Among them, XIAP is the causative gene for the immunodeficiency phenotype seen in the patient. The remaining genes displayed specific expression in the fetal brain and have known biologically relevant functions in brain development, suggesting putative candidate genes for neurodevelopmental phenotypes. This study demonstrates the application of NGS technologies in mapping individual gene disruptions in ABCR as a resource for deciphering candidate genes in human neurodevelopmental disorders (NDDs).Delineating candidate genes at the chromosomal breakpoint regions in the apparently balanced chromosome rearrangements (ABCR) has been shown to be more effective with the emergence of next-generation sequencing (NGS) technologies. We employed a large-insert (7–11 kb) paired-end tag sequencing technology (DNA-PET) to systematically analyze genome of four patients harbouring cytogenetically defined ABCR with neurodevelopmental symptoms, including developmental delay (DD) and speech disorders. We characterized structural variants (SVs) specific to each individual, including those matching the chromosomal breakpoints. Refinement of these regions by Sanger sequencing resulted in the identification of five disrupted genes in three individuals: guanine nucleotide binding protein, q polypeptide  DMD in Dystrophin for Duchenne Muscular Dystrophy DISC1 in Schizophrenia ATP7A in Menkes disease. in situ hybridization (FISH) mapping across the breakpoints for more detailed delineation is restricted to the investigated region. Microarrays technologies are able to identify copy number changes at considerable resolution but fail to identify copy number neutral rearrangements. Apparently balanced chromosomal rearrangements (ABCR) occur sporadically in the population or segregate within families, with a frequency of 1 in every 2000 live births. ABCR have been largely associated with infertility, ovarian failure and intellectual disability (ID) when detected during pre- and post-natal investigation or genetic counselling. de novo inversion), and prior aCGH analysis did not reveal chromosomal imbalances. Our study shows that NGS technology enables rapid identification of individual gene disruptions and potential candidate genes in ABCR. We also demonstrated the correlation of disrupted gene XIAP in an inversion breakpoint to be causative for the patient’s immunodeficiency phenotype.We have established a NGS pipeline, referred to as a DNA-PET sequencing, By using patients’ genomic DNA as starting material, libraries were generated and sequenced using a SOLiD platform, and we obtained an average of 35 million non-redundant paired-end reads . To evaluate the potential implication of the disrupted genes, we assessed the occurrence of pre-existing Copy Number Variations (CNVs) in DD cases from published studies and the DECIPHER Consortium RBFOX3 and UNC5D, suggesting that altered gene dosage in these genes might have a role in NDDs.From these extensive analyses, we identified five disrupted genes in three patients: d TMEM47 . Among tGNAQ at intron 5 on chromosome 9 and RBFOX3 at intron 2 on chromosome 17. Both disrupted genes shared the same orientation, breakpoints lie within introns, and the resulting fusion is predicted to be in frame. However, RT-PCR analysis on the patient’s lymphoblastoid cell line did not reveal any fusion transcript expression as neither gene is expressed in these cells, reflecting the importance of relevant cell type for validation of brain-specific genes. We checked the mRNA expression of both genes in human tissue panel and found that both genes are highly expressed in the fetal brain and the cerebellum . These five SVs overlapped with known CNV regions  reported in healthy individuals or were in intergenic regions  and were therefore excluded for further analysis  and DD during his childhood, and his two sons (CD21 and CD22) displayed absence of speech, DD and autistic disorder . The trarebellum . GNAQ enUNC5D at intron 5 on chromosome 8, while no coding genes were disrupted on chromosome 6 , the causative effect of the t balanced translocation is unclear, due to variable expression of phenotype in the translocation carriers; his mother is asymptomatic and his younger sibling displayed schizencephaly and DD (CD11) . DNA-PETmosome 6 . UNC5D erebellum , thus exrebellum . Analysirebellum .The third patient (CD8), carried a maternal-derived pericentric inversion at chromosome X, between Xp22 and Xq26 . His monXIAP and TMEM47. The latter encodes a member of PMP22/EMP/Claudin protein family is ubiquitously expressed in human tissues including adult and fetal brain , which has been associated with X-linked lymphoproliferative syndrome type 1 (XLP-1 [MIM: 308240]). This gene was initially suspected as the primary cause of immunodeficiency phenotype seen in the patient, although it was rejected due to normal SAP expression in patient’s blood lysates (see Patients and Methods). ODZ1 is the second gene affected by this rearrangement. It encodes an Odd Oz/Ten-M homolog 1 of Drosophila pair-rule gene involved in post-segmentation processes, including embryonic development of the central nervous system (CNS), eyes, and limbs in Drosophila This complex rearrangement disrupted two genes: al brain . Two othXIAP1 and TMEM47 and a moderate to normal expression for SH2D1A and ODZ1 in patient’s fibroblast cell line compared to sex-matched controls, supporting the absence of functional XIAP1 and TMEM47 genes in the male patient  , sequencing analysis identified two paired-inversion clusters corresponding to the large inversion on chromosome 5. This inversion spans 53 Mb on chromosome 5q22.2  and 5q34 , with 1 bp deletion, and microhomologies of 3 bp between paired breakpoints. No gene was disrupted at the breakpoint junctions  with a diagnostic yield of 16%, comparable to the CNV burden obtained in copy number studies In the past decade, we have seen substantial progresses for identification of novel candidate genes in NDDs with the recent development in technologies. Two studies of large cohorts of patients with developmental delay described the enrichment of large CNVs in 15% of the cases Recent studies have described the feasibility of using NGS technologies to map the ABCR breakpoints in patients with neurodevelopmental abnormalities Our work described the use of a genome paired end tag (DNA-PET) sequencing with larger insert sizes between 7–11 kb GNAQ, RBFOX3, UNC5D, XIAP, and TMEM47), and we observed various complications in attempting to correlate these genes to the expressed phenotypes. For one case (Patient CD8), the complex inversion pattern seen in chromosome X resembles chromothripsis, phenomenon commonly found in cancer and recently, in congenital diseases, which resulted from localized shattering of one or few chromosomes and assembly of chromosomal pieces by non-homologous end-joining (NHEJ), and thus appeared as complex chromosome rearrangements, involving three or more breakpoints XIAP gene, which is the primary cause of a rare form of XLP2 TMEM47 is among the potentially interesting candidate, with total absence of transcript expression in patient’s cell line and expression in fetal to adult brain. We proposed the need to perform mutational screening of TMEM47 in X-linked ID patients for future studies.In this study, five genes were identified within the ABCR-breakpoint regions in three patients (For one patient (Patient CD9), there were no disrupted genes or regulatory elements at the breakpoint regions, neither potential other interesting SVs, showing the limitation for candidate gene detection merely through ABCR breakpoint cloning methods. Alternatively, exome sequencing of parent-offspring trio can be considered as a method of choice to further investigate possible causal variants especially in cases where there is a lack of association between ABCR and disease.GNAQ and RBFOX3 genes in a t translocation in three affected members of a family (Patient CD5) with different level of neurological symptoms (mild to severe) and UNC5D disruption in a family harbouring a t translocation carried by two affected siblings (Patient CD10) and his mother. The translocation carriers in latter family displayed a broad range of clinical presentations; an asymptomatic mother, her first child with mild DD, and her second child presenting schizencephaly, polymicrogyria and LD, suggesting additional etiological factor underlying these features apart from the t translocation. Besides the large phenotypic variability between translocation carriers seen in both families, we observed an enrichment of CNV counts for UNC5D and RBFOX3 in NDDs-associated cases For the two remaining familial translocations, we identified fusion gene at the genomic level, although unverifiable at the transcript level due to lack of expression in available cell lines between GNAQ, RBFOX3, UNC5D and TMEM47) in brain development, further functional validations are required to establish a correlation between these disrupted genes and patients’ clinical phenotype. Furthermore, reporting such ABCR-disrupted genes is crucial to determine the clinical relevance of newly identified CNVs or SNVs encompassing these genes.Despite the potential functions of four identified genes  between both NGS-and array-based technologies shown in this study emphasizes the importance of employing a single technique that can provide high coverage of genome information with consistent validation rate as a routine clinical diagnosis tool. The stringency of our filtering pipeline can be optimized and adjusted depending on the sequencing platforms, and this would revolutionize the characterization of individual genomes of patients without prior karyotypic observation. For the implementation in the clinical settings, unaffected parents or siblings should be included for genome investigation to reduce the number of familial, non-pathogenic SVs. Whilst the technology continues to improve, validation with longer read depth by Sanger sequencing is still necessary for better SVs annotation. Our study complements the existing application of NGS technology in unexplained NDDs patients for better characterization of chromosomal rearrangements and discovery of potential candidate genes.All samples and information were collected after written informed consent from patient’s parents was obtained and in accordance with local institutional review board approved protocols from National University of Singapore in Singapore, Children’s Hospital Westmead in Sydney, Australia and Centre Hospitalier Regional in Orleans, France. DNA samples were obtained from peripheral blood lymphocytes and cultured skin fibroblasts obtained from patients seen at the participating institutes.This is a familial balanced translocation presenting variable degree of DD and autistic features. The first son displayed an autistic behavior and global DD at three years of age with an absence of speech, feeding and sleeping difficulties, habit disorders, and stereotypic movements. At 4 years, there is an improvement in communication and speech seen in the first son. Chromosome analysis revealed a translocation t see , which iThe patient was born at term by lower segment caesarean section, due to breech presentation. Delay in developmental milestones was noted at 18 months of age affecting both walking and speech. At 9 years old, comprehension and behavioural difficulties were noted at school. Karyotype analysis revealed a balanced translocation t see . MetabolThe patient was one of monozygotic twins. At the age of three years, he had an acute EBV infection with prolonged hepatosplenomegaly and abnormality of liver function tests. Immunological investigations showed reduced IgM, decreased CD4 T-helper cells and decreased natural killer (NK) cell function. Patient was suspected for XLP-1, but western blot analysis on patient’s whole blood cell lysates showed normal expression of SAP. At the age of five years, mild ID was diagnosed, as well as a specific language disorder affecting his receptive and expressive skills. Karyotype analysis revealed a complex inversion inv(X) see , derivedThe patient was born by normal delivery after an uneventful pregnancy. Mild delay in early motor and speech developmental milestones was reported. At age twelve, he was found to have average intellectual ability, an expressive language disorder, and specific learning difficulties in maths, spellings and reading; as well as attention deficit disorder. Chromosome analysis revealed a paracentric inversion of chromosome 5 see . Both paKaryotypes were determined from G-banding analysis using standard protocol according to the ISCN nomenclature. FISH analysis was carried out using protocols as described elsewhere. Genomic DNA from lymphoblastoid cell lines, fibroblast, or blood from the patient was extracted by Qiagen Blood and Cell Culture DNA Kits (Qiagen), according to manufacturer’s instruction. lymphoblastoid and fibroblast cell lines were maintained to a minimum number of passages prior to DNA extraction. Quality and quantity of the extracted DNA were measured using NanoDrop 1000 Spectrophotometer and agarose gel electrophoresis.aCGH was performed in all patient samples using the SurePrint G3 Human 2 x 400 k aCGH Microarray (Agilent Technologies Inc. Santa Clara) according to the manufacturer’s instruction. The microarray slides were scanned on an Agilent Microarray Scanner. Data were processed by Genomic Workbench software, standard edition 5.0.14 (Agilent). We used the Aberration Detection Method (ADM-2) algorithm to identify DNA copy number variations (CNV). The ADM-2 algorithm identifies all aberrant intervals in a given sample with consistently high or low log ratio based on the statistical score. We applied a filtering option of minimum of three probes in region and centralization threshold of 6. We used NCBI Build 36 as a reference genome. For smaller CNVs that were identified by DNA-PET, we compared with the aCGH raw data and interpreted the copy number change by looking at the individual probe ratio, using a cutoff of 0.1 .We constructed the DNA-PET libraries for four patients according to Method described in Hillmer et al. 5 The remaining portion of the PETs mapped discordantly to the reference genome (discordant PET or dPETs), classified as those with incorrect paired-tag orientation and incorrect genomic distances. These dPETs provided information to search for genomic rearrangements; with specific criteria for different types of SVs according to PET mapping orientation and genomic region as described by Yao et al. The majority of the PET sequences mapped accordingly to the reference genome (concordant PETs or cPETs) with expected mapping orientation (5′ tag to 3′ tag) and expected mapping distance (according to the selected fragment size) The distribution of cPET in the genome was used to retrieve copy number information.Comparison of clusters across different genomes was performed as described by Ng et al. We used the CNVs map from published data by Cooper et al. with 15,767 cases of DD and 8,329 adult controls to screen for deletions or duplications in our candidate genes The hg18 coordinates of genomic regions from each patient’s SVs list were converted to hg19 in LiftOver from UCSC genome browser. SVs coincide within introns or intergenic regions were assessed for the probability score of functional regulatory regions in RegulomeDB Primers were designed by Primer3 program, and the amplicons spanning the breakpoint were predicted by dPET clusters according to human genome assembly NCBI Build 36. PCR was carried out with JumpStart REDAccuTaq LA polymerase  in a 50 µl reaction volume and with 500 ng of genomic DNA as a template. The following program was used: 1) Initial denaturation at 96°C for 30 sec, 2) 40 cycles of 15 sec at 94°, 30 sec at 58°C, 10 min at 68°, 3) 68°C for 10 min. PCR products showing single bands were purified by Gel Extraction Kit (Qiagen) and used as templates for sequencing in both directions by Sanger sequencing. The sequences of junction fragments were aligned to the human genome reference sequence using Blat ΔΔCt method, and normalized against control sample with human ACTB. Each measurement was performed in triplicate. The controls used in this study are derived from EBV-LCL or skin fibroblast cells of 4 normal individuals.Total RNA was extracted from patient’s EBV-LCL or fibroblasts using RNAeasy kit (Qiagen). Reverse transcription of 2 µg RNA derived from patients, 2 lymphoblast controls, 2 fibroblast controls and commercially available human tissue panel RNA (Clontech) was performed in 20 µl of SuperSCript III Reverse Transcriptase reaction buffer (Life Technologies) using random hexamer primers. Quantitative real-time PCR (qRT-PCR) reactions were performed in the ABI PRISM 7500 HT system (Life Technologies) with five-fold dilution of cDNA, 200 nM of each primer using the SybrGreen PCR Master Mix (Life Technologies). Data were analyzed using 2Information S1SupportingInformation_S1.pdf contains additional information to the manuscript. It consists of 9 pages, 1 Figure and 7 tables.The file (DOCX)Click here for additional data file.


Gentrepid uses a multiple-locus-based approach, drawing on protein pathway- or domain-based data to make predictions. Known disease genes may be used as additional information (seeded method) or predictions can be based entirely on GWAS single nucleotide polymorphisms (SNPs) (ab initio method). We looked in detail at specific predictions made by Gentrepid for CAD and compared these with known genetic data and the scientific literature. Gentrepid was able to extract known disease genes from the candidate search space and predict plausible novel disease genes from both known and novel WTCCC-implicated loci. The disease gene candidates are consistent with known biological information. The results demonstrate that this computational approach is feasible and a valuable discovery tool for geneticists.Current single-locus-based analyses and candidate disease gene prediction methodologies used in genome-wide association studies (GWAS) do not capitalize on the wealth of the underlying genetic data, nor functional data available from molecular biology. Here, we analyzed GWAS data from the Wellcome Trust Case Control Consortium (WTCCC) on coronary artery disease (CAD).  Coronary artery disease (CAD) is the leading cause of death and disability in the world  with the true variant, as SNP chips contain only a selection of common variants and have incomplete coverage of the genome. Second, even with knowledge of the true variant, its functional significance may not be obvious as the genetic architecture of the genome still remains unclear. GWAS typically report the nearest neighboring gene to the disease-associated SNP/locus, but this assumption may not hold for all reported associations. For instance, long range regulation and distal control elements suggest the disease gene may be near the significant SNP but may not be the closest gene to it  data; along with a novel functional approach whereby protein domains parsed in sequences are used to infer function. Although previous pathway analyses of the Wellcome Trust Case Control Consortium (WTCCC)   study da (WTCCC) , no domaGentrepid looks at interactions and similarities between loci, it is particularly apt for analyzing the multiple loci suggested by GWAS data. By looking at the GWAS data holistically and incorporating protein information, interactions and common features between loci can be detected, thereby improving candidate disease gene prediction outcomes. Gentrepid was originally benchmarked , a moderately high significant set , a moderately weak significant set , and a weakly significant set . SNPs in close proximity (within 50 Kbp) were merged into a single locus.We selected four SNP sets by iteratively lowering the stringency threshold of the Cochran-Armitage For each of the four significant SNP sets, we created six gene search spaces, three based on SNP gene proximity, labeled the “nearest neighbor” (NN) approach , and three based on SNP gene distance labeled the “bystander” (BY) approach . For the NN approach, the resident set includes only genes with significant SNPs within the gene boundary. The nearest set contains the closest gene to each SNP. In the adjacent set, a gene is selected upstream and downstream of each SNP on both strands of DNA, resulting mostly in four genes for each SNP. For the BY approach, genes were pooled from an interval around each SNP of window sizes of 1 Mbp, 0.5 Mbp, and 0.1 Mbp, respectively. As most SNPs on the chip used by the WTCCC are in noncoding regions, creating several different search spaces ensured that likely genes were included in the analysis. The methods are fully described in the protocol development paper ; and one that uses only genes within the search space (ab initio). For seeded mode, we used 13 genes already associated with the disease listed in OMIM , searches for genes in the implicated loci that bind transcription factors. Regulatory information was sourced from oRegAnno  looks for genes among the implicated loci that are common miRNA targets and in regulatory hubs. Dysfunction of miRNAs is believed to play a role in diseases of the heart, central nervous system, and immune system , the data had 410 implicated loci, with ∼49% overlap with previously implicated regions from OMIM  receptor 1 CX3CR1 (MIM 601470) in 3p22.1-3, a chemokine involved in LDL signaling pathways; and CD36 (MIM 173510) and insulin receptor substrate 1 IRS1 (MIM 147545), which are both receptors in the adipocytokine signaling pathway. A fourth gene, nitric oxide synthase 3, NOS3 (MIM 163729) in 7q21.11, though only supported by SNPs in the WS set, was predicted through CPS seeded. Lipoprotein A , also only supported by weak SNP association signals, was predicted through CMP  and a phosphorylase MTAP (MIM 156540), associated with a single highly significant locus (9p21), were predicted via common metabolic pathways along with a modestly associated dehydrogenase MTHFD1L . The metalloproteinase ADAMTS7 (MIM 605009), implicated by a modest association (15q24), had common domains with another metalloproteinase in the gene search spaces.Of the five reported WTCCC genes of interest, all five genes were predicted by MP Table  and wereCX3CR1 (MIM 601470) and IRS1 (MIM 147545) have previously been identified as the disease genes. Predictions within previously determined loci for which a known gene has not been determined were based on very weak genetic signals, typically one, or at most two WS SNPs. They include tyrosine kinase 2 TYK2 (MIM 176541) in 19q13, regulatory factor X RFX5 (MIM 601863) in 1q21.2, fragile histidine triad FHIT (MIM 601153) in 3p14.2, and the tumor necrosis factor receptors TNFRSF10A-D  in 8p21.2.Our next assessment was to analyze the candidate disease gene predictions that had not been previously reported at the time of the generation of the data. First, we looked at the candidates predicted in loci that were previously implicated but had no known or candidate disease gene, as not all the loci listed in OMIM have candidates. Of 15 previously determined disease loci, all contained WS SNPs. Only five loci contained MWS SNPs and one locus contained MHS SNPs. None of the previously implicated loci were detected at the HS level. The largest numbers of SNPs were associated with 2q36.3 and 3p22-p21 in which IKBKAP (MIM 603722) in 9q31.2 was recently shown to be differentially regulated in patients with acute myocardial infarction compared to controls .In addition, 15 alternative gene predictions were made for loci with previously determined disease genes. Of these, the IκB kinase complex-associated protein Gentrepid CPS in loci novel to the WTCCC study were diabetes related. In the “Type II diabetes mellitus” pathway , three genes in three novel loci  were implicated in addition to the known disease gene IRS1 (MIM 147545). In the MWS set, the “Insulin signaling pathway” was the most significant . Patients with T2D are known to have a higher risk of CAD. The possible commonality of pathways underlying CAD and T2D was raised by Torkamani et al. (HIF1A (MIM 603348) as a candidate. Overall, 56 novel pathways were predicted in ab initio mode across all the gene search spaces (Table S2).We then looked at the novel candidates from each of the modules for the regions implicated by our novel methodology and SNP/gene mappings. Two of the most significant pathways predicted by i et al.  based onab initio and seeded module (Table S1) predicted three of those genes: RAC1 , PDGFRA , and PLD1  . WASF1 , and the enzyme LIM domain kinase  were not in the Framingham study but were predicted by our method. LIMK1 (MIM 601329) may be involved in cardiovascular disease through its interactions with BMP type II receptor   in the gene search spaces; two genes homologous to the lipoprotein carrier LPA (MIM 152200): PLG (MIM 173350) and LPAL2 (MIM 611682); and a matrix metalloproteinase  similar to MMP3 (MIM 185250), involved in extracellular matrix (ECM) breakdown (Table S3). Many plausible candidates were predicted by CMP ab initio (Table S4). Cell–cell and ECM adhesion, as well as their remodeling, featured prominently. Genes with the strongest genetic support are the vascular adhesion factors SEZ6L  and CSMD2 . Adhesion between the cell and the ECM is implicated by multiple integrins and matrix metalloproteases as well as by transforming growth factor TGFBI (MIM 601692) and periostin POSTN (MIM 608777). TGFBI (MIM 601692) binds to type I, II, and IV collagens. Other adhesion genes predicted were adhesion G-protein coupled receptors. Lipid signaling was also implicated by phospholipases, DAG kinases, and protein kinase C-like genes (Table S4).Protein domains are highly conserved globular structures each with their own biochemical function  that interact with known disease gene CCL2 (MIM 158105), and LIMK1 (MIM 601329) that interacts with ABCA1 (MIM 600046)  and less stringent significant thresholds (WS and MWS) had the greatest number of predictions in both ab initio module are listed in Tables S6, S7. Visualizations of the interactions  or the ubiquitin-specific processing protease USP7 (MIM 602519). The ubiquitin–proteasome complex and proper protein degradation are involved in cardiovascular physiology and disease with roles in endothelial function and atherosclerosis  gene was the sole prediction. For the MIR module, IRS1 (MIM 147545) was the only significant prediction in seeded mode. The miRNAs that implicated this gene were mir-126, known to be involved in angiogenesis . The homeobox CDX2 (MIM 600297), transcription factor GATA6 (MIM 601656) and axin interactor AIDA (MIM 612375) are regulated by the mir-181 family. Mir-181 target genes are involved in myogenesis, muscle regeneration  mutations are known to cause congenital heart defects  is highly expressed in the heart and skeletal muscle, also making it an interesting candidate  by their common regulator mir-148 are interesting predictions, as mir-148a also promotes skeletal muscle differentiation , mitogen-activated protein kinase kinase kinase 4  and guanylate cyclase soluble subunit beta-1 . GUCY1B3 (MIM 139397) is known to interact with the endothelial NOS , a gene associated with ischemic heart disease and hypertension  gene may be a false-positive result. However, the OMIM validation set contained genes that were selected by our system, thereby validating the technique to some extent. Also, by demonstrating that Gentrepid selected at least half of the candidates from the CARDIoGRAMplusC4D, the system appears competitive with meta-analysis methods used by researchers to determine candidates. The system also made alternate predictions which might be of interest too. Another point to note is the low concordance of the pathway enrichment results with the Framingham study  that a result is a false positive. From our methods paper , no prior disease gene information is required, allowing for the discovery of novel pathways and regulatory elements that may be important in the disease that were not previously considered, along with protein domains attributing novel functions to the mechanisms behind the disease. The few functionally annotated miRNAs that produced predictions had relevant biological functionality in the phenotype and it would be very interesting to prod further into these, in particular genes GATA6 (MIM 601656) and AIDA (MIM 612375). MicroRNA research is expanding and will produce motivating hypotheses relevant to disease. Further to this, genes such as RAC1 (MIM 602048), LIMK1 (MIM 601329), SEZ6L (MIM 607021), and CSMD2 (MIM 608398) also warrant further investigation as they were predicted by multiple methods or had strong genetic support.Biological filtering improves the amount of knowledge extracted from the study. Using the blind method . Registration is free and data are stored remotely and securely. Each of the methods highlighted here can be performed with the exception of the regulatory modules CRT and MIR under development. The only input required is a list of SNPs or markers and an optional phenotype for the seeded mode.Although there are alternate methods that capitalize on gene and pathway enrichment analysis (Raychaudhuri et al. As in all candidate disease gene methods, it is still difficult to perform a fair assessment of the results without further biochemical functional studies. Overall, we believe our pipeline is a suitable methodology for generating plausible hypotheses from GWAS. The study demonstrates that using existing knowledge and a holistic multiple loci approach provides insight into what is a very complex disease.


Meconium fatty acid ethyl esters (FAEE) constitute a biomarker of heavy fetal ethanol exposure. Our objective was to measure meconium FAEE in fetal sheep following daily, relatively moderate-dose ethanol exposure in late gestation, and to evaluate their utility in identifying fetal organ-system injury.n = 14) or saline (n = 8) via 1-h IV infusion daily during the third trimester equivalent, while additional pregnant sheep served as untreated controls (n = 6). The daily ethanol regimen produced similar maximal maternal and fetal plasma ethanol concentrations of 0.11–0.12 g/dL. Ewes and fetuses were euthanized shortly before term, and meconium was collected and analyzed for FAEE .Pregnant ewes received ethanol  were classified according to meconium FAEE concentration, FAEE-positive and FAEE-negative groups frequently differed with respect to previously examined pathological endpoints, including nephron endowment, lung collagen deposition, cardiomyocyte maturation, and tropoelastin gene expression in cerebral vessels. Furthermore, in all studied animals as a group (ethanol-exposed and controls combined), meconium FAEE concentration was correlated with many of these pathological endpoints in fetal organs.in-utero ethanol exposure that does not result in overt physical signs of ethanol teratogenicity.We conclude that, in fetal sheep, meconium FAEE could serve as a biomarker of daily ethanol exposure in late gestation and could identify fetuses with subtle ethanol-induced toxic effects in various organs. This study illustrates the potential for using meconium FAEE to identify neonates at risk for dysfunction of major organs following  Alcohol (ethanol) is a well established human teratogen that can result in a range of physical defects, and cognitive and behavioral deficits, known collectively as fetal alcohol spectrum disorders (FASD) A major challenge in diagnosing milder forms of FASD is the absence of clear external markers of ethanol teratogenicity such as the craniofacial characteristics, which are present only in the most severe cases. In the absence of such overt dysmorphology, it is necessary to confirm prenatal alcohol exposure to make a diagnosis Fatty acid ethyl esters (FAEE) are produced by non-oxidative ethanol biotransformation involving the conjugation of ethanol to endogenous free fatty acids or fatty-acyl-CoA Recently, a comprehensive study was conducted on the effects of repeated maternal administration of relatively moderate-dose ethanol in pregnant sheep during the third-trimester-equivalent on several fetal organs. In this study, pregnant ewes received one-hour daily ethanol infusion during the third-trimester-equivalent that produced maximal maternal and fetal plasma ethanol concentrations (PEC) of 0.11–0.12 g/dL, which approximated blood ethanol concentration measured in non-pregnant women drinking socially  All animal experiments were approved by the Animal Ethics Committee of Monash University where the animal experimentation was performed (permit number: PHYS/2005/31) in accordance with the National Health and Medical Research Council of Australia guidelines. All surgery was performed under anesthesia as described below, and all efforts were made to minimize suffering.2-N2O (50∶50 vol/vol). Catheters were aseptically inserted into a maternal carotid artery for blood sampling and into a jugular vein for ethanol or saline infusion. Six additional sheep served as untouched controls. From 95–124 DGA, 14 randomly chosen surgically-instrumented pregnant ewes received daily, 1-h intravenous infusion of 0.75 g ethanol/kg maternal body weight of 40% (v/v) ethanol-saline solution, and 8 pregnant ewes received 1-h intravenous infusion of an equivalent volume of saline. Pregnant ewes had free access to food and water, and animals in the saline cohort were provided with about 120 g  of additional food to compensate for calories derived from ethanol metabolism. At 126 DGA, pregnant ewes  were anesthetized, and catheters were inserted aseptically into the fetal brachial artery for fetal blood collection. During fetal surgery twins were observed in one ethanol and one control animal, and only one of the twins was catheterized. After recovery from surgery, daily maternal ethanol infusion resumed on 128 DGA and continued until 133 DGA. Paired fetal and maternal arterial blood samples were collected from 131–134 DGA and it was determined that this ethanol regimen produced maximal maternal and fetal PEC of 0.12±0.01 g/dL and 0.11±0.01 g/dL (mean +/− SEM), respectively, at the end of the 1-h ethanol infusion Experimental animal preparation, ethanol dosing, and study design have been described previously in detail T (cycle threshold) method. Values were expressed relative to the mean mRNA expression level in control animals for each gene. This protocol was previously used and reported by our group Table S1).Pregnant ewes and their fetuses were euthanized with an intravenous dose of pentobarbital sodium  administered to the ewe at 134 DGA. Each fetus was immediately removed from the uterus and weighed, and then major fetal organs  were collected and weighed. Several (2–3) placental cotyledons were also collected. Fetal large intestine was removed, cut into proximal (colonic) and distal , and meconium was collected by squeezing the intestine to push out the contents from one end. The samples were then frozen at –80°C, until shipping (on dry ice) to the Hospital for Sick Children, Toronto, Canada for analysis. The subsequent investigations of ethanol-induced pathology in the kidneys 2 gas. The extracted FAEE were quantified using HS-SPME GC-MS with their respective deuterated ethyl esters used as internal standards (prepared as described previously 2) ranged from 0.98 to 1.00. The limit of detection (LOD) and limit of quantification (LOQ) of this method for each ester in sheep meconium were determined by construction of a calibration curve in the low concentration range (2.5–40 ng FAEE). The LOD was calculated as three times the ratio between the SD of the linear regression line and the slope of the calibration curve (LOD = 3·SD/slope) and the LOQ was determined as ten times the same ratio (LOQ = 10·SD/slope). The LOD for the four quantified FAEE ranged from 3.682 to 4.941 ng FAEE/g meconium, and the LOQ was in the range of 12.274 to 16.470 ng FAEE/g meconium. The intra-day coefficient of variation (CV) ranged from 0.5–10.4%, and the inter-day CV ranged from 2–11.3%. The FAEE concentration data were expressed as nanomoles FAEE per gram meconium.Four FAEE  were extracted from the individual meconium samples  and quantified by headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME GC-MS) using an established methodology Figure S1 in supplement), the average meconium FAEE concentrations  were used in all subsequent analyses. Meconium FAEE concentrations were compared between treatment groups using the Kruskal-Wallis test with Dunn’s multiple comparison test, and between all ethanol-exposed , where more information on the statistical tests used can be found. Since there was no difference in meconium FAEE concentrations between colonic and rectal meconium samples as assessed by the Wilcoxon signed rank test for matched pairs , trace levels (above LOD but below LOQ) of mostly one or two measured FAEE species were frequently detectable in at least one of the two collected meconium samples  from each fetus, but only several fetuses had meconium with quantifiable amounts of any FAEE species (>LOQ), and this was ethyl oleate in all cases 3. In coFigure S1 in supplement), average meconium concentrations for each fetus were used in comparisons of meconium FAEE content between treatment groups. Meconium FAEE concentrations were significantly higher in the two ethanol-exposed groups (1 and 3) compare to the two control (non-exposed) groups (2 and 4), and there were no statistically significant differences in FAEE concentrations between untouched and saline controls (groups 2 and 4) or between surgically instrumented and non-instrumented fetuses exposed to ethanol (groups 1 and 3) (p<0.0001)  Figure 1The ability of meconium FAEE concentration to identify the ethanol exposure regimen used in this study was determined by ROC analysis using data from all ethanol-exposed and control  fetuses and using data from instrumented fetuses only   Table 5.In the fetal brain, ethanol exposure increased mRNA levels of tropoelastin and collagen I α1 in small cerebral vessels (unpublished observations), and three out of eight ethanol-exposed fetuses examined for brain pathology exhibited small subarachnoid hemorrhages in the cerebrum and/or cerebellar parenchyma associated with focal cortical neuronal cell death and gliosis In the placenta, ethanol exposure elevated mRNA level of tumor necrosis factor (TNF)-α, suggesting a persistent inflammatory response in this tissue p = 1.000).In instrumented fetuses exposed to ethanol  as an assessment of its ability to identify ethanol-affected newborns. The late gestation ethanol regimen is relevant because it has been shown that women may be more likely to drink in the third trimester than throughout pregnancy in response to the commonly-held misconception that alcohol consumption is less harmful to the fetus later in pregnancy The fetal sheep is a suitable model for studying ethanol teratogenicity and validation of biomarkers because of similarities in organ development and comparable ethanol disposition with the human fetus. In both sheep and humans, ethanol readily distributes across the placenta and the pharmacokinetics of maternal-fetal distribution and elimination are similar The present study demonstrated that, in sheep, fetal meconium FAEE concentration can serve as a biomarker of chronic ethanol exposure in the third trimester that results in peak PEC frequently observed in non-pregnant women drinking socially In the present study, we evaluated the four FAEE species the sum of which has been shown to have the highest sensitivity and specificity for detecting prenatal ethanol exposure in humans vs. Merino x Border Leicester) and/or diet differences leading to variation in endogenous ethanol levels and thus, variable baseline FAEE concentration. Differences also could be due to the use of different FAEE analytical procedures; the less selective GC-flame ionization detector method used by Littner and colleagues may be more prone to “false positives” compared with the GC-MS procedure used in the present study One previous study investigated meconium FAEE concentration in fetal sheep prenatally exposed to ethanol Maternal and fetal instrumentation, which was necessary for ethanol/saline administration and PEC measurements in this study, was not a likely confounding factor with regard to meconium FAEE concentrations. Since groups 1 and 3 were exposed to the same ethanol dosing regimen, we did not expect and did not find differences in meconium FAEE concentrations between the two groups. Similarly, we did not find differences in baseline FAEE concentrations between groups 2 and 4 , none of which received ethanol– the primary determinant of FAEE levels in tissues. In contrast, the difference in meconium FAEE concentrations between groups that received ethanol and those that did not, was significant. Additionally, we confirmed that inclusion of non-instrumented fetuses did not affect the results of ROC curve sensitivity/specificity analysis and yielded essentially the same optimal cut- off values, thus further confirming that instrumentation was not a likely confounding factor with respect to meconium FAEE concentrations.Using meconium FAEE as a biomarker has numerous advantages. Meconium is a cumulative matrix that provides a record of xenobiotic exposure over a relatively long time; it can be collected non-invasively; and sensitive methods have been developed to measure FAEE in meconium There are some challenges to using meconium FAEE as a biomarker of fetal ethanol exposure. Since meconium begins to form in the human fetal intestine in the second trimester (15–20 weeks gestation) and accumulates mostly in the latter part of pregnancy We conclude that meconium FAEE concentration can serve as a reliable biomarker of fetal ethanol exposure in late ovine gestation, and can be used as an indicator of abnormalities in fetal organ development. These findings are encouraging as they suggest that meconium FAEE may identify newborns at risk of multi-organ dysfunction, who may not be displaying overt physical signs of ethanol teratogenicity. Whether it will be possible to set a lower positive cut-off value in the human than the current 2 nmol FAEE/g meconium to identify individuals at risk for more subtle ethanol-related disabilities remains to be determined through human studies.Figure S1Effect of daily ethanol exposure in late gestation on FAEE concentration in fetal rectal and colonic meconium. Bars depict FAEE concentration (sum of four) in meconium collected from the rectal and colonic portions of the large intestine, as well as their average, in ethanol-exposed (groups 1 and 3) and control (groups 2 and 4) fetuses. Data are presented as mean +/− SEM, n = 15 per treatment group. There was no statistically significant difference in meconium FAEE concentration between colonic and rectal meconium samples as assessed by the Wilcoxon signed rank test for matched pairs (p>0.05). Kruskal-Wallis test with Dunn’s multiple comparison test, which was used to compare FAEE concentrations in colonic and rectal samples between ethanol exposed and non-exposed fetuses, showed significantly higher FAEE concentrations in both colonic and rectal meconium of ethanol exposed fetuses compare to controls . Mann-Whitney U test was used to compare the average meconium FAEE concentration  between the two treatment groups and yielded the same result (†††p<0.001).(TIF)Click here for additional data file.Table S1Forward and reverse primer sequences (5′-3′) used for qPCR to amplify Collagen Iα1 and tropoelastin.(DOC)Click here for additional data file.


Mycobacterium avium spp. paratuberculosis (MAP). The hMRS are designed to bind to a unique genomic sequence found in the MAP genome, causing significant changes in the sample’s magnetic resonance signal. Clinically relevant samples, including tissue and blood, were screened with hMRS and results were compared with traditional PCR analysis. Within less than an hour, the hMRS identified MAP-positive samples in a library of laboratory cultures, clinical isolates, blood and homogenized tissues. Comparison of the hMRS with culture methods in terms of prediction of disease state revealed that the hMRS outperformed established culture methods, while being significantly faster (1 hour vs 12 weeks). Additionally, using a single instrument and one nanoparticle preparation we were able to detect the intracellular bacterial target in clinical samples at the genomic and epitope levels. Overall, since the nanoparticles are robust in diverse environmental settings and substantially more affordable than PCR enzymes, the potential clinical and field-based use of hMRS in the multiplexed identification of microbial pathogens and other disease-related biomarkers via a single, deployable instrument in clinical and complex environmental samples is foreseen.Bacterial infections are still a major global healthcare problem. The quick and sensitive detection of pathogens responsible for these infections would facilitate correct diagnosis of the disease and expedite treatment. Of major importance are intracellular slow-growing pathogens that reside within peripheral leukocytes, evading recognition by the immune system and detection by traditional culture methods. Herein, we report the use of hybridizing magnetic nanosensors (hMRS) for the detection of an intracellular pathogen,  Mycobacterium tuberculosis among others, relies on the survival of the microorganism within host cells, such as macrophages and dendritic cells. This mode of infection with or without clinical symptomology hampers bacterial detection, preventing correct diagnosis. Consequently, the physician cannot proceed to the assignment of an appropriate treatment course, delaying the clearance of the pathogen from the body. Serological immunoassays cannot effectively detect intracellular pathogens in biological fluids  as the microorganism is hidden away within the immune cells. For this reason, isolation of the infected cells and extraction of their DNA, followed by detection of specific bacterial genomic markers via Polymerase Chain Reaction (PCR), is needed to facilitate identification of the pathogen. Although highly specific and sensitive, PCR methods are generally laborious, time-consuming and typically require homogeneous and pure DNA samples Pathogenesis caused by intracellular pathogens, such as 2) occur that correlate with the target concentration in solution. By measuring the changes in T2 relaxation times upon target interaction and correlating the intensity of the change with the target concentration, one can develop a sensitive detection method, therefore the acronym of magnetic relaxation nanosensors (MRS).It has been predicted that nanotechnology will have a major impact in medicine, agriculture and biotechnology among other fields 2 increases upon target binding 2. Kinetic studies also revealed that the binding of MRS was faster and more sensitive than the MRS clustering, while achieving lower detection thresholds. In addition, nanoparticle valency – the amount of a targeting ligand on the surface of the nanoparticle – plays a role in the magnetic relaxation response. Nanoparticle valency controls the trend of the change in the spin-spin relaxation time (ΔΤ2). Specifically, it has been found that low valency results in decreases in the ΔΤ2 as the target concentration increases, while at high valency conditions the ΔΤ2 increases as the target concentration increases.In contrast to other methods that utilize magnetic nanoparticles as labels and directly measure an intrinsic physical property, such as a magnetic field 2) upon binding to a unique bacterial genomic marker and tested their performance in clinical samples. The hMRS were designed to bind to a specific genomic marker in Mycobacterium avium spp. paratuberculosis (MAP); an intracellular pathogen known to cause Johne’s disease in cattle Hence considering the faster detection kinetics of MRS binding-based detection assays and the higher sensitivity of the MRS low-valency nanoparticle system, we developed hybridizing MRS (hMRS) that switched (increased) the sample’s water proton relaxation times  than the nPCR methodologies. Specifically, hMRS outperformed the 12-week-long culture-based nPCR in clinical prognosis, as well as the least reliable direct nPCR, which are currently used in clinical and research studies. Overall, we show for the first time the use and validation of a novel translational nanotechnology, where the unique water relaxation properties of iron oxide nanoparticles and the dynamics of the nanoparticle/target interaction were harnessed to achieve a sensitive genomic biomarker detection system for an intracellular pathogen in samples directly obtained from human patients.Attracted by MAP’s slow growing characteristics, intracellular residence and clinical and agricultural relevance, we used hMRS for the identification of a highly conserved MAP genomic element (IS900). This unique DNA insertion sequence has been previously used to identify MAP in clinical samples and cultures by PCR, as it has not been found in other microorganisms or mycobacteria ATGTGGTTGCTGTGT) complementary to the IS900 sequence in MAP is conjugated to facilitate binding and detection. The resulting MAP specific hMRS had a diameter of 78 ± 3 nm, zeta potential value of –35 mV, an average of 55 oligonucleotides per nanoparticle, with R1 and R2 relaxivities (at 0.47T) of 45 mM-1s-1 and 60 mM-1s-1 respectively. We anticipated that hMRS could utilize crude DNA, obtained from isolated buffy coats that have been subjected to heating  > T2(control)). Within 30 minutes after removing the samples from heat, significant and reproducible changes were observed prompting us to set this time-point as the hMRS readout point . Based on the observation that the sample’s T2 times were higher than those of the sterile control throughout the experimental time course, we deduced that the hMRS first bound to the relaxed circular MAP DNA that was heated, while the hMRS still remained bound to the cooled DNA as it was returning to its supercoil form. These results were corroborated by dynamic light scattering studies that showed no clustering of the nanoparticle suspension upon addition of the isolated MAP genome, since the nanoparticle diameter was found to be 81 ± 5 nm. Likely, this may be attributed to the fact that during sample cooling the DNA returned to its supercoiled conformation, which can be in the order of a few nanometers, and as a result nominally altering the overall size of the hybridizing nanoparticle – DNA complex.The detection of MAP’s IS900 genomic marker relies on DNA isolation and subsequent amplification and detection via PCR. Typically, DNA from peripheral leukocytes found in the buffy coat (Direct nPCR) or leukocyte-derived cultured bacteria (Culture-based nPCR) is first isolated in a multistep protocol 2 signal.To determine the specificity of the hMRS, we incubated the hMRS (6 µg Fe/mL) with pure DNA extracts from various mycobacteria and monitored changes in T2+ signal, being attributed to the hMRS – MAP DNA binding . The observed MAP concentration response pattern exhibited a high magnetic resonance signal (ΔΤ2+) at low target concentration levels and low signals at higher DNA concentrations. This suggests that the binding between the hMRS and MAP DNA was facilitated via low valency interaction, as previously described in other models 2+ signal at low numbers of MAP. Based on the findings that both pure and crude MAP DNA exhibited similar hMRS quantification patterns, it was deduced that the hMRS assay can detect IS900 independent of the sample’s DNA extraction methodology. Although higher changes were recorded in pure MAP DNA samples, both extraction methodologies had equivalent detection thresholds, reaching low femtogram (10–15) levels. Potentially the differences in the magnitude of the ΔΤ2+ from pure and crude MAP DNA might be attributed to the sample’s unique characteristics. It is plausible that the high purity of the pure DNA extracts is associated with marginal protein and lipid levels, resulting in lower viscosity. Alternatively, a crude MAP DNA sample may have substantially higher content of proteins and lipids, thus higher viscosity, which may be reflected in lower ΔΤ2+ values upon IS9000 – hMRS hybridization We then examined whether nested PCR (nPCR) can achieve comparable sensitivity using crude MAP DNA. We decided to compare our hMRS method with nPCR as opposed to reverse transcriptase PCR (RT-PCR), due to the fact that MAP is an intracellular pathogen that grows very slowly. Similar to other intracellular microorganisms, it can be dormant and reduce its metabolism, suppressing transcriptional and translational activity Figure S2). This demonstrates the sensitivity and robustness of hMRS to rapidly detect their target even under conditions of interference, which prevent the use of traditional enzyme-based methods. In line with this, recent studies have demonstrated that nucleic-acid-decorated nanostructures can be highly stable even in serum-containing media and avoid non-specific protein and nuclease interactions, due to the steric inaccessibility and high local salt concentration introduced by the nanoparticles’ DNA strands , Table S2). This translates to a total readout time for nPCR of more than 6 hours, with the need of more reagents, such as primers and enzymes, as well as labor and equipment time. Intriguingly though, the hMRS had a single genome-copy detection threshold when crude MAP DNA was used, without compromising their sensitivity in this minimally processed sample . Overall, these data demonstrate that hMRS can achieve fast single MAP genome detection even in crude DNA samples, outperforming nPCR in readout time, sensitivity and robustness.In subsequent studies, we determined if the hMRS could quantify MAP DNA. MAP DNA was isolated using either an elaborate multistep extraction protocol to obtain pure DNA, or a fast 30-minute boiling-based methodology to obtain crude DNA. After a 30-minute incubation period, the hMRS were able to detect MAP DNA in both pure and crude DNA preparations, exhibiting concentration-dependent ΔΤ2+  =  1.3 ± 0.2 ms), establishing this magnetic signal change as the hMRS negative control baseline. Further studies indicated that the hMRS were able to rapidly detect MAP in several clinical isolates with good correlation to the culture-based nPCR, indicating that the hMRS can quickly detect the IS900 biomarker in minimally processed cultured clinical isolates . Out of the ten cultures screened, one sample from a culture of an ileal biopsy (GN2’) was identified as positive by the hMRS, yet negative by nPCR. A false negative result with culturing methods can occur when either low amounts of the pathogen are present in the collected sample or when the pathogen is not viable to properly grow in culture. This prompted us to analyze the culture sample from the corresponding patient’s blood (GN2). We reasoned that viable MAP could be present in the patient’s leucocytes, granting correct diagnosis. Indeed, culture from the blood sample GN2 was identified as positive in agreement with the hMRS diagnosis and clinical sympthology. Therefore, nPCR may have failed to detect IS900 in ileal biopsy GN2’ perhaps due to either low levels of the pathogen or the presence of interferences in the biopsy sample that inhibited growth of the pathogen in culture. Overall, these results suggest that the high sensitivity of hMRS facilitates the positive identification of MAP in blood and biopsy samples, whereas PCR techniques were not always successful in identifying MAP.Since the hMRS detected MAP DNA in samples from pure bacterial cultures, we investigated if the nanosensors could identify their targets in more complex samples, such as cultured clinical isolates and homogenized tissue preparations. We first screened cultures of clinical isolates from Crohn’s disease patients with the hMRS and compared these results with nPCR. Specifically, we used crude DNA for the hMRS and pure DNA for the nPCR, since nPCR cannot utilize crude DNA. Following a single-step procedure, crude DNA obtained from cultured isolates of healthy individuals yielded a magnetic signal change that was comparable to that of the sterile culture medium (ΔΤ1  =  48 mM-1s-1 and R2  =  57 mM-1s-1) that identifies a conserved MAP bacterial surface antigen vs antibody), binding site topology and target characteristics (DNA vs surface epitope), both MRS provided a ΔT2+ signal for all samples with clinical symptomology , which served as the hMRS reference point. Interestingly, results obtained with hMRS and magnetic relaxation detection using crude DNA samples from patients within Cohort 1 were in complete agreement with results obtained with direct nPCR that used pure DNA samples . Addition of a synthetic target complementary to hMRS led to signal abrogation in the samples that had been identified as MAP positive, confirming the specificity of the assay. This indicates that the hMRS can detect their target in minimally processed blood samples, while eliminating lengthy sample preparation procedures.Acknowledging previous reports that identified MAP in the blood of Crohn’s disease patients, we sought to screen blood samples from healthy individuals and Crohn’s disease patients for the presence of IS900 (n = 34). For this, we chose to screen MAP’s IS900 genomic marker, rather than a protein (epitope) marker, since the later involves the use of anti-MAP antibodies that are difficult to reproducibly generate while avoiding antibody batch variabilities. In addition, a DNA-based hybridizing MRS would be more robust and selective than an antibody-based MRS. Peripheral blood was collected and DNA was directly isolated from white blood cells, via either a 30-minutes crude DNA extraction protocol for hMRS or a multistep high purity and quality DNA isolation procedure for direct nPCR screening. Direct nPCR for the MAP IS900 biomarker provided conclusive results about the pathogen’s presence in these samples, hence these samples established the first peripheral blood cohort (Cohort 1). In this cohort, positive samples were defined as those samples where a prominent band corresponding to IS900 was observed and negative samples were those where no visible band was observed at all. Control and Cohort 1 samples were screened with hMRS, after a 30-minutes crude DNA extraction protocol from white blood cells. Crude DNA extracted from control samples induced small changes in the magnetic signal (ΔΤvs negative) nested PCR assays  in MAP’s genome, and not in other microorganisms. Through additional studies, we identified that the sensitivity of the nanosensors was equal to one genome copy even in minimally processed samples, allowing detection of MAP in clinical isolates, tissue homogenates and blood. Compared to nPCR, hMRS had improved performance and yielded faster results. Consequently, this translates to lower costs, since the magnetic nanosensors are fairly easy and cheap to manufacture in larger quantities, and the assay does not require multiple oligonucleotide primer pairs, expensive enzymes and nucleotides. Detection was performed using a simple table-top relaxometer that measured the increase in the water proton relaxation time  that occurred after the hMRS probes bound to their genomic target. The unprecedented detection limit of this technique is due to the build-in water relaxation amplification that results upon a single binding event. The TM. tuberculosis, its detection is difficult, due to the pathogen’s residence within an infected leukocyte’s organelles. Thus contemporary diagnostic methodologies rely on cell isolation, cell lysis and nucleic acid isolation, in order to yield high purity DNA. This renders MAP’s detection problematic since the microorganism has to be isolated from highly complex samples, such as blood or severely inflamed tissue biopsies, before amplification and detection via PCR analysis. Apart from DNA quality, the quantity of MAP DNA can affect the outcome of PCR. For instance, low MAP DNA levels from a clinical sample may not be successfully amplified through direct nPCR or biological matrix components can affect the polymerase activity, yielding false-negative or dubious results. In our studies, a significant number of samples that were identified as MAP dubious by direct nPCR were found to be positive by culture-based nPCR and hMRS (Cohort 2), indicating that the pathogen was originally present in the patient’s blood  and successfully grew in culture at adequate levels.hMRS can be used for the detection of diverse targets in complex media, including turbid and opaque clinical samples The culture-based nPCR, relies on the availability of viable bacteria in the clinical sample that can be grown in culture in sufficient amounts before nPCR. Most clinical microbial diagnostics rely on the growth of the pathogen in culture, before performing the appropriate immunological or PCR tests Herein, we have also demonstrated that MAP DNA can be detected in various tissues from Johne’s disease animals, as well as in biopsies and blood from Crohn’s disease patients, at the epitope and genomic biomarker levels. Furthermore, our results indicate that hMRS can detect the presence of MAP in Crohn’s disease patients more reliably than direct or cultured-based nPCR. As a matter of fact, hMRS is a better predictor of the state of the disease, correlating the clinical state of a patient with the presence of MAP in blood samples Figure 65′-ATGTGGTTGCTGTGT-3′ was achieved via “click” chemistry, as previously described 3 buffer . To this, 200 µL of 10 mM TCEP (tris(2-carboxyethyl)phosphane hydrochloride, Sigma) were added as a reducing agent. Twenty µL of 6.3 M oligo were diluted in 80 µL NaHCO3 buffer  and added to the nanoparticle solution. The reaction was initiated with the dropwise addition of 150 µL Cu(I)-TBTA complex (tris(benzyltriazolylmethyl) amine, 10 mM, Sigma), which was previously prepared in DI water and tbutanol (9∶1). The reaction was incubated at room temperature under continuous mixing for 3 hours, followed by overnight incubation at 4°C under constant mixing. The resulting IS900 hMRS were dialyzed against DI water using a 6,000 – 8,000 MWCO membrane (Spectrum), followed by magnetic separation with an LS25 MACS column (Miltenyi). The epitope-sensing MRS were formulated as previously reported, and the MAP antibody was obtained from Dr. Naser’s lab 1 and R2 relaxivities were determined after determination of the nanoparticle’s iron content and relaxation studies on a compact relaxometer . Determination of the hMRS oligonucleotide concentration was achieved by monitoring the absorbance at 260 and 305 nm (background), using a Nanodrop 1000 spectrophotometer (Thermo Scientific), as previously described The IS900-specific hMRS were prepared from propargylated polyacrylic-acid-coated iron oxide nanoparticles, utilizing literature available protocols Proteus vulgaris # 8427, Staphylococcus aureus #33862, Pseudomonas aeruginosa # 27853, Enterococcus faecalis, Escherichia coli # 8739, and Serratia marcensis were grown in culture tubes with nutrient broth, and bacterial growth was monitored spectrophotometrically. Heat inactivation of these bacteria was performed by autoclaving the culture tubes for 10 minutes. Upon inactivation, all bacterial stocks were placed in a Fisher Isotemp freezer (Fisher Scientific), until further use. Clinical isolates and tissue specimens from animals with Johne’s disease were obtained from tissue collections stored in Dr. Naser’s laboratory.Lab strains of MAP and clinical isolates were grown in 12B* BACTEC bottles (Becton Dickinson) as previously described DNA was extracted from cultured bacteria in a class II biosafety cabinet, according to literature-available procedures 5′-TCCTTCGGCCATCCAACACAGCAACCACAT-3′), 1 µL of the target (1.1 M) was added to the hMRS mycobacterial samples containing the same DNA concentration (3.5 ng/µL). Similar DNA concentration was used for the specificity experiment utilizing other Gram-positive and Gram-negative bacteria and hMRS (3 µg Fe/mL). For the sensitivity experiments, serial dilutions of pure and crude MAP DNA were prepared from pure cultures of MAP grown in 12B* BACTEC bottles, which had been processed and quantified as described in the preceding section (DNA extraction methodology). Nested PCR (nPCR) was performed as previously described Serial dilutions of bacterial DNA were prepared in TE buffer. For the relaxation-mediated experiments, 200 µL of the nanoparticle suspension  were incubated with 1 µL of bacterial DNA or control (TE buffer). The samples were heated twice for 3 minutes at 95°C. After this, the samples were transferred to relaxometer tubes and cooled down at room temperature, while taking relaxation measurements on a magnetic relaxometer . For the specificity assays using a complementary synthetic target . Isolation of white blood cells was achieved via centrifugation at 3,000 rpm for 10 minutes at room temperature, as previously described 2) were measured using a 0.47 T mq20 NMR analyzer . T2 values were obtained before and after addition of the sample, and through the time course of the study. All T2 measurements were performed using a CPMG pulse-echo train with a 1.5 ms interpulse spacing . The change in magnetic resonance signal (ΔΤ2+) was defined as the sample’s T2 minus the magnetic signal of the corresponding negative control. All experiments and measurements were carried out in triplicate and data were expressed as mean ± standard error, unless otherwise denoted.Spin-spin relaxation times . ROC analysis was performed based on hMRS determination of the sample’s MAP load. This was achieved through the correlation of the ΔΤ2+ signal to bacterial genome copies, using a crude extracted DNA standard curve . For nPCR, the presence of a band corresponding to IS900 was marked as positive , whereas its absence was assigned as negative . The overall ROC methodology was performed as previously reported in literature vs healthy), whereas the output curve was fitted using logistic regression.Clinical diagnosis and sample collection was performed at the College of Medicine, University of Florida, based on clinical and endoscopic criteria The use of human subjects was approved by the University of Florida’s Institutional Review Board (protocol number 354–2007). A signed individual written informed consent agreement was obtained from each subject or in the case of children from their parent or legally authorized representative before enrolment in the study.Figure S1Kinetics for MAP’s IS900 genomic marker detection with hMRS. After heating the samples to facilitate DNA stand separation and hMRS hybridization, the changes in the T2 magnetic resonance signal were recorded over time, with marked changes occurring within less than an hour (Means±SE).(TIF)Click here for additional data file.Figure S2Nested PCR (nPCR) cannot quantify crude MAP DNA. – Ctrl: negative control (dH2O) of the first nPCR round, – Ctrl: negative control (dH2O) of the second nPCR round, TE: TE buffer, + Ctrl: two controls of pure extracted MAP DNA.(TIF)Click here for additional data file.Figure S3Genome-copy-based quantification of MAP with hMRS and crude extracted DNA. Samples with known amounts of crude DNA from cultured MAP were utilized to correlate the changes in the T2 signal (ΔT2+) and the number of bacteria originally present in the sample, using the MAP’s genome size as a reference. (TIF)Click here for additional data file.Table S1Spin-spin relaxation times (T2) of crude MAP DNA samples. Serial dilutions of crude MAP DNA samples have been utilized to assess the sensitivity of the hMRS method in minimally processed bacterial cultures. The averages of each independent experiment are listed and the studies’ mean.(PDF)Click here for additional data file.Table S2Spin-spin relaxation times (T2) of pure MAP DNA samples with known genome copies. Three independent experiments were performed on pure DNA samples obtained from cultured MAP. Correlation between DNA levels and bacterial populations was achieved by quantifying DNA spectrophotometrically and using the MAP genome size as a reference.(PDF)Click here for additional data file.Table S3Demographics of cultured clinical isolates that were screened with hMRS and nPCR. (PDF)Click here for additional data file.Table S4Clinical data and hMRS results of Cohort 1 blood samples. Samples from Crohn’s disease (CD) patients, healthy individuals or asymptomatic carriers were analyzed by direct nPCR and hMRS. Quantification of the MAP genome copies was achieved in positive (+) samples using a training standard curve for crude extracted MAP DNA.(PDF)Click here for additional data file.


Nature Medicine, we have reported the specific mutation of the molecular chaperone HSP110 in MSI CRCs and how the presence of this mutant may constitute a first step towards the understanding of their particular clinical characteristics [Colorectal cancer (CRC) is the second cause of cancer-related death worldwide. Surgery constitutes the primary therapy for these tumors, together with chemotherapy that is usually recommended in patients with metastatic primary CRC. Although molecularly distinct entities arising from different physiopathogenic mechanisms - microsatellite (MSI) and chromosomal instability  - have been characterized in CRC, there is still no specific therapeutic approach that takes into account disease's molecular heterogeneity . MSI is eristics .17 mononucleotide repeat located in intron 8 of HSP110 was systematically mutated in MSI CRC cell lines and primary tumors [HSP110 transcript due to exon 9 skipping, to the detriment of wild-type HSP110 mRNA. As a result, a truncated HSP110 mutant protein (HSP110DE9) accumulated in MSI tumors. Strikingly, we demonstrated that HSP110DE9 acts as a dominant negative mutant that binds to HSP110 abrogating its chaperone activity and cytoprotective function. In colon tumors, HSPs including HSP110 have been clearly shown to promote cancer cell survival, protecting oncogenic proteins and inhibiting apoptosis [17 located in HSP110 intron 8 constitute hot spots for mutations in MSI tumors due to the MMR deficiency. Our hypothesis is that when these mutations are endowed with a biological anti-cancer activity, as it is the case with HSP110DE9, they can represent an Achilles' heel in the MSI-driven tumorigenic process. Further studies are now necessary to determine the exact role of HSP110DE9 during MSI tumor progression and to understand the contribution of HSP110DE9 in the more favorable prognosis of CRC MSI compared to MSS patients.It is now well established that MMR deficiency is not in itself a direct transforming event and that the development of these tumors is MSI-driven. This distinctive MSI pathway is characterized by somatic mutational events affecting short coding repeated sequences that, when having an oncogenic effect, provide selective pressure during tumor progression . We showy tumors . The shopoptosis -6. It isIn vitro, HSP110DE9 expression sensitized colon cancer cells to anticancer agents such as oxaliplatin and 5-fluorouracil, which are routinely prescribed in the adjuvant treatment of patients with CRC [Nature Medicine [“it is fascinating to speculate that such studies might show a lack of response to 5-FU confined to the MSI CRCs with low levels of HSP110DE9”.with CRC . In linewith CRC . TherefoMedicine , “it is In tumor samples, MSI phenotype can be determined by PCR according to international criteria or by immunohistochemistry studying mismatch repair (MMR) protein expression affecting MLH1, MSH2, MSH6 or PMS2. Our findings highlight that routine evaluation of the MSI phenotype together with investigation of HSP110 status could be of clinical interest in CRC diagnosis. Note worthily, HSP110DE9 is the first HSP mutant identified in a cancer so far. Developing inhibitors of HSP110 that mimic the anti-cancer chemosensitizing effect of HSP110DE9 is also a promising perspective.


Objective: To determine the prevalence of PTSD induced by ACS and to quantify the association between ACS-induced PTSD and adverse clinical outcomes using systematic review and meta-analysis. Data Sources: Articles were identified by searching Ovid MEDLINE, PsycINFO, and Scopus, and through manual search of reference lists.Acute coronary syndromes  can induce posttraumatic stress disorder (PTSD), and ACS-induced PTSD may increase patients’ risk for subsequent cardiac events and mortality. Observational cohort studies that assessed PTSD with specific reference to an ACS event at least 1 month prior. We extracted estimates of the prevalence of ACS-induced PTSD and associations with clinical outcomes, as well as study characteristics. We identified 56 potentially relevant articles, 24 of which met our criteria (N = 2383). Meta-analysis yielded an aggregated prevalence estimate of 12%  for clinically significant symptoms of ACS-induced PTSD in a random effects model. Individual study prevalence estimates varied widely (0%–32%), with significant heterogeneity in estimates explained by the use of a screening instrument  vs a clinical diagnostic interview . The aggregated point estimate for the magnitude of the relationship between ACS-induced PTSD and clinical outcomes  across the 3 studies that met our criteria (N = 609) suggested a doubling of risk  in ACS patients with clinically significant PTSD symptoms relative to patients without PTSD symptoms.This meta-analysis suggests that clinically significant PTSD symptoms induced by ACS are moderately prevalent and are associated with increased risk for recurrent cardiac events and mortality. Further tests of the association of ACS-induced PTSD and clinical outcomes are needed. In recent decades, survival rates after acute coronary syndrome  have steadily increased Diagnostic and Statistical Manual of Mental Disorders  In the It is also unclear whether ACS-induced PTSD, like other psychosocial vulnerabilities such as depression, is associated with worse survival after ACS. However, epidemiological and observational evidence suggests that PTSD due to traumatic events other than ACS is related to increased risk of incident cardiovascular disease ACS-induced PTSD, not PTSD due to any other type of traumatic event. Potentially relevant articles were identified by searching the biomedical electronic databases Ovid MEDLINE, PsycINFO and Scopus. Dates searched were 1948 to July Week 2, 2011, and the search was conducted on July 21, 2011. All relevant subject headings and free-text terms were used to represent PTSD and ACS and the sets of terms were combined with AND. Terms for MEDLINE were: exp Stress Disorders, Traumatic/OR ptsd.tw. OR (post-traumatic OR (post adj traumatic)).tw or posttraumatic.tw. OR acute stress disorder$.tw. OR asd.tw, exp Acute Coronary Syndrome/OR acute coronary.tw. OR acs.tw OR exp Myocardial Infarction/OR myocardial infarct$.tw. OR (mi or ami).tw. OR (heart adj attack$).tw. OR ((post adj mi) or postmi).tw. OR (stemi or nstemi).tw. OR ((preinfarction or unstable) adj angina$).tw. These terms were adapted for the other databases. Additional records were identified by scanning the reference lists of relevant studies and reviews and by employing the Related Articles feature in PubMed and the Cited Reference Search in ISI Web of Science.We sought to identify all studies that reported a valid estimate of the prevalence of ACS-induced PTSD. To be included, studies must have been observational cohorts and must have assessed PTSD with specific reference to an ACS event that had occurred at least 1 month prior to the PTSD assessment. Further, studies must have used a self-report PTSD screening instrument or clinical interview designed or specifically altered to query about only To determine the studies to be assessed further, two authors  independently read the, title or abstract of every record retrieved. All potentially relevant articles were investigated as full text. Where differences in opinion existed, they were resolved by consensus.ACS-induced PTSD due to the index ACS event were included in PTSD prevalence rates. Where multiple publications from the same cohort were identified, we chose the publication with the largest sample size and the earliest post-ACS assessment of PTSD  served as the statistical platform for completing all statistical tests and associated graphic results. To summarize the prevalence findings, we computed prevalence point estimates using these formulas:Logit Event Rate  =  Log [Event Rate/(1 − Event Rate)].Event Rate Standard Error  =  √(1/(Event Rate · Sample Size)/(1/[(1−Event Rate) · Sample Size]),We computed 95% confidence intervals (CIs) using this formula:Lower Limit  =  Logit Event Rate − (1.96 · Logit Event Rate Standard Error).Upper Limit  =  Logit Event Rate + (1.96 · Logit Event Rate Standard Error).For meta-analytic tests of ACS-induced PTSD prevalence, in addition to the overall random-effects model, we used sensitivity analyses to assess evidence of moderator effects for the prevalence results across a number of methodological factors. These factors included the method used to assess ACS-induced PTSD, the location of the study (United States vs other), and whether studies explicitly included patients with NSTEMI and UA. Further, meta-regression analysis was used to test the association between study prevalence rates and timing of the PTSD assessment (number of months after ACS), study publication date, and sample age and sex composition. Heterogeneity assessments preceded all meta-analytic tests concerning PTSD prevalence and clinical outcomes. There was statistically significant heterogeneity, so random-effects models were used to estimate and test effects.We calculated an aggregated point estimate for the risk ratio associated with a positive screen for PTSD on clinical outcome categories including cardiac hospitalization and a combined endpoint of cardiac re-hospitalization and all-cause mortality. Log-transformed risk ratios and 95% CIs were calculated for each study using the reported effect size and estimates of the SE of each effect drawn from data reported in the article. When articles reported multiple models, we selected the model with the highest level of covariate adjustment. For meta-analytic tests of the association of ACS-induced PTSD and clinical outcomes, there were too few studies to test for moderator effects. To address the issue of publication bias, we calculated a fail-safe N.We initially identified 548 articles, and 2 coders agreed that 48 articles required full reading. Of these 48 potentially relevant articles plus another 8 identified from references, 24 met our criteria for inclusion see Figure 1Q23 = 120.09; P<.001; I2 = 80.85). There was no evidence of publication bias , as shown in Q16 = 43.89; P<.001; I2 = 63.55), compared with 4% in the 7 studies in which patients were assessed with clinical interviews .The PTSD prevalence rates varied significantly by type of assessment (P = .29).The mean number of months between the event and PTSD assessment across studies was 10.24; however, 1 study P = .01).Later publication date was related to lower estimates of PTSD prevalence and accounted for 5% of the variance in estimates .We compared prevalence rates by whether studies were conducted in the United States (7 studies) P<.001). The proportion of the study sample that was male varied from 56% to 100% but was unrelated to PTSD prevalence in the 21 studies in which data on sex were reported (P = .87). There were not enough studies with racial/ethnic diversity to conduct meta-analytic tests.The mean age of the participants varied from 53 to 67 years. A younger mean age was significantly related to a greater prevalence rate in the 21 studies in which data on age were reported .Two studies explicitly included patients with NSTEMI or UA , and their aggregated prevalence estimate did not differ significantly from those of studies that did not explicitly include NSTEMI or UA patients  was unrelated to prevalence estimates, advances in treatment may well be responsible for slightly better post-ACS psychological outcomes in recent years. Timing of PTSD assessment, study location, and sex of the sample were unrelated to prevalence estimates.Our analyses indicated that ACS-induced PTSD symptoms are associated with an approximate doubling of risk for recurrent cardiac events or mortality. Two of the 3 studies included in the analysis Important caveats to this review include the fact that only studies with relatively small sample sizes have addressed ACS-induced PTSD, many of which were marginally powered to detect associations with outcomes over relatively short intervals. Furthermore, no study to date has been sufficiently powered to test for potential mechanisms or effect modifiers of the relationship between ACS-induced PTSD and adverse clinical outcomes. Thus, although this meta-analysis was able to quantify more precisely the magnitude of the increased risk of clinical events due to ACS-induced PTSD symptoms, a clear need for additional research remains.Caveats aside, there is now considerable evidence for a link between PTSD due to various life stressors and subsequent heart disease The present review has several limitations. First, since we did not include unpublished articles or articles from non–peer-reviewed journals, we are more likely to have excluded negative findings We were able to locate only one PTSD treatment study in ACS patients: a phase I safety trial of cognitive behavioral therapy for ACS-induced PTSD While little research exists on treatment of ACS-induced PTSD, a growing body of literature suggests risk factors for PTSD diagnosis and symptom severity after ACS. These risk factors include intense fear In conclusion, across published studies concerning the prevalence of ACS-induced PTSD and its associations with clinical outcomes, a few general conclusions can be drawn: (1) ACS is a traumatic event for many, and ACS-induced PTSD is relatively common, with approximately 12% experiencing clinically significant PTSD symptoms and 4% meeting full diagnostic criteria for the disorder, and (2) based on a small number of studies, clinically significant symptoms of ACS-induced PTSD appear to double patients’ risk of recurrent cardiac events and mortality. These results identify areas requiring further research; in particular, studies testing treatments for ACS-induced PTSD are needed.


People living with HIV (PLWH) who have positive tuberculin skin tests (TST) benefit from isoniazid preventive therapy (IPT) whereas those testing TST-negative do not. Revised World Health Organization guidelines explicitly state that assessment of TST is not a requirement for initiation of IPT. However, it is not known what proportions of patients will benefit from IPT if implemented without targeting according to TST status. We therefore determined the proportions of PLWH who test TST-positive.We systematically reviewed the literature published between January 1990 and February 2012 to determine the proportions of patients without active tuberculosis attending HIV care services in low and middle-income countries who tested TST-positive (≥5 mm induration). Proportions were also determined for different CD4 count strata. Data from 19 studies with 9,478 PLWH from sub-Saharan Africa, Asia and Central and South America were summarized. The vast majority were not receiving antiretroviral therapy (ART). A sub-analysis was conducted of 5 studies  from high TB prevalence countries of PLWH with negative TB screens attending HIV care and treatment settings for whom CD4 stratified data were available. The median proportion of PLWH testing TST-positive overall was 22.8% . The median (range) proportions with CD4 cell counts of <200, 200–499 or ≥500 cells/µL who tested positive were 12.4% (8.2–15.3%), 28.4% (20.1–36.9%) and 37.4% (31.3–56.3%), respectively. Heterogeneity in the data precluded calculation of pooled summary estimates.In most settings, if IPT is administered to PLWH pre-ART without assessment of TST status, only a minority of those treated are likely to benefit, especially among those with the lowest CD4 cell counts. This may be inefficient use of resources and cost-effectiveness analyses should take this into account. Local knowledge of TST response rates may help inform policies. New simple means of identifying those who will benefit from IPT are needed to permit appropriate targeting of this intervention. HIV-associated tuberculosis (TB) remains a substantial challenge to global health A meta-analysis of placebo-controlled trials reported that IPT in people living with HIV (PLWH) conferred an overall risk reduction of 33%  With low uptake of IPT and recognition that the process of TST assessment was a major programmatic stumbling block, the 2011 revision of the WHO IPT guidelines made a strong explicit recommendation that “TST is not a requirement for initiating IPT in people living with HIV” although it “can be used where feasible” The proportion of PLWH testing TST-positive is likely to be associated with the prevalence of TB in the local community, the prevailing force of infection, social mixing patterns and the clinical population sampled. Prevalence also increases with age We conducted the search and review process according to a predefined protocol and the study conformed to the PRISMA statement checklist Citations identified through the search process were compiled into Endnote X4 and duplicates removed. Titles and abstracts were screened for eligibility according to pre-defined inclusion and exclusion criteria and full-texts were reviewed for eligibility by ADK and SDL. The authors of eligible studies were contacted as required to clarify or modify the stratification of their data by CD4 cell count.A study was eligible for inclusion if it was conducted in a low-income or middle-income country (as defined by the World Bank list of economies current on 1st July 2011) Data from eligible studies were abstracted directly into a structured Microsoft Excel spreadsheet. Study characteristics recorded included title, authors, year of publication, study period, study location, mean or median patient age, bacille Calmette et Guerin (BCG) scar status, clinical setting, ART status, TST methodology, inclusion of special populations or sub-groups and the total number of PLWH with TST-positive and TST-negative results stratified by CD4 counts .For inclusion in a sub-analysis and potential meta-analysis, we selected those studies in high TB prevalence settings which were of sufficient size, were conducted among PLWH accessing HIV care and treatment services and in whom active TB had been excluded. This selection reflects the PLWH who are specified in WHO guidelines as being the key targets for IPT Two reviewers (ADK and SDL) assessed the quality of studies using a graded checklist . A studyAll statistical analyses were conducted using STATA 10. The proportions of PLWH testing TST-positive were calculated with corresponding 95% confidence intervals (95% CI). These data were stratified by geographic region, country TB prevalence  and CD4 cell count . Findings were presented using forest plots. The median and corresponding range of proportions of patients testing TST-positive were calculated. I-squared statistics ). Ten publications initially met inclusion criteria and nine others met inclusion criteria after supplementary data were provided by study authors. Ten others remained ineligible despite seeking supplementary data , five were from sub-Saharan Africa, four from South-East Asia, four from the Americas, three from the Eastern Mediterranean two from the Western Pacific and one from Europe . A majorThe proportions of PLWH receiving ART at the time of TST assessment was reported by ten studies and two other studies pre-dated national ART implementation and these were therefore assumed to be ART-naïve populations. Among these twelve studies, the proportion of patients receiving ART ranged from 0 to 84% with a median of 5.4% .The studies included a total of 9,478 subjects and 2,820 (29.8%) of these had CD4 cell counts <200 cells/µL and 6,658 (70.3%) had counts of >200 cells/µL. In 16 studies the data were further stratified, providing data from 4,034 subjects with CD4 counts of 200–499 cells/µL and 2,231 with counts of ≥500 cells/µL.). The overall median proportion testing TST positive was 26.0% . The proportions testing positive stratified according to geographical region , 5 also fulfilled the additional criteria for inclusion in the meta-analysis . Four stThe median proportion testing TST-positive in these 5 studies was 22.8% . These data are shown in a forest plot stratified by CD4 cell count <200 cells/µL, 200–499 cells/µL and ≥500 cells/µL . The proIn this systematic review, we summarized data from 19 studies that included 9,478 PLWH from low- and middle-income countries in sub-Saharan Africa, Asia and Central and South America. The median proportion of PLWH testing TST-positive was 26.0% overall but with substantial heterogeneity . Five of these studies  were selected for inclusion in a sub-analysis on the basis of high country TB prevalence, adequate study size and quality and inclusion of PLWH without TB attending HIV care and treatment settings. Among these studies, the median proportions of patients with CD4 cell count <200 cells/µL, 200–499 cells/µL and ≥500 cells/µL testing TST-positive were 12.4% , 28.4%  and 37.4% , respectively. Data heterogeneity, however, precluded calculation of pooled summary estimates. These data indicate that in most settings, implementation of IPT among PLWH without assessment of TST status is likely to benefit a minority of patients across all CD4 count categories.In resource-limited settings, assessment of TST status is the only recommended means of identifying patients who are likely to benefit from IPT Removal of the requirement for TST assessment simplifies implementation of IPT and may thereby help catalyze scale-up of IPT. However, this study suggests that overall only approximately 1 in 4 PLWH stand to benefit from IPT if implemented in an untargeted way. Although this would save resources needed to assess TSTs, this would be wasteful of both patient and scarce health care resources used in treating and following-up large numbers of PLWH who would derive no benefit. Cost-effectiveness analyses suggest that use of IPT either in a targeted or untargeted way is cost-effective compared to not implementing IPT at all Data from southern Africa increasingly suggest that because of high prevailing rates of community TB transmission, benefit from IPT is largely limited to the period within which it is taken Concerns have been expressed about the risks of widespread use of isoniazid monotherapy fuelling development of drug resistance. There is no evidence of such an effect from data generated during carefully conducted randomized controlled trials Isoniazid is well known to be associated with low rates of hepatotoxicity and occasional deaths There was great heterogeneity between studies in the proportions testing TST positive. This observation suggests that knowledge of TST responses in specific countries or settings may help local decisions on the pros and cons of targeted or untargeted IPT delivery. This heterogeneity may reflect both the local TB epidemiology as well as study-related factors. The latter may include study design, methodology used for TST assessment (including dose of PPD), the accuracy with which this was done, potential biases associated with non-return of patients for TST assessment, the rigour with which TB was excluded and the characteristics of the patients tested. While we have attempted to broadly summarize these variables, it was not possible to precisely define the role of each. Importantly, however, the mean or median ages of PLWH included in the meta-analysis all fell within a narrow range (29–37 years). It was notable that country TB prevalence did not appear from the forest plots to be a more dominant factor. This may be related to imprecision of TB prevalence estimates, differences between national estimates and prevalence in the specific communities studied or the fact that TB exposure is related to cumulative exposure over the life-time of the individual rather than current prevalence.The proportions of positive TST results stratified by CD4 cell count observed among the total group of 19 studies were very similar to those found in the 5 studies that were selected for further analysis, providing reassurance that study selection did not introduce substantial bias. Moreover, many of the other studies that were excluded from the systematic review also report that between 22–25% of PLWH tested TST-positive These data help to rationalize prevention strategies that need to be tailored according to subjects’ CD4 cell counts. As CD4 cell counts decrease during the natural history of HIV infection, risk of TB progressively increases Although it is being recommended that ART be started at increasingly high CD4 cell counts, IPT might be prioritized for PLWH prior to ART eligibility. Ideally HIV would be identified early in the course of disease and all those testing TST-positive would receive a course of IPT prior to becoming eligible for ART. However, even in those with CD4 cell counts of ≥500 cells/µL, less than two in five PLWH (37.4%) tested TST-positive. Use of TST assessment  in such patients would permit appropriate targeting of therapy and could substantially reduce the numbers unnecessarily treated.Strengths of this study include a comprehensive search strategy which identified studies from diverse geographic regions with a range of TB prevalence estimates. Large numbers of PLWH overall and with a broad range of CD4 cell counts were included. Weaknesses include the paucity of data from PLWH receiving ART, the lack of availability of data from other large as yet unpublished studies and the inability to precisely define the impact of other potential variables. In addition, the reliability of TST assessment could not be assessed and the methods for excluding TB were not standardized. Heterogeneity in the data precluded calculation of pooled summary estimates.In conclusion, we found that a minority PLWH were TST-positive, even in the highest TB prevalence settings, and that the proportion testing positive was strongly associated with CD4 cell counts. This factor may undermine the cost-effectiveness of use of untargeted IPT. Local knowledge of TST response rates may help inform policies. Operational research to improve TST implementation as well as development of new, simple means to identify those who will benefit from IPT are urgently needed Table S1Overview of search strategy.(DOCX)Click here for additional data file.Table S2Graded quality assessment checklist.(DOCX)Click here for additional data file.Table S3Summary of studies meeting criteria for contacting authors for further data but for which insufficient secondary data was provided for study inclusion.(DOCX)Click here for additional data file.