, tissue biopsy and clinical correlation; however, we aim to use the knowledge obtained to improve upon the accuracy and timing of diagnosing the tissue of origin in patients with CUP in a noninvasive manner in the future. Patients with poorly differentiated carcinomas may pose the greatest challenge in identifying the primary both in terms of biopsy of the metastatic site(s) and the CTCs. If this small clinical study is successful, further implementation will require prospective clinical trials. We estimate that if a simple CUP-CTC blood test can facilitate diagnosis in as few as 10-20% of patients with CUP, such relatively noninvasive and inexpensive assay would have wide appeal for clinical use especially in the outpatient setting.The following antibodies and fluorophores were used: CK7 (Dako), CK20 (Dako), TTF1 (Invitrogen), ER clone 16660 (Abcam), PSA  and AFP (Cell Marque). DyLight 594, DyLight 488 (Pierce), Q-Dot 605 and Q-Dot 655 (Life Technologies).Blood (∼7.5mL) collected in an EDTA tube was filtered through a single FMSA device within 4hrs of collection. The device was gently moved to a glass microscope slide and fixed with Cytofix/Cytoperm (BD Biosciences) for 30-45 minutes at 4 degrees Celsius, washed in PBS  and incubated in PBS with 0.3% Triton-X-100/0.1% BSA (modified PBT) for 10 minutes. The samples were subsequently blocked for > 1hr with 1:1 mixture of serum from the same species as the secondary antibody and modified PBT. The first set of antibodies are diluted in modified PBT overnight at 4 degrees Celsius in a humidified chamber with paraffin covering the device resting on a glass slide. After washing with PBS, the secondary antibody diluted in PBT was added at RT for 2hrs. After gentle washing with PBT and PBS, the device was either washed with deionized water for nuclear staining or incubated with conjugated antibody/antibodies diluted in PBT for 5.5 to 6hr incubation at 4 degrees Celsius followed by washing in PBT, PBS and deionized water. DAPI or Hoechst freshly diluted in deionized water was added for 1 minute and washed with deionized water. After adding mounting media and a coverslip, the slides were viewed. The same protocol was used for the CellSieve (Creatv Microtech) device, which may be retained longer in the filter holder assembly before moving it to a glass slide. The CellSieve device (7micron pore diameter) is used with a syringe pump for gently pushing the blood through the device and washing with PBS.Plated cells positive and negative for all the markers were also evaluated for the presence of the marker proteins on the same day as the patient sample for the metastatic ER/PR positive breast cancer patient on both days that blood was drawn for CTC enrichment. MCF7 (ER positive control) and SKBR3 (CK7 positive control) cells were spiked into normal donor blood and captured on the CellSieve device the same day as the patient sample for this patient's first on-study blood draw. The antibodies were diluted for the positive/negative control plated cells, spiked positive controls and patient samples in one tube before aliquoting to ensure that results from patient samples cannot be attributed to the experimental execution.Blood (7.5mL) collected in a CellSave tube was used run through the Janssen (Veridex) CellSearch system using an EpCAM-based Circulating Epithelial Cell Kit within 72hrs of collection.Post-CellSearch immunofluorescence was performed after collecting cells by flushing the CellSearch cassette with PBS and cytospin of the cells onto a glass slide.i inverted microscope and NIS Elements software or to evaluate Q-dots CRi Nuance software for multispectral imaging (El-Deiry laboratory). Some automated imaging was conducted using a DeltaVision Elite microscope system, plus an upright fluorescence microscope with a Retiga Exi high-speed CCD camera with QED software for image acquisition and Huygens software (Penn State Hershey Imaging Core).Imaging was primarily performed on a Nikon Eclipse T