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Sample records for detecting disease-associated genotype

  1. Disease associated balanced chromosome rearrangements: a resource for large scale genotype-phenotype delineation in man

    PubMed Central

    Bugge, M.; Bruun-Petersen, G.; Brondum-Nielsen, K.; Friedrich, U.; Hansen, J.; Jensen, G.; Jensen, P.; Kristoffersson, U.; Lundsteen, C.; Niebuhr, E.; Rasmussen, K.; Rasmussen, K.; Tommerup, N.

    2000-01-01

    Disease associated balanced chromosomal rearrangements (DBCRs), which truncate, delete, or otherwise inactivate specific genes, have been instrumental for positional cloning of many disease genes. A network of cytogenetic laboratories, Mendelian Cytogenetics Network (MCN), has been established to facilitate the identification and mapping of DBCRs. To get an estimate of the potential of this approach, we surveyed all cytogenetic archives in Denmark and southern Sweden, with a population of ~6.6 million. The nine laboratories have performed 71 739 postnatal cytogenetic tests. Excluding Robertsonian translocations and chromosome 9 inversions, we identified 216 DBCRs (~0.3%), including a minimum estimate of 114 de novo reciprocal translocations (0.16%) and eight de novo inversions (0.01%). Altogether, this is six times more frequent than in the general population, suggesting a causal relationship with the traits involved in most of these cases. Of the identified cases, only 25 (12%) have been published, including 12 cases with known syndromes and 13 cases with unspecified mental retardation/congenital malformations. The remaining DBCRs were associated with a plethora of traits including mental retardation, dysmorphic features, major congenital malformations, autism, and male and female infertility. Several of the unpublished DBCRs defined candidate breakpoints for nail-patella, Prader-Willi, and Schmidt syndromes, ataxia, and ulna aplasia. The implication of the survey is apparent when compared with MCN; altogether, the 292 participating laboratories have performed >2.5 million postnatal analyses, with an estimated ~7500 DBCRs stored in their archives, of which more than half might be causative mutations. In addition, an estimated 450-500 novel cases should be detected each year. Our data illustrate that DBCRs and MCN are resources for large scale establishment of phenotype-genotype relationships in man.


Keywords: translocations; inversions; abnormal

  2. KIR Genotypic Diversity Can Track Ancestries in Heterogeneous Populations: A Potential Confounder for Disease Association Studies

    PubMed Central

    Singh, Komal Manpreet; Phung, Yume T.; Kohla, Mohamed S.; Lan, Billy Y-A; Chan, Sharon; Suen, Diana L.; Murad, Sahar; Rheault, Shana; Davidson, Peter; Evans, Jennifer; Singh, Manpreet; Dohil, Sofie; Osorio, Robert W.; Wakil, Adil E.; Page, Kimberly; Feng, Sandy; Cooper, Stewart L.

    2014-01-01

    Killer cell immunoglobulin-like receptors (KIR) are encoded by highly polymorphic genes that regulate the activation of natural killer (NK) cells and other lymphocyte subsets, and likely play key roles in innate and adaptive immunity. Association studies increasingly implicate KIR in disease predisposition and outcome but could be confounded by unknown KIR genetic structure in heterogeneous populations. To examine this we characterized the diversity of 16 KIR genes in 712 Northern Californians (NC) stratified by selfassigned ethnicities, and compared the profiles of KIR polymorphism with other US and global populations using a reference database. Sixty-eight distinct KIR genotypes were characterized: 58 in 457 Caucasians (NCC); 17 in 47 African Americans (NCAA); 21 in 80 Asians (NCA); 20 in 74 Hispanics (NCH) and 18 in 54 “other” ethnicities (NCO). KIR genotype patterns and frequencies in the 4 defined ethnicities were compared with each other and with 34 global populations by phylogenetic analysis. Although there were no population-specific genotypes, the KIR genotype frequency patterns faithfully traced the ancestry of NCC, NCAA and NCA but not of NCH whose ancestries are known to be more heterogeneous. KIR genotype frequencies can therefore track ethnic ancestries in modern urban populations. Our data emphasize the importance of selecting ethnically matched controls in KIR based studies to avert spurious associations. PMID:21898189

  3. Precise genotyping and recombination detection of Enterovirus

    PubMed Central

    2015-01-01

    Enteroviruses (EV) with different genotypes cause diverse infectious diseases in humans and mammals. A correct EV typing result is crucial for effective medical treatment and disease control; however, the emergence of novel viral strains has impaired the performance of available diagnostic tools. Here, we present a web-based tool, named EVIDENCE (EnteroVirus In DEep conception, http://symbiont.iis.sinica.edu.tw/evidence), for EV genotyping and recombination detection. We introduce the idea of using mixed-ranking scores to evaluate the fitness of prototypes based on relatedness and on the genome regions of interest. Using phylogenetic methods, the most possible genotype is determined based on the closest neighbor among the selected references. To detect possible recombination events, EVIDENCE calculates the sequence distance and phylogenetic relationship among sequences of all sliding windows scanning over the whole genome. Detected recombination events are plotted in an interactive figure for viewing of fine details. In addition, all EV sequences available in GenBank were collected and revised using the latest classification and nomenclature of EV in EVIDENCE. These sequences are built into the database and are retrieved in an indexed catalog, or can be searched for by keywords or by sequence similarity. EVIDENCE is the first web-based tool containing pipelines for genotyping and recombination detection, with updated, built-in, and complete reference sequences to improve sensitivity and specificity. The use of EVIDENCE can accelerate genotype identification, aiding clinical diagnosis and enhancing our understanding of EV evolution. PMID:26678286

  4. Precise genotyping and recombination detection of Enterovirus.

    PubMed

    Lin, Chieh-Hua; Wang, Yu-Bin; Chen, Shu-Hwa; Hsiung, Chao Agnes; Lin, Chung-Yen

    2015-01-01

    Enteroviruses (EV) with different genotypes cause diverse infectious diseases in humans and mammals. A correct EV typing result is crucial for effective medical treatment and disease control; however, the emergence of novel viral strains has impaired the performance of available diagnostic tools. Here, we present a web-based tool, named EVIDENCE (EnteroVirus In DEep conception, http://symbiont.iis.sinica.edu.tw/evidence), for EV genotyping and recombination detection. We introduce the idea of using mixed-ranking scores to evaluate the fitness of prototypes based on relatedness and on the genome regions of interest. Using phylogenetic methods, the most possible genotype is determined based on the closest neighbor among the selected references. To detect possible recombination events, EVIDENCE calculates the sequence distance and phylogenetic relationship among sequences of all sliding windows scanning over the whole genome. Detected recombination events are plotted in an interactive figure for viewing of fine details. In addition, all EV sequences available in GenBank were collected and revised using the latest classification and nomenclature of EV in EVIDENCE. These sequences are built into the database and are retrieved in an indexed catalog, or can be searched for by keywords or by sequence similarity. EVIDENCE is the first web-based tool containing pipelines for genotyping and recombination detection, with updated, built-in, and complete reference sequences to improve sensitivity and specificity. The use of EVIDENCE can accelerate genotype identification, aiding clinical diagnosis and enhancing our understanding of EV evolution. PMID:26678286

  5. Detection of the disease associated form of the prion protein in biological samples

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transmissible spongiform encephalopathies (TSEs) or prion diseases are neurodegenerative diseases that occur in a variety of mammals. In these diseases, a chromosomally encoded protein (PrP**c) undergoes a conformational change to the disease associated form (PrP**d), and PrP**d is capable inducing ...

  6. Sherlock: Detecting Gene-Disease Associations by Matching Patterns of Expression QTL and GWAS

    PubMed Central

    He, Xin; Fuller, Chris K.; Song, Yi; Meng, Qingying; Zhang, Bin; Yang, Xia; Li, Hao

    2013-01-01

    Genetic mapping of complex diseases to date depends on variations inside or close to the genes that perturb their activities. A strong body of evidence suggests that changes in gene expression play a key role in complex diseases and that numerous loci perturb gene expression in trans. The information in trans variants, however, has largely been ignored in the current analysis paradigm. Here we present a statistical framework for genetic mapping by utilizing collective information in both cis and trans variants. We reason that for a disease-associated gene, any genetic variation that perturbs its expression is also likely to influence the disease risk. Thus, the expression quantitative trait loci (eQTL) of the gene, which constitute a unique “genetic signature,” should overlap significantly with the set of loci associated with the disease. We translate this idea into a computational algorithm (named Sherlock) to search for gene-disease associations from GWASs, taking advantage of independent eQTL data. Application of this strategy to Crohn disease and type 2 diabetes predicts a number of genes with possible disease roles, including several predictions supported by solid experimental evidence. Importantly, predicted genes are often implicated by multiple trans eQTL with moderate associations. These genes are far from any GWAS association signals and thus cannot be identified from the GWAS alone. Our approach allows analysis of association data from a new perspective and is applicable to any complex phenotype. It is readily generalizable to molecular traits other than gene expression, such as metabolites, noncoding RNAs, and epigenetic modifications. PMID:23643380

  7. Use of Oligonucleotide Microarrays for Rapid Detection and Serotyping of Acute Respiratory Disease-Associated Adenoviruses

    PubMed Central

    Lin, Baochuan; Vora, Gary J.; Thach, Dzung; Walter, Elizabeth; Metzgar, David; Tibbetts, Clark; Stenger, David A.

    2004-01-01

    The cessation of the adenovirus vaccination program for military trainees has resulted in several recent acute respiratory disease (ARD) outbreaks. In the absence of vaccination, rapid detection methods are necessary for the timely implementation of measures to prevent adenovirus transmission within military training facilities. To this end, we have combined a fluorogenic real-time multiplex PCR assay with four sets of degenerate PCR primers that target the E1A, fiber, and hexon genes with a long oligonucleotide microarray capable of identifying the most common adenovirus serotypes associated with adult respiratory tract infections (serotypes 3, 4, 7, 16, and 21) and a representative member of adenovirus subgroup C (serotype 6) that is a common cause of childhood ARD and that often persists into adulthood. Analyses with prototype strains demonstrated unique hybridization patterns for representative members of adenovirus subgroups B1, B2, C, and E, thus allowing serotype determination. Microarray-based sensitivity assessments revealed lower detection limits (between 1 and 100 genomic copies) for adenovirus serotype 4 (Ad4) and Ad7 cell culture lysates, clinical nasal washes, and throat swabs and purified DNA from clinical samples. When adenovirus was detected from coded clinical samples, the results obtained by this approach demonstrated an excellent concordance with those obtained by the more established method of adenovirus identification as well as by cell culture with fluorescent-antibody staining. Finally, the utility of this method was further supported by its ability to detect adenoviral coinfections, contamination, and, potentially, recombination events. Taken together, the results demonstrate the usefulness of the simple and rapid diagnostic method developed for the unequivocal identification of ARD-associated adenoviral serotypes from laboratory or clinical samples that can be completed in 1.5 to 4.0 h. PMID:15243087

  8. Kullback-Leibler divergence for detection of rare haplotype common disease association.

    PubMed

    Lin, Shili

    2015-11-01

    Rare haplotypes may tag rare causal variants of common diseases; hence, detection of such rare haplotypes may also contribute to our understanding of complex disease etiology. Because rare haplotypes frequently result from common single-nucleotide polymorphisms (SNPs), focusing on rare haplotypes is much more economical compared with using rare single-nucleotide variants (SNVs) from sequencing, as SNPs are available and 'free' from already amassed genome-wide studies. Further, associated haplotypes may shed light on the underlying disease causal mechanism, a feat unmatched by SNV-based collapsing methods. In recent years, data mining approaches have been adapted to detect rare haplotype association. However, as they rely on an assumed underlying disease model and require the specification of a null haplotype, results can be erroneous if such assumptions are violated. In this paper, we present a haplotype association method based on Kullback-Leibler divergence (hapKL) for case-control samples. The idea is to compare haplotype frequencies for the cases versus the controls by computing symmetrical divergence measures. An important property of such measures is that both the frequencies and logarithms of the frequencies contribute in parallel, thus balancing the contributions from rare and common, and accommodating both deleterious and protective, haplotypes. A simulation study under various scenarios shows that hapKL has well-controlled type I error rates and good power compared with existing data mining methods. Application of hapKL to age-related macular degeneration (AMD) shows a strong association of the complement factor H (CFH) gene with AMD, identifying several individual rare haplotypes with strong signals. PMID:25735482

  9. Optical detection of nanoparticle-enhanced human papillomavirus genotyping microarrays.

    PubMed

    Li, Xue Zhe; Kim, Sookyung; Cho, Wonhyung; Lee, Seung-Yop

    2013-02-01

    In this study, we propose a new detection method of nanoparticle-enhanced human papillomavirus genotyping microarrays using a DVD optical pick-up with a photodiode. The HPV genotyping DNA chip was labeled using Au/Ag core-shell nanoparticles, prepared on a treatment glass substrate. Then, the bio information of the HPV genotyping target DNA was detected by measuring the difference of the optical signals between the DNA spots and the background parts for cervical cancer diagnosis. Moreover the approximate linear relationship between the concentration of the HPV genotyping target DNA and the optical signal depending on the density of Au/Ag core-shell nanoparticles was obtained by performing a spot finding algorithm. It is shown that the nanoparticle-labeled HPV genotyping target DNA can be measured and quantified by collecting the low-cost photodiode signal on the treatment glass chip, replacing high-cost fluorescence microarray scanners using a photomultiplier tube. PMID:23413051

  10. SNP marker detection and genotyping in tilapia.

    PubMed

    Van Bers, N E M; Crooijmans, R P M A; Groenen, M A M; Dibbits, B W; Komen, J

    2012-09-01

    We have generated a unique resource consisting of nearly 175 000 short contig sequences and 3569 SNP markers from the widely cultured GIFT (Genetically Improved Farmed Tilapia) strain of Nile tilapia (Oreochromis niloticus). In total, 384 SNPs were selected to monitor the wider applicability of the SNPs by genotyping tilapia individuals from different strains and different geographical locations. In all strains and species tested (O. niloticus, O. aureus and O. mossambicus), the genotyping assay was working for a similar number of SNPs (288-305 SNPs). The actual number of polymorphic SNPs was, as expected, highest for individuals from the GIFT population (255 SNPs). In the individuals from an Egyptian strain and in individuals caught in the wild in the basin of the river Volta, 197 and 163 SNPs were polymorphic, respectively. A pairwise calculation of Nei's genetic distance allowed the discrimination of the individual strains and species based on the genotypes determined with the SNP set. We expect that this set will be widely applicable for use in tilapia aquaculture, e.g. for pedigree reconstruction. In addition, this set is currently used for assaying the genetic diversity of native Nile tilapia in areas where tilapia is, or will be, introduced in aquaculture projects. This allows the tracing of escapees from aquaculture and the monitoring of effects of introgression and hybridization. PMID:22524158

  11. Multiplex Detection and SNP Genotyping in a Single Fluorescence Channel

    PubMed Central

    Fu, Guoliang; Miles, Andrea; Alphey, Luke

    2012-01-01

    Probe-based PCR is widely used for SNP (single nucleotide polymorphism) genotyping and pathogen nucleic acid detection due to its simplicity, sensitivity and cost-effectiveness. However, the multiplex capability of hydrolysis probe-based PCR is normally limited to one target (pathogen or allele) per fluorescence channel. Current fluorescence PCR machines typically have 4–6 channels. We present a strategy permitting the multiplex detection of multiple targets in a single detection channel. The technique is named Multiplex Probe Amplification (MPA). Polymorphisms of the CYP2C9 gene (cytochrome P450, family 2, subfamily C, polypeptide 9, CYP2C9*2) and human papillomavirus sequences HPV16, 18, 31, 52 and 59 were chosen as model targets for testing MPA. The allele status of the CYP2C9*2 determined by MPA was entirely concordant with the reference TaqMan® SNP Genotyping Assays. The four HPV strain sequences could be independently detected in a single fluorescence detection channel. The results validate the multiplex capacity, the simplicity and accuracy of MPA for SNP genotyping and multiplex detection using different probes labeled with the same fluorophore. The technique offers a new way to multiplex in a single detection channel of a closed-tube PCR. PMID:22272339

  12. Multiplex detection and SNP genotyping in a single fluorescence channel.

    PubMed

    Fu, Guoliang; Miles, Andrea; Alphey, Luke

    2012-01-01

    Probe-based PCR is widely used for SNP (single nucleotide polymorphism) genotyping and pathogen nucleic acid detection due to its simplicity, sensitivity and cost-effectiveness. However, the multiplex capability of hydrolysis probe-based PCR is normally limited to one target (pathogen or allele) per fluorescence channel. Current fluorescence PCR machines typically have 4-6 channels. We present a strategy permitting the multiplex detection of multiple targets in a single detection channel. The technique is named Multiplex Probe Amplification (MPA). Polymorphisms of the CYP2C9 gene (cytochrome P450, family 2, subfamily C, polypeptide 9, CYP2C9*2) and human papillomavirus sequences HPV16, 18, 31, 52 and 59 were chosen as model targets for testing MPA. The allele status of the CYP2C9*2 determined by MPA was entirely concordant with the reference TaqMan® SNP Genotyping Assays. The four HPV strain sequences could be independently detected in a single fluorescence detection channel. The results validate the multiplex capacity, the simplicity and accuracy of MPA for SNP genotyping and multiplex detection using different probes labeled with the same fluorophore. The technique offers a new way to multiplex in a single detection channel of a closed-tube PCR. PMID:22272339

  13. High-resolution HLA-DRB1 genotyping in an Australian inclusion body myositis (s-IBM) cohort: an analysis of disease-associated alleles and diplotypes.

    PubMed

    Rojana-udomsart, Arada; James, Ian; Castley, Alison; Needham, Merrilee; Scott, Adrian; Day, Timothy; Kiers, Lynette; Corbett, Alastair; Sue, Carolyn; Witt, Campbell; Martinez, Patricia; Christiansen, Frank; Mastaglia, Frank

    2012-09-15

    We performed high-resolution (4-digit) HLA-DRB1 genotyping in an Australian cohort of 105s-IBM patients and 189 controls. Our findings showed that whilst the strongest association was with the HLA-DRB1*03:01 allele and the HLA-DRB1*03:01/*01:01 diplotype, HLA-DRB1*01:01 and HLA-DRB1*13:01 are also risk alleles. A number of other alleles, HLA-DRB1*04:01, *04:04, *07:01, *09:01, *11:01 and *15:01, as well as the HLA-DRB1*03:01/*04:01 and HLA-DRB1*03:01/*07:01 diplotypes were reduced in s-IBM cases and may be protective. The HLA-DRB1*03:01 and HLA-DRB1*13:01 alleles also appear to have an influence on the age at onset of the disease and severity of muscle weakness. Our findings indicate that the influence of HLA-DRB1 in s-IBM is complex and that epistatic interactions at the HLA-DRB1 locus contribute both to disease susceptibility and to the clinical phenotype. PMID:22633068

  14. Detection of genotype recycling fraud in U.S. immigrants.

    PubMed

    Wenk, Robert E

    2011-01-01

    Relationship testing laboratories provide genetic evidence to support or refute claims of kinship between U.S. citizen petitioners and potential immigrant beneficiaries. One female beneficiary presented a male amelogenin type and alleles at 15 autosomal loci that were identical to an alleged brother's. Laboratory records showed that her alleged father had petitioned to have 15 children emigrate from Ghana. The petitioner's 15 paternity indices exceeded 10⁵, but the children shared only four short tandem repeat (STR) profiles, suggesting fraudulent reuse of genotypes in this alleged pedigree (AP). To determine the extent of this "genotype recycling," I examined the laboratory's 555 APs from Ghana and 532 control APs from Nigeria. Seventeen Ghanaian APs (3.1%) but no Nigerian APs showed genotype recycling. Of 90 tested people in the 17 APs, 56 shared identical STR profiles with others in their AP. Of these 56 people, 10 were petitioners with unexpectedly high parentage indices. Seven of 56 had amelogenin types that disagreed with their declared genders. Database searches for identical multilocus genotypes in allegedly different people would best detect this fraud. PMID:20950312

  15. Development of an improved genotyping assay for the detection of hepatitis C virus genotypes and subtypes in Pakistan.

    PubMed

    Idrees, Muhammad

    2008-06-01

    A new genotyping system was established for the specific detection of HCV genotypes 1a, 1b, 1c, 2a, 2b, 2c, 3a, 3b, 3c, 4a-h, 5a and 6a during the course of this study. The system is based on entire core region and a part of 5' noncoding region (5'NCR) with genotype-specific primers. Genotype-specific primers were designed on the basis of 114 HCV isolates. Serum samples with known genotypes were used as positive controls to validate the assay developed and to generate PCR band patterns. Band patterns generated from the clinical serum samples from HCV patients were compared to the patterns produced from these control samples. In addition, the type-specific bands were sequenced from the test patients and control clinical samples to validate further the test results. To determine sensitivity and specificity of the assay, a total 260 samples were analyzed simultaneously by this HCV genotyping method and that developed by Ohno and Murex HCV Serotyping 1-6 Assay. The system showed 79.2% concordance with Ohno's system and 65.38% with serotyping system. Samples with discordant results were sequenced and their genotypes were determined by molecular evolutionary analysis. The data indicate that the method described in this study may offer better sensitivity and specificity for the detection directly of HCV genotypes present at low levels in HCV patient samples. PMID:18423633

  16. Detection of a novel genotype of Cryptosporidium in Antarctic pinnipeds.

    PubMed

    Rengifo-Herrera, Claudia; Ortega-Mora, Luis Miguel; Gómez-Bautista, Mercedes; García-Peña, Francisco Javier; García-Párraga, Daniel; Pedraza-Díaz, Susana

    2013-01-16

    A study was conducted to investigate the presence of Cryptosporidium and Giardia in Antarctic marine mammals. A total of 270 faecal samples from different species of pinnipeds from different locations in the South Shetland Islands and Antarctic Peninsula were analysed by immunofluorescence microscopy and PCR. Cryptosporidium was detected by PCR in three samples from Southern elephant seals (Mirounga leonina) and 2 Weddell seals (Leptonychotes weddellii). However, no oocysts were observed in any of the samples by immunofluorescence microscopy. Molecular characterisation of the isolates, using the 18S rDNA, the HSP70 and the COWP loci, revealed the presence of a Cryptosporidium sp., previously reported from an Antarctic Southern elephant seal, in the elephant seals and a novel genotype in Weddell seals. Giardia could not be detected in any of the samples analysed. PMID:23021408

  17. Wilson's Disease Association International

    MedlinePlus

    ... Connect with Wilson Disease Association Send Email Physician Contacts List of Physicians and Institutions in Your Area View Contacts Support Contacts Individuals who can offer Support and Information View ...

  18. Detection of the disease-associated isoform of the prion protein in formalin-fixed tissues by Western blot

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clinical signs of prion disease are not pathognomonic and include a variety of differential diagnoses. Specific immune responses have not been detected in affected organisms, serological tests to obtain evidence for the presence of the infectious agent are not available, and nucleic acid-based detec...

  19. Detection of disease-associated prion protein in the optic nerve and the adrenal gland of cattle with bovine spongiform encephalopathy by using highly sensitive immunolabeling procedures.

    PubMed

    Okada, Hiroyuki; Iwamaru, Yoshifumi; Fukuda, Shigeo; Yokoyama, Takashi; Mohri, Shirou

    2012-04-01

    A sensitive immunohistochemical procedure, the tyramide signal amplification (TSA) system, was applied to detect the localization of immunolabeled disease-associated prion protein (PrP(Sc)) in cattle affected with bovine spongiform encephalopathy (BSE). In this procedure, immunolabeling could be visualized in the optic nerve and the adrenal medulla. In the optic nerve, the dual immunofluorescent technique showed that the granular PrP(Sc) was occasionally detected in the astrocytes, microglia, and myelin sheath adjacent to the axon. Clustered PrP(Sc) was also scattered in association with microglial cells and astrocytes of the optic nerve. In the adrenal gland, PrP(Sc) immunolabeling was confined within the sympathetic nerve fibers and endings. The results suggest that (1) PrP(Sc) might centrifugally spread within and between glial cells and/or the non-axonal (also known as ad-axonal) region of nerve fibers, rather than the axonal and/or extracellular space pathway in the optic nerve, and (2) the sympathetic innervations might be important for the trafficking of BSE agent in the adrenal glands of cattle. This study also suggests that tyramide-based immunochemical analysis should be performed to detect immunolabeled PrP(Sc) in the extracerebral tissues of BSE-affected cattle. PMID:22260993

  20. Rapid genotypic detection of rifampin- and isoniazid-resistant Mycobacterium tuberculosis directly in clinical specimens.

    PubMed

    Bang, Didi; Bengård Andersen, Ase; Thomsen, Vibeke Østergaard

    2006-07-01

    A multiplex PCR DNA strip assay (Genotype MTBDR) designed to detect rifampin (rpoB) and high-level isoniazid (katG) resistance mutations in Mycobacterium tuberculosis isolates was optimized for clinical specimens. Successful genotypic results were achieved with 36 of 38 (95%) smear-positive respiratory specimens, allowing rapid therapeutic adjustments in transmittable drug-resistant tuberculosis. PMID:16825393

  1. Detection of genotype-specific Ehrlichia canis exposure in Brazilian dogs by TRP36 peptide ELISA.

    PubMed

    Aguiar, Daniel M; Zhang, Xiaofeng; Braga, Isis A; Taques, Isis I G G; McBride, Jere W

    2016-02-01

    We recently characterized a novel genotype of Ehrlichia canis based on the tandem repeat (TR) sequence of the TRP36 gene in Brazil. The TR amino acid sequence of the Brazilian (Br) genotype (ASVVPEAE) was divergent from the previously described US genotype (TEDSVSAPA) of E. canis. In this study, we developed an ELISA based on TRP36 TR synthetic peptides from both Br and US E. canis TRP36 genotypes to serologically detect and distinguish infections caused by these genotypes. Sera from 30 Brazilian dogs naturally infected with E. canis, sera from dogs experimentally infected E. canis (Jake and Cuiabá #1 strains) and E. chaffeensis (Arkansas strain) and 12 seronegative E. canis dogs were evaluated. Fifteen naturally infected Brazilian dogs had antibodies that reacted with the US TRP36 (n=9) or Br TRP36 (n=6) only, and 13 dogs had antibodies that reacted with both TPR36 peptides suggesting that these dogs were exposed to both genotypes. Most dogs (n=28) had antibodies that reacted with the highly conserved E. canis TRP19 peptide; however, two dogs had antibodies to E. canis TRP19, but did not have TRP36 antibodies, raising the possibility that another novel TRP36 genotype is circulating in Brazil. Our results demonstrate that synthetic peptides based on the TR region of E. canis TRP36 can be used to serologically distinguish infections or identify coinfections by different genotypes, and to determine the seroprevalence of various E. canis genotypes in Brazil. PMID:26482949

  2. Universal Primers for Detection and Sequencing of Hepatitis B Virus Genomes across Genotypes A to G

    PubMed Central

    Chook, Jack Bee; Teo, Woon Li; Ngeow, Yun Fong; Tee, Kok Keng; Ng, Kee Peng

    2015-01-01

    Hepatitis B virus (HBV) has been divided into 10 genotypes, A to J, based on an 8% nucleotide sequence divergence between genotypes. The conventional practice of using a single set of primers to amplify a near-complete HBV genome is hampered by its low analytical sensitivity. The current practice of using overlapping conserved primer sets to amplify a complete HBV genome in a clinical sample is limited by the lack of pan-primers to detect all HBV genotypes. In this study, we designed six highly conserved, overlapping primer sets to cover the complete HBV genome. We based our design on the sequences of 5,154 HBV genomes of genotypes A to I downloaded from the GenBank nucleotide database. These primer sets were tested on 126 plasma samples from Malaysia, containing genotypes A to D and with viral loads ranging from 20 to >79,780,000 IU/ml. The overall success rates for PCR amplification and sequencing were >96% and >94%, respectively. Similarly, there was 100% amplification and sequencing success when the primer sets were tested on an HBV reference panel of genotypes A to G. Thus, we have established primer sets that gave a high analytical sensitivity for PCR-based detection of HBV and a high rate of sequencing success for HBV genomes in most of the viral genotypes, if not all, without prior known sequence data for the particular genotype/genome. PMID:25788548

  3. Universal Primers for Detection and Sequencing of Hepatitis B Virus Genomes across Genotypes A to G.

    PubMed

    Chook, Jack Bee; Teo, Woon Li; Ngeow, Yun Fong; Tee, Kok Keng; Ng, Kee Peng; Mohamed, Rosmawati

    2015-06-01

    Hepatitis B virus (HBV) has been divided into 10 genotypes, A to J, based on an 8% nucleotide sequence divergence between genotypes. The conventional practice of using a single set of primers to amplify a near-complete HBV genome is hampered by its low analytical sensitivity. The current practice of using overlapping conserved primer sets to amplify a complete HBV genome in a clinical sample is limited by the lack of pan-primers to detect all HBV genotypes. In this study, we designed six highly conserved, overlapping primer sets to cover the complete HBV genome. We based our design on the sequences of 5,154 HBV genomes of genotypes A to I downloaded from the GenBank nucleotide database. These primer sets were tested on 126 plasma samples from Malaysia, containing genotypes A to D and with viral loads ranging from 20 to >79,780,000 IU/ml. The overall success rates for PCR amplification and sequencing were >96% and >94%, respectively. Similarly, there was 100% amplification and sequencing success when the primer sets were tested on an HBV reference panel of genotypes A to G. Thus, we have established primer sets that gave a high analytical sensitivity for PCR-based detection of HBV and a high rate of sequencing success for HBV genomes in most of the viral genotypes, if not all, without prior known sequence data for the particular genotype/genome. PMID:25788548

  4. [Hepatitis B virus genotype E infection in Turkey: the detection of the first case].

    PubMed

    Sayan, Murat; Sanlıdağ, Tamer; Akçalı, Sinem; Arıkan, Ayşe

    2014-10-01

    Hepatitis B virus (HBV) infection is a global major health problem. Currently, 10 genotypes (A-J) of hepatitis B virus (HBV) are identified based on the nucleic acid sequence heterogeneity, and these genotypes have been shown to have distinct geographic distribution. Reports of the previous studies indicated that the genotype D is the predominant type among hepatitis B patients in different regions of Turkey. However, recent studies indicated that other HBV genotypes are also seen with an increasing rate. Although epidemiological and clinical information on genotype E infection is currently limited, it is known that genotype E infection is common in West and Central Africa. In this report, the first case of HBV genotype E infection in Turkey was presented. A 22-year-old Nigerian male employee who resided in Manisa for five years was admitted to Celal Bayar University Hospital Manisa, Turkey, for his routine check-up. Since HBsAg was found positive, other HBV markers were tested with a repeated serum sample. Laboratory findings were as follows; HBsAg (+), anti-HBs (-), HBeAg (-), anti-HBe (+), anti-HBc (+), anti-HCV (-), anti-HIV (-), ALT: 44 U/L and AST: 45 U/L. HBV-DNA level was detected as 700 IU/ml by real-time PCR (Artus HBV QS RGQ Qiagen, Germany). HBV-DNA isolated from the serum sample of the patient was amplified by PCR and polymerase gene segment of HBV was directly sequenced. UPGMA method was used for phylogenetic analysis and Inno-LIPA HBV genotyping method (Innogenetics, Belgium) was performed to determine multiple HBV genotype infection. On the basis of those methods the genotype of the virus was identified as genotype E. The partial sequences of the HBV polymerase gene were loaded to the international DNA data bank (GenBank) for contribution to the global HBV surveillance. This report emphasized that besides genotype D the other HBV genotypes could be found in Turkey. Since the patient was an inactive HBsAg carrier before his residence in Turkey, this

  5. Development and evaluation of a new PCR assay for detection of Pseudomonas aeruginosa D genotype.

    PubMed

    Lødeng, A G G; Ahlén, C; Lysvand, H; Mandal, L H; Iversen, O J

    2006-08-01

    This report describes a new PCR-based assay for the detection of Pseudomonas aeruginosa genotype D in occupational saturation diving systems in the North Sea. This genotype has persisted in these systems for 11 years (1993-2003) and represents 18% of isolates from infections analysed during this period. The new PCR assay was based on sequences obtained after randomly amplified polymorphic DNA (RAPD)-PCR analysis of a group of isolates related to diving that had been identified previously by pulsed-field gel electrophoresis (PFGE). The primer set for the D genotype targets a gene that codes for a hypothetical class 4 protein in the P. aeruginosa PAO1 genome. A primer set able to detect P. aeruginosa at the species level was also designed, based on the 23S-5S rDNA spacer region. The two assays produced 382-bp and 192-bp amplicons, respectively. The PCR assay was evaluated by analysing 100 P. aeruginosa isolates related to diving, representing 28 PFGE genotypes, and 38 clinical and community P. aeruginosa isolates and strains from other species. The assay identified all of the genotype D isolates tested. Two additional diving-relevant genotypes (TP2 and TP27) were also identified, as well as three isolates of non-diving origin. It was concluded that the new PCR assay is a useful tool for early detection and prevention of infections with the D genotype. PMID:16842571

  6. Molecular detection of bovine Noroviruses in Argentinean dairy calves: Circulation of a tentative new genotype.

    PubMed

    Ferragut, Fátima; Vega, Celina G; Mauroy, Axel; Conceição-Neto, Nádia; Zeller, Mark; Heylen, Elisabeth; Uriarte, Enrique Louge; Bilbao, Gladys; Bok, Marina; Matthijnssens, Jelle; Thiry, Etienne; Badaracco, Alejandra; Parreño, Viviana

    2016-06-01

    Bovine noroviruses are enteric pathogens detected in fecal samples of both diarrheic and non-diarrheic calves from several countries worldwide. However, epidemiological information regarding bovine noroviruses is still lacking for many important cattle producing countries from South America. In this study, three bovine norovirus genogroup III sequences were determined by conventional RT-PCR and Sanger sequencing in feces from diarrheic dairy calves from Argentina (B4836, B4848, and B4881, all collected in 2012). Phylogenetic studies based on a partial coding region for the RNA-dependent RNA polymerase (RdRp, 503 nucleotides) of these three samples suggested that two of them (B4836 and B4881) belong to genotype 2 (GIII.2) while the third one (B4848) was more closely related to genotype 1 (GIII.1) strains. By deep sequencing, the capsid region from two of these strains could be determined. This confirmed the circulation of genotype 1 (B4848) together with the presence of another sequence (B4881) sharing its highest genetic relatedness with genotype 1, but sufficiently distant to constitute a new genotype. This latter strain was shown in silico to be a recombinant: phylogenetic divergence was detected between its RNA-dependent RNA polymerase coding sequence (genotype GIII.2) and its capsid protein coding sequence (genotype GIII.1 or a potential norovirus genotype). According to this data, this strain could be the second genotype GIII.2_GIII.1 bovine norovirus recombinant described in literature worldwide. Further analysis suggested that this strain could even be a potential norovirus GIII genotype, tentatively named GIII.4. The data provides important epidemiological and evolutionary information on bovine noroviruses circulating in South America. PMID:26940636

  7. Influence of breed and genotype on the onset and distribution of infectivity and disease-associated prion protein in sheep following oral infection with the bovine spongiform encephalopathy agent.

    PubMed

    McGovern, G; Martin, S; Jeffrey, M; Bellworthy, S J; Spiropoulos, J; Green, R; Lockey, R; Vickery, C M; Thurston, L; Dexter, G; Hawkins, S A C; González, L

    2015-01-01

    The onset and distribution of infectivity and disease-specific prion protein (PrP(d)) accumulation was studied in Romney and Suffolk sheep of the ARQ/ARQ, ARQ/ARR and ARR/ARR prion protein gene (Prnp) genotypes (where A stands for alanine, R for arginine and Q for glutamine at codons 136, 154 and 171 of PrP), following experimental oral infection with cattle-derived bovine spongiform encephalopathy (BSE) agent. Groups of sheep were killed at regular intervals and a wide range of tissues taken for mouse bioassay or immunohistochemistry (IHC), or both. Bioassay results for infectivity were mostly coincident with those of PrP(d) detection by IHC both in terms of tissues and time post infection. Neither PrP(d) nor infectivity was detected in any tissues of BSE-dosed ARQ/ARR or ARR/ARR sheep or of undosed controls. Moreover, four ARQ/ARQ Suffolk sheep, which were methionine (M)/threonine heterozygous at codon 112 of the Prnp gene, did not show any biological or immunohistochemical evidence of infection, while those homozygous for methionine (MARQ/MARQ) did. In MARQ/MARQ sheep of both breeds, initial PrP(d) accumulation was identified in lymphoreticular system (LRS) tissues followed by the central nervous system (CNS) and enteric nervous system (ENS) and finally by the autonomic nervous system and peripheral nervous system and other organs. Detection of infectivity closely mimicked this sequence. No PrP(d) was observed in the ENS prior to its accumulation in the CNS, suggesting that ENS involvement occurred simultaneously to that of, or followed centrifugal spread from, the CNS. The distribution of PrP(d) within the ENS further suggested a progressive spread from the ileal plexus to other ENS segments via neuronal connections of the gut wall. Differences between the two breeds were noted in terms of involvement of LRS and ENS tissues, with Romney sheep showing a more delayed and less consistent PrP(d) accumulation than Suffolk sheep in such tissues. Whether this

  8. Detection of Rotavirus Genotypes in Korea 5 Years after the Introduction of Rotavirus Vaccines.

    PubMed

    Chung, Ju-Young; Kim, Min-Sung; Jung, Tae Woong; Kim, Seong Joon; Kang, Jin-Han; Han, Seung Beom; Kim, Sang Yong; Rhim, Jung Woo; Kim, Hwang-Min; Park, Jae Hong; Jo, Dae Sun; Ma, Sang Hyuk; Jeong, Hye-Sook; Cheon, Doo-Sung; Kim, Jong-Hyun

    2015-10-01

    Rotavirus (RV) is one of the most important viral etiologic agents of acute gastroenteritis (AGE) in children. Although effective RV vaccines (RVVs) are now used worldwide, novel genotypes and outbreaks resulting from rare genotype combinations have emerged. This study documented RV genotypes in a Korean population of children with AGE 5 yr after the introduction of RVV and assessed potential genotype differences based on vaccination status or vaccine type. Children less than 5-yr-old diagnosed with AGE between October 2012 and September 2013 admitted to 9 medical institutions from 8 provinces in Korea were prospectively enrolled. Stool samples were tested for RV by enzyme immunoassay and genotyped by multiplex reverse-transcription polymerase chain reaction. In 346 patients, 114 (32.9%) were RV-positive. Among them, 87 (76.3%) patients were infected with RV alone. Eighty-six of 114 RV-positive stool samples were successfully genotyped, and their combinations of genotypes were G1P[8] (36, 41.9%), G2P[4] (12, 14.0%), and G3P[8] (6, 7.0%). RV was detected in 27.8% of patients in the vaccinated group and 39.8% in the unvaccinated group (P=0.035). Vaccination history was available for 67 of 86 cases with successfully genotyped RV-positive stool samples; RotaTeq (20, 29.9%), Rotarix (7, 10.4%), unvaccinated (40, 59.7%). The incidence of RV AGE is lower in the RV-vaccinated group compared to the unvaccinated group with no evidence of substitution with unusual genotype combinations. PMID:26425045

  9. A sensitive method for detecting and genotyping Cryptosporidium parvum oocysts

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cryptosporidium parvum oocysts represent a considerable health risk to humans and animals because the parasite has a low infectious dose and usually exists in low numbers in environmental samples, which makes detection problematic. The purpose of this study was to evaluate Cryspovirus as a target f...

  10. Genotypic and phenotypic detection of efflux pump in Rhodococcus equi

    PubMed Central

    Gressler, Letícia Trevisan; de Vargas, Agueda Castagna; da Costa, Mateus Matiuzzi; Pötter, Luciana; da Silveira, Bibiana Petri; Sangioni, Luis Antônio; de Avila Botton, Sônia

    2014-01-01

    The req_39680 gene, associated to a putative efflux system, was detected in 60% (54/90) of R. equi isolates by PCR. The phenotypic expression of efflux mechanism was verified in 20% of the isolates using ethidium bromide. For the first time, the expression of efflux mechanism was demonstrated in R. equi. PMID:25242956

  11. Detection and Genotyping of Leuconostoc spp. in a Sausage Processing Plant.

    PubMed

    Padilla-Frausto, J J; Cepeda-Marquez, L G; Salgado, L M; Iturriaga, M H; Arvizu-Medrano, S M

    2015-12-01

    Some Leuconostoc spp. have the ability to produce slime and undesirable compounds in cooked sausage. The objectives of this research were to identify Leuconostoc sources in a Vienna-type sausage processing plant and to evaluate the genetic diversity of the isolated strains. Three hundred and two samples of sausage batter, sausages during processing, spoiled sausage, equipment surfaces, chilling brine, workers' gloves and aprons, and used casings were collected (March to November 2008 and February to April 2010) from a sausage processing plant. Lactic acid bacteria (LAB) were quantified, and Leuconostoc were detected using PCR. Strains were isolated and identified in Leuconostoc-positive samples. Leuconostoc strains were genotyped using randomly amplified polymorphic DNA and pulsed-field gel electrophoresis. LAB content of nonspoiled and spoiled sausage ranged from <0.8 to 4.4 log CFU/g and from 4.9 to 8.3 log CFU/g, respectively. LAB levels on equipment surfaces ranged from <1.3 to 4.8 log CFU/100 cm(2). Leuconostoc was detected in 35% of the samples, and 88 Leuconostoc spp. strains were isolated and genotyped. The main Leuconostoc spp. isolated were L. mesenteroides (37 genotypes), L. fallax (29 genotypes), and L. lactis (6 genotypes). Some strains of Leuconostoc isolated from equipment surfaces and sausages showed the same genotype. One L. lactis genotype included strains isolated from spoiled sausages analyzed in April 2008 and March to April 2010. Equipment and conveyor belts constitute Leuconostoc contamination sources. Leuconostoc persistence in the sausage processing environment and in the final product suggests the existence of microbial reservoirs, possibly on equipment surfaces. PMID:26613911

  12. Detection of rare and possibly carcinogenic human papillomavirus genotypes as single infections in invasive cervical cancer.

    PubMed

    Geraets, Daan; Alemany, Laia; Guimera, Nuria; de Sanjose, Silvia; de Koning, Maurits; Molijn, Anco; Jenkins, David; Bosch, Xavier; Quint, Wim

    2012-12-01

    The contribution of carcinogenic human papillomavirus (HPV) types to the burden of cervical cancer has been well established. However, the role and contribution of phylogenetically related HPV genotypes and rare variants remains uncertain. In a recent global study of 8977 HPV-positive invasive cervical carcinomas (ICCs), the genotype remained unidentified in 3.7% by the HPV SPF10 PCR-DEIA-LiPA25 (version 1) algorithm. The 331 ICC specimens with unknown genotype were analysed by a novel sequence methodology, using multiple selected short regions in L1. This demonstrated HPV genotypes that have infrequently or never been detected in ICC, ie HPV26, 30, 61, 67, 68, 69, 73 and 82, and rare variants of HPV16, 18, 26, 30, 34, 39, 56, 67, 68, 69, 82 and 91. These are not identified individually by LiPA25 and only to some extent by other HPV genotyping assays. Most identified genotypes have a close phylogenetic relationship with established carcinogenic HPVs and have been classified as possibly carcinogenic by IARC. Except for HPV85, all genotypes in α-species 5, 6, 7, 9 and 11 were encountered as single infections in ICCs. These species of established and possibly carcinogenic HPV types form an evolutionary clade. We have shown that the possibly carcinogenic types were detected only in squamous cell carcinomas, which were often keratinizing and diagnosed at a relatively higher mean age (55.3 years) than those associated with established carcinogenic types (50.9 years). The individual frequency of the possibly carcinogenic types in ICCs is low, but together they are associated with 2.25% of the 8338 included ICCs with a single HPV type. This fraction is greater than seven of the established carcinogenic types individually. This study provides evidence that possibly carcinogenic HPV types occur as single infections in invasive cervical cancer, strengthening the circumstantial evidence of a carcinogenic role. PMID:22711526

  13. Identification and Characterization of a Peptide Mimetic That May Detect a Species of Disease-Associated Anticardiolipin Antibodies in Patients With the Antiphospholipid Syndrome

    PubMed Central

    Visvanathan, Sudha; Scott, Jamie K.; Hwang, Kwan-Ki; Banares, Michelle; Grossman, Jennifer M.; Merrill, Joan T.; FitzGerald, John; Chukwuocha, Reginald U.; Tsao, Betty P.; Hahn, Bevra H.; Chen, Pojen P.

    2007-01-01

    Objective To test the feasibility of applying a mimetic (specific for a patient-derived prothrombotic anticardiolipin antibody [aCL]) to study the homologous, disease-associated aCL in patients with antiphospholipid syndrome (APS). Methods We used the CL15 monoclonal aCL to screen 17 phage-display peptide libraries. Peptides (corresponding to recurrent peptide sequences) and their derivatives were synthesized and analyzed for binding to CL15 and for their abilities to inhibit CL15 from binding to cardiolipin. A peptide was chosen and used to study CL15-like IgG aCL in plasma samples from patients with APS, patients with systemic lupus erythematosus (SLE) but without APS, and normal healthy donors. Results Library screening with CL15 yielded 4 recurrent peptide sequences. Analyses of peptides showed that peptide CL154C reacted with antibody CL15 and inhibited binding of CL15 to cardiolipin, indicating that peptide CL154C may be a peptide mimetic for the CL15 aCL. Initial studies with plasma samples revealed that CL154C-reactive IgG was present (positivity defined as the mean + 3 SD optical density of the 25 normal controls) in 15 of 21 APS patients and 1 of 12 SLE patients. Conclusion These findings suggest that it is feasible to develop a specific enzyme-linked immunosorbent assay for each immunologically and functionally distinct disease-associated aCL. Additional testing of CL154C with a larger number of APS patients and SLE patients, as well as identification of peptide mimetics for each distinct aCL, will reveal the diagnostic potential of CL154C and other mimetics in identifying patients with aCL who are at risk of developing life-threatening thrombosis. PMID:12632428

  14. Human population structure detection via multilocus genotype clustering

    PubMed Central

    Gao, Xiaoyi; Starmer, Joshua

    2007-01-01

    Background We describe a hierarchical clustering algorithm for using Single Nucleotide Polymorphism (SNP) genetic data to assign individuals to populations. The method does not assume Hardy-Weinberg equilibrium and linkage equilibrium among loci in sample population individuals. Results We show that the algorithm can assign sample individuals highly accurately to their corresponding ethnic groups in our tests using HapMap SNP data and it is also robust to admixed populations when tested with Perlegen SNP data. Moreover, it can detect fine-scale population structure as subtle as that between Chinese and Japanese by using genome-wide high-diversity SNP loci. Conclusion The algorithm provides an alternative approach to the popular STRUCTURE program, especially for fine-scale population structure detection in genome-wide association studies. This is the first successful separation of Chinese and Japanese samples using random SNP loci with high statistical support. PMID:17592628

  15. Electronic hybridization detection in microarray format and DNA genotyping

    PubMed Central

    Blin, Antoine; Cissé, Ismaïl; Bockelmann, Ulrich

    2014-01-01

    We describe an approach to substituting a fluorescence microarray with a surface made of an arrangement of electrolyte-gated field effect transistors. This was achieved using a dedicated blocking of non-specific interactions and comparing threshold voltage shifts of transistors exhibiting probe molecules of different base sequence. We apply the approach to detection of the 35delG mutation, which is related to non-syndromic deafness and is one of the most frequent mutations in humans. The process involves barcode sequences that are generated by Tas-PCR, a newly developed replication reaction using polymerase blocking. The barcodes are recognized by hybridization to surface attached probes and are directly detected by the semiconductor device. PMID:24569823

  16. Electronic hybridization detection in microarray format and DNA genotyping

    NASA Astrophysics Data System (ADS)

    Blin, Antoine; Cissé, Ismaïl; Bockelmann, Ulrich

    2014-02-01

    We describe an approach to substituting a fluorescence microarray with a surface made of an arrangement of electrolyte-gated field effect transistors. This was achieved using a dedicated blocking of non-specific interactions and comparing threshold voltage shifts of transistors exhibiting probe molecules of different base sequence. We apply the approach to detection of the 35delG mutation, which is related to non-syndromic deafness and is one of the most frequent mutations in humans. The process involves barcode sequences that are generated by Tas-PCR, a newly developed replication reaction using polymerase blocking. The barcodes are recognized by hybridization to surface attached probes and are directly detected by the semiconductor device.

  17. DEVELOPMENT AND EVALUATION OF A MICROARRAY APPROACH TO DETECT AND GENOTYPE NOROVIRUSES IN WATER

    EPA Science Inventory

    Noroviruses are the leading cause of nonbacterial gastroenteritis outbreaks in the United States, some of which are caused by the ingestion of contaminated water. These viruses are usually detected and genotyped using reverse transcription-polymerase chain reaction (RT-PCR) base...

  18. Semiquantitative Multiplexed Tandem PCR for Detection and Differentiation of Four Theileria orientalis Genotypes in Cattle

    PubMed Central

    Perera, Piyumali K.; Gasser, Robin B.; Firestone, Simon M.; Smith, Lee; Roeber, Florian

    2014-01-01

    Oriental theileriosis is an emerging, tick-borne disease of bovines in the Asia-Pacific region and is caused by one or more genotypes of the Theileria orientalis complex. This study aimed to establish and validate a multiplexed tandem PCR (MT-PCR) assay using three distinct markers (major piroplasm surface protein, 23-kDa piroplasm membrane protein, and the first internal transcribed spacer of nuclear DNA), for the simultaneous detection and semiquantification of four genotypes (Buffeli, Chitose, Ikeda, and type 5) of the T. orientalis complex. Analytical specificity, analytical sensitivity, and repeatability of the established MT-PCR assay were assessed in a series of experiments. Subsequently, the assay was evaluated using 200 genomic DNA samples collected from cattle from farms on which oriental theileriosis outbreaks had occurred, and 110 samples from a region where no outbreaks had been reported. The results showed the MT-PCR assay specifically and reproducibly detected the expected genotypes (i.e., genotypes Buffeli, Chitose, Ikeda, and type 5) of the T. orientalis complex, reliably differentiated them, and was able to detect as little as 1 fg of genomic DNA from each genotype. The diagnostic specificity and sensitivity of the MT-PCR were estimated at 94.0% and 98.8%, respectively. The MT-PCR assay established here is a practical and effective diagnostic tool for the four main genotypes of T. orientalis complex in Australia and should assist studies of the epidemiology and pathophysiology of oriental theileriosis in the Asia-Pacific region. PMID:25339402

  19. Easy and fast detection and genotyping of high-risk human papillomavirus by dedicated DNA microarrays.

    PubMed

    Albrecht, Valérie; Chevallier, Anne; Magnone, Virginie; Barbry, Pascal; Vandenbos, Fanny; Bongain, André; Lefebvre, Jean-Claude; Giordanengo, Valérie

    2006-11-01

    Persistent cervical high-risk human papillomavirus (HPV) infection is correlated with an increased risk of developing a high-grade cervical intraepithelial lesion. A two-step method was developed for detection and genotyping of high-risk HPV. DNA was firstly amplified by asymmetrical PCR in the presence of Cy3-labelled primers and dUTP. Labelled DNA was then genotyped using DNA microarray hybridization. The current study evaluated the technical efficacy of laboratory-designed HPV DNA microarrays for high-risk HPV genotyping on 57 malignant and non-malignant cervical smears. The approach was evaluated for a broad range of cytological samples: high-grade squamous intraepithelial lesions (HSIL), low-grade squamous intraepithelial lesions (LSIL) and atypical squamous cells of high-grade (ASC-H). High-risk HPV was also detected in six atypical squamous cells of undetermined significance (ASC-US) samples; among them only one cervical specimen was found uninfected, associated with no histological lesion. The HPV oligonucleotide DNA microarray genotyping detected 36 infections with a single high-risk HPV type and 5 multiple infections with several high-risk types. Taken together, these results demonstrate the sensitivity and specificity of the HPV DNA microarray approach. This approach could improve clinical management of patients with cervical cytological abnormalities. PMID:16879879

  20. Close genotypic relationship between Enterocytozoon bieneusi from humans and pigs and first detection in cattle.

    PubMed

    Rinder, H; Thomschke, A; Dengjel, B; Gothe, R; Löscher, T; Zahler, M

    2000-02-01

    The reservoirs and the routes of transmission of Enterocytozoon bieneusi are still unknown. In humans, it is the most commonly found microsporidial species. It has also been found repeatedly in pigs, too. The first detection of E. bieneusi in cattle is reported herein. Two distinct genotypes were characterized and compared with 4 other genotypes from humans, 6 from pigs, and 1 from a cat. From these 13 E. bieneusi genotypes known to date, 25 polymorphic sites could be identified in the internal transcribed spacer of the rRNA gene. The spectrum of polymorphisms within and between each of the 4 host species indicates a close relationship between E. bieneusi strains from humans and pigs, whereas those from cattle are more distantly related. The data suggest the absence of a transmission barrier between pigs and humans for this pathogen. PMID:10701590

  1. Detecting and Genotyping Escherichia coli O157:H7 using multiplexed PCR and nucleic acid microarrays

    SciTech Connect

    Call, Douglas R.; Brockman, Fred J. ); Chandler, Darrell P.

    2000-12-01

    Rapid detection and characterization of food borne pathogens such as Escherichia coli O157:H7 is crucial for epidemiological investigations and food safety surveillance. As an alternative to conventional technologies, we examined the sensitivity and specificity of nucleic acid microarrays for detecting and genotyping E. coli O157:H7. The array was composed of oligonucleotide probes (25-30 mer) complementary to four virulence loci (intimin, Shiga-like toxins I and II, and hemolysin A). Target DNA was amplified from whole cells or from purified DNA via single or multiplexed polymerase chain reaction (PCR), and PCR products were hybridized to the array without further modification or purification. The array was 32-fold more sensitive than gel electrophoresis and capable of detecting amplification products from < 1 cell equivalent of genomic DNA (1 fg). Immunomagnetic capture, PCR and a microarray were subsequently used to detect 55 CFU ml-1 (E. coli O157:H7) from chicken rinsate without the aid of pre-enrichment. Four isolates of E. coli O157:H7 and one isolate of O91:H2, for which genotypic data were available, were unambiguously genotyped with this array. Glass based microarrays are relatively simple to construct and provide a rapid and sensitive means to detect multiplexed PCR products and the system is amenable to automation.

  2. Detecting and genotyping Escherichia coli O157:H7 using multiplexed PCR and nucleic acid microarrays

    SciTech Connect

    Call, Douglas R.; Brockman, Fred J.; Chandler, Darrell P.

    2001-07-05

    Rapid detection and characterization of food borne pathogens such as Escherichia coli O157:H7 is crucial for epidemiological investigations and food safety surveillance. As an alternative to conventional technologies, we examined the sensitivity and specificity of nucleic acid microarrays for detecting and genotyping E. coli O157:H7. The array was composed of oligonucleotide probes (25-30 mer) complementary to four virulence loci (intimin, Shiga-like toxins I and II, and hemolysin A). Target DNA was amplified from whole cells or from purified DNA via single or multiplexed polymerase chain reaction (PCR), and PCR products were hybridized to the array without further modification or purification. The array was 32-fold more sensitive than gel electrophoresis and capable of detecting amplification products from < 1 cell equivalent of genomic DNA (1 fg). Immunomagnetic capture, PCR and a microarray were subsequently used to detect 55 CFUs ml-1 (E. coli O157:H7) from chicken rinsate without the aid of pre-enrichment. Four isolates of E. coli O157:H7 and one isolate of O91:H2, for which genotypic data were available, were unambiguously genotyped with this array. Glass based microarrays are relatively simple to construct and provide a rapid and sensitive means to detect multiplexed PCR products and the system is amenable to automation.

  3. Toxoplasma gondii in free-ranging wild small felids from Brazil: molecular detection and genotypic characterization.

    PubMed

    Cañón-Franco, W A; Araújo, F A P; López-Orozco, N; Jardim, M M A; Keid, L B; Dalla-Rosa, C; Cabral, A D; Pena, H F J; Gennari, S M

    2013-11-01

    Brazil harbors the largest number of wild Neotropical felid species, with ten of the twelve species recorded in the American continent. Although these animals are considered to be definitive hosts for Toxoplasma gondii, there are few descriptions of the parasite in these species. Here, we performed a molecular detection of T. gondii by amplification of the marker ITS-1 from tissue samples obtained from 90 free-ranging wild small Neotropical felids from Rio Grande do Sul - Brazil. Of the sampled animals, 34.4% (n=31) were positive including the species Puma yagouaroundi - jaguarondi (9/22), Leopardus geoffroyi - Geoffroy's cat (6/22), Leopardus tigrinus - oncilla (8/28), Leopardus wiedii - margay (6/10), Leopardus pardalis - ocelot (1/1) and Leopardus colocolo - Pampas cat (1/7). Toxoplasma DNA was detected with a frequency of 14.6% (63/433) in primary samples of tongue (16/56), brain (8/43), skeletal muscle (15/83), heart (7/63), diaphragm (3/56), vitreous humor (2/44), eye muscle (6/44) and eyeball (6/44). Multilocus PCR-RFLP genotyping of eleven small Neotropical felids using the molecular markers SAG1, 5'3'SAG2, alt. SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico and CS3 allowed the partial characterization of eight genotypes. We fully characterized two new genotypes that have not been described previously in Brazil (Lw#31Tn from L. wiedii and Py#21Sm from P. yagouaroundi) and one genotype Py#56Br from P. yagouaroundi that has been described previously in isolates from cats, dogs and capybaras from São Paulo state. This study constitutes the first detection and genotypic characterization of T. gondii in free-ranging felids in Brazil, demonstrating the occurrence of the parasite in wild populations and suggesting its potential transmissibility to humans and other domestic and wild animals. PMID:23932730

  4. Detection of Hepatitis E Virus Genotype 1 Among Blood Donors From Southwest of Iran

    PubMed Central

    Parsa, Rahil; Adibzadeh, Setare; Behzad Behbahani, Abbas; Farhadi, Ali; Yaghobi, Ramin; Rafiei Dehbidi, Gholam Reza; Hajizamani, Saeideh; Rahbar, Sanaz; Nikouyan, Negin; Okhovat, Mohammad Ali; Naderi, Samaneh; Salehi, Saeede; Alizadeh, Marzieh; Ranjbaran, Reza; Zarnegar, Golnoosh; Alavi, Parnian

    2016-01-01

    Background Infection with hepatitis E virus (HEV) is endemic in developing countries and reveals significant regional differences. Several studies have reported virus transmission via blood transfusion. To date, however, no cases of HEV RNA detection in blood donors have been reported from Iran. Objectives The aim of this study was to determine the presence of HEV RNA in plasma samples of blood donors referred to a blood transfusion center in Shiraz in the southwest of Iran. The HEV genotypes were also investigated using nucleotide sequencing. Patients and Methods Blood samples were collected from 700 blood donors who were referred to Fars blood transfusion organization from January to March 2014. Plasma samples were screened for the presence of HEV IgG and IgM antibodies by standard enzyme immunoassay. Samples seroreactive to anti-HEV were further tested for the presence of HEV RNA using nested polymerase chain reaction (PCR) with universal primers for detection of all four HEV genotypes. Positive PCR samples were then subjected to DNA sequencing for further analysis. Results Fifty (50, 7.1%) out of 700 plasma samples tested positive for anti-HEV antibodies. HEV RNA was detected in 7/50 (12%) of the antibody-positive samples, the majority of which were IgM positive. Sequence analysis of seven isolates of the HEV RNA ORF 2 gene region revealed > 80% similarity with genotype 1. Conclusions The analysis indicates that the HEV isolated from blood donors in the southwest of Iran belongs to genotype 1. However, more samples from other geographic regions of Iran are needed to confirm these findings. Because transmission of HEV by administration of blood or blood components is likely to occur, it may be sensible to screen donor blood for HEV to eliminate transfusion-transmitted HEV infection when the recipient is immunocompromised.

  5. Development of a rapid HRM genotyping method for detection of dog-derived Giardia lamblia.

    PubMed

    Tan, Liping; Yu, Xingang; Abdullahi, Auwalu Yusuf; Wu, Sheng; Zheng, Guochao; Hu, Wei; Song, Meiran; Wang, Zhen; Jiang, Biao; Li, Guoqing

    2015-11-01

    Giardia lamblia is a zoonotic flagellate protozoan in the intestine of human and many mammals including dogs. To assess a threat of dog-derived G. lamblia to humans, the common dog-derived G. lamblia assemblages A, C, and D were genotyped by high-resolution melting (HRM) technology. According to β-giardin gene sequence, the qPCR-HRM primers BG5 and BG7 were designed. A series of experiments on the stability, sensitivity, and accuracy of the HRM method were also tested. Results showed that the primers BG5 and BG7 could distinguish among three assemblages A, C, and D, which Tm value differences were about 1 °C to each other. The melting curves of intra-assay reproducibility were almost coincided, and those of inter-assay reproducibility were much the same shape. The lowest detection concentration was about 5 × 10(-6)-ng/μL sample. The genotyping results from 21 G. lamblia samples by the HRM method were in complete accordance with sequencing results. It is concluded that the HRM genotyping method is rapid, stable, specific, highly sensitive, and suitable for clinical detection and molecular epidemiological survey of dog-derived G. lamblia. PMID:26212101

  6. Heminested PCR assay for detection of six genotypes of rabies and rabies-related viruses.

    PubMed Central

    Heaton, P R; Johnstone, P; McElhinney, L M; Cowley, R; O'Sullivan, E; Whitby, J E

    1997-01-01

    A heminested reverse transcriptase PCR (hnRT-PCR) protocol which is rapid and sensitive for the detection of rabies virus and rabies-related viruses is described. Sixty isolates from six of the seven genotypes of rabies and rabies-related viruses were screened successfully by hnRT-PCR and Southern blot hybridization. Of the 60 isolates, 93% (56 of 60) were positive by external PCR, while all isolates were detected by heminested PCR and Southern blot hybridization. We also report on a comparison of the sensitivity of the standard fluorescent-antibody test (FAT) for rabies antigen and that of hnRT-PCR for rabies viral RNA with degraded tissue infected with a genotype 1 virus. Results indicated that FAT failed to detect viral antigen in brain tissue that was incubated at 37 degrees C for greater than 72 h, while hnRT-PCR detected viral RNA in brain tissue that was incubated at 37 degrees C for 360 h. PMID:9350729

  7. QuantiSNP: an Objective Bayes Hidden-Markov Model to detect and accurately map copy number variation using SNP genotyping data.

    PubMed

    Colella, Stefano; Yau, Christopher; Taylor, Jennifer M; Mirza, Ghazala; Butler, Helen; Clouston, Penny; Bassett, Anne S; Seller, Anneke; Holmes, Christopher C; Ragoussis, Jiannis

    2007-01-01

    Array-based technologies have been used to detect chromosomal copy number changes (aneuploidies) in the human genome. Recent studies identified numerous copy number variants (CNV) and some are common polymorphisms that may contribute to disease susceptibility. We developed, and experimentally validated, a novel computational framework (QuantiSNP) for detecting regions of copy number variation from BeadArray SNP genotyping data using an Objective Bayes Hidden-Markov Model (OB-HMM). Objective Bayes measures are used to set certain hyperparameters in the priors using a novel re-sampling framework to calibrate the model to a fixed Type I (false positive) error rate. Other parameters are set via maximum marginal likelihood to prior training data of known structure. QuantiSNP provides probabilistic quantification of state classifications and significantly improves the accuracy of segmental aneuploidy identification and mapping, relative to existing analytical tools (Beadstudio, Illumina), as demonstrated by validation of breakpoint boundaries. QuantiSNP identified both novel and validated CNVs. QuantiSNP was developed using BeadArray SNP data but it can be adapted to other platforms and we believe that the OB-HMM framework has widespread applicability in genomic research. In conclusion, QuantiSNP is a novel algorithm for high-resolution CNV/aneuploidy detection with application to clinical genetics, cancer and disease association studies. PMID:17341461

  8. Detection of genotypic clarithromycin-resistant Helicobacter pylori by string tests

    PubMed Central

    Wu, Jeng-Yih; Wang, Sophie S W; Lee, Yi-Chern; Yamaoka, Yoshio; Graham, David Y; Jan, Chang-Ming; Wang, Wen-Ming; Wu, Deng-Chyang

    2014-01-01

    AIM: To evaluate the utility of the string test to detect genotypic clarithromycin-resistant Helicobacter pylori (H. pylori) by polymerase chain reaction (PCR)-restriction fragment length polymorphism. METHODS: Patients undergoing endoscopic examinations were enrolled in the present study. String tests were done on the next day of endoscopy. Segments of 23S rRNA were amplified from DNA obtained from string tests. PCR-restriction fragment length polymorphism was accomplished by restriction enzymes BbsI and BsaI recognizing the mutation site A to G at 2143 or at 2142 of 23S rRNA domain V, respectively. RESULTS: One hundred and thirty-four patients with H. pylori infection underwent string tests. To compare phenotypic resistance, 43 isolates were successfully cultured in 79 patients in whom 23S rRNA was successfully amplified. Of five patients with clarithromycin-resistant H. pylori, 23S rRNA of H. pylori isolates from four patients could be digested by BsaI. In 38 susceptible isolates, 23S rRNA of H. pylori isolates from 36 patients could not be digested by either BsaI or BbsI. The sensitivity and specificity of the string test to detect genotypic clarithromycin resistance were 66.7% and 97.3%, respectively. Positive and negative predictive values were 80% and 94.7%, respectively. CONCLUSION: String test with molecular analysis is a less invasive method to detect genotypic resistance before treatment. Further large-scale investigations are necessary to confirm our results. PMID:24695835

  9. Molecular Detection and Genotyping of Chlamydia psittaci in Captive Psittacines from Costa Rica

    PubMed Central

    Sheleby-Elías, Jessica; Solórzano-Morales, Ántony; Romero-Zuñiga, Juan José

    2013-01-01

    Oropharyngeal and cloacal swabs from 117 captive psittacine birds presented at veterinary clinics (88) and from shelters/rescue centers of wildlife (29) were collected to determine the prevalence of C. psittaci in captive birds in Costa Rica. Samples were collected during 2009 from a total of 19 different species of parrots, with Ara macao (33), Amazona autumnalis (24), Amazona ochrocephala (21), and Ara ararauna (8) being the most representative species sampled. C. psittaci was detected in four (3.4%) birds using molecular detection (PCR). The positive samples belonged to birds presented at veterinary clinics; three of them were Ara macao and one Amazona ochrocephala. Three birds were adults; all positive birds showed no symptoms of illness and lived in homes with other birds, two in San José and two in Heredia. Sequencing was used to confirm the PCR positive results, showing that two samples of C. psittaci belonged to genotype A, representing the first report of the presence of this genotype in Costa Rica. The detection of this bacterium in captive psittacine birds shows that there is a potential risk for people living or having contact with them and that there is a possibility of infecting other birds. PMID:24163776

  10. High-throughput polymorphism detection and genotyping in Brassica napus using next-generation RAD sequencing

    PubMed Central

    2012-01-01

    Background The complex genome of rapeseed (Brassica napus) is not well understood despite the economic importance of the species. Good knowledge of sequence variation is needed for genetics approaches and breeding purposes. We used a diversity set of B. napus representing eight different germplasm types to sequence genome-wide distributed restriction-site associated DNA (RAD) fragments for polymorphism detection and genotyping. Results More than 113,000 RAD clusters with more than 20,000 single nucleotide polymorphisms (SNPs) and 125 insertions/deletions were detected and characterized. About one third of the RAD clusters and polymorphisms mapped to the Brassica rapa reference sequence. An even distribution of RAD clusters and polymorphisms was observed across the B. rapa chromosomes, which suggests that there might be an equal distribution over the Brassica oleracea chromosomes, too. The representation of Gene Ontology (GO) terms for unigenes with RAD clusters and polymorphisms revealed no signature of selection with respect to the distribution of polymorphisms within genes belonging to a specific GO category. Conclusions Considering the decreasing costs for next-generation sequencing, the results of our study suggest that RAD sequencing is not only a simple and cost-effective method for high-density polymorphism detection but also an alternative to SNP genotyping from transcriptome sequencing or SNP arrays, even for species with complex genomes such as B. napus. PMID:22726880

  11. Molecular Detection and Genotyping of Chlamydia psittaci in Captive Psittacines from Costa Rica.

    PubMed

    Sheleby-Elías, Jessica; Solórzano-Morales, Antony; Romero-Zuñiga, Juan José; Dolz, Gaby

    2013-01-01

    Oropharyngeal and cloacal swabs from 117 captive psittacine birds presented at veterinary clinics (88) and from shelters/rescue centers of wildlife (29) were collected to determine the prevalence of C. psittaci in captive birds in Costa Rica. Samples were collected during 2009 from a total of 19 different species of parrots, with Ara macao (33), Amazona autumnalis (24), Amazona ochrocephala (21), and Ara ararauna (8) being the most representative species sampled. C. psittaci was detected in four (3.4%) birds using molecular detection (PCR). The positive samples belonged to birds presented at veterinary clinics; three of them were Ara macao and one Amazona ochrocephala. Three birds were adults; all positive birds showed no symptoms of illness and lived in homes with other birds, two in San José and two in Heredia. Sequencing was used to confirm the PCR positive results, showing that two samples of C. psittaci belonged to genotype A, representing the first report of the presence of this genotype in Costa Rica. The detection of this bacterium in captive psittacine birds shows that there is a potential risk for people living or having contact with them and that there is a possibility of infecting other birds. PMID:24163776

  12. Using identity by descent estimation with dense genotype data to detect positive selection.

    PubMed

    Han, Lide; Abney, Mark

    2013-02-01

    Identification of genomic loci and segments that are identical by descent (IBD) allows inference on problems such as relatedness detection, IBD disease mapping, heritability estimation and detection of recent or ongoing positive selection. Here, employing a novel statistical method, we use IBD to find signals of selection in the Maasai from Kinyawa, Kenya (MKK). In doing so, we demonstrate the advantage of statistical tools that can probabilistically estimate IBD sharing without having to thin genotype data because of linkage disequilibrium (LD), and that allow for both inbreeding and more than one allele to be shared IBD. We use our novel method, GIBDLD, to estimate IBD sharing between all pairs of individuals at all genotyped SNPs in the MKK, and, by looking for genomic regions showing excess IBD sharing in unrelated pairs, find loci that are known to have undergone recent selection (eg, the LCT gene and the HLA region) as well as many novel loci. Intriguingly, those loci that show the highest amount of excess IBD, with the exception of HLA, also show a substantial number of unrelated pairs sharing all four of their alleles IBD. In contrast to other IBD detection methods, GIBDLD provides accurate probabilistic estimates at each locus for all nine possible IBD sharing states between a pair of individuals, thus allowing for consanguinity, while also modeling LD, thus removing the need to thin SNPs. These characteristics will prove valuable for those doing genetic studies, and estimating IBD, in the wide variety of human populations. PMID:22781100

  13. Rapid and label-free amplification and detection assay for genotyping of cancer biomarker.

    PubMed

    Shin, Yong; Soo, Ross A; Yoon, Jaeyun; Perera, Agampodi Promoda; Yoon, Yong-Jin; Park, Mi Kyoung

    2015-06-15

    As understanding of the molecular pathways that drive malignancy in human cancer improves, personalized genotype-based therapy in combination with the predictive biomarker for the efficacy of targeted therapy is becoming more popular in cancer management. Sanger sequencing, that has been the gold standard for mutation analysis in cancer since the 1970s, suffers from low sensitivity, complexity, and time-consuming and labor-intensive procedure. Although several PCR based molecular testing methods are being emerged, there is no universal assay available for genotyping of cancer biomarkers. Here we present a rapid, simple and sensitive assay for the detection of epidermal growth factor receptor (EGFR) mutation in non-small cell lung cancers (NSCLCs). The assay employs a novel double mis-matched primer (DMP) set to improve the detection ability of isothermal solid-phase amplification/detection (ISAD) based on silicon microring biosensor. We show that the EGFR-DMP can detect EGFR gene mutations within 20 min in a label-free and real-time manner. The EGFR-DMP was able to detect a mutation in a sample containing only 1% of the mutant cells in a mixture of wild-type cells. Furthermore, to validate the proposed assay for potential applications in clinical diagnostics, we examined paraffin-embedded tissue samples from 10 NSCLC patients for the presence of EGFR mutations by performing EGFR-DMP and direct sequencing. The EGFR-DMP assay was able to rapidly detect the mutation, with high sensitivity and specificity. The EGFR-DMP assay offers a robust and sensitive approach for the rapid identification of the EGFR mutation. The high sensitivity and specificity and rapidity of this approach may make it useful for predicting the clinical response to targeted EGFR TKIs as a companion diagnostic. PMID:25569872

  14. Genotyping of Pseudomonas aeruginosa isolates from lung transplant recipients and aquatic environment-detected in-hospital transmission.

    PubMed

    Johansson, Ewa; Welinder-Olsson, Christina; Gilljam, Marita

    2014-02-01

    Lung infection with Pseudomonas aeruginosa is common in lung transplant recipients and may lead to severe complications. Bacteriological surveillance aims to detect transmission of microbes between hospital environment and patients. We sought to determine whether genotyping of P. aeruginosa isolates could improve identifications of pathways of infection. From 2004 to 2009, we performed genotyping with multiple-locus variable number of tandem repeats analysis (MLVA) and pulsed-field gel electrophoresis (PFGE) of P. aeruginosa isolates cultured from lung transplant recipients at Sahlgrenska University Hospital, Gothenburg. During a small outbreak in 2008, cultivation and genotyping of isolates from sink and drains samples from the hospital ward were performed. Pseudomona aeruginosa from 11/18 patients were genotyped to unique strains. The remaining seven patients were carriers of a P. aeruginosa strain of cluster A genotype. Pseudomona aeruginosa was isolated in 4/8 water samples, typed by MLVA also as cluster A genotype and confirmed by PFGE to be similar or identical to the isolates from four transplanted patients. In conclusion, genotyping of isolates revealed a clonal relationship between patient and water isolates, indicating in-hospital transmission of P. aeruginosa. We suggest genotyping with MLVA for rapid routine surveillance, with the PFGE method used for extended, confirmatory analyses. PMID:24450429

  15. Protocol for the use of a bead array for the multiple detection of genotype of Chlamydia trachomatis.

    PubMed

    Huang, Chung-Te; Li, Shu-Ying

    2012-01-01

    The identification of Chlamydia trachomatis genotypes is important for both molecular epidemiology and infection control such as contact tracing and identification of high-risk groups. Currently, at least 19 human serovars have been recognized by using polyclonal and monoclonal antibodies against the major outer membrane protein. In sexually transmitted diseases, multiple pathogens or genotype infections are not uncommon. Hence, detection of multiple gene targets in one reaction is becoming increasingly important. Here, we describe the multiplex detection of eight genotypes of C. trachomatis by a combination of a PCR amplification with a multiplex bead array detection. The bead array system comprises distinct bead sets, which are color coded by different fluorescent intensities and a dual-laser flow cytometer analyzer to identify the identity of the bead and the intensity of the reporter dye that binds to the target molecules. The DNA sequences of the variable segments (VS2 or VS1-VS2) in outer membrane protein (omp1) gene are PCR amplified and biotin labeled and used as a gene target for the genotyping of C. trachomatis. Genotype-specific probes coupled to beads are used for capturing the labeled target amplicons through specific hybridization. Thus, multiple genotypes are detected and differentiated simultaneously by yielding quantitative data. PMID:22782819

  16. Performance of a Polymer-Based DNA Chip Platform in Detection and Genotyping of Human Papillomavirus in Clinical Samples▿

    PubMed Central

    Schenk, T.; Brandstetter, T.; zur Hausen, A.; Alt-Mörbe, J.; Huzly, D.; Rühe, J.

    2009-01-01

    Human papillomavirus (HPV) plays a key role in the development of cervical and laryngeal cancers. The aim of our study was to compare the performance of a new hydrogel-based HPV genotyping biochip assay (Biochip) to a commercially available and CE-marked conventional PCR followed by reverse hybridization (GenID-PCR). One hundred twenty-three samples were available for the study. Of these samples, 101/123 were gynecological swabs, 8/123 were swabs or biopsy samples of genital warts, 7/123 were biopsy samples of otorhinolaryngeal lesions, 5/123 were samples of skin warts, and 2/123 were samples of orolabial abnormalities. These molecular methods for HPV genotyping showed comparable sensitivity and specificity. However, 19/123 of the results were discrepant. Specifically, Biochip showed better performance in the detection of multiple infections, especially when more than one high-risk genotype was present. Due to the different probe configurations used in the two assays, GenID-PCR achieves only group-specific detection of many HPV genotypes, whereas Biochip allows for specific identification. Overall, the newly developed HPV chip system (Biochip) proved to be a suitable tool for HPV detection and genotyping; it also proved to be superior for establishing HPV genotyping methods. PMID:19279180

  17. Detection of disease-associated prion protein in the posterior portion of the small intestine involving the continuous Peyer's patch in cattle orally infected with bovine spongiform encephalopathy agent.

    PubMed

    Okada, H; Iwamaru, Y; Imamura, M; Masujin, K; Matsuura, Y; Murayama, Y; Mohri, S; Yokoyama, T

    2011-08-01

    Twenty-eight calves were exposed to 5 g of homogenized brainstems confirmed as bovine spongiform encephalopathy (BSE) agents. Two to five animals were sequentially killed for post-mortem analyses 20 months post-inoculation (MPI) at intervals of 6 or 12 months. Samples from animals challenged orally with BSE agents were examined by Western blot and immunohistochemical analyses. Immunolabelled, disease-associated prion protein (PrPsc) was detected in a small portion of follicles in the continuous Peyer's patch from the posterior portion of the small intestine involving the entire ileum and the posterior jejunum but not in the discrete Peyer's patches in the remaining jejunum in preclinical animals at 20, 36, and 48 MPI. The PrPsc-positive cells corresponded to tingible body macrophages on double immunofluorescence labelling. In addition, PrPsc accumulated in 7 of 14 animals in the central nervous system (CNS) after 34 MPI, and five of them developed clinical signs and were killed at 34, 46, 58, and 66 MPI. Two preclinical animals killed at 36 and 48 MPI presented the earliest detectable and smallest deposition of immunolabelled PrPsc in the dorsal motor nucleus of the vagus nerve, the spinal trigeminal nucleus of the medulla oblongata at the obex region, and/or the intermediolateral nucleus of the 13th thoracic segment of the spinal cord. Based on serial killing, no PrPsc was detectable in the CNS, including the medulla oblongata at the obex level, before 30 MPI, by Western blot and immunohistochemical analyses. These results are important for understanding the pathogenesis of BSE. PMID:21320296

  18. Phenotypic- and Genotypic-Resistance Detection for Adaptive Resistance Management in Tetranychus urticae Koch

    PubMed Central

    Kwon, Deok Ho; Kang, Taek-Jun; Kim, Young Ho; Lee, Si Hyeock

    2015-01-01

    Rapid resistance detection is necessary for the adaptive management of acaricide-resistant populations of Tetranychus urticae. Detection of phenotypic and genotypic resistance was conducted by employing residual contact vial bioassay (RCV) and quantitative sequencing (QS) methods, respectively. RCV was useful for detecting the acaricide resistance levels of T. urticae, particularly for on-site resistance detection; however, it was only applicable for rapid-acting acaricides (12 out of 19 tested acaricides). QS was effective for determining the frequencies of resistance alleles on a population basis, which corresponded to 12 nonsynonymous point mutations associated with target-site resistance to five types of acaricides [organophosphates (monocrotophos, pirimiphos-methyl, dimethoate and chlorpyrifos), pyrethroids (fenpropathrin and bifenthrin), abamectin, bifenazate and etoxazole]. Most field-collected mites exhibited high levels of multiple resistance, as determined by RCV and QS data, suggesting the seriousness of their current acaricide resistance status in rose cultivation areas in Korea. The correlation analyses revealed moderate to high levels of positive relationships between the resistance allele frequencies and the actual resistance levels in only five of the acaricides evaluated, which limits the general application of allele frequency as a direct indicator for estimating actual resistance levels. Nevertheless, the resistance allele frequency data alone allowed for the evaluation of the genetic resistance potential and background of test mite populations. The combined use of RCV and QS provides basic information on resistance levels, which is essential for choosing appropriate acaricides for the management of resistant T. urticae. PMID:26545209

  19. Investigating the Epidemiology of Repeat Chlamydia trachomatis Detection after Treatment by Using C. trachomatis OmpA Genotyping

    PubMed Central

    Kapil, Richa; Press, Christen G.; Hwang, M. Lisa; Brown, LaDraka

    2014-01-01

    Repeat Chlamydia trachomatis detection frequently occurs within months after C. trachomatis infection treatment. The origins of such infection (persistence versus reinfection from untreated or new partners) are varied and difficult to determine. C. trachomatis strains can be differentiated by sequencing the ompA gene encoding the outer membrane protein A (OmpA). We used OmpA genotyping to investigate the epidemiology of repeat C. trachomatis detection after treatment in C. trachomatis-infected subjects seen at a sexually transmitted diseases clinic. Subjects were enrolled, tested for C. trachomatis, treated with azithromycin, and scheduled for a 6-month follow-up for repeat C. trachomatis testing. OmpA genotyping was performed on C. trachomatis-positive urogenital specimens obtained from patients at enrollment and follow-up. The enrollment visit OmpA genotypes for C. trachomatis were determined for 162 subjects (92% female, 94% African American). C. trachomatis was detected at follow-up in 39 subjects (24%). The OmpA genotype distribution at enrollment did not differ in those with versus those without repeat C. trachomatis detection. Of the 35 subjects with C. trachomatis strains genotyped at enrollment and follow-up, 7 (20%) had the same ompA sequence at both visits, while 28 (80%) had discordant sequences. A new sexual partner was reported more often in subjects with discordant C. trachomatis strains than in those with concordant strains (13 [46%] versus 1 [14%]; P = 0.195). Half of the subjects with discordant C. trachomatis strains who reported sexual activity since treatment denied a new sexual partner; 62% of these subjects reported that their partner was treated. Our study demonstrates that most repeat C. trachomatis detections after treatment were new infections with a different C. trachomatis strain rather than reinfection with the same strain. OmpA genotyping can be a useful tool in understanding the origins of repeat C. trachomatis detection after

  20. Toxoplasma gondii and Neospora caninum in wild small mammals: Seroprevalence, DNA detection and genotyping.

    PubMed

    Machačová, Tereza; Ajzenberg, Daniel; Žákovská, Alena; Sedlák, Kamil; Bártová, Eva

    2016-06-15

    Generally, rodents and other small mammals are considered as one of the sources of Toxoplasma gondii or Neospora caninum infection for cats and dogs as the definitive hosts of these two parasites, respectively. The aim of the study was to find out the prevalence of these two parasites in wild small mammals from the Czech Republic and to characterize T. gondii isolates by methods of molecular biology. A total of 621 wild small mammals were caught in the Czech Republic during years 2002-2014. Antibodies to T. gondii were detected by latex agglutination test in six (2.5%) of 240 small mammals (in two A. agrarius and four A. flavicollis). Antibodies to N. caninum were detected by commercially available competitive-inhibition enzyme-linked immunosorbent assay in one A. flavicolis (0.4%). Three of 427 (0.7%) liver samples were positive for T. gondii by PCR while negative for N. caninum. All embryo samples (n=102) were negative for both T. gondii and N. caninum. The three liver samples positive for T. gondii DNA (two from A. flavicollis and one from A. sylvaticus) were genotyped by 15 microsatellite markers and characterized as type II. To our knowledge, this is the first information about genetic characterization of T. gondii isolates in small mammals from Europe and the first detection of N. caninum antibodies in wild rodents from the Czech Republic. PMID:27198782

  1. Diversity of human parechoviruses in Bulgaria, 2011: Detection of rare genotypes 8 and 10.

    PubMed

    Mladenova, Zornitsa; Dikova, Antoaneta; Thongprachum, Aksara; Petrov, Petar; Pekova, Liliq; Komitova, Radka; Iturriza-Gomara, Miren; Ushijima, Hiroshi

    2015-12-01

    Human parechovirus (HPeV) infections are commonly asymptomatic but are also found in association with symptoms of the gastrointestinal and respiratory tract, or central nervous system. In order to study their distribution and genetic diversity in Bulgaria, specimens from 229 children aged <5years old hospitalized due to neurological manifestations (n=104) and acute gastroenteritis (n=125) were analyzed. Stool samples were tested using reverse transcription followed by real-time polymerase chain reaction toward the 5'UTR region, and the HPeVs detected were identified by PCR directed to VP1 followed by sequencing. HPeV infection rates of 1.9% and 7.2% were found in children presented with neurological symptoms or with acute diarrhea, respectively. Four different HPeV genotypes, HPeV-3 (n=2), HPeV-5 (n=2), HPeV-8 (n=1) and HPeV-10 (n=1) were identified. All but two HPeVs were detected in acute diarrheal cases, while a single HPeV-3 strain and an HPeV-8 strain were detected in association with facial palsy and encephalitis, respectively. This is the first report of HPeV-8 and HPeV-10 in Europe. PMID:26453770

  2. Evaluation of a Novel Chlamydia trachomatis Microsphere Suspension Assay for Detection and Genotyping of the Different Serovars in Clinical Samples

    PubMed Central

    Quint, Koen D.; Geraets, Daan T.; van den Munckhof, Henk A.M.; de Koning, Maurits N.C.; Smelov, Vitaly; Melchers, Willem J.G.; de Vries, Henry J.C.; Morré, Servaas A.; Meijer, Chris J.M.; van Alewijk, Dirk C.J.G.; van Doorn, Leen-Jan; Quint, Wim G.V.

    2011-01-01

    A novel Chlamydia trachomatis (Ct) microsphere suspension (MS) assay was evaluated for identification of the different serovars, using the same PCR primer set established for the Ct Detection and genoTyping assay. Both assays can detect and identify all 14 major serovars (A, B/Ba, C, D/Da, E, F, G/Ga, H, I/Ia, J, K, L1, L2/L2a, and L3) and one genovariant of serovar J. The probe specificity for the Ct-MS assay was determined using 14 Ct reference strains and 1 clinical isolate from a genovariant of serovar J. Also, the Ct-MS assay and the Ct detection and genoTyping assay were compared in 712 Ct-positive clinical samples. The Ct-MS assay showed a highly specific reaction for all probes with the amplicons of the reference strains, giving a very low background median fluorescence intensity signal (median fluorescence intensity ≤ 10). An excellent overall agreement in the Ct detection (kappa = 0.947, 95% confidence interval, 0.89 to 0.999; McNemar's test, P = 1.000) and the Ct genotyping (kappa = 0.993, 95% confidence interval, 0.977 to 1.000; McNemar's test, P = 0.053) was observed between the Ct detection and genoTyping (DT) assay and the Ct-MS assay. In conclusion, the novel Ct-MS assay permits simultaneous detection and genotyping of Ct serovars, making the Ct-MS assay an excellent high throughput method. PMID:21354049

  3. Detection of Human Papillomavirus Genotypes and Major BRCA Mutations in Familial Breast Cancer.

    PubMed

    Mohtasebi, Parinaz; Rassi, Hossein; Maleki, Fatemeh; Hajimohammadi, Sameh; Bagheri, Zahra; Fakhar Miandoab, Malihe; Naserbakht, Mahdieh

    2016-06-01

    Breast cancer is a multistep disease and infection with a DNA virus could play a role in one or more of the steps in this pathogenic process. High-risk human papillomaviruses (HPVs) are the causative agents of several cancers. In this study, we investigated HPV genotypes associated with breast cancer and its relationship with BRCA mutation for the detection of familial breast cancer. We analyzed 84 formalin-fixed, paraffin-embedded tissue blocks from 38 familial breast cancer and 46 nonfamilial breast cancer samples by multiplex polymerase chain reaction and clinical parameters. Overall prevalence of HPV infection was 27 of 84: 10 (37.03%) HPV-16, 9 (29.62%) HPV-18, 4 (14.81%) HPV-11, 1 (3.7%) HPV-31, 1 (3.7%) HPV-33, and 2 (7.4%) HPV35. Furthermore, 17 mtDNA4977 deletions and 5 5382insC mutations were detected from 38 familial breast cancer samples. Our results demonstrate that infection with HPV was prevalent among Iranian women with familial breast cancer and the testing of mtDNA4977 deletions and 5382insC mutations in combination with clinical parameters as major risk factors can serve in the identification of familial breast cancer. PMID:27186947

  4. Detection of hepatitis A virus genotype IB variants in clams from Maputo Bay, Mozambique.

    PubMed

    Nenonen, Nancy P; Hernroth, Bodil; Chauque, Arlindo A; Hannoun, Charles; Bergström, Tomas

    2006-07-01

    Clams provide an important source of food and income for the population of Maputo, Mozambique, where conditions of poor water supply and inadequate sanitation favor endemic infection with hepatitis A virus (HAV). To determine the role of bivalves in an endemic area, clams gathered from Maputo Bay were bought from market and examined for HAV. Four batches, total 150 clams, were sampled over the year. RNA extracted from individual digestive glands was assayed by nested RT-PCR and sequencing of HAV 5' noncoding region (5' NCR). Specific HAV signals were detected in one batch, 23 of 34 clams (67%) testing positive. Phylogenetic analyses of VP3/VP1, VP1/P2A, and 5' NCR determined clustering of clam strains as genotype I, subtype B. In addition to identifying HAV IB strains with predicted conserved amino acid sequence, IB variants exhibiting novel amino acid substitutions at the VP1/P2A junction were detected. HAV strains from clams showed 93%-99% homology with wild-type IB strains from South African outbreaks and from a panel of HAV IgM positive Swedish patients. DNA from enteric human adenovirus 40/41 was found in a limited number of clams from two batches, 6/34 (17%) and 4/35 (11%). Detection of HAV subgenotype IB in bivalves provided indirect evidence of the strains circulating in a densely populated coastal region where HAV is presumed to be hyperendemic. The results suggest that clams may be an important source of HAV in Maputo region, and indicate the need for further molecular study of strains circulating in the indigenous population. PMID:16721847

  5. Use of immuno-dominant epitope derived from genotype 4 as a diagnostic reagent for detecting the antibodies against Hepatitis E Virus

    PubMed Central

    2013-01-01

    Background Despite the genotype 4 has become the dominant cause of hepatitis E disease in China, none antigen derived from genotype 4 of hepatitis E virus (HEV) was used in current commercial anti-HEV immunoassay, and the serological reactivity of antigen derive from genotype 4 is not well-charactered. Methods We expressed and purified the 4 main immuno-dominant epitopes derived from genotype 1 and 4 including ORF2 (410-621aa) of genotype 4, ORF3 (47-114aa) of genotype 4, ORF2 (396-606aa) of genotype 1 and ORF3 (56-123aa) of genotype 4. Results The ORF2 of genotype 4 displayed good diagnostics performance according to ROC analysis using in-house panel, and the immunoassays based the ORF2 of genotype 4 was then developed to detect the anti-HEV IgG antibodies and evaluated further in 530 anti-HEV IgG positive specimens and 380 negative specimens. The sensitivity and the specificity is 98.1% (520/530) and 94.7% (360/380) for immunoassay based on ORF2 of genotype 4, 96.6% (512/530) and 92.6% (352/380) for commercial immunoassay based on genotype 1. It is noted that all of the positive samples will be detected by combing two assays together. The anti-HEV immunoassays based on genotype 4 are in accordance with Chinese anti-HEV national standard,and show an good agreement of 95.8% with commercial assay (kappa=0.913, P=0.014). Conclusions The immunoassay based on ORF2G4 displays good performance, and combining assay based on genotype 1 together with genotype 4 will benefit the HEV diagnosis in large scale samples. PMID:23618011

  6. DNA probe for detection of the Leptospira interrogans serovar hardjo genotype hardjo-bovis.

    PubMed Central

    LeFebvre, R B

    1987-01-01

    A DNA probe is described for the diagnostic and taxonomic identification of the North American cattle pathogen Leptospira interrogans genotype hardjo-bovis. The probe is specific for this genotype and does not hybridize to genomic DNA of any other leptospire pathogen commonly found in North America. Images PMID:2826538

  7. Seroprevalence, detection of DNA in blood and milk, and genotyping of Toxoplasma gondii in a goat population in Italy.

    PubMed

    Mancianti, Francesca; Nardoni, Simona; D'Ascenzi, Carlo; Pedonese, Francesca; Mugnaini, Linda; Franco, Filomena; Papini, Roberto

    2013-01-01

    Toxoplasma gondii is the causative agent of a major zoonosis with cosmopolitan distribution and is known to be transmitted mainly by the ingestion of undercooked or raw animal products. Drinking unpasteurized goat's milk is a risk factor associated with human toxoplasmosis. However, very little is known about the excretion of DNA in goat milk. Aim of the present study was to determine the seroprevalence of T. gondii infection using a modified agglutination test (MAT), to detect T. gondii DNA by nested-PCR (n-PCR) in samples of blood and milk from seropositive goats, and to genotype DNA isolates using 11 molecular markers in 127 adult lactating goats from 6 farms in Italy. Positive MAT results were found in 60.6% of goats while 13% of blood and milk samples from seropositive goats were positive to n-PCR. A kappa coefficient of 1 indicated a perfect agreement between blood and milk n-PCR. Genetic characterization of isolates revealed the occurrence of genotype III (n = 7), genotype I (n = 1), and atypical genotypes with hints for genotype I (n = 2). Our results suggest that the risk of excretion of Toxoplasma tachyzoites might frequently occur in milk of seropositive goats testing positive to n-PCR on blood. PMID:24093106

  8. A new sieving matrix for DNA sequencing, genotyping and mutation detection and high-throughput genotyping with a 96-capillary array system

    SciTech Connect

    Gao, David

    1999-11-08

    Capillary electrophoresis has been widely accepted as a fast separation technique in DNA analysis. In this dissertation, a new sieving matrix is described for DNA analysis, especially DNA sequencing, genetic typing and mutation detection. A high-throughput 96 capillary array electrophoresis system was also demonstrated for simultaneous multiple genotyping. The authors first evaluated the influence of different capillary coatings on the performance of DNA sequencing. A bare capillary was compared with a DB-wax, an FC-coated and a polyvinylpyrrolidone dynamically coated capillary with PEO as sieving matrix. It was found that covalently-coated capillaries had no better performance than bare capillaries while PVP coating provided excellent and reproducible results. The authors also developed a new sieving Matrix for DNA separation based on commercially available poly(vinylpyrrolidone) (PVP). This sieving matrix has a very low viscosity and an excellent self-coating effect. Successful separations were achieved in uncoated capillaries. Sequencing of M13mp18 showed good resolution up to 500 bases in treated PVP solution. Temperature gradient capillary electrophoresis and PVP solution was applied to mutation detection. A heteroduplex sample and a homoduplex reference were injected during a pair of continuous runs. A temperature gradient of 10 C with a ramp of 0.7 C/min was swept throughout the capillary. Detection was accomplished by laser induced fluorescence detection. Mutation detection was performed by comparing the pattern changes between the homoduplex and the heteroduplex samples. High throughput, high detection rate and easy operation were achieved in this system. They further demonstrated fast and reliable genotyping based on CTTv STR system by multiple-capillary array electrophoresis. The PCR products from individuals were mixed with pooled allelic ladder as an absolute standard and coinjected with a 96-vial tray. Simultaneous one-color laser-induced fluorescence

  9. Two distinct human parainfluenza virus type 1 genotypes detected during the 1991 Milwaukee epidemic.

    PubMed Central

    Henrickson, K J; Savatski, L L

    1996-01-01

    The extent of genetic and antigenic variation found in a population of human parainfluenza virus type 1 (HPIV-1) during a single local epidemic was investigated. Fifteen HPIV-1 strains isolated from children in 1991 were analyzed. Nucleotide sequence variation in the hemagglutinin-neuraminidase protein (HN) gene demonstrated two distinct genotypes (genotypes C and D). Unique patterns were identified involving 62 nucleotide and 10 amino acid positions. These patterns represented 40% of all mutations within the HN gene. The remaining mutations were randomly distributed, and 74% involved only one (55%) or two isolates. Genotypes were statistically different from each other at both the nucleotide (P = 0.001) and amino acid (P = 0.001) levels and demonstrated unique potential N-linked glycosylation patterns. Thirty-eight monoclonal antibodies (MAbs) made to four different viral proteins (22 HN, 2 fusion [F], 1 phosphoprotein, and 13 nucleoprotein) (originating from two different genotypes [genotypes A and D]) were compared for their ability to bind to the clinical isolates in enzyme-linked immunosorbent assays (ELISAs) and hemagglutinin-inhibition (HI) assays. Twenty-one MAbs bound well to all clinical isolates in ELISAs and HI assays. The remaining 17 MAbs showed variation in all four structural proteins. Three HN MAbs demonstrated genotype C- and D-specific antigenic and neutralization differences. Evolutionary analysis using parsimony methods confirmed the differences between the two genotypes. No differences in either clinical presentation or disease severity between the two genotypes were found. Geographically localized HPIV-1 epidemics can be caused by at least two distinct genotypes with minor but specific antigenic changes. The clinical and immunologic roles of HPIV-1 genotypes have not been determined. PMID:8904440

  10. Comparative Analyses of Phenotypic and Genotypic Methods for Detection of Enterotoxigenic Escherichia coli Toxins and Colonization Factors▿

    PubMed Central

    Sjöling, Å.; Wiklund, G.; Savarino, S. J.; Cohen, D. I.; Svennerholm, A.-M.

    2007-01-01

    Enterotoxigenic Escherichia coli (ETEC) is one of the main causes of childhood diarrhea in developing countries and in travelers. However, this pathogen has often not been reported in surveys of diarrheal pathogens, due to lack of simple standardized methods to detect ETEC in many laboratories. ETEC expresses one or both of two different enterotoxin subtypes: heat-stable toxins, a heat-labile toxin (LT), and more than 22 different colonization factors (CFs) that mediate adherence to the intestinal cell wall. Here we compare established phenotypic and genotypic detection methods and newly developed PCR detection methods with respect to sensitivity, specificity, positive predictive value, and ease of performance. The methods include GM1-enzyme-linked immunosorbent assay and dot blot techniques using specific monoclonal antibodies (MAbs) for phenotypic detection of the toxins and CFs, respectively, as well as different PCR and DNA/DNA hybridization techniques, including new PCR assays, for genotypic identification of the toxin and CF genes, respectively. We found very good general agreement in results derived from genotypic and phenotypic methods. In a few strains, LT and CFs were identified genetically but not phenotypically. Based on our analyses, we recommend initial screening for ETEC in clinical samples by multiplex toxin gene PCR. Toxin-positive strains may then be analyzed by dot blot tests for detection of the CFs expressed on the bacterial surface and by PCR for determination of additional CFs for which MAbs are currently lacking as well as for strains that harbor silent CF genes. PMID:17687011

  11. Valvular disease associated with systemic illness.

    PubMed

    Roldan, C A

    1998-08-01

    clinical and prognostic implications of valvular disease associated with the connective tissue diseases, incomplete data are available about pathogenesis, relation to clinical features of the primary disease, evolution, and effect of steroid or cytotoxic therapy. Echocardiography, especially TEE, has the potential to redefine the prevalence rates and to characterize better the valve abnormalities associated with these conditions. Finally, future large cross-sectional and longitudinal studies using clinical and echocardiographic data may help to define better the presence, evolution, and therapy of the valvular disease associated with the connective tissue diseases. PMID:9742329

  12. Prevalence of Primary HIV Drug Resistance in Thailand Detected by Short Reverse Transcriptase Genotypic Resistance Assay

    PubMed Central

    Kiertiburanakul, Sasisopin; Pinsai, Subencha; Chantratita, Wasun; Pasomsub, Ekawat; Leechawengwongs, Manoon; Thipmontree, Wilawan; Siriyakorn, Nirada; Sungkanuparph, Somnuek

    2016-01-01

    Background HIV drug resistance (HIVDR) is the major cause of treatment failure after scaling up of antiretroviral therapy (ART). HIVDR testing prior to ART initiation is not routinely performed in resource-limited settings. We aimed to assess the prevalence of primary HIVDR by short reverse transcriptase (RT) genotypic resistance assay and evaluate of the impact of the mutations on the treatment outcomes. Methods A prospective cohort study was conducted in treatment-naïve HIV-infected patients. Fourteen major mutations of codon 99–191 on the RT gene were selected (K103N, V106A/M, V108I, Q151M, Y181C/I, M184V/I, Y188C/L/H, and G190S/A) at a cost of testing of 35 USD. The association between the presence of primary HIVDR and undetectable HIV RNA (<50 copies/mL) after 6 months of ART was determined. Results A total of 265 HIV-infected patients were included, with a median age of 35.2 (range, 16.8–75.2) years; 62.6% were males. The median (interquartile range) CD4 cell count at ART initiation was 216 (77–381) cells/mm3. The overall prevalence of primary HIVDR was 7.9%. The prevalence of each HIVDR mutation were K103N 6.0%, V106I 1.1%, V108I 0.4%, Y181C 2.3%, Y181I 0.7%, Y181V 0.4%, M184V 3.0%, M184I 1.5%, and G190A 2.3%. No associated factor of having primary HIVDR was determined. By multiple stepwise logistic regression, factors associated with undetectable HIV RNA after 6 months of ART were: having M184V/I (odds ratio [OR] 0.11; 95% confidence interval [CI] 0.02–0.62, p = 0.013), condom use (OR 2.38; 95% CI 1.12–5.06, p = 0.024), and adherence per 5% increase (OR 1.16; 95% CI 1.00–1.35, p = 0.044). Conclusions The prevalence of primary HIVDR is approximately 8%; it is associated with detectable HIV RNA at 6 months after ART initiation. Routine “short RT” genotypic resistance assay should be considered in resource-limited settings to maximize treatment outcome. PMID:26828876

  13. Copy number variation detection using SNP genotyping arrays in three Chinese pig breeds.

    PubMed

    Dong, K; Pu, Y; Yao, N; Shu, G; Liu, X; He, X; Zhao, Q; Guan, W; Ma, Y

    2015-04-01

    We performed genome-wide CNV detection based on SNP genotyping data of 96 Chinese-native Tibetan, Dahe and Wuzhishan pigs. These pigs are particularly interesting because of their excellent adaptation to hypoxia or small body size, which facilitates the use of them as models of different human diseases in addition to valuable agricultural animals. A total of 105 CNV regions (CNVRs) were identified, encompassing 16.71 Mb of the pig genome. Seven of 10 (70%) CNVRs selected randomly were validated by quantitative real-time PCR. Comparison with previous studies revealed 25 (23.81%) novel CNVRs, indicating that CNV coverage of the pig genome is still incomplete and there exists large diversity between pig breeds. Functional analysis of genes located in these CNVRs confirmed the high representation of genes involved in sensory perception, neurological system processes and other basic metabolic processes. In addition, the majority of these CNVRs were detected to span reported pig QTL that affect various traits, which highlighted three biologically interesting genes with copy number changes (i.e., ANKRD34B, FAM110B and ABCG1). These genes may have economic importance in pig breeding and are worth being further investigated. We also obtained some CNVRs harboring genes that had human orthologs involved in human diseases such as cardiovascular disease and Alzheimer's disease. The findings of this study are a significant extension of the coverage of CNVRs in the pig genome and provide valuable resources for follow-up-associated studies of CNVs in pig complex traits as well as important implications of human diseases. PMID:25590996

  14. Comparative sensitivity of commercial tests for hepatitis E genotype 3 virus antibody detection.

    PubMed

    Avellon, Ana; Morago, Lucia; Garcia-Galera del Carmen, Maria; Munoz, Milagros; Echevarría, Jose-Manuel

    2015-11-01

    Hepatitis E virus (HEV) acute infection is often diagnosed only by anti-HEV IgM ELISA methods, whose sensitivity varies, according to different reports. Reports assessing the specificity of commercial assays for anti-HEV IgG testing are scarce, and estimates of sensitivity and specificity are both controversial. The aim of this work is to assess the sensitivity of different commercial techniques for HEV genotype 3 antibody (anti-HEV) IgM and IgG detection in entirely specific sample panels including both high and low antibody concentrations. The anti-HEV IgM and IgG ELISA methods compared were: DSI, Mikrogen, Wantai, Euroimmun, MP, and Dia.pro. The rapid test All Diag was also included in the anti-HEV IgM comparison. Our results show that low anti-HEV IgM concentrations were better detected by DSI, Mikrogen, and All Diag, these tests being the most sensitive in our study. Euroimmun, MP and Dia.pro gave concordant results, showing lower sensitivity than the others. Regarding anti-HEV IgG our results revealed similar anti-HEV IgG sensitivity. Furthermore, there was a striking overall lack of concordance among the results. We present a thorough review of previous comparative reports, with particular reference to the anti-HEV IgG comparison, since published results differ from ours. This discrepancy may be related to the improved versions of the tests for MP and Dia.pro that we employed. PMID:25959136

  15. HPV genotypes detected in the oropharyngeal mucosa of HIV-infected men who have sex with men in Northern Italy.

    PubMed

    Martinelli, M; Mazza, F; Frati, E R; Fasolo, M M; Colzani, D; Bianchi, S; Fasoli, E; Amendola, A; Orlando, G; Tanzi, E

    2016-09-01

    The aim of this study was to investigate the epidemiological profile of HPV oropharyngeal infections in HIV-infected men who have sex with men. A total of 135 subjects were enrolled at the L. Sacco University Hospital (Milan, Italy) to evaluate their HPV oropharyngeal infection status at baseline and at a follow-up visit at least 12 months later. HPV DNA was detected from oropharyngeal swabs using an in-house nested PCR that amplifies a segment of the L1 gene. The PCR products were then sequenced and genotyped. A greater percentage of high-risk genotypes was identified compared to low-risk genotypes (13·7% vs. 6·9%, P < 0·05), and two uncommon alpha-HPV genotypes were detected, i.e. HPV-102 and HPV-114. HPV infection prevalence was 24·4% and the cumulative incidence was 24·1%. During the follow-up period, one case of HPV infection (HPV-33) persisted, while the overall rate of infection clearance was 58·3%. HPV oropharyngeal infection was widespread in the cohort examined, and most of the infections were transient and cleared within 12 months. These results may help to clarify the role of HPV in the oropharynx and may also improve our understanding of the need to implement preventive strategies in at-risk populations. PMID:27267944

  16. Evaluation of the GenoType MTBDR assay for detection of rifampicin and isoniazid resistance in Mycobacterium tuberculosis complex isolates.

    PubMed

    Saglik, I; Oz, Y; Kiraz, N

    2014-01-01

    Detection of drug resistance plays a critical role in tuberculosis treatment. The aim of this study was to evaluate the performance of GenoType Mycobacteria Drug Resistance (MTBDR) assay (Hain Lifescience, Germany) and to compare it with radiometric BACTEC 460 TB system (Becton Dickinson, USA) for the detection of rifampicin (RIF) and isoniazid (INH) resistance in 84 Mycobacterium tuberculosis complex (MTBC) isolates. RIF resistance was identified in 6 of 7 (85.7%) isolates and INH resistance was identified in 8 of 14 (57.1%) isolates by the GenoType MTBDR assay. Compared with BACTEC system, the sensitivity, specificity, positive predictive value and negative predictive values were 85.7%, 98.7%, 85.7% and 98.7% for RIF resistance; and 57.1%, 100%, 100% and 92.1% for INH resistance, respectively. GenoType MTBDR assay is reliable when tested specimen is resistant to the tested drugs. Although test was more successful in the detection of RIF resistance, it exhibited low sensitivity for the detection of INH resistance. PMID:25008829

  17. Comparison of methods to detect copy number alterations in cancer using simulated and real genotyping data

    PubMed Central

    2012-01-01

    Background The detection of genomic copy number alterations (CNA) in cancer based on SNP arrays requires methods that take into account tumour specific factors such as normal cell contamination and tumour heterogeneity. A number of tools have been recently developed but their performance needs yet to be thoroughly assessed. To this aim, a comprehensive model that integrates the factors of normal cell contamination and intra-tumour heterogeneity and that can be translated to synthetic data on which to perform benchmarks is indispensable. Results We propose such model and implement it in an R package called CnaGen to synthetically generate a wide range of alterations under different normal cell contamination levels. Six recently published methods for CNA and loss of heterozygosity (LOH) detection on tumour samples were assessed on this synthetic data and on a dilution series of a breast cancer cell-line: ASCAT, GAP, GenoCNA, GPHMM, MixHMM and OncoSNP. We report the recall rates in terms of normal cell contamination levels and alteration characteristics: length, copy number and LOH state, as well as the false discovery rate distribution for each copy number under different normal cell contamination levels. Assessed methods are in general better at detecting alterations with low copy number and under a little normal cell contamination levels. All methods except GPHMM, which failed to recognize the alteration pattern in the cell-line samples, provided similar results for the synthetic and cell-line sample sets. MixHMM and GenoCNA are the poorliest performing methods, while GAP generally performed better. This supports the viability of approaches other than the common hidden Markov model (HMM)-based. Conclusions We devised and implemented a comprehensive model to generate data that simulate tumoural samples genotyped using SNP arrays. The validity of the model is supported by the similarity of the results obtained with synthetic and real data. Based on these results and

  18. Bias Characterization in Probabilistic Genotype Data and Improved Signal Detection with Multiple Imputation.

    PubMed

    Palmer, Cameron; Pe'er, Itsik

    2016-06-01

    Missing data are an unavoidable component of modern statistical genetics. Different array or sequencing technologies cover different single nucleotide polymorphisms (SNPs), leading to a complicated mosaic pattern of missingness where both individual genotypes and entire SNPs are sporadically absent. Such missing data patterns cannot be ignored without introducing bias, yet cannot be inferred exclusively from nonmissing data. In genome-wide association studies, the accepted solution to missingness is to impute missing data using external reference haplotypes. The resulting probabilistic genotypes may be analyzed in the place of genotype calls. A general-purpose paradigm, called Multiple Imputation (MI), is known to model uncertainty in many contexts, yet it is not widely used in association studies. Here, we undertake a systematic evaluation of existing imputed data analysis methods and MI. We characterize biases related to uncertainty in association studies, and find that bias is introduced both at the imputation level, when imputation algorithms generate inconsistent genotype probabilities, and at the association level, when analysis methods inadequately model genotype uncertainty. We find that MI performs at least as well as existing methods or in some cases much better, and provides a straightforward paradigm for adapting existing genotype association methods to uncertain data. PMID:27310603

  19. Bias Characterization in Probabilistic Genotype Data and Improved Signal Detection with Multiple Imputation

    PubMed Central

    Palmer, Cameron; Pe’er, Itsik

    2016-01-01

    Missing data are an unavoidable component of modern statistical genetics. Different array or sequencing technologies cover different single nucleotide polymorphisms (SNPs), leading to a complicated mosaic pattern of missingness where both individual genotypes and entire SNPs are sporadically absent. Such missing data patterns cannot be ignored without introducing bias, yet cannot be inferred exclusively from nonmissing data. In genome-wide association studies, the accepted solution to missingness is to impute missing data using external reference haplotypes. The resulting probabilistic genotypes may be analyzed in the place of genotype calls. A general-purpose paradigm, called Multiple Imputation (MI), is known to model uncertainty in many contexts, yet it is not widely used in association studies. Here, we undertake a systematic evaluation of existing imputed data analysis methods and MI. We characterize biases related to uncertainty in association studies, and find that bias is introduced both at the imputation level, when imputation algorithms generate inconsistent genotype probabilities, and at the association level, when analysis methods inadequately model genotype uncertainty. We find that MI performs at least as well as existing methods or in some cases much better, and provides a straightforward paradigm for adapting existing genotype association methods to uncertain data. PMID:27310603

  20. Detection and genotyping of Toxoplasma gondii DNA in the blood and milk of naturally infected donkeys (Equus asinus)

    PubMed Central

    2014-01-01

    Background Toxoplasma gondii is a worldwide zoonotic protozoan. Consumption of raw milk from infected animals is considered a risk factor for acquiring toxoplasmosis in humans. Recently, donkey milk has been indicated for therapeutic and nutritional purposes and T. gondii infection is common in donkeys. The purpose of the present paper was to detect the presence of parasite DNA in milk of T. gondii positive donkeys. Findings Antibodies to T. gondii were found in 11 out of 44 healthy lactating donkeys by IFAT. T. gondii DNA was detected by PCR in blood of 6 and milk of 3 seropositive jennies. Results of limited RFLP-PCR genotyping indicated the presence of T. gondii genotype II or III, commonly found in Europe. Conclusions The occurrence of T. gondii DNA in milk suggests that the consumption of raw milk from seropositive donkeys could be a potential source of human infection. PMID:24708691

  1. Genotype–phenotype associations: substitution models to detect evolutionary associations between phenotypic variables and genotypic evolutionary rate

    PubMed Central

    O'Connor, Timothy D.; Mundy, Nicholas I.

    2009-01-01

    Motivation: Mapping between genotype and phenotype is one of the primary goals of evolutionary genetics but one that has received little attention at the interspecies level. Recent developments in phylogenetics and statistical modelling have typically been used to examine molecular and phenotypic evolution separately. We have used this background to develop phylogenetic substitution models to test for associations between evolutionary rate of genotype and phenotype. We do this by creating hybrid rate matrices between genotype and phenotype. Results: Simulation results show our models to be accurate in detecting genotype–phenotype associations and robust for various factors that typically affect maximum likelihood methods, such as number of taxa, level of relevant signal, proportion of sites affected and length of evolutionary divergence. Further, simulations show that our method is robust to homogeneity assumptions. We apply the models to datasets of male reproductive system genes in relation to mating systems of primates. We show that evolution of semenogelin II is significantly associated with mating systems whereas two negative control genes (cytochrome b and peptidase inhibitor 3) show no significant association. This provides the first hybrid substitution model of which we are aware to directly test the association between genotype and phenotype using a phylogenetic framework. Availability: Perl and HYPHY scripts are available upon request from the authors. Contact: to252@cam.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online. PMID:19478022

  2. Rapid Detection of Rare Deleterious Variants by Next Generation Sequencing with Optional Microarray SNP Genotype Data

    PubMed Central

    Watson, Christopher M.; Crinnion, Laura A.; Gurgel‐Gianetti, Juliana; Harrison, Sally M.; Daly, Catherine; Antanavicuite, Agne; Lascelles, Carolina; Markham, Alexander F.; Pena, Sergio D. J.; Bonthron, David T.

    2015-01-01

    ABSTRACT Autozygosity mapping is a powerful technique for the identification of rare, autosomal recessive, disease‐causing genes. The ease with which this category of disease gene can be identified has greatly increased through the availability of genome‐wide SNP genotyping microarrays and subsequently of exome sequencing. Although these methods have simplified the generation of experimental data, its analysis, particularly when disparate data types must be integrated, remains time consuming. Moreover, the huge volume of sequence variant data generated from next generation sequencing experiments opens up the possibility of using these data instead of microarray genotype data to identify disease loci. To allow these two types of data to be used in an integrated fashion, we have developed AgileVCFMapper, a program that performs both the mapping of disease loci by SNP genotyping and the analysis of potentially deleterious variants using exome sequence variant data, in a single step. This method does not require microarray SNP genotype data, although analysis with a combination of microarray and exome genotype data enables more precise delineation of disease loci, due to superior marker density and distribution. PMID:26037133

  3. A multiplex PCR for the simultaneous detection and genotyping of the Echinococcus granulosus complex.

    PubMed

    Boubaker, Ghalia; Macchiaroli, Natalia; Prada, Laura; Cucher, Marcela A; Rosenzvit, Mara C; Ziadinov, Iskender; Deplazes, Peter; Saarma, Urmas; Babba, Hamouda; Gottstein, Bruno; Spiliotis, Markus

    2013-01-01

    Echinococcus granulosus is characterized by high intra-specific variability (genotypes G1-G10) and according to the new molecular phylogeny of the genus Echinococcus, the E. granulosus complex has been divided into E. granulosus sensu stricto (G1-G3), E. equinus (G4), E. ortleppi (G5), and E. canadensis (G6-G10). The molecular characterization of E. granulosus isolates is fundamental to understand the spatio-temporal epidemiology of this complex in many endemic areas with the simultaneous occurrence of different Echinococcus species and genotypes. To simplify the genotyping of the E. granulosus complex we developed a single-tube multiplex PCR (mPCR) allowing three levels of discrimination: (i) Echinococcus genus, (ii) E. granulosus complex in common, and (iii) the specific genotype within the E. granulosus complex. The methodology was established with known DNA samples of the different strains/genotypes, confirmed on 42 already genotyped samples (Spain: 22 and Bulgaria: 20) and then successfully applied on 153 unknown samples (Tunisia: 114, Algeria: 26 and Argentina: 13). The sensitivity threshold of the mPCR was found to be 5 ng Echinoccoccus DNA in a mixture of up to 1 µg of foreign DNA and the specificity was 100% when template DNA from closely related members of the genus Taenia was used. Additionally to DNA samples, the mPCR can be carried out directly on boiled hydatid fluid or on alkaline-lysed frozen or fixed protoscoleces, thus avoiding classical DNA extractions. However, when using Echinococcus eggs obtained from fecal samples of infected dogs, the sensitivity of the mPCR was low (<40%). Thus, except for copro analysis, the mPCR described here has a high potential for a worldwide application in large-scale molecular epidemiological studies on the Echinococcus genus. PMID:23350011

  4. Detection of bovine leukemia virus and identification of its genotype in Mongolian cattle.

    PubMed

    Ochirkhuu, Nyamsuren; Konnai, Satoru; Odbileg, Raadan; Nishimori, Asami; Okagawa, Tomohiro; Murata, Shiro; Ohashi, Kazuhiko

    2016-04-01

    Epidemiological studies have indicated that bovine leukemia virus (BLV) infection is globally distributed. However, no information regarding the disease and genetic diversity of the virus in the cattle of Mongolia is currently available. In this study, the prevalence of BLV was assessed using PCR, and the genetic diversity was analyzed through DNA sequencing. Of the 517 samples tested, 20 positives were identified. Phylogenetic analysis showed that six, one, and four isolates were classified into genotype 4, 7, and 1, respectively. Most isolates were clustered with isolates from Eastern Europe and Russia. This study is the first to investigate the BLV genotype in Mongolia. PMID:26711456

  5. Detection of mumps virus genotype H in two previously vaccinated patients from Mexico City.

    PubMed

    Del Valle, Alberto; García, Alí A; Barrón, Blanca L

    2016-06-01

    Infections caused by mumps virus (MuV) have been successfully prevented through vaccination; however, in recent years, an increasing number of mumps outbreaks have been reported within vaccinated populations. In this study, MuV was genotyped for the first time in Mexico. Saliva samples were obtained from two previously vaccinated patients in Mexico City who had developed parotitis. Viral isolation was carried out in Vero cells, and the SH and HN genes were amplified by RT-PCR. Amplicons were sequenced and compared to a set of reference sequences to identify the MuV genotype. PMID:26935913

  6. Multi-allele genotyping platform for the simultaneous detection of mutations in the Wilson disease related ATP7B gene.

    PubMed

    Amvrosiadou, Maria; Petropoulou, Margarita; Poulou, Myrto; Tzetis, Maria; Kanavakis, Emmanuel; Christopoulos, Theodore K; Ioannou, Penelope C

    2015-12-01

    Wilson's disease is an inherited disorder of copper transport in the hepatocytes with a wide range of genotype and phenotype characteristics. Mutations in the ATP7B gene are responsible for the disease. Approximately, over 500 mutations in the ATP7B gene have been described to date. We report a method for the simultaneous detection of the ten most common ATP7B gene mutations in Greek patients. The method comprises 3 simple steps: (i) multiplex PCR amplification of fragments in the ATP7B gene flanking the mutations (ii) multiplex primer extension reaction of the unpurified amplification products using allele-specific primers and (iii) visual detection of the primer extension reaction products within minutes by means of dry-reagent multi-allele dipstick assay using anti-biotin conjugated gold nanoparticles. Optimization studies on the efficiency and specificity of the PEXT reaction were performed. The method was evaluated by genotyping 46 DNA samples of known genotype and 34 blind samples. The results were fully concordant with those obtained by reference methods. The method is simple, rapid, cost-effective and it does not require specialized instrumentation or highly qualified personnel. PMID:26580967

  7. Immunohistochemical Detection of Hepatitis C Virus (Genotype 4) in B-cell NHL in an Egyptian Population

    PubMed Central

    Gouda, Iman; Nada, Ola; Ezzat, Sameera; Eldaly, Mai; Loffredo, Christopher; Taylor, Clive; Abdel-Hamid, Mohamed

    2013-01-01

    Background and Aim Retrospective evaluation of hepatitis C virus (HCV) prevalence in lymphoma tissues has important applications in clarifying the contribution of viral factors to the pathogenesis. Trials for detection of HCV at the cellular level in lymphoma tissues are, so far, minimal with unsatisfactory results. We aimed to study the detection and localization of HCV in the tissues of B-cell non-Hodgkin lymphoma (NHL) patients. Design We performed immunohistochemistry to detect the HCV nonstructural 3 protein in paraffin-embedded tissue specimens of B-cell NHL patients, in 39 serum HCV-RNA positive samples and 35 serum HCV-RNA negative samples as controls. The serum analysis was carried out for HCV antibodies using enzyme-linked immunoassay and for HCV-RNA using reverse transcription-polymerase chain reaction. Reverse transcription-polymerase chain reaction was used to detect the HCV-RNA in tissues in immunohistochemically positive cases. We correlated the results with the clinicopathologic characteristics of the patients. Results A diffuse cytoplasmic immunohistochemical staining for HCV in the lymphoid cells was detected in 8 of 39 serum positive cases (20.5%), all of which were genotype 4, which is the most prevalent HCV genotype in Egypt. Only 2 out of 35 serum negative control samples showed positive staining and in 1 of them HCV-RNA was detected in tissue. No significant correlation was detected between HCV positive cases and the clinicopathologic features of the patients. Conclusions Immunohistochemical detection of HCV proteins in lymphoma tissues supports a potential role of viral replication in lymphomagenesis. The low number of cases showing expression of viral proteins may represent a low viral load in lymphoid tissue and/or restriction of HCV protein expression to certain subtypes of B-cell NHL. Immunohistochemistry can be used as a complementary tool for specific HCV detection in the paraffin-embedded material of lymphoma tissues not suitable for

  8. HBV vaccine efficacy and detection and genotyping of vaccineé asymptomatic breakthrough HBV infection in Egypt

    PubMed Central

    Abushady, Eman AE; Gameel, Magda MA; Klena, John D; Ahmed, Salwa F; Abdel-Wahab, Kouka SE; Fahmy, Sanya M

    2011-01-01

    AIM: To evaluate the impact of mass vaccination against the hepatitis B virus (HBV) in Egypt, and to search for vaccinee asymptomatic breakthrough HBV infection and its genotype. METHODS: Seven hundred serum samples from vaccinated children and adults (aged 2-47 years) were used for quantitative and qualitative detection of HBsAb by ELISA. Three hundred and sixty serum samples representing undetectable or low or high HBsAb were screened for markers of active HBV infection (HBsAg, HBcAb (IgG) and HBeAb by ELISA, plus HBsAg by AxSYM) and HBV-DNA genotyping by nested multiplex PCR and by DNA sequencing. RESULTS: It was found that 65% of children aged 2-4 years, and 20.5% aged 4-13 years, as well as 45% adults were good responders to HBV vaccination mounting protective level HBsAb. Poor responders were 28%, 59.5% and 34%, and non-responders were 7%, 20% and 21% respectively, in the three studied groups. Markers of asymptomatic HBV infections were HBsAg detected by ELISA in 2.5% vs 11.39% by AxSYM. Other markers were HBcAb (IgG) in 1.38%, HBeAb in 0.83%, and HBV-DNA in 7.8%. All had HBV genotype E infection. CONCLUSION: It is concluded that HBV vaccine is efficient in controlling HBV infection among children and adults. The vaccine breakthrough infection was by HBV genotype E. A booster dose of vaccine is recommended, probably four years after initial vaccination. PMID:21860674

  9. Detection of east/central/south African genotype of chikungunya virus in Myanmar, 2010.

    PubMed

    Tun, Mya Myat Ngwe; Thant, Kyaw Zin; Inoue, Shingo; Nabeshima, Takeshi; Aoki, Kotaro; Kyaw, Aung Kyaw; Myint, Tin; Tar, Thi; Maung, Kay Thwe Thwe; Hayasaka, Daisuke; Morita, Kouichi

    2014-08-01

    In 2010, chikungunya virus of the East Central South African genotype was isolated from 4 children in Myanmyar who had dengue-like symptoms. Phylogenetic analysis of the E1 gene revealed that the isolates were closely related to isolates from China, Thailand, and Malaysia that harbor the A226V mutation in this gene. PMID:25062511

  10. Detection of East/Central/South African Genotype of Chikungunya Virus in Myanmar, 2010

    PubMed Central

    Tun, Mya Myat Ngwe; Thant, Kyaw Zin; Inoue, Shingo; Nabeshima, Takeshi; Aoki, Kotaro; Kyaw, Aung Kyaw; Myint, Tin; Tar, Thi; Maung, Kay Thwe Thwe; Hayasaka, Daisuke

    2014-01-01

    In 2010, chikungunya virus of the East Central South African genotype was isolated from 4 children in Myanmyar who had dengue-like symptoms. Phylogenetic analysis of the E1 gene revealed that the isolates were closely related to isolates from China, Thailand, and Malaysia that harbor the A226V mutation in this gene. PMID:25062511

  11. SEQCHIP: a powerful method to integrate sequence and genotype data for the detection of rare variant associations

    PubMed Central

    Liu, Dajiang J.; Leal, Suzanne M.

    2012-01-01

    Motivation: Next-generation sequencing greatly increases the capacity to detect rare-variant complex-trait associations. However, it is still expensive to sequence a large number of samples and therefore often small datasets are used. Given cost constraints, a potentially more powerful two-step strategy is to sequence a subset of the sample to discover variants, and genotype the identified variants in the remaining sample. If only cases are sequenced, directly combining sequence and genotype data will lead to inflated type-I errors in rare-variant association analysis. Although several methods have been developed to correct for the bias, they are either underpowered or theoretically invalid. We proposed a new method SEQCHIP to integrate genotype and sequence data, which can be used with most existing rare-variant tests. Results: It is demonstrated using both simulated and real datasets that the SEQCHIP method has controlled type-I errors, and is substantially more powerful than all other currently available methods. Availability: SEQCHIP is implemented in an R-Package and is available at http://linkage.rockefeller.edu/suzanne/seqchip/Seqchip.htm Contacts: dajiang@umich.edu or sleal@bcm.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:22556370

  12. [Interstitial lung diseases associated with smoking].

    PubMed

    Nová, Markéta; Hornychová, Helena; Matěj, Radoslav

    2016-01-01

    There are many different interstitial lung diseases associated with smoking. This short review describes officially recognized disorders (desquamative interstitial pneumonia, respiratory bronchiolitis and pulmonary Langerhans´cells histiocytosis) and entities with uncertain relationship to smoking, which have recently been published in the literature. Histopathological pictures and differential diagnosis of smoking-related diseases of the lungs are discussed. PMID:27223588

  13. Simultaneous Detection, Genotyping, and Quantification of Human Papillomaviruses by Multicolor Real-Time PCR and Melting Curve Analysis

    PubMed Central

    Liao, Yiqun; Zhou, Yulin; Guo, Qiwei; Xie, Xiaoting; Luo, Ena

    2013-01-01

    Long-term infection with high-risk human papillomavirus (HPV) is the leading cause of cervical cancer, while infection with low-risk HPV is the major reason for condylomata acuminata. An accurate, rapid, and convenient assay that is able to simultaneously detect, genotype, and quantify HPV would be of great clinical value yet remains to be achieved. We developed a three-color real-time PCR assay that is able to analyze 30 predominant HPV types in three reactions. The amplification curves indicated the presence of HPV, melting curve analysis identified the HPV genotype, and the quantification cycle value determined the quantity. We applied this assay to 647 cervical swab samples, and the results were compared with those obtained with a commercial genotyping system. The proposed assay had a limit of detection of 5 to 50 copies per reaction and a dynamic range of 5 × 101 to 5 × 106 copies per reaction. A comparison study showed that the overall sample concordance with the comparison method was 91.6% and the type agreement was greater than 98.7%. The quantification study demonstrated that the loads of HPV type 16 in 30 samples with cervical intraepithelial neoplasia grade III (CIN III) lesions were significantly higher than those in samples with CIN I lesions or CIN II lesions, and the results were concordant with those of the comparison method. The increased information content, high throughput, and low cost would facilitate the use of this real-time PCR-based assay in a variety of clinical settings. PMID:23175255

  14. Detection and genotype analysis of AmpC β-lactamase in Klebsiella pneumoniae from tertiary hospitals

    PubMed Central

    LIU, XIANG-QUN; LIU, YONG-RUI

    2016-01-01

    The aim of the present study was to investigate the phenotype and genotype of plasmid-mediated AmpC (pAmpC) β-lactamase in Klebsiella pneumoniae and its antibiotic resistance. A total of 130 non-repetitive clinical isolates of Klebsiella pneumoniae, obtained from tertiary hospitals, were phenotypically screened for pAmpC β-lactamase production with the cefoxitin disk diffusion test. β-lactamase genes in the screened isolates were detected using multiplex polymerase chain reaction (PCR); carbapenemase genes in pAmpC β-lactamase-producing isolates that were resistant to imipenem were detected using PCR. Out of the 130 isolates of Klebsiella pneumoniae, 62 strains (47.7%) were resistant to cefoxitin, including 14 strains (10.8%) positive for pAmpC β-lactamase (DHA type), among which 12 strains (85.7%) were susceptible to imipenem, and 2 strains, which were carrying Klebsiella pneumoniae carbapenemase (KPC)-2 gene, were resistant to imipenem. The pAmpC β-lactamase-producing Klebsiella pneumoniae isolates from the tertiary hospitals were mainly of DHA-1 genotype, and the majority were susceptible to carbapenems; drug-resistant strains were associated with KPC-2 expression. PMID:27347082

  15. Multiplex detection and genotyping of pathogenic bacteria on paper-based biosensor with a novel universal primer mediated asymmetric PCR.

    PubMed

    Liu, Fang; Liu, Hongxing; Liao, Yuhui; Wei, Jitao; Zhou, Xiaoming; Xing, Da

    2015-12-15

    Traditionary multiplex asymmetric polymerase chain reaction (PCR) can be applied to detect multiplex target organisms simultaneously, but complex optimizations of primer concentrations and staggered additions of primers are required to achieve equal amplification of multiplex genes. To overcome this shortcoming, we propose a novel method based on multiplex asymmetric PCR and paper-based nucleic acid diagnostics (PBNAD). In the asymmetric PCR, a universal primer was introduced to break the bottlenecks of low sensitivity and self-inhibition among different sets of primers. Amplification using the novel multiplex asymmetric PCR boosted the quantity of single-stranded amplicons, and the amplified products contained the same sequence at the 5' end. Therefore, only one gold nanoparticle-based signal probe was needed for the simultaneous detection of three genes using the PBNAD platform, and the detection signals could be observed with the naked eye. With this highly efficient, novel multiplex asymmetric PCR, as little as 1 pg/μL genomic DNA can be detected. This method can also be applied to genotyping for reliable epidemiological investigations. This proof-of-concept study highlights the potential of the PBNAD platform for cost- and labor-effective applications in the detection of pathogenic bacteria. PMID:26226347

  16. Compressed Genotyping

    PubMed Central

    Erlich, Yaniv; Gordon, Assaf; Brand, Michael; Hannon, Gregory J.; Mitra, Partha P.

    2011-01-01

    Over the past three decades we have steadily increased our knowledge on the genetic basis of many severe disorders. Nevertheless, there are still great challenges in applying this knowledge routinely in the clinic, mainly due to the relatively tedious and expensive process of genotyping. Since the genetic variations that underlie the disorders are relatively rare in the population, they can be thought of as a sparse signal. Using methods and ideas from compressed sensing and group testing, we have developed a cost-effective genotyping protocol to detect carriers for severe genetic disorders. In particular, we have adapted our scheme to a recently developed class of high throughput DNA sequencing technologies. The mathematical framework presented here has some important distinctions from the ’traditional’ compressed sensing and group testing frameworks in order to address biological and technical constraints of our setting. PMID:21451737

  17. GenoType MTBDRplus Assay for Rapid Detection of Multidrug Resistance in Mycobacterium tuberculosis: A Meta-Analysis

    PubMed Central

    Bai, Yuanyuan; Wang, Yueling; Shao, Chunhong; Hao, Yingying; Jin, Yan

    2016-01-01

    Background There is an urgent demand for rapid and accurate drug-susceptibility testing for the detection of multidrug-resistant tuberculosis. The GenoType MTBDRplus assay is a promising molecular kit designed for rapid identification of resistance to first-line anti-tuberculosis drugs, isoniazid and rifampicin. The aim of this meta-analysis was to evaluate the diagnostic accuracy of GenoType MTBDRplus in detecting drug resistance to isoniazid and rifampicin in comparison with the conventional drug susceptibility tests. Methods We searched PubMed, EMBASE, and Cochrane Library databases to identify studies according to predetermined criteria. A total of 40 studies were included in the meta-analysis. QUADAS-2 was used to assess the quality of included studies with RevMan 5.2. STATA 13.0 software was used to analyze the tests for sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, diagnostic odds ratio, and area under the summary receiver operating characteristic curves. Heterogeneity in accuracy measures was tested with Spearman correlation coefficient and Chi-square. Results Patient selection bias was observed in most studies. The pooled sensitivity (95% confidence intervals were 0.91 (0.88–0.94) for isoniazid, 0.96 (0.95–0.97) for rifampicin, and 0.91(0.86–0.94) for multidrug-resistance. The pooled specificity (95% CI) was 0.99 (0.98–0.99) for isoniazid, 0.98 (0.97–0.99) for rifampicin and 0.99 (0.99–1.00) for multidrug-resistance, respectively. The area under the summary receiver operating characteristic curves ranged from 0.99 to 1.00. Conclusion This meta-analysis determined that GenoType MTBDRplus had good accuracy for rapid detection of drug resistance to isoniazid and/or rifampicin of M. tuberculosis. MTBDRplus method might be a good alternative to conventional drug susceptibility tests in clinical practice. PMID:26934724

  18. Newly Identified Enterovirus C Genotypes, Identified in the Netherlands through Routine Sequencing of All Enteroviruses Detected in Clinical Materials from 2008 to 2015

    PubMed Central

    Poelman, Randy; Borger, Renze; Niesters, Hubert G. M.

    2016-01-01

    Enteroviruses (EVs) are a group of human and animal viruses that are capable of causing a variety of clinical syndromes. Different genotypes classified into species can be distinguished on the basis of sequence divergence in the VP1 capsid-coding region. Apparently new genotypes are discovered regularly, often as incidental findings in studies investigating respiratory syndromes or as part of poliovirus surveillance. Recently, some EVs have become recognized as significant respiratory pathogens, and a number of new genotypes belonging to species C have been identified. The circulation of these newly identified species C EVs, such as EV-C104, EV-C105, EV-C109, and EV-C117, nevertheless appears to be limited. In this report, we show the results of routine genotyping of all enteroviruses detected in our tertiary care hospital between January 2008 and April 2015. We detected 365 EVs belonging to 40 genotypes. Interestingly, several newly identified species C EVs were detected during the study period. Sequencing of the 5′ untranslated region (5′ UTR) of these viruses shows divergence in this region, which is a target region in many detection assays. PMID:27358467

  19. Newly Identified Enterovirus C Genotypes, Identified in the Netherlands through Routine Sequencing of All Enteroviruses Detected in Clinical Materials from 2008 to 2015.

    PubMed

    Van Leer-Buter, Coretta C; Poelman, Randy; Borger, Renze; Niesters, Hubert G M

    2016-09-01

    Enteroviruses (EVs) are a group of human and animal viruses that are capable of causing a variety of clinical syndromes. Different genotypes classified into species can be distinguished on the basis of sequence divergence in the VP1 capsid-coding region. Apparently new genotypes are discovered regularly, often as incidental findings in studies investigating respiratory syndromes or as part of poliovirus surveillance. Recently, some EVs have become recognized as significant respiratory pathogens, and a number of new genotypes belonging to species C have been identified. The circulation of these newly identified species C EVs, such as EV-C104, EV-C105, EV-C109, and EV-C117, nevertheless appears to be limited. In this report, we show the results of routine genotyping of all enteroviruses detected in our tertiary care hospital between January 2008 and April 2015. We detected 365 EVs belonging to 40 genotypes. Interestingly, several newly identified species C EVs were detected during the study period. Sequencing of the 5' untranslated region (5' UTR) of these viruses shows divergence in this region, which is a target region in many detection assays. PMID:27358467

  20. Detection of Porphyromonas gingivalis fimA Type I Genotype in Gingivitis by Real-Time PCR–A Pilot Study

    PubMed Central

    Krishnan, Mahalakshmi; Chandrasekaran, S.C.

    2016-01-01

    Introduction Published literature till date reveals a high prevalence of Porphyromonas gingivalis fimA type I genotype among healthy subjects. Quite a few studies have reported its prevalence also in periodontitis patients. Nevertheless incidence of this genotype in gingivitis is lacking in adult population. Aim The present study was chosen to detect P. gingivalis fimA type I genotype among chronic gingivitis patients. Materials and Methods A total of 46 subgingival plaque samples collected from chronic marginal gingivitis (n=23) and chronic periodontitis subjects (control group) (n=23) were subjected to Real-Time Polymerase Chain Reaction to detect the P. gingivalis fimA type I gene. Statistical analysis was performed using chi-square test. Results Prevalence of P. gingivalis fimA type I gene among chronic periodontitis and chronic gingivitis patients were 8.7% and 30.4% respectively. P. gingivalis fimA type I genotype prevalence was found to be statistically insignificant between the two study groups (p=0.135). Conclusion The avirulent P. gingivalis fimA type I genotype, occurred in high prevalence among chronic gingivitis patients, while its presence was low in chronic periodontitis patients. Presence of this avirulent genotype in chronic marginal gingivitis signifies its reversible condition. PMID:27504406

  1. Detection and Genotyping of Human Papillomavirus DNA in Formalin-Fixed Paraffin-Embedded Specimens with the HPV Direct Flow CHIP System

    PubMed Central

    Herraez-Hernandez, Elsa; Preda, Ovidiu; Alonso, Sonia; Pardo, Rosario Serrano; Olmo, Asuncion

    2013-01-01

    The novel HPV Direct Flow CHIP commercial system for Human Papillomavirus (HPV) genotyping is based on rapid PCR and automatic reverse dot blot hybridization to genotype-specific probes, allowing the detection of 36 HPV genotypes. This study examined the performance of HPV Direct Flow CHIP in formalin-fixed paraffin-embedded (FFPE) samples (n= 99). Each sample was analyzed both by Direct PCR, using crude cell extracts without DNA purification, and by conventional PCR, using purified DNA. Pair-wise analysis of the results demonstrated strong concordance between the results obtained with the two protocols, although a slightly higher rate of multiple infections was detected by conventional PCR. In summary, HPV Direct Flow CHIP achieves effective HPV detection from FFPE samples with both Direct PCR and Conventional PCR protocols. PMID:24222806

  2. Advances in alloimmune thrombocytopenia: perspectives on current concepts of human platelet antigens, antibody detection strategies, and genotyping

    PubMed Central

    Hayashi, Tomoya; Hirayama, Fumiya

    2015-01-01

    Alloimmunisation to platelets leads to the production of antibodies against platelet antigens and consequently to thrombocytopenia. Numerous molecules located on the platelet surface are antigenic and induce immune-mediated platelet destruction with symptoms that can be serious. Human platelet antigens (HPA) cause thrombocytopenias, such as neonatal alloimmune thrombocytopenia, post-transfusion purpura, and platelet transfusion refractoriness. Thirty-four HPA are classified into 28 systems. Assays to identify HPA and anti-HPA antibodies are critically important for preventing and treating thrombocytopenia caused by anti-HPA antibodies. Significant progress in furthering our understanding of HPA has been made in the last decade: new HPA have been discovered, antibody-detection methods have improved, and new genotyping methods have been developed. We review these advances and discuss issues that remain to be resolved as well as future prospects for preventing and treating immune thrombocytopenia. PMID:26057488

  3. Detection and genetic characterization of hepatitis E virus (HEV) genotype 3 subtype c in wild boars in Italy.

    PubMed

    Di Profio, Federica; Melegari, Irene; Sarchese, Vittorio; Robetto, Serena; Marruchella, Giuseppe; Bona, Maria Cristina; Orusa, Riccardo; Martella, Vito; Marsilio, Fulvio; Di Martino, Barbara

    2016-10-01

    Hepatitis E virus (HEV) was detected in stools collected from wild boars in Italy, with an overall prevalence of 1.5 % (3/196). The sequence of a ~3.0-kb portion at the 3' end of the genome of one such strain, HEV/WB/P6-15/ITA, was determined. In the full-length ORF2, which encodes the capsid protein, the virus was genetically closest to wild boar and human HEV strains currently classified as genotype 3 subtype c. Interestingly, the 3' end of ORF2 of the WB/P6-15/ITA matched the 340-nucleotide (nt) sequence (94.0 % nt identity) of the human strain PeGe, identified in 2015 from a patient with acute hepatitis E in Genoa, Italy, suggesting that similar HEV strains are circulating in the same geographical setting in humans and animals. PMID:27393602

  4. Detection of Leptospira spp. in wildlife reservoir hosts in Ontario through comparison of immunohistochemical and polymerase chain reaction genotyping methods.

    PubMed

    Shearer, Karen E; Harte, Michael J; Ojkic, Davor; Delay, Josepha; Campbell, Douglas

    2014-03-01

    A total of 460 kidney samples from wildlife (beavers, coyotes, deer, foxes, opossums, otters, raccoons, skunks) were obtained from road-kill and hunter/trapper donations in Ontario between January 2010 and November 2012. The objectives of the study were to detect Leptospira spp. by immunohistochemistry and polymerase chain reaction (PCR), to map presence of leptospires in wildlife relative to livestock and human populations, and to characterize positive samples by sequencing and comparison to leptospires known to affect domestic animals and humans. The proportion of samples that tested positive ranged from 0% to 42%, with the highest rates in skunks and raccoons. Leptospira spp. were present in kidneys of wildlife across Ontario, particularly in areas of high human density, and areas in which livestock populations are abundant. The PCR was too weak in most samples to permit genotyping and examination of the relationship between the leptospires found in this study and those affecting domestic animals and humans. PMID:24587507

  5. Detection of Self Incompatibility Genotypes in Prunus africana: Characterization, Evolution and Spatial Analysis.

    PubMed

    Nantongo, Judith Ssali; Eilu, Gerald; Geburek, Thomas; Schueler, Silvio; Konrad, Heino

    2016-01-01

    In flowering plants, self-incompatibility is an effective genetic mechanism that prevents self-fertilization. Most Prunus tree species exhibit a homomorphic gametophytic self-incompatibility (GSI) system, in which the pollen phenotype is encoded by its own haploid genome. To date, no identification of S-alleles had been done in Prunus africana, the only member of the genus in Africa. To identify S-RNase alleles and hence determine S-genotypes in African cherry (Prunus africana) from Mabira Forest Reserve, Uganda, primers flanking the first and second intron were designed and these amplified two bands in most individuals. PCR bands on agarose indicated 26 and 8 different S-alleles for second and first intron respectively. Partial or full sequences were obtained for all these fragments. Comparison with published S-RNase data indicated that the amplified products were S-RNase alleles with very high interspecies homology despite the high intraspecific variation. Against expectations for a locus under balancing selection, frequency and spatial distribution of the alleles in a study plot was not random. Implications of the results to breeding efforts in the species are discussed, and mating experiments are strongly suggested to finally prove the functionality of SI in P. africana. PMID:27348423

  6. Genotyping by multiplex polymerase chain reaction for detection of endemic hepatitis B virus transmission.

    PubMed Central

    Repp, R; Rhiel, S; Heermann, K H; Schaefer, S; Keller, C; Ndumbe, P; Lampert, F; Gerlich, W H

    1993-01-01

    A nested polymerase chain reaction (PCR) protocol was developed for rapid genotyping of hepatitis B virus (HBV). During the first PCR round, a universal HBV primer pair was used to amplify the entire pre-S region of the HBV genome. Within the pre-S region, many nucleotide exchanges are observed. These are partly correlated to the serological hepatitis B surface antigen subtypes. Five additional subtype-specific primers were selected from that region which, together with two universal non-group-specific primers, generated specific combinations of two to four DNA fragments of defined sizes. By this approach, 55 hepatitis B surface antigen-positive patients from a pediatric oncology unit in Germany were analyzed. Fifty-four patients who had been infected within 2 years had an identical pattern in the multiplex PCR, suggesting a common source of infection and person-to-person transmission within the unit. One child who was infected 5 years later had a different PCR pattern and, therefore, must have been infected from a different source. Furthermore, 109 serum samples taken from pregnant Cameroonian women and 25 serum samples from their babies taken 6 months after birth were analyzed. In one case, mother-to-infant transmission of the virus was demonstrated. Apart from its role in epidemiological studies on HBV, multiplex PCR may also be a useful tool for rapid genetic analysis in other fields if there is a moderate degree of sequence variation which enables the design of specific primers. Images PMID:8501209

  7. Detection of Self Incompatibility Genotypes in Prunus africana: Characterization, Evolution and Spatial Analysis

    PubMed Central

    Nantongo, Judith Ssali; Eilu, Gerald; Geburek, Thomas; Schueler, Silvio; Konrad, Heino

    2016-01-01

    In flowering plants, self-incompatibility is an effective genetic mechanism that prevents self-fertilization. Most Prunus tree species exhibit a homomorphic gametophytic self-incompatibility (GSI) system, in which the pollen phenotype is encoded by its own haploid genome. To date, no identification of S-alleles had been done in Prunus africana, the only member of the genus in Africa. To identify S-RNase alleles and hence determine S-genotypes in African cherry (Prunus africana) from Mabira Forest Reserve, Uganda, primers flanking the first and second intron were designed and these amplified two bands in most individuals. PCR bands on agarose indicated 26 and 8 different S-alleles for second and first intron respectively. Partial or full sequences were obtained for all these fragments. Comparison with published S-RNase data indicated that the amplified products were S-RNase alleles with very high interspecies homology despite the high intraspecific variation. Against expectations for a locus under balancing selection, frequency and spatial distribution of the alleles in a study plot was not random. Implications of the results to breeding efforts in the species are discussed, and mating experiments are strongly suggested to finally prove the functionality of SI in P. africana. PMID:27348423

  8. Accurate detection of Neisseria gonorrhoeae ciprofloxacin susceptibility directly from genital and extragenital clinical samples: towards genotype-guided antimicrobial therapy

    PubMed Central

    Pond, Marcus J.; Hall, Catherine L.; Miari, Victoria F.; Cole, Michelle; Laing, Ken G.; Jagatia, Heena; Harding-Esch, Emma; Monahan, Irene M.; Planche, Timothy; Hinds, Jason; Ison, Catherine A.; Chisholm, Stephanie; Butcher, Philip D.; Sadiq, Syed Tariq

    2016-01-01

    Introduction Increasing use of nucleic acid amplification tests (NAATs) as the primary means of diagnosing gonococcal infection has resulted in diminished availability of Neisseria gonorrhoeae antimicrobial susceptibility data. We conducted a prospective diagnostic assessment of a real-time PCR assay (NGSNP) enabling direct detection of gonococcal ciprofloxacin susceptibility from a range of clinical sample types. Methods NGSNP, designed to discriminate an SNP associated with ciprofloxacin resistance within the N. gonorrhoeae genome, was validated using a characterized panel of geographically diverse isolates (n = 90) and evaluated to predict ciprofloxacin susceptibility directly on N. gonorrhoeae-positive NAAT lysates derived from genital (n = 174) and non-genital (n = 116) samples (n = 290), from 222 culture-confirmed clinical episodes of gonococcal infection. Results NGSNP correctly genotyped all phenotypically susceptible (n = 49) and resistant (n = 41) panel isolates. Ciprofloxacin-resistant N. gonorrhoeae was responsible for infection in 29.7% (n = 66) of clinical episodes evaluated. Compared with phenotypic susceptibility testing, NGSNP demonstrated sensitivity and specificity of 95.8% (95% CI 91.5%–98.3%) and 100% (95% CI 94.7%–100%), respectively, for detecting ciprofloxacin-susceptible N. gonorrhoeae, with a positive predictive value of 100% (95% CI 97.7%–100%). Applied to urogenital (n = 164), rectal (n = 40) and pharyngeal samples alone (n = 30), positive predictive values were 100% (95% CI 96.8%–100%), 100% (95% CI 87.2%–100%) and 100% (95% CI 82.4%–100%), respectively. Conclusions Genotypic prediction of N. gonorrhoeae ciprofloxacin susceptibility directly from clinical samples was highly accurate and, in the absence of culture, will facilitate use of tailored therapy for gonococcal infection, sparing use of current empirical treatment regimens and enhancing acquisition of susceptibility data for

  9. Sensitivity in detecting facial displays of emotion: Impact of maternal depression and oxytocin receptor genotype.

    PubMed

    Burkhouse, Katie L; Woody, Mary L; Owens, Max; McGeary, John E; Knopik, Valerie S; Gibb, Brandon E

    2016-01-01

    The current study examined sensitivity in detecting emotional faces among children of depressed and non-depressed mothers. A second goal was to examine the potential moderating role of the oxytocin receptor gene (OXTR rs53576), which has been linked to emotion recognition in the past. Participants included 247 children (ages 8-14). Children completed a forced choice emotion identification task. Maternal history of major depressive disorder during children's lives was associated with children's sensitivity in detecting emotional faces among children homozygous for the OXTR rs53576 G allele, but not among carriers of the A allele. Among G homozygotes, children of depressed mothers exhibited increased sensitivity in detecting sad faces, and reduced sensitivity in detecting happiness, compared to children of non-depressed mothers. PMID:25622005

  10. Rifampin Heteroresistance in Mycobacterium tuberculosis Cultures as Detected by Phenotypic and Genotypic Drug Susceptibility Test Methods

    PubMed Central

    Folkvardsen, Dorte Bek; Thomsen, Vibeke Ø.; Rigouts, Leen; Rasmussen, Erik Michael; Bang, Didi; Bernaerts, Gertjan; Werngren, Jim; Toro, Juan Carlos; Hoffner, Sven; Hillemann, Doris

    2013-01-01

    Tuberculosis patients may harbor both drug-susceptible and -resistant bacteria, i.e., heteroresistance. We used mixtures of rifampin-resistant and -susceptible Mycobacterium tuberculosis strains to simulate heteroresistance in patient samples. Molecular tests can be used for earlier discovery of multidrug resistance (MDR), but the sensitivity to detect heteroresistance is unknown. Conventional phenotypic drug susceptibility testing was the most sensitive, whereas two line probe assays and sequencing were unable to detect the clinically important 1% resistant bacteria. PMID:24068005

  11. Rapid detection of duck hepatitis A virus genotype C using reverse transcription loop-mediated isothermal amplification.

    PubMed

    Li, Chuanfeng; Chen, Zongyan; Meng, Chunchun; Liu, Guangqing

    2014-02-01

    A one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was used and optimized to develop a rapid and sensitive detection system for duck hepatitis A virus genotype C (DHAV-C) RNA. A set of four specific primers was designed against highly conserved sequences located within the 3D gene from DHAV (strain GX1201). Under optimal reaction conditions, the sensitivity of DHAV-C-specific RT-LAMP was 100-fold higher than that of reverse transcriptase-polymerase chain reaction (RT-PCR), with a detection limit of 0.3pg (6.59×10(4) copies) per reaction. No cross-reactivity was observed from the samples of other duck viruses, which is in good accordance with RT-PCR. Furthermore, a positive reaction can be visually inspected by observing turbidity or color change after the addition of SYBR green I dye. The DHAV-C-specific RT-LAMP assay was applied to the samples and compared with RT-PCR. The positive-sample ratios were 26.7% (12 of 45) by RT-LAMP and 20% (9 of 45) by RT-PCR. Therefore, the newly developed RT-LAMP assay is a rapid, specific, sensitive, and cost-effective method of DHAV-C detection. This assay has potential applications in both clinical diagnosis and field surveillance of DHAV-C infection. PMID:24291148

  12. AN EVALUATION OF CRYPTOSPORIDIUM PARVUM GENOTYPING

    EPA Science Inventory

    We evaluated the specificity and sensitivity of 11 previously described species differentiation and genotyping PCR protocols for detection of Cryptosporidium parasites. Genomic DNA from three species of Crytosporidium parasites (genotype 1 and genotype 2 of C. parvum, C. muris, a...

  13. International collaborative study to compare reverse transcriptase PCR assays for detection and genotyping of noroviruses.

    PubMed

    Vinjé, Jan; Vennema, Harry; Maunula, Leena; von Bonsdorff, Carl-Henrik; Hoehne, Marina; Schreier, Eckart; Richards, Alison; Green, Jon; Brown, David; Beard, Suzanne S; Monroe, Stephan S; de Bruin, Erwin; Svensson, Lennart; Koopmans, Marion P G

    2003-04-01

    To allow more rapid and internationally standardized assessment of the spread of noroviruses (previously called Norwalk-like viruses [NLVs]) as important food-borne pathogens, harmonization of methods for their detection is needed. Diagnosis of NLVs in clinical diagnostic laboratories is usually performed by reverse transciptase PCR (RT-PCR) assays. In the present study, the performance of five different RT-PCR assays for the detection of NLVs was evaluated in an international collaborative study by five laboratories in five countries with a coded panel of 91 fecal specimens. The assays were tested for their sensitivity, detection limit, and ease of standardization. In total, NLVs could be detected by at least one RT-PCR assay in 69 (84%) of the samples that originally tested positive. Sensitivity ranged from 52 to 73% overall and from 54 to 100% and 58 to 85% for genogroup I and II viruses, respectively. In all, 64% of the false-negative results were obtained with a set of diluted stools (n = 20) that may have lost quality upon storage. Sensitivity was improved when these samples were excluded from analysis. No one single assay stood out as the best, although the p1 assay demonstrated the most satisfactory overall performance. To promote comparability of data, this assay will be recommended for newly starting groups in future collaborative studies. PMID:12682125

  14. Detection of two porcine circovirus type 2 genotypic groups in United States swine herds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In late 2005, sporadic cases of an acute onset disease of high mortality were observed in 10- to 16-week-old growing pigs among several swine herds of the United States. Tissues from the affected pigs in Kansas, Iowa, and North Carolina were examined and porcine circovirus type 2 (PCV2) was detected...

  15. Sensitive detection of multiple hepatitis A virus genotypes with a single polony-based assay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Hepatitis A virus (HAV) is one of the major causes of non-bacterial gastroenteritis in humans worldwide. HAV is mostly transmitted via direct person-to-person contact, or by consumption of contaminated foods and water. Since only a few viral particles may cause disease, detection of low levels of HA...

  16. A new molecular diagnostic tool for quantitatively detecting and genotyping “Candidatus Liberibacter species”

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A new molecular diagnostic method was developed for quantitative detection of “Candidatus Liberibacter” species associated with citrus Huanglongbing (“Ca. Liberibacter asiaticus”, “Ca. Liberibacter africanus” and “Ca. Liberibacter americanus”) and potato zebra chip disorder (“Ca. Liberibacter solana...

  17. Type-specific detection of human papillomaviruses in Kazakh esophageal squamous cell carcinoma by genotyping both E6 and L1 genes with MALDI-TOF mass spectrometry

    PubMed Central

    Dong, Hong-Chao; Cui, Xiao-Bin; Wang, Liang-Hai; Li, Man; Shen, Yao-Yuan; Zhu, Jian-Bo; Li, Cheng-Fang; Hu, Jian-Ming; Li, Shu-Gang; Yang, Lei; Zhang, Wen-Jie; Chen, Yun-Zhao; Li, Feng

    2015-01-01

    Background: Many studies have suggested a relationship between human papillomavirus (HPV) infection and the risk of esophageal squamous cell carcinoma (ESCC). However, findings are inconclusive, potentially because of geographic heterogeneity and variations in detection methods. Objectives: We sought to further investigate the prevalence of HPV with a new detection method, the MassARRAY Sequenom technique, in esophageal squamous cell carcinomas occurring in patients belonging to Kazakh populations in Xinjiang, China. Study design: In the present study, a novel genotyping method for detecting 30 HPV genotypes, specifically by genotyping both the HPV E6 and L1 genes with multiplex PCR using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (PCR-MS) was first adopted to evaluate HPV genotypes in 89 esophageal cancer samples and 49 matched adjacent normal esophageal tissues. Results: Six HPV genotypes (HPV6, HPV16, HPV33, HPV39, HPV51, and HPV82) were present in at least 51.7% of the esophageal carcinoma tissues, which was significantly greater than 28.6% prevalence among controls (P < 0.05). HPV16 was the most common of all the genotypes investigated (HPV16 prevalence in carcinoma tissue: 49.4%; odds ratio 3.02, 95% confidence interval 1.39-6.53). HPV-positive ESCC patients were generally younger than HPV-negative patients (P = 0.04). In addition, HPV infection was more common in cases of well-differentiated and shallower invasive depth. Conclusions: Based on this new detection method, our findings reiterate the possibility that HPV infection (especially HPV16) may be involved in the etiology of esophageal carcinoma in the Kazakh populations and that HPV E6 gene positivity may be associated with prognosis of patients. PMID:26722514

  18. Biomedical Information Extraction: Mining Disease Associated Genes from Literature

    ERIC Educational Resources Information Center

    Huang, Zhong

    2014-01-01

    Disease associated gene discovery is a critical step to realize the future of personalized medicine. However empirical and clinical validation of disease associated genes are time consuming and expensive. In silico discovery of disease associated genes from literature is therefore becoming the first essential step for biomarker discovery to…

  19. Molecular detection and genotyping of Japanese Encephalitis Virus in mosquitoes during a 2010 outbreak in the Republic of Korea

    USGS Publications Warehouse

    Seo, Hyun-Ji; Kim, Heung Chul; Klein, Terry A.; Ramey, Andrew M.; Lee, Ji-Hyee; Kyung, Soon-Goo; Park, Jee-Yong; Cho, In-Soo; Yeh, Jung-Yong

    2013-01-01

    Japanese encephalitis virus (JEV), a mosquito-borne zoonotic pathogen, is one of the major causes of viral encephalitis. To reduce the impact of Japanese encephalitis among children in the Republic of Korea (ROK), the government established a mandatory vaccination program in 1967. Through the efforts of this program only 0-7 (mean 2.1) cases of Japanese encephalitis were reported annually in the ROK during the period of 1984-2009. However, in 2010 there was an outbreak of 26 confirmed cases of Japanese encephalitis, including 7 deaths. This represented a >12-fold increase in the number of confirmed cases of Japanese encephalitis in the ROK as compared to the mean number reported over the last 26 years and a 3.7-fold increase over the highest annual number of cases during this same period (7 cases). Surveillance of adult mosquitoes was conducted during the 2010 outbreak of Japanese encephalitis in the ROK. A total of 6,328 culicine mosquitoes belonging to 12 species from 5 genera were collected at 6 survey sites from June through October 2010 and assayed by reverse-transcription polymerase chain reaction (RT-PCR) for the presence of JEV. A total of 34/371 pooled samples tested positive for JEV (29/121 Culex tritaeniorhynchus, 4/64 Cx. pipiens, and 1/26 Cx. bitaeniorhynchus) as confirmed by sequencing of the pre-membrane and envelope protein coding genes. The maximum likelihood estimates of JEV positive individuals per 1,000 culicine vectors for Cx. tritaeniorhynchus, Cx. pipiens, and Cx. bitaeniorhynchus were 11.8, 5.6, and 2.8, respectively. Sequences of the JEV pre-membrane and envelope protein coding genes amplified from the culicine mosquitoes by RT-PCR were compared with those of JEV genotypes I-V. Phylogenetic analyses support the detection of a single genotype (I) among samples collected from the ROK in 2010.

  20. Molecular Detection and Genotyping of Japanese Encephalitis Virus in Mosquitoes during a 2010 Outbreak in the Republic of Korea

    PubMed Central

    Klein, Terry A.; Ramey, Andrew M.; Lee, Ji-Hye; Kyung, Soon-Goo; Park, Jee-Yong; Cho, Yun Sang; Cho, In-Soo; Yeh, Jung-Yong

    2013-01-01

    Japanese encephalitis virus (JEV), a mosquito-borne zoonotic pathogen, is one of the major causes of viral encephalitis. To reduce the impact of Japanese encephalitis among children in the Republic of Korea (ROK), the government established a mandatory vaccination program in 1967. Through the efforts of this program only 0–7 (mean 2.1) cases of Japanese encephalitis were reported annually in the ROK during the period of 1984–2009. However, in 2010 there was an outbreak of 26 confirmed cases of Japanese encephalitis, including 7 deaths. This represented a >12-fold increase in the number of confirmed cases of Japanese encephalitis in the ROK as compared to the mean number reported over the last 26 years and a 3.7-fold increase over the highest annual number of cases during this same period (7 cases). Surveillance of adult mosquitoes was conducted during the 2010 outbreak of Japanese encephalitis in the ROK. A total of 6,328 culicine mosquitoes belonging to 12 species from 5 genera were collected at 6 survey sites from June through October 2010 and assayed by reverse-transcription polymerase chain reaction (RT-PCR) for the presence of JEV. A total of 34/371 pooled samples tested positive for JEV (29/121 Culex tritaeniorhynchus, 4/64 Cx. pipiens, and 1/26 Cx. bitaeniorhynchus) as confirmed by sequencing of the pre-membrane and envelope protein coding genes. The maximum likelihood estimates of JEV positive individuals per 1,000 culicine vectors for Cx. tritaeniorhynchus, Cx. pipiens, and Cx. bitaeniorhynchus were 11.8, 5.6, and 2.8, respectively. Sequences of the JEV pre-membrane and envelope protein coding genes amplified from the culicine mosquitoes by RT-PCR were compared with those of JEV genotypes I-V. Phylogenetic analyses support the detection of a single genotype (I) among samples collected from the ROK in 2010. PMID:23390520

  1. [Basedow disease associated with Evans syndrome].

    PubMed

    Kuroda, Hiroyuki; Kida, Masaya; Watanabe, Hideki; Matsunaga, Takuya; Niitsu, Yoshiro; Matsumoto, Masanori

    2005-10-01

    A 60-year-old woman was admitted to a hospital complaining of dizziness and general fatigue in October, 2004. Because of heart failure and severe anemia, she was referred to our hospital. Based on a positive direct Coombs test and an elevated level of platelet-associated IgG (PAIgG), the patient was diagnosed as having autoimmune hemolytic anemia (AIHA) associated with idiopathic thrombocytopenic purpura (ITP), i.e., Evans syndrome. Basedow disease was also diagnosed due to hyperthyroidism with an elevation of anti-thyroid stimulating hormone (TSH) receptor antibodies. Both the Evans syndrome and Basedow disease were considerably ameliorated with plasma exchange, corticosteroid and thiamazole therapy. Although Basedow disease is known to be associated with hematological disorders such as AIHA or ITP, the combination of Basedow disease and Evans syndrome is rare. We report here a case of Basedow disease associated with Evans syndrome. PMID:16440774

  2. Detection and Genotyping of Coxiella burnetii and Coxiella-Like Bacteria in Horses in South Korea.

    PubMed

    Seo, Min-Goo; Lee, Seung-Hun; VanBik, Dorene; Ouh, In-Ohk; Yun, Sun-Hee; Choi, Eunsang; Park, Yong-Soo; Lee, Sang-Eun; Kim, Jong Wan; Cho, Gil-Jae; Kwon, Oh-Deog; Kwak, Dongmi

    2016-01-01

    Coxiella burnetii and Coxiella-like bacteria (CLB) are genetically and ecologically distinct despite some genetic similarities. Furthermore, CLB are exceptionally diverse and widespread in ticks, but rarely detected in domestic animals. Since Coxiella bacteria can be transmitted from infected horses by inhalation or by coming in contact with ticks during activities such as horseback riding, it is necessary to study their prevalence. To the best of our knowledge, this is the first large-scale nationwide investigation of the prevalence of C. burnetii and CLB among horses reared in South Korea. Of 816 blood samples collected between 2007 and 2013, 11 (1.3%) were identified as C. burnetii by ELISA, and six (0.7%) as CLB by 16S rRNA sequencing. While a sequence from Jeju Island was similar (97.9-100%) to those within clade B, five sequences obtained from the northern region were categorized into a new clade, indicating the sequence diversity of the genus Coxiella. Studies until date had detected CLB only in ticks; here, we describe their detection in mammals. Given their zoonotic potential, strategic monitoring and appropriate control programs for Coxiella species need to be established. PMID:27244230

  3. Detection and Genotyping of Coxiella burnetii and Coxiella-Like Bacteria in Horses in South Korea

    PubMed Central

    Seo, Min-Goo; Lee, Seung-Hun; VanBik, Dorene; Ouh, In-Ohk; Yun, Sun-Hee; Choi, Eunsang; Park, Yong-Soo; Lee, Sang-Eun; Kim, Jong Wan; Cho, Gil-Jae; Kwon, Oh-Deog

    2016-01-01

    Coxiella burnetii and Coxiella-like bacteria (CLB) are genetically and ecologically distinct despite some genetic similarities. Furthermore, CLB are exceptionally diverse and widespread in ticks, but rarely detected in domestic animals. Since Coxiella bacteria can be transmitted from infected horses by inhalation or by coming in contact with ticks during activities such as horseback riding, it is necessary to study their prevalence. To the best of our knowledge, this is the first large-scale nationwide investigation of the prevalence of C. burnetii and CLB among horses reared in South Korea. Of 816 blood samples collected between 2007 and 2013, 11 (1.3%) were identified as C. burnetii by ELISA, and six (0.7%) as CLB by 16S rRNA sequencing. While a sequence from Jeju Island was similar (97.9–100%) to those within clade B, five sequences obtained from the northern region were categorized into a new clade, indicating the sequence diversity of the genus Coxiella. Studies until date had detected CLB only in ticks; here, we describe their detection in mammals. Given their zoonotic potential, strategic monitoring and appropriate control programs for Coxiella species need to be established. PMID:27244230

  4. Detection of Anaplasma phagocytophilum genotypes that are potentially virulent for human in wild ruminants and Ixodes ricinus in Central Italy.

    PubMed

    Di Domenico, M; Pascucci, I; Curini, V; Cocco, A; Dall'Acqua, F; Pompilii, C; Cammà, C

    2016-07-01

    Human granulocytic anaplasmosis (HGA) is an emerging tick-borne zoonosis worldwide. As is the case for many tick-borne diseases, the epidemiological cycle is associated to the environmental conditions, including the presence of wild vertebrate reservoir hosts, vectors, climate and vegetation. In this study a total number of 87 spleen samples of wild ruminants carcasses from Central Italy, and 77 Ixodes ricinus collected from the same dead animals were screened for Anaplasma phagocytophilum by using Real Time PCR. A. phagocytophilum DNA was detected in 75%, 66.7% and 54.2% of the spleen samples from red deer (Cervus elaphus), Apennine chamois (Rupicapra pyrenaica ornata) and roe deer (Capreolus capreolus) respectively, whereas it was detected in the 31.2% of I. ricinus. A total of 27 positive samples were characterized by sequencing a portion of the groEL gene. Two A. phagocytophilum lineages could clearly be delineated from the phylogenetic tree. Four sequences from red deer, 2 from I. ricinus and 1 from Apennine chamois clustered into lineage I together with those previously described as virulent genotypes related to HGA. The presence of A. phagocytophilum DNA in the Apennine chamois represents the first report for this Italian endemic subspecies. PMID:27020736

  5. Agreement for HPV genotyping detection between self-collected specimens on a FTA cartridge and clinician-collected specimens

    PubMed Central

    Guan, YaoYao; Gravitt, Patti E.; Howard, Roslyn; Eby, Yolanda J.; Wang, Shaoming; Li, Belinda; Feng, Changyan; Qiao, You-Lin; Castle, Philip E.

    2016-01-01

    The current method of transporting self-collected cervicovaginal specimen for HPV DNA testing relies on liquid based medium, which is challenging and expensive to transport. A novel, dry storage and transportation device, Whatman indicating FTA™ Elute Cartridge, avoids some of the pitfalls of liquid-based medium. This method has been shown to be comparable to liquid-based collection medium, but relative performance of self-collected (SC) and clinician-collected (CC) samples onto FTA cards has not been reported. The objective of this study is to compare the analytic performance of self- and clinician-collected samples onto FTA cartridges for the detection of carcinogenic HPV using Linear Array. There was a 91% agreement, 69% positive agreement, and kappa of 0.75 between the clinician-collected and self-collected specimens for detection of any carcinogenic HPV genotype. When the HPV results were categorized hierarchically according to cervical cancer risk, there was no difference in the distribution of the HPV results for the clinician- and self-collected specimens (p = 0.7). This study concludes that FTA elute cartridge is a promising method of specimen transport for cervical cancer screening programs considering using self-collected specimen and HPV testing. Larger studies with clinical endpoints are now needed to assess the clinical performance. PMID:23370404

  6. Phenotypic and genotypic detection of methicillin-resistant Staphylococcus aureus in hunting dogs in Maiduguri metropolitan, Borno State, Nigeria

    PubMed Central

    Mustapha, Muhammad; Bukar-Kolo, Yachilla Maryam; Geidam, Yaqub Ahmed; Gulani, Isa Adamu

    2016-01-01

    Aim: To determine the presence of MRSA in hunting dogs in Maiduguri metropolitan. Materials and Methods: Phenotypic methods used includes microscopic technique, colony morphology study, catalase-coagulase tests, and the use of mannitol salt agar test, oxacillin resistance screening agar base, and antibiotic susceptibility testing methods. Genotypic approach was used for deoxyribonucleic acid extraction, and the presence of nuc and mecA gene was detected using polymerase chain reaction (PCR) techniques. Results: Examination of 416 swab samples from nasal and perineal region of dogs revealed a total of 79.5% of S. aureus, where 62.5% of the isolates were MRSA. Molecular analysis revealed that 7nuc genes specific for S. aureus from 20 presumptive MRSA assay were all mecA PCR negative. The isolates were sensitive to gentamicin and ciprofloxacin but proved resistant to cefoxitin and oxacillin. Conclusion: High isolation rate of MRSA was found in hunting dogs. Significant level (p<0.05) of MRSA was isolated in the nasal cavity of hunting dogs than its perineum. Only nuc genes were detected from the MRSA isolates. PMID:27284227

  7. A finite mixture model for genotype and environment interactions: Detecting latent population heterogeneity

    PubMed Central

    Gillespie, Nathan A.; Neale, Michael C.

    2013-01-01

    Approaches such as DeFries-Fulker extremes regression (LaBuda et al., 1986) are commonly used in genetically informative studies to assess whether familial resemblance varies as a function of the scores of pairs of twins. While useful for detecting such effects, formal modelling of differences in variance components as a function of pairs' trait scores is rarely attempted. We therefore present a finite mixture model which specifies that the population consists of latent groups which may differ in i) their means, and ii) the relative impact of genetic and environmental factors on within-group variation and covariation. This model may be considered as a special case of a factor mixture model, which combines the features of a latent class model with those of a latent trait model. Various models for the class membership of twin pairs may be employed, including additive genetic, common environment, specific environment or major locus (QTL) factors. Simulation results based on variance components derived from Turkheimer and colleagues (2003), illustrate the impact of factors such as the difference in group means and variance components on the feasibility of correctly estimating the parameters of the mixture model. Model-fitting analyses estimated group heritability as .49, which is significantly greater than heritability for the rest of the population in early childhood. These results suggest that factor mixture modelling is sufficiently robust for detecting heterogeneous populations even when group mean differences are modest. PMID:16790151

  8. Helicobacter pylori resistance to antibiotics in 2014 in France detected by phenotypic and genotypic methods.

    PubMed

    Ducournau, A; Bénéjat, L; Sifré, E; Bessède, E; Lehours, P; Mégraud, F

    2016-08-01

    A large survey of antimicrobial resistance of Helicobacter pylori was performed in France in 2014: 984 patients were enrolled by 75 gastroenterologists all over the country. Among the 783 patients who had never received eradication treatment before, 266 (33.9%) were H. pylori positive. The strains showed a high rate of clarithromycin resistance (22.2%), moderate rate of resistance to levofloxacin (15.4%) and high rate of resistance to metronidazole (45.9%). In all, 187 patients had received previous treatment, of which 115 were H. pylori positive with very high resistance to clarithromycin (73.9%) and metronidazole (78.3%). None of the patients receiving PYLERA (Bismuth salt-Tetracycline HCl-Metronidazole) proton-pump inhibitor developed resistance to tetracycline. A real-time PCR applied to gastric biopsy specimens detected all the cases that were positive by culture as well as 30 additional cases. A good correlation was found between the clarithromycin resistance detected by phenotypic methods and the associated mutations for clarithromycin resistance, which has continued to increase in the last decade but at a lower rate than previously observed. PMID:27345177

  9. Oligoclonal bands in the cerebrospinal fluid of amyotrophic lateral sclerosis patients with disease-associated mutations

    PubMed Central

    Mencacci, Niccolò E.; Morelli, Claudia; Doretti, Alberto; Rusconi, Daniela; Colombrita, Claudia; Sangalli, Davide; Verde, Federico; Finelli, Palma; Messina, Stefano; Ratti, Antonia; Silani, Vincenzo

    2014-01-01

    In amyotrophic lateral sclerosis (ALS) cerebrospinal fluid (CSF) analysis is usually performed to exclude inflammatory processes of the central nervous system. Although in a small subset of patients an intrathecal synthesis of IgG is detectable, usually there is no clear explanation for this evidence. This study investigates the occurrence of oligoclonal bands (OCBs) in the CSF of a large series of ALS patients, attempting a correlation with genotype data. CSF was collected from 259 ALS patients. CSF parameters were measured according to standard procedures, and detection of OCBs performed by isoelectric focusing. The patients were screened for mutations in SOD1, FUS, TARDBP, ANG, OPTN, and C9ORF72. We observed the presence of OCBs in the CSF of 9/259 ALS patients (3.5 %), and of disease-associated mutations in 12 cases. OCBs were significantly more frequent in mutation carriers compared to the remaining cohort (3/12 vs 6/247; p < 0.01). Among patients with OCBs, two patients had the TARDBP p.A382T mutation (one of which in homozygous state), and one the ANG p.P-4S variant. Both patients carrying the p.A382T mutation had an atypical phenotype, one of them manifesting signs suggestive of a cerebellar involvement, and the other presenting neuroradiological findings suggestive of an inflammatory disorder of the central nervous system. Our results suggest that ALS patients with OCBs may harbor mutations in disease-causing genes. We speculate that mutations in both TARDBP and ANG genes may disrupt the blood–brain barrier (BBB), promoting local immune responses and neuroinflammation. The role of mutant TARDBP and ANG genes on BBB integrity of ALS patients warrants further investigation. PMID:22752089

  10. Cladograms with Path to Event (ClaPTE): A novel algorithm to detect associations between genotypes or phenotypes using phylogenies

    PubMed Central

    Handelman, Samuel K; Aaronson, Jacob M.; Seweryn, Michal; Voronkin, Igor; Kwiek, Jesse J.; Sadee, Wolfgang; Verducci, Joseph S.; Janies, Daniel A.

    2015-01-01

    Background Associations between genotype and phenotype provide insight into the evolution of pathogenesis, drug resistance, and the spread of pathogens between hosts. However, common ancestry can lead to apparent associations between biologically unrelated features. The novel method Cladograms with Path to Event (ClaPTE) detects associations between character-pairs (either a pair of mutations or a mutation paired with a phenotype) while adjusting for common ancestry, using phylogenetic trees. Methods ClaPTE tests for character-pairs changing close together on the phylogenetic tree, consistent with an associated character-pair. ClaPTE is compared to three existing methods (independent contrasts, mixed model, and likelihood ratio) to detect character-pair associations adjusted for common ancestry. Comparisons utilize simulations on gene trees for: HIV Env, HIV promoter, and bacterial DnaJ and GuaB; and case studies for Oseltamavir resistance in Influenza, and for DnaJ and GuaB. Simulated data include both true-positive/associated character-pairs, and true-negative/not-associated character-pairs, used to assess type I (frequency of p-values in true-negatives) and type II (sensitivity to true-positives) error control. Results and conclusions ClaPTE has competitive sensitivity and better type I error control than existing methods. In the Influenza/Oseltamavir case study, ClaPTE reports no new permissive mutations but detects associations between adjacent (in primary sequence) amino acid positions which other methods miss. In the DnaJ and GuaB case study, ClaPTE reports more frequent associations between positions both from the same protein family than between positions from different families, in contrast to other methods. In both case studies, the results from ClaPTE are biologically plausible. PMID:25577610

  11. UDP-glucuronosyltransferase 2B17 genotyping in Japanese athletes and evaluation of the current sports drug testing for detecting testosterone misuse.

    PubMed

    Okano, Masato; Ueda, Toshihiko; Nishitani, Yasunori; Kano, Hiroko; Ikekita, Ayako; Kageyama, Shinji

    2013-03-01

    Ethnicity has been found to influence urinary testosterone glucuronide to epitestosterone glucuronide (T/E) ratios among athletes. Uridine diphospho-glucuronosyltransferase 2B17 (UGT2B17) is the most active enzyme in testosterone glucuronidation. UGT2B17 polymorphism analysis is rarely performed in Japanese athletes, and the influence of testosterone administration on steroid profiles and carbon isotope ratios, according to gene polymorphisms, in Asians remains unknown. The prevalence of UGT2B17 genotypes and urinary androgenic steroid profiles, classified according to UGT2B17 genotypes, was investigated in Japanese athletes (255 male and 256 female). Testosterone enanthate (100 mg) was administered intramuscularly to Japanese female volunteers (del/del: n = 6, del/ins: n = 3, ins/ins: n = 1). The distribution rates of the UGT2B17 del/del genotype in Japanese male and female athletes were 74.5% and 60.2%, respectively. The ins/ins genotype was detected in only three male (1.2%) and seven female (2.7%) athletes. The prevalence of the UGT2B17 deletion genotype was extremely high in Japanese athletes. The T/E ratio in the del/del group was significantly lower than that in the other groups. After testosterone was administered to female volunteers, the T/E ratios for the del/del individuals failed to reach the positivity criterion of 4. By contrast, in all of the del/del subjects, the gas chromatography/combustion/isotope ratio mass spectrometry (GC-C-IRMS) analysis successfully fulfilled the positivity criterion. The overall result has demonstrated the limited effectiveness of population-based T/E ratios in screening tests for testosterone use. Subject-based steroid profiling with UGT2B17 genotyping will be an effective strategy for detecting testosterone misuse. PMID:22887913

  12. HLA and Disease Associations in Koreans

    PubMed Central

    Ahn, Stephen; Choi, Hee-Back

    2011-01-01

    The human leukocyte antigen (HLA), the major histocompatibility complex (MHC) in humans has been known to reside on chromosome 6 and encodes cell-surface antigen-presenting proteins and many other proteins related to immune system function. The HLA is highly polymorphic and the most genetically variable coding loci in humans. In addition to a critical role in transplantation medicine, HLA and disease associations have been widely studied across the populations world-wide and are found to be important in prediction of disease susceptibility, resistance and of evolutionary maintenance of genetic diversity. Because recently developed molecular based HLA typing has several advantages like improved specimen stability and increased resolution of HLA types, the association between HLA alleles and a given disease could be more accurately quantified. Here, in this review, we have collected HLA association data on some autoimmune diseases, infectious diseases, cancers, drug responsiveness and other diseases with unknown etiology in Koreans and attempt to summarize some remarkable HLA alleles related with specific diseases. PMID:22346771

  13. Renal disease associated with colic in horses.

    PubMed

    Seanor, J W; Byars, T D; Boutcher, J K

    1984-05-01

    Renal dysfunction secondary to GI disorders may be relatively common in horses. Persistent dehydration of 8-10% of body weight can lead to prerenal azotemia, which may result in renal ischemia and renal disease if uncorrected. Dehydrated azotemic horses with a urine specific gravity less than 1.018 may have renal disease. Urine specific gravity readings greater than 1.025 usually indicate normal kidney function. A urine Na level less than 20 mEq/L and a urine/plasma creatinine ratio greater than or equal to 20:1 indicate prerenal problems. Use of nephrotoxic drugs should be avoided in septicemic or dehydrated horses. Salmonellosis and proximal enteritis often lead to renal complications. Renal disease associated with DIC warrants a poor prognosis. Treatment of acute renal failure is aimed at eliminating the underlying cause and correcting metabolic abnormalities. Use of IV fluids, dopamine, prostaglandin inhibitors, fresh and electrolyte-spiked water ad libitum, water-soluble vitamins and high-P diets is beneficial. Success of therapy should be judged by laboratory results rather than clinical impressions. PMID:6738502

  14. Detection of parasitizing coccidia and determination of host crane species, sex and genotype by faecal DNA analysis.

    PubMed

    Honma, H; Suyama, Y; Nakai, Y

    2011-11-01

    In Japan, the three main crane species are the endangered red-crowned crane (Grus japonensis) inhabiting Hokkaido, the northernmost island of Japan; the vulnerable hooded crane (Grus monacha); and the vulnerable white-naped crane (Grus vipio). Both the hooded and white-naped cranes migrate in winter to Izumi in Kyushu, the southern island of Japan. In this study, we investigated the cranes and their coccidian parasites, through a targeted molecular approach using faecal DNA to develop a noninvasive method for infectious disease research. To determine the origin of noninvasively collected faecal samples, host species were identified by sequencing a region of approximately 470 bp of the mitochondrial 16S ribosomal RNA gene in the faecal DNA. Furthermore, to avoid sample redundancy, individual determination was performed by fragment analysis using microsatellite and sex-linked markers. For microsatellite genotyping, previously reported markers and markers isolated in this study were examined, and seven loci for red-crowned cranes, eight for hooded cranes and six for white-naped cranes displayed polymorphisms. A low error rate was demonstrated by comparing microsatellite data generated from faecal DNA samples with that generated from feather DNA samples, indicating a high reliability. Polymerase chain reaction-based capillary electrophoresis (PCR-CE), employing genetic markers in the second internal transcribed spacer (ITS2) of nuclear ribosomal DNA, was employed to detect crane coccidia. The sensitivity of detection of PCR-CE using faecal DNA was inferior to that with traditional microscopy; however, our results suggest that PCR-CE can depict crane coccidia diversity with higher resolution and it is a useful tool to characterize community composition of coccidia in detail. PMID:21791031

  15. Development of a Real-Time, TaqMan Reverse Transcription-PCR Assay for Detection and Differentiation of Lyssavirus Genotypes 1, 5, and 6

    PubMed Central

    Wakeley, P. R.; Johnson, N.; McElhinney, L. M.; Marston, D.; Sawyer, J.; Fooks, A. R.

    2005-01-01

    Several reverse transcription-PCR (RT-PCR) methods have been reported for the detection of rabies and rabies-related viruses. These methods invariably involve multiple transfers of nucleic acids between different tubes, with the risk of contamination leading to the production of false-positive results. Here we describe a single, closed-tube, nonnested RT-PCR with TaqMan technology that distinguishes between classical rabies virus (genotype 1) and European bat lyssaviruses 1 and 2 (genotypes 5 and 6) in real time. The TaqMan assay is rapid, sensitive, and specific and allows for the genotyping of unknown isolates concomitant with the RT-PCR. The assay can be applied quantitatively and the use of an internal control enables the quality of the isolated template to be assessed. Despite sequence heterogeneity in the N gene between the different genotypes, a universal forward and reverse primer set has been designed, allowing for the simplification of previously described assays. We propose that within a geographically constrained area, this assay will be a useful tool for the detection and differentiation of members of the Lyssavirus genus. PMID:15956398

  16. Development and Evaluation of a Real-Time RT-qPCR for Detection of Crimean-Congo Hemorrhagic Fever Virus Representing Different Genotypes

    PubMed Central

    Kallio-Kokko, Hannimari; Ozkul, Aykut; Bodur, Hurrem; Korukruoglu, Gulay; Mousavi, Mehrdad; Pranav, Patel; Vaheri, Antti; Mirazimi, Ali; Vapalahti, Olli

    2014-01-01

    Abstract Crimean–Congo hemorrhagic fever (CCHF) is a zoonotic disease caused by a nairovirus belonging to family Bunyaviridae. The CCHF virus (CCHFV) can be transmitted to humans by Hyalomma ticks as well as by direct contact with infected body fluids or tissues from viremic livestock or humans. Our aim was to set up a fast RT-qPCR for detection of the different CCHFV genotypes in clinical samples, including an inactivation step to make the sample handling possible in lower biosafety levels (BSL) than BSL-4. This method was evaluated against commercial reference assays and international External Quality Assessment (EQA) samples. The analytical limit of detection for the developed CCHFV-S RT-qPCR was 11 CCHFV genomes per reaction. After exclusion of four dubious samples, we studied 38 CCHFV-positive samples (using reference tests) of which 38 were found positive by CCHFV-S RT-qPCR, suggesting a sensitivity of 100%. CCHFV-S RT q-PCR detected all eight different CCHFV strains representing five different CCHFV genotypes. In conclusion, the CCHFV-S RT-qPCR described in this study was evaluated using various sources of CCHFV samples and shown to be an accurate tool to detect human CCHFV infection caused by different genotypes of the virus. PMID:25514124

  17. Genotyping of 27 human papillomavirus types by using L1 consensus PCR products by a single-hybridization, reverse line blot detection method.

    PubMed

    Gravitt, P E; Peyton, C L; Apple, R J; Wheeler, C M

    1998-10-01

    Amplification of human papillomavirus (HPV) DNA by L1 consensus primer systems (e.g., MY09/11 or GP5(+)/6(+)) can detect as few as 10 to 100 molecules of HPV targets from a genital sample. However, genotype determination by dot blot hybridization is laborious and requires at least 27 separate hybridizations for substantive HPV-type discrimination. A reverse blot method was developed which employs a biotin-labeled PCR product hybridized to an array of immobilized oligonucleotide probes. By the reverse blot strip analysis, genotype discrimination of multiple HPV types can be accomplished in a single hybridization and wash cycle. Twenty-seven HPV probe mixes, two control probe concentrations, and a single reference line were immobilized to 75- by 6-mm nylon strips. Each individual probe line contained a mixture of two bovine serum albumin-conjugated oligonucleotide probes specific to a unique HPV genotype. The genotype spectrum discriminated on this strip includes the high-risk, or cancer-associated, HPV genotypes 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 55, 56, 58, 59, 68 (ME180), MM4 (W13B), MM7 (P291), and MM9 (P238A) and the low-risk, or non-cancer-associated, genotypes 6, 11, 40, 42, 53, 54, 57, 66, and MM8 (P155). In addition, two concentrations of beta-globin probes allowed for assessment of individual specimen adequacy following amplification. We have evaluated the performance of the strip method relative to that of a previously reported dot blot format (H. M. Bauer et al., p. 132-152, in C. S. Herrington and J. O. D. McGee (ed.), Diagnostic Molecular Pathology: a Practical Approach, (1992), by testing 328 cervical swab samples collected in Digene specimen transport medium (Digene Diagnostics, Silver Spring, Md.). We show excellent agreement between the two detection formats, with 92% concordance for HPV positivity (kappa = 0.78, P < 0.001). Nearly all of the discrepant HPV-positive samples resulted from weak signals and can be attributed to sampling error from

  18. Development of a SYBR-Green Ⅰ quantitative PCR assay for the detection and genotyping of different hantaviruses.

    PubMed

    Liu, Ziyu; Wang, Fang; Yuan, Lijuan; Zhang, Xiaoxiao; Ying, Qikang; Yu, Lan; Zhang, Liang; Cheng, Linfeng; Zhang, Fanglin; Lu, Jianguo; Wu, Xing'an

    2016-09-01

    Hemorrhagic fever with renal syndrome (HFRS) is a severe, viral zoonotic disease which occurs worldwide, particularly in Asia and Europe. In China, the Hantaan virus (HTNV) and the Seoul virus (SEOV) are known to be the most prevalent causative agents of HFRS. Since no protective vaccines or effective treatments are available for human use, accurate and reliable diagnostic methods are essential for disease surveillance. In the present study, the viral loads in cell culture supernatant, infected mice blood and clinical serum samples were quantified using the SYBR‑Green I-based reverse transcription-quantitiative polymerase chain reaction (RT-qPCR) assay, which targeted the S gene sequence of the HTNV and SEOV genomes. The cRNA of these two viruses were synthesized as a positive control and 10-fold serially diluted from 1x105 to 1x100 copies/µl. Standard curves were generated by plotting the mean cycle threshold (Ct) values versus copy numbers. The standard curve of HTNV had a correlation coefficient (R2) of 0.994, efficiency of amplification (E) of 101.9%, and the slope of -3.278, whereas that of SEOV had an R2 of 0.993, E of 104.8%, and the slope of -3.212. The minimum detection limit of the RT-qPCR assay for HTNV and SEOV was 101 copies/µl. Two qPCR assays were successfully established for the detection of HTNV and SEOV, respectively. Taken together, these findings demonstrate that using the SYBR‑Green I-based RT-qPCR assay, the HTNV and SEOV may be genotyped precisely without cross-reactivity. Furthermore, viral RNA may be detected and quantified in cells, mice and infected individuals, which may be useful in epidemiological studies as well as for early monitoring and further preventative treatment against SEOV and HTNV-induced diseases. PMID:27430149

  19. Genotype MTBDRplus for Direct Detection of Mycobacterium tuberculosis and Drug Resistance in Strains from Gold Miners in South Africa

    PubMed Central

    Chihota, Violet N.; Lewis, James J.; van der Meulen, Minty; Mathema, Barun; Beylis, Natalie; Fielding, Katherine L.; Grant, Alison D.; Churchyard, Gavin J.

    2012-01-01

    GenoType MTBDRplus is a molecular assay for detection of Mycobacterium tuberculosis and drug resistance. Assay performance as applied directly to consecutive unselected sputum samples has not been established. The objective of this study was to determine the accuracy of the MTBDRplus test for direct detection of M. tuberculosis (in sputum) and for drug resistance in consecutively submitted sputum samples. In this cross-sectional study in South Africa, one sputum specimen from each person suspected of having pulmonary tuberculosis was tested by smear microscopy, direct MTBDRplus, and Mycobacterial Growth Indicator Tube (MGIT) culture with MGIT drug susceptibility testing. MGIT results were the reference standard. We tested 2,510 sputum samples, and 529 (21.1%) were positive for M. tuberculosis by MGIT. Direct MTBDRplus identified M. tuberculosis in 256 of 529 specimens (sensitivity, 48.4%; 95% confidence interval [CI], 44.1, 52.7). The sensitivity of MTBDRplus for M. tuberculosis detection by sputum smear status was as follows: smear negative, 13.7% (95% CI, 9.8, 18.4); smear scanty, 46.2% (95% CI, 19.2, 74.9); smear 1+, 69.1% (95% CI, 55.2, 80.9); smear 2+, 86.3% (95% CI, 73.7, 94.3); smear 3+, 89.8% (95% CI, 83.7, 94.2). Direct MTBDRplus testing was negative for 1,594/1,612 sputum samples that were culture negative for M. tuberculosis (specificity, 98.9%; 95% CI, 98.2, 99.3). For specimens positive for M. tuberculosis by MTBDRplus, this assay's sensitivity and specificity for rifampin resistance were 85.7% (95% CI, 57.2, 98.2) and 96.6% (95% CI, 93.2, 98.6) and for isoniazid resistance they were 62.1% (95% CI, 42.3, 79.3) and 97.9% (95% CI, 94.8, 99.4). For sputum testing, the sensitivity of MTBDRplus is directly related to the specimen's bacillary burden. Our results support recommendations that the MTBDRplus test not be used for direct testing of smear-negative or paucibacillary sputum samples. PMID:22238443

  20. Phenotypic assays and sequencing are less sensitive than point mutation assays for detection of resistance in mixed HIV-1 genotypic populations.

    PubMed

    Van Laethem, K; Van Vaerenbergh, K; Schmit, J C; Sprecher, S; Hermans, P; De Vroey, V; Schuurman, R; Harrer, T; Witvrouw, M; Van Wijngaerden, E; Stuyver, L; Van Ranst, M; Desmyter, J; De Clercq, E; Vandamme, A M

    1999-10-01

    The sensitivity and discriminatory power of the 151 and 215 amplification refractory mutation system (ARMS) were evaluated, and their performance for the detection of drug resistance in mixed genotypic populations of the reverse transcription (RT) gene of HIV-1 were compared with T7 sequencing, cycle sequencing, the line probe assay (LiPA) HIV-1 RT test, and the recombinant virus assay (RVA). ARMS and the LiPA HIV-1 RT test were shown to be able to detect minor variants that in particular cases comprised only 1%. T7 sequencing on an ALF semiautomated sequencer could correctly score mixtures only when variants were present at 50%. Cycle sequencing on an ABI PRISM 310 improved the sensitivity for mixtures to about 25%. Using RVA, it was shown that at least 50% of the virus population needed to carry the resistance mutation at codon 184 to afford phenotypic resistance against lamivudine. The two point mutation assays therefore proved to be more sensitive methods than sequencing and RVA to reliably determine a gradual shift in HIV-1 drug resistance mutations in follow-up of patients infected with HIV-1. In 4 of 5 treated patients who were followed by ARMS, a gradual shift in resistant genotypic populations was observed during a period of 6 to 19 months. For 1 patient, a shift from wild to mutant type at position 151 occurred within 2 months, without mixed genotypic intermediate type's being detected. PMID:10843523

  1. Detection of rotavirus species A, B and C in domestic mammalian animals with diarrhoea and genotyping of bovine species A rotavirus strains.

    PubMed

    Otto, Peter H; Rosenhain, Stefanie; Elschner, Mandy C; Hotzel, Helmut; Machnowska, Patrycja; Trojnar, Eva; Hoffmann, Kathrin; Johne, Reimar

    2015-09-30

    Rotaviruses (RVs) are a major cause of neonatal diarrhoea in humans and animals worldwide. In this study, 425 faecal samples were collected between 1999 and 2013 from diarrhoeic livestock and companion animals at different locations in Germany and tested for RVs. A previously published real-time RT-PCR assay was optimized for detection of a larger variety of RV species A (RVA) strains, and real-time RT-PCR assays for detection of RV species B (RVB) and C (RVC) were newly developed. The detection limits of the assays were 1.54×10(2), 3.95×10(2) and 3.60×10(3) genome copies for RVA, RVB and RVC, respectively. RVA was identified in 85.2% of bovine samples, 51.2% of porcine samples, 50.0% of feline samples, 43.2% of equine samples and 39.7% of canine samples. RVB was found in 3.0% of bovine samples, 2.7% of equine samples and 1.6% of porcine samples. RVC was detected in 31.0% of porcine samples, 21.7% of feline samples, 9.0% of canine samples and 6.0% of bovine samples. For genotyping, 101 RVA-positive bovine samples were further analysed by semi-nested RT-PCR. Genotype combination G6P[5] was most frequently detected (67.3% of samples), followed by G6P[11] (13.9%), G10P[5] (4.0%), G8P[11] (3.0%), G6P[1] (1.0%), and G10P[11] (1.0%). Mixed RVA infections were detected in 5.9% of samples; no or incomplete typing was possible in 4.0% of the samples. This first overview on RV species and RVA genotypes in diarrhoeic livestock and companion animals from Germany indicates a broad circulation of a large variety of RVs. PMID:26223422

  2. Detection of a knockdown resistance mutation associated with permethrin resistance in the body louse Pediculus humanus corporis by use of melting curve analysis genotyping.

    PubMed

    Drali, Rezak; Benkouiten, Samir; Badiaga, Sékéné; Bitam, Idir; Rolain, Jean Marc; Brouqui, Philippe

    2012-07-01

    Louse-borne diseases are prevalent in the homeless, and body louse eradication has thus far been unsuccessful in this population. We aim to develop a rapid and robust genotyping method usable in large field-based clinical studies to monitor permethrin resistance in the human body louse Pediculus humanus corporis. We assessed a melting curve analysis genotyping method based on real-time PCR using hybridization probes to detect the M815I-T917I-L920F knockdown resistance (kdr) mutation in the paraorthologous voltage-sensitive sodium channel (VSSC) α subunit gene, which is associated with permethrin resistance. The 908-bp DNA fragment of the VSSC gene, encoding the α subunit of the sodium channel and encompassing the three mutation sites, was PCR sequenced from 65 lice collected from a homeless population. We noted a high prevalence of the 3 indicated mutations in the body lice collected from homeless people (100% for the M815I and L920F mutations and 56.73% for the T917I mutation). These results were confirmed by melting curve analysis genotyping, which had a calculated sensitivity of 100% for the M815I and T917I mutations and of 98% for the L920F mutation. The specificity was 100% for M815I and L920F and 96% for T917I. Melting curve analysis genotyping is a fast, sensitive, and specific tool that is fully compatible with the analysis of a large number of samples in epidemiological surveys, allowing the simultaneous genotyping of 96 samples in just over an hour (75 min). Thus, it is perfectly suited for the epidemiological monitoring of permethrin resistance in human body lice in large-scale clinical studies. PMID:22573588

  3. A comparison of human papillomavirus genotype-specific DNA and E6/E7 mRNA detection to identify anal precancer among HIV-infected men who have sex with men

    PubMed Central

    Castle, Philip E.; Follansbee, Stephen; Borgonovo, Sylvia; Tokugawa, Diane; Schwartz, Lauren M.; Lorey, Thomas S.; LaMere, Brandon; Gage, Julia C.; Fetterman, Barbara; Darragh, Teresa M.; Rodriguez, Ana Cecilia; Wentzensen, Nicolas

    2012-01-01

    Background Human papillomavirus (HPV) RNA detection is reportedly more specific for the detection of anogenital precancer than HPV DNA but it is unknown whether this is due to detection of RNA or due to HPV genotype restriction. Materials and Methods 363 human immunodeficiency virus (HIV)-positive men who have sex with men had two anal cytology samples taken and were evaluated using high-resolution anoscopy and biopsies of visible lesions. Anal specimens were tested for E6/E7 RNA for 5 carcinogenic HPV genotypes (HPV16, 18, 31, 33, and 45) and tested for the DNA of 13 carcinogenic HPV genotypes. Results DNA testing was more likely to be positive than RNA testing (53% vs. 48%, p = 0.02) for the same 5 HPV genotypes in aggregate. When restricted to 5 HPV genotypes targeted by the RNA test, the sensitivity to detect anal precancer was the same for DNA and RNA (81%) while RNA was more specific than DNA (65% vs. 58%, p = 0.007). By comparison, DNA detection of all 13 carcinogenic HPV genotypes was more sensitive (96% vs. 81%, p = 0.001) but much less specific (65% vs. 33%, p < 0.001) compared to RNA detection of the 5 HPV genotypes. Conclusion After controlling for HPV genotypes, RNA was only slightly more specific than DNA detection for anal precancer. Impact DNA or RNA testing for a subset of the most carcinogenic HPV genotypes may be useful for distinguishing between those HPV-positive men at higher and lower risk of anal precancer and cancer. PMID:23155136

  4. Detection of Resistance to Second-Line Antituberculosis Drugs by Use of the Genotype MTBDRsl Assay: a Multicenter Evaluation and Feasibility Study

    PubMed Central

    Ignatyeva, Olga; Kontsevaya, Irina; Kovalyov, Alexander; Balabanova, Yanina; Nikolayevskyy, Vladislav; Toit, Kadri; Dragan, Anda; Maxim, Daniela; Mironova, Svetlana; Kummik, Tiina; Muntean, Ionela; Koshkarova, Ekaterina

    2012-01-01

    The rate of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) has been steadily increasing in countries of the former USSR. The availability of rapid and reliable methods for the detection of drug resistance to second-line drugs is vital for adequate patient management. We evaluated the performance of the Genotype MTBDRsl assay compared to that of phenotypic drug susceptibility testing (Becton Dickinson Bactec MGIT 960 system) with a test panel of 200 Mycobacterium tuberculosis isolates at four sites in Eastern Europe. The interpretability of the Genotype MTBDRsl assay was over 95%. The sensitivity for the detection of resistance to fluoroquinolones, ethambutol, amikacin, and capreomycin varied between 77.3% and 92.3%; however, it was much lower for kanamycin (42.7%). The sensitivity for the detection of XDR TB was 22.6%. The test specificity was over 82% for all drugs. The assay presents a good screening tool for the rapid detection of resistance to individual second-line drugs and can be recommended for use in countries with a high burden of MDR/XDR TB. The sensitivity for the detection of kanamycin resistance needs improvement. PMID:22378910

  5. Characterization of Solenopsis invicta (Hymenoptora: Formicidae) populations in Virginia: Social form genotyping and pathogen/parasitoid detection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Red imported fire ant, Solenopsis invicta Buren, workers were sampled from 26 colonies in Virginia during the 2007-2008 time period. Polymerase chain reaction (PCR) assays were used to determine colony social form (monogyny or polygyny) by genotyping ants at the Gp-9 locus. Twenty of the colonies ...

  6. A novel species within the Fusarium graminearum complex from Ethiopia detected by a multilocus genotyping assay and molecular phylogenetics

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Twenty isolates resembling members of the Fusarium graminearum species complex (Fg complex; O’Donnell et al., Fungal Genet. Biol. 41:600-623, 2004) were isolated from ground wheat samples collected in two different geographic areas in Ethiopia. Results of a multilocus genotyping (MLGT) assay (Ward ...

  7. Insights into epidemiology of human parvovirus B19 and detection of an unusual genotype 2 variant, Bulgaria, 2004 to 2013.

    PubMed

    Ivanova, Stefka Krumova; Mihneva, Zafira Georgieva; Toshev, Andon Krumov; Kovaleva, Valentina Pavlova; Andonova, Lubena Georgieva; Muller, Claude P; Hübschen, Judith M

    2016-01-01

    The present study aimed to determine the role of human parvovirus В19 (B19V) as an aetiological agent in measles and rubella negative fever/rash patients from Bulgaria between 2004 and 2013. A total of 1,266 sera from all over the country were tested for B19V IgM antibodies and all positives were further investigated by polymerase chain reaction (PCR). Overall, 280 sera (22%) were B19V IgM positive and 227 of these (81%) were also PCR positive. The highest number of IgM positives was found among five to nine year-old children (27%). Eight infected women gave birth to healthy children; one fetus was aborted with hydrops fetalis. Of the 55 genetic sequences obtained, 54 belonged to genotype 1a and one grouped as a genotype 2 outlier. Phylogenetic analysis of all available genotype 2 sequences covering the 994 nucleotide non-structural protein 1(NS1)/capsid viral protein 1 (VP1) unique region junction, showed that only one other sequence grouped with the outlier strain, forming a clearly distinct and well-supported cluster of genotype 2 (between-group genetic distance: 3.32%). In accordance with B19V nomenclature, this cluster may represent a new subgenotype 2b. The study showed that B19V infections may be falsely identified as rubella or measles in ca 22% of cases, emphasising the need for laboratory confirmation. PMID:26847955

  8. Toward fully automated genotyping: Allele assignment, pedigree construction, phase determination, and recombination detection in Duchenne muscular dystrophy

    SciTech Connect

    Perlin, M.W.; Burks, M.B.; Hoop, R.C.; Hoffman, E.P.

    1994-10-01

    Human genetic maps have made quantum leaps in the past few years, because of the characterization of >2,000 CA dinucleotide repeat loci: these PCR-based markers offer extraordinarily high PIC, and within the next year their density is expected to reach intervals of a few centimorgans per marker. These new genetic maps open new avenues for disease gene research, including large-scale genotyping for both simple and complex disease loci. However, the allele patterns of many dinucleotide repeat loci can be complex and difficult to interpret, with genotyping errors a recognized problem. Furthermore, the possibility of genotyping individuals at hundreds or thousands of polymorphic loci requires improvements in data handling and analysis. The automation of genotyping and analysis of computer-derived haplotypes would remove many of the barriers preventing optimal use of dense and informative dinucleotide genetic maps. Toward this end, we have automated the allele identification, genotyping, phase determinations, and inheritance consistency checks generated by four CA repeats within the 2.5-Mbp, 10-cM X-linked dystrophin gene, using fluorescein-labeled multiplexed PCR products analyzed on automated sequencers. The described algorithms can deconvolute and resolve closely spaced alleles, despite interfering stutter noise; set phase in females; propagate the phase through the family; and identify recombination events. We show the implementation of these algorithms for the completely automated interpretation of allele data and risk assessment for five Duchenne/Becker muscular dystrophy families. The described approach can be scaled up to perform genome-based analyses with hundreds or thousands of CA-repeat loci, using multiple fluorophors on automated sequencers. 16 refs., 5 figs., 1 tab.

  9. Evaluation of an Array-Based Method for Human Papillomavirus Detection and Genotyping in Comparison with Conventional Methods Used in Cervical Cancer Screening▿

    PubMed Central

    García-Sierra, Nerea; Martró, Elisa; Castellà, Eva; Llatjós, Mariona; Tarrats, Antoni; Bascuñana, Elisabet; Díaz, Rosana; Carrasco, María; Sirera, Guillem; Matas, Lurdes; Ausina, Vicente

    2009-01-01

    Cervical cancer is the second-most prevalent cancer in young women around the world. Infection with human papillomavirus (HPV), especially high-risk HPV types (HR-HPV), is necessary for the development of this cancer. HPV-DNA detection is increasingly being used in cervical cancer screening programs, together with the Papanicolau smear test. We evaluated the usefulness of introducing this new array-based HPV genotyping method (i.e., Clinical Arrays Papillomavirus Humano) in the cervical cancer screening algorithm in our center. The results obtained using this method were compared to those obtained by the hybrid capture II high-risk HPV DNA test (HC-II) and Papanicolau in a selected group of 408 women. The array-based assay was performed in women that were HC-II positive or presented cytological alterations. Among 246 array-positive patients, 123 (50%) presented infection with ≥2 types, and HR-HPV types were detected in 206 (83.7%), mainly HPV-16 (24.0%). Up to 132 (33.2%) specimens were classified as ASCUS (for atypical squamous cells of undetermined significance), and only 48 (36.4%) of them were HPV-DNA positive by either assay; however, 78.7% of these cases were caused by HR-HPV types. The agreement between both HPV-DNA detection techniques was fairly good (n = 367). Screening with Papanicolau smear and HC-II tests, followed by HPV detection and genotyping, provided an optimal identification of women at risk for the development of cervical cancer. Furthermore, with the identification of specific genotypes, either in single or multiple infections, a better prediction of disease progression was achieved. The array method also made allowed us to determine the possible contribution of the available vaccines in our setting. PMID:19439534

  10. An Improved PCR-RFLP Assay for Detection and Genotyping of Asymptomatic Giardia lamblia Infection in a Resource-Poor Setting

    PubMed Central

    Hawash, Yoursry; Ghonaim, M. M.; Al-Shehri, S. S.

    2016-01-01

    Laboratory workers, in resource-poor countries, still consider PCR detection of Giardia lamblia more costly and more time-consuming than the classical parasitological techniques. Based on 2 published primers, an in-house one-round touchdown PCR-RFLP assay was developed. The assay was validated with an internal amplification control included in reactions. Performance of the assay was assessed with DNA samples of various purities, 91 control fecal samples with various parasite load, and 472 samples of unknown results. Two cysts per reaction were enough for PCR detection by the assay with exhibited specificity (Sp) and sensitivity (Se) of 100% and 93%, respectively. Taking a published small subunit rRNA reference PCR test results (6%; 29/472) as a nominated gold standard, G. lamblia was identified in 5.9% (28/472), 5.2%, (25/472), and 3.6% (17/472) by PCR assay, RIDA® Quick Giardia antigen detection test (R-Biopharm, Darmstadt, Germany), and iodine-stained smear microscopy, respectively. The percent agreements (kappa values) of 99.7% (0.745), 98.9% (0.900), and 97.7% (0.981) were exhibited between the assay results and that of the reference PCR, immunoassay, and microscopy, respectively. Restriction digestion of the 28 Giardia-positive samples revealed genotype A pattern in 12 and genotype B profile in 16 samples. The PCR assay with the described format and exhibited performance has a great potential to be adopted in basic clinical laboratories as a detection tool for G. lamblia especially in asymptomatic infections. This potential is increased more in particular situations where identification of the parasite genotype represents a major requirement as in epidemiological studies and infection outbreaks. PMID:26951972

  11. An Improved PCR-RFLP Assay for Detection and Genotyping of Asymptomatic Giardia lamblia Infection in a Resource-Poor Setting.

    PubMed

    Hawash, Yoursry; Ghonaim, M M; Al-Shehri, S S

    2016-02-01

    Laboratory workers, in resource-poor countries, still consider PCR detection of Giardia lamblia more costly and more time-consuming than the classical parasitological techniques. Based on 2 published primers, an in-house one-round touchdown PCR-RFLP assay was developed. The assay was validated with an internal amplification control included in reactions. Performance of the assay was assessed with DNA samples of various purities, 91 control fecal samples with various parasite load, and 472 samples of unknown results. Two cysts per reaction were enough for PCR detection by the assay with exhibited specificity (Sp) and sensitivity (Se) of 100% and 93%, respectively. Taking a published small subunit rRNA reference PCR test results (6%; 29/472) as a nominated gold standard, G. lamblia was identified in 5.9% (28/472), 5.2%, (25/472), and 3.6% (17/472) by PCR assay, RIDA(®) Quick Giardia antigen detection test (R-Biopharm, Darmstadt, Germany), and iodine-stained smear microscopy, respectively. The percent agreements (kappa values) of 99.7% (0.745), 98.9% (0.900), and 97.7% (0.981) were exhibited between the assay results and that of the reference PCR, immunoassay, and microscopy, respectively. Restriction digestion of the 28 Giardia-positive samples revealed genotype A pattern in 12 and genotype B profile in 16 samples. The PCR assay with the described format and exhibited performance has a great potential to be adopted in basic clinical laboratories as a detection tool for G. lamblia especially in asymptomatic infections. This potential is increased more in particular situations where identification of the parasite genotype represents a major requirement as in epidemiological studies and infection outbreaks. PMID:26951972

  12. EUROarray human papillomavirus (HPV) assay is highly concordant with other commercial assays for detection of high-risk HPV genotypes in women with high grade cervical abnormalities.

    PubMed

    Cornall, A M; Poljak, M; Garland, S M; Phillips, S; Machalek, D A; Tan, J H; Quinn, M A; Tabrizi, S N

    2016-06-01

    The purpose of this study was to evaluate the performance of the EUROIMMUN EUROArray HPV genotyping assay against the Roche Cobas 4800, Roche HPV Amplicor, Roche Linear Array and Qiagen Hybrid Capture 2 assays in the detection of high-risk HPV (HR-HPV) from liquid based cervical cytology samples collected from women undergoing follow-up for abnormal cervical cytology results. Cervical specimens from 404 women undergoing management of high-grade cytological abnormality were evaluated by EUROarray HPV for detection of HR-HPV genotypes and prediction of histologically-confirmed cervical intraepithelial neoplasia grade 2 or higher (≥CIN2). The results were compared to Hybrid Capture 2, Cobas 4800 HPV, Amplicor and Linear Array HPV. Positivity for 14 HR-HPV types was 80.0 % for EUROarray (95 % CI; 75.7-83.8 %). Agreement (κ, 95 % CI) between the EUROarray and other HPV tests for detection of HR-HPV was good to very good [Hybrid Capture κ = 0.62 (0.54-0.71); Cobas κ = 0.81 (0.74-0.88); Amplicor κ = 0.68 (0.60-0.77); Linear Array κ = 0.77 (0.70-0.85)]. For detection of HR-HPV, agreement with EUROarray was 87.90 % (Hybrid Capture), 93.58 % (Cobas), 92.84 % (Amplicor) and 92.59 % (Linear Array). Detection of HR-HPV was not significantly different between EUROarray and any other test (p < 0.001). EUROarray was concordant with other assays evaluated for detection of high-risk HPV and showed sensitivity and specificity for detection of ≥ CIN2 of 86 % and 71 %, respectively. PMID:27048314

  13. Development of a Novel Genus-Specific Real-Time PCR Assay for Detection and Differentiation of Bartonella Species and Genotypes

    PubMed Central

    Bai, Ying; Malania, Lile; Winchell, Jonas M.; Kosoy, Michael Y.

    2012-01-01

    The genus Bartonella includes numerous species with varied host associations, including several that infect humans. Development of a molecular diagnostic method capable of detecting the diverse repertoire of Bartonella species while maintaining genus specificity has been a challenge. We developed a novel real-time PCR assay targeting a 301-bp region of the ssrA gene of Bartonella and demonstrated specific amplification in over 30 Bartonella species, subspecies, and strains. Subsequent analysis of ssrA sequences was sufficient to discriminate Bartonella species and provided phylogenetic data consistent with that of gltA, a commonly used gene for differentiating Bartonella genotypes. Using this assay, we identified Bartonella DNA in 29% and 47% of blood specimens from elk in Wyoming and cattle in the Republic of Georgia, respectively. Sequence analysis of a subset of genotypes from elk specimens revealed a cluster most closely related to Bartonella capreoli, and genotypes from cattle were identified as Bartonella bovis, both Bartonella species commonly found in wild and domestic ruminants. Considering the widespread geographic distribution and infectivity potential to a variety of hosts, this assay may be an effective diagnostic method for identification of Bartonella infections in humans and have utility in Bartonella surveillance studies. PMID:22378904

  14. A real-time PCR genotyping assay to detect FAD2A SNPs in peanuts (Arachis hypogaea L.)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The high oleic (C18:1) phenotype in peanuts has been previously demonstrated to result from a homozygous recessive genotype (ol1ol1ol2ol2) in two homeologous fatty acid desaturase genes (FAD2A and FAD2B) with two key SNPs. These mutant SNPs, specifically G448A in FAD2A and 442insA in FAD2B, signifi...

  15. Human papillomavirus genotyping, human papillomavirus mRNA expression, and p16/Ki-67 cytology to detect anal cancer precursors in HIV-infected MSM

    PubMed Central

    Wentzensen, Nicolas; Follansbee, Stephen; Borgonovo, Sylvia; Tokugawa, Diane; Schwartz, Lauren; Lorey, Thomas S.; Sahasrabuddhe, Vikrant V.; Lamere, Brandon; Gage, Julia C.; Fetterman, Barbara; Darragh, Teresa M.; Castle, Philip E.

    2014-01-01

    Objective Anal cancer incidence is high in HIV-infected MSM. Screening for anal intraepithelial lesions and cancers is performed at specialized clinics and relies on high-resolution anoscopy (HRA) and anal cytology. Both approaches have limited reproducibility and sensitivity for detecting anal cancer precursors. We evaluated biomarkers for human papillomavirus (HPV)-related disease in a population of HIV-infected MSM. Methods A cross-sectional screening study with passive follow-up included 363 MSM followed at a HIV/AIDS clinic. All men had anal cytology samples taken and were evaluated using HRA and anal biopsies. Using a composite endpoint of biopsy results and cytology, we compared the performance of HPV16/18 genotyping, HPVE6/E7 mRNA expression, and p16/Ki-67 cytology to detect high-grade anal intraepithelial neoplasias (AINs). Results For all biomarkers analyzed, there was a significant trend of increasing percentage of men testing positive with increasing severity of disease (P< 0.001). HPV DNA testing had the highest sensitivity for anal intraepithelial neoplasia grade 2 and anal intraepithelial neoplasia grade 3 (AIN3), followed by p16/Ki-67, HPVE6/E7 mRNA testing, and HPV16/18 genotyping. The highest Youden's index was observed for HPVE6/E7 mRNA testing, followed by HPV16/18 genotyping, p16/Ki-67 cytology, and HPV DNA testing. Increasing the threshold for positivity of p16/Ki-67 to five or more positive cells led to significantly higher specificity, but unchanged sensitivity for detecting AIN3. Conclusion Molecular features of anal disease categories are similar to those of corresponding cervical lesions. Biomarkers evaluated for cervical cancer screening may be used for primary anal cancer screening or to decide who should require immediate treatment vs. expectant management. PMID:23018436

  16. Population differences in the human arsenic (+ 3 oxidation state) methyltransferase (AS3MT) gene polymorphism detected by using genotyping method

    SciTech Connect

    Fujihara, Junko; Kunito, Takashi; Agusa, Tetsuro; Yasuda, Toshihiro; Iida, Reiko; Fujii, Yoshimi; Takeshita, Haruo

    2007-12-15

    Arsenic poisoning from drinking groundwater is a serious problem, particularly in developing Asian countries. Human arsenic (+ 3 oxidation state) methyltransferase (AS3MT) is known to catalyze the methylation of arsenite. Recently, a single nucleotide polymorphism (SNPs; rs17885947, M287T (T860C)) in the AS3MT gene was shown to be related to enzyme activity and considered to be related to genetic susceptibility to arsenic. In the present study, a useful genotyping method for M287T was developed using the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) technique. Applying this method, the genotype distribution of M287T in Ovambo (n = 185), Turkish (n = 191), Mongolian (n = 233), Korean (n = 200), and Japanese (n = 370) populations were investigated. The mutation frequencies in Asian populations were relatively lower than those of African and Caucasian populations, including those from previous studies: the frequencies of mutation in the Mongolian, Korean, and Japanese populations were 0.040, 0.010, and 0.010, respectively. In the course of this study, a PCR-based genotyping method that is inexpensive and does not require specialized equipment was developed. This method could be applied to a large number of residents at risk for arsenic poisoning.

  17. Ontogenetic change in relative performance of allozyme genotypes influences detection of heterosis in the earthworm Eisenia andrei.

    PubMed

    McElroy, T C; Diehl, W J

    2005-02-01

    The effect of ontogeny on relationships between allozyme genotypes and fresh weight was measured weekly throughout the life history of the earthworm Eisenia andrei to test the hypothesis that there is an ontogenetic component to variation in such relationships. Two of six allozyme loci showed a significant increase in apparent heterosis with ontogeny, while one locus showed a significant decrease in apparent heterosis. Three loci showed a significant decrease in the performance of common homozygotes with ontogeny. Patterns of relative genotypic performance varied among loci, but the cumulative effect was an increase in apparent allozyme heterosis later in ontogeny coinciding with a series of positive relationships between multilocus heterozygosity and fresh weight. The results could not be used to determine whether these patterns were caused by selection acting on the loci directly or on loci tightly linked to allozyme loci. However, because the same individuals were used throughout this study and thus allele frequencies and heterozygote deficiency were constant, the presence of both ontogenetic effects and differences in such patterns among loci is not compatible with a general inbreeding effect. Examining relative genotypic performance repetitively using the same individuals through ontogeny or in different environments is a very powerful experimental design for testing the effects of inbreeding or other populational factors. PMID:15523505

  18. Population differences in the human arsenic (+3 oxidation state) methyltransferase (AS3MT) gene polymorphism detected by using genotyping method.

    PubMed

    Fujihara, Junko; Kunito, Takashi; Agusa, Tetsuro; Yasuda, Toshihiro; Iida, Reiko; Fujii, Yoshimi; Takeshita, Haruo

    2007-12-15

    Arsenic poisoning from drinking groundwater is a serious problem, particularly in developing Asian countries. Human arsenic (+3 oxidation state) methyltransferase (AS3MT) is known to catalyze the methylation of arsenite. Recently, a single nucleotide polymorphism (SNPs; rs17885947, M287T (T860C)) in the AS3MT gene was shown to be related to enzyme activity and considered to be related to genetic susceptibility to arsenic. In the present study, a useful genotyping method for M287T was developed using the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) technique. Applying this method, the genotype distribution of M287T in Ovambo (n=185), Turkish (n=191), Mongolian (n=233), Korean (n=200), and Japanese (n=370) populations were investigated. The mutation frequencies in Asian populations were relatively lower than those of African and Caucasian populations, including those from previous studies: the frequencies of mutation in the Mongolian, Korean, and Japanese populations were 0.040, 0.010, and 0.010, respectively. In the course of this study, a PCR-based genotyping method that is inexpensive and does not require specialized equipment was developed. This method could be applied to a large number of residents at risk for arsenic poisoning. PMID:17889916

  19. The Implicitome: A Resource for Rationalizing Gene-Disease Associations.

    PubMed

    Hettne, Kristina M; Thompson, Mark; van Haagen, Herman H H B M; van der Horst, Eelke; Kaliyaperumal, Rajaram; Mina, Eleni; Tatum, Zuotian; Laros, Jeroen F J; van Mulligen, Erik M; Schuemie, Martijn; Aten, Emmelien; Li, Tong Shu; Bruskiewich, Richard; Good, Benjamin M; Su, Andrew I; Kors, Jan A; den Dunnen, Johan; van Ommen, Gert-Jan B; Roos, Marco; 't Hoen, Peter A C; Mons, Barend; Schultes, Erik A

    2016-01-01

    High-throughput experimental methods such as medical sequencing and genome-wide association studies (GWAS) identify increasingly large numbers of potential relations between genetic variants and diseases. Both biological complexity (millions of potential gene-disease associations) and the accelerating rate of data production necessitate computational approaches to prioritize and rationalize potential gene-disease relations. Here, we use concept profile technology to expose from the biomedical literature both explicitly stated gene-disease relations (the explicitome) and a much larger set of implied gene-disease associations (the implicitome). Implicit relations are largely unknown to, or are even unintended by the original authors, but they vastly extend the reach of existing biomedical knowledge for identification and interpretation of gene-disease associations. The implicitome can be used in conjunction with experimental data resources to rationalize both known and novel associations. We demonstrate the usefulness of the implicitome by rationalizing known and novel gene-disease associations, including those from GWAS. To facilitate the re-use of implicit gene-disease associations, we publish our data in compliance with FAIR Data Publishing recommendations [https://www.force11.org/group/fairgroup] using nanopublications. An online tool (http://knowledge.bio) is available to explore established and potential gene-disease associations in the context of other biomedical relations. PMID:26919047

  20. The Implicitome: A Resource for Rationalizing Gene-Disease Associations

    PubMed Central

    van der Horst, Eelke; Kaliyaperumal, Rajaram; Mina, Eleni; Tatum, Zuotian; Laros, Jeroen F. J.; van Mulligen, Erik M.; Schuemie, Martijn; Aten, Emmelien; Li, Tong Shu; Bruskiewich, Richard; Good, Benjamin M.; Su, Andrew I.; Kors, Jan A.; den Dunnen, Johan; van Ommen, Gert-Jan B.; Roos, Marco; ‘t Hoen, Peter A.C.; Mons, Barend; Schultes, Erik A.

    2016-01-01

    High-throughput experimental methods such as medical sequencing and genome-wide association studies (GWAS) identify increasingly large numbers of potential relations between genetic variants and diseases. Both biological complexity (millions of potential gene-disease associations) and the accelerating rate of data production necessitate computational approaches to prioritize and rationalize potential gene-disease relations. Here, we use concept profile technology to expose from the biomedical literature both explicitly stated gene-disease relations (the explicitome) and a much larger set of implied gene-disease associations (the implicitome). Implicit relations are largely unknown to, or are even unintended by the original authors, but they vastly extend the reach of existing biomedical knowledge for identification and interpretation of gene-disease associations. The implicitome can be used in conjunction with experimental data resources to rationalize both known and novel associations. We demonstrate the usefulness of the implicitome by rationalizing known and novel gene-disease associations, including those from GWAS. To facilitate the re-use of implicit gene-disease associations, we publish our data in compliance with FAIR Data Publishing recommendations [https://www.force11.org/group/fairgroup] using nanopublications. An online tool (http://knowledge.bio) is available to explore established and potential gene-disease associations in the context of other biomedical relations. PMID:26919047

  1. Comparison of the f-HPV typing™ and Hybrid Capture II® assays for detection of high-risk HPV genotypes in cervical samples.

    PubMed

    Cañadas, María-Paz; Cirigliano, Vincenzo; Darwich, Laila; Sirera, Guillermo; Coll, Josep; Clotet, Bonaventura; Videla, Sebastian

    2012-07-01

    Human papillomavirus genotyping is being considered in cervical screening programs and for monitoring the effectiveness of HPV vaccination. Both approaches require access to fast, easy and high-throughput technology. The aim of this study was to compare a new commercial assay (f-HPV typing™) with the Hybrid Capture II® (HC2) to detect HPV infection. The F-HPV typing is a multiplex fluorescent PCR method recognizing E6 and E7 regions of 13 high-risk (HR) HPV types, the same set of HR-types targeted HC2 test. A subset of 157 cervical samples was tested with both assays. The percentage of positive HR-HPV DNA samples was 24% (37/155) by HC2 and 33% (49/155) by f-HPV typing. Concordant results were found in 133/155 (overall agreement, 85.8%; Cohen's kappa=0.65). The analytical sensitivity and specificity of f-HPV were 97.6 and 93, respectively. In conclusion, this study shows that the f-HPV assay provides a good alternative to HC2 to detect HPV infection, allowing simple and rapid HPV genotyping and detecting multiple infections. PMID:22449759

  2. Real-time PCR assays for detection of Brucella spp. and the identification of genotype ST27 in bottlenose dolphins (Tursiops truncatus).

    PubMed

    Wu, Qingzhong; McFee, Wayne E; Goldstein, Tracey; Tiller, Rebekah V; Schwacke, Lori

    2014-05-01

    Rapid detection of Brucella spp. in marine mammals is challenging. Microbiologic culture is used for definitive diagnosis of brucellosis, but is time consuming, has low sensitivity and can be hazardous to laboratory personnel. Serological methods can aid in diagnosis, but may not differentiate prior exposure versus current active infection and may cross-react with unrelated Gram-negative bacteria. This study reports a real-time PCR assay for the detection of Brucella spp. and application to screen clinical samples from bottlenose dolphins stranded along the coast of South Carolina, USA. The assay was found to be 100% sensitive for the Brucella strains tested, and the limit of detection was 0.27fg of genomic DNA from Brucella ceti B1/94 per PCR volume. No amplification was detected for the non-Brucella pathogens tested. Brucella DNA was detected in 31% (55/178) of clinical samples tested. These studies indicate that the real-time PCR assay is highly sensitive and specific for the detection of Brucella spp. in bottlenose dolphins. We also developed a second real-time PCR assay for rapid identification of Brucella ST27, a genotype that is associated with human zoonotic infection. Positive results were obtained for Brucella strains which had been identified as ST27 by multilocus sequence typing. No amplification was found for other Brucella strains included in this study. ST27 was identified in 33% (18/54) of Brucella spp. DNA-positive clinical samples. To our knowledge, this is the first report on the use of a real-time PCR assay for identification of Brucella genotype ST27 in marine mammals. PMID:24632518

  3. NTSMDA: prediction of miRNA-disease associations by integrating network topological similarity.

    PubMed

    Sun, Dongdong; Li, Ao; Feng, Huanqing; Wang, Minghui

    2016-06-21

    Recently, accumulating studies have indicated that microRNAs (miRNAs) play an important role in exploring the pathogenesis of various human diseases at the molecular level and may result in the design of specific tools for diagnosis, treatment evaluation and prevention. Experimental identification of disease-related miRNAs is time-consuming and labour-intensive. Hence, there is a stressing need to propose efficient computational methods to detect more potential miRNA-disease associations. Currently, several computational approaches for identifying disease-related miRNAs on the miRNA-disease network have gained much attention by means of integrating miRNA functional similarities and disease semantic similarities. However, these methods rarely consider the network topological similarity of the miRNA-disease association network. Here, in this paper we develop an improved computational method named NTSMDA that is based on known miRNA-disease network topological similarity to exploit more potential disease-related miRNAs. We achieve an AUC of 89.4% by using the leave-one-out cross-validation experiment, demonstrating the excellent predictive performance of NTSMDA. Furthermore, predicted highly ranked miRNA-disease associations of breast neoplasms, lung neoplasms and prostatic neoplasms are manually confirmed by different related databases and literature, providing evidence for the good performance and potential value of the NTSMDA method in inferring miRNA-disease associations. The R code and readme file of NTSMDA can be downloaded from . PMID:27153230

  4. A new pyrosequencing assay for rapid detection and genotyping of Shiga toxin, intimin and O157-specific rfbE genes of Escherichia coli.

    PubMed

    Goji, Noriko; Mathews, Amit; Huszczynski, George; Laing, Chad R; Gannon, Victor P J; Graham, Morag R; Amoako, Kingsley K

    2015-02-01

    Shiga toxin (stx)-producing Escherichia coli (STEC) contamination in food and water is one of the most recognized concerns and a major financial burden in human hygiene control worldwide. Rapid and highly reliable methods of detecting and identifying STEC causing gastroenteric illnesses are crucial to prevent foodborne outbreaks. A number of tests have been developed and commercialized to detect STEC using molecular microbiology techniques. Most of these are designed to identify virulence factors such as Shiga toxin and intimin as well as E. coli O and H antigen serotype specific genes. In order to screen pathogenic STEC without relying on O:H serotyping, we developed a rapid detection and genotyping assay for STEC virulence genes using a PCR-pyrosequencing application. We adapted the PyroMark Q24 Pyrosequencing platform for subtyping 4 major virulence genes, Shiga toxin 1 and 2 (stx1 and stx2), intimin (eae) and O157-antigen gene cluster target rfbE, using Single Nucleotide Polymorphism (SNP) analysis. A total of 224 E. coli strains including isolates from Canadian environment, food and clinical cases were examined. Based on the multiple alignment analysis of 30-80 base nucleotide pyrogram reads, three alleles of the Shiga toxin 1a gene (stx1a) (stx1a-I, stx1a-II, stx1a-III) were identified. Results of the stx1, stx2, eae and rfbE genotyping revealed that each group of O:H serotype shares distinctive characteristics that could be associated with the virulence of each genotype. O157:H7/NM carries stx1a-II (94%), stx2a (82%), λ/γ1-eae (100%) and rfbE type-H7/NM (100%). Whereas isolates of the "Top-6" serotypes (O26, O45, O103, O111, O121, O145) had a high incidence of stx1a-I (90%) and stx2a (100%). stx1a-III (60%) was only observed in non Top-7 (Top-6 plus O157) STEC and Shigella spp. The entire assay, from extracting DNA from colonies on a plate to the generation of sequence information, can be completed in 5h. The method of profiling these 4 STEC pathogenic

  5. Detection of EGFR mutations in plasma DNA from lung cancer patients by mass spectrometry genotyping is predictive of tumor EGFR status and response to EGFR inhibitors

    PubMed Central

    Brevet, Marie; Johnson, Melissa L.; Azzoli, Christopher G.; Ladanyi, Marc

    2012-01-01

    Aims EGFR mutations now guide the clinical use of EGFR-targeted therapy in lung cancer. However, standard EGFR mutation analysis requires a minimum amount of tumor tissue, which may not be available in certain situations. In this study, we combined a mass spectrometry genotyping assay (Sequenom) with a mutant-enriched PCR (ME-PCR) to detect EGFR mutations in free plasma DNA from patients with lung cancer. Method DNAs were extracted from 31 plasma samples from 31 patients and analyzed by both methods for EGFR exon 19 deletion and EGFR L858R mutation. Results in plasma DNA samples were compared with EGFR mutation status obtained in tumor DNA (18/31 EGFR mutant). The relationship of EGFR mutation status in tumor and/or plasma samples to overall survival was assessed. Results The EGFR mutation status in plasma DNA was identical to the primary tumor in 61% of patients (19/31). By mass spectrometry genotyping, the plasma samples contained mutant DNA corresponding to 5/14 EGFR exon 19 deletions and 3/4 EGFR L858R mutations previously diagnosed in the matched tumors. Two samples were positive in plasma DNA but negative in primary tumor tissue. Results were similar for ME-PCR. For patients treated with erlotinib, overall survival was correlated with the presence of EGFR mutation in plasma and/or tumor tissue (p=0.002), with the two patients positive only in plasma DNA showing responses and favorable outcomes. Conclusion The detection of EGFR mutations in plasma DNA samples by mass spectrometry genotyping and ME-PCR is feasible. A positive EGFR result in plasma DNA has a high predictive value for tumor EGFR status and for favorable clinical course on EGFR-targeted therapy and could therefore be useful in guiding clinical decisions in patients with insufficient or unavailable tumor specimens. PMID:21130517

  6. Granuloma annulare: Pathogenesis, disease associations and triggers, and therapeutic options.

    PubMed

    Piette, Evan W; Rosenbach, Misha

    2016-09-01

    Granuloma annulare (GA) represents a cutaneous reaction pattern of unknown cause with a variety of previously described potential disease associations and triggers. This review attempts to synthesize the available data regarding potential etiopathogenesis, reviews the available data on potential GA disease associations and work-up indicated for patients with GA, and discusses potential inciting triggers. In the final part, this article describes the available treatments options and supporting data, and provides a framework for approaching management of patients with GA. The previous accompanying article provided a comprehensive overview of the available information known about the clinical variants, epidemiology, genetics, and histology of GA. PMID:27543210

  7. Detection of the frequency, antimicrobial susceptibility, and genotypic discrimination of Aeromonas strains isolated from municipally treated tap water samples by cultivation and AP-PCR.

    PubMed

    Emekdas, Gurol; Aslan, Gonul; Tezcan, Seda; Serin, Mehmet Sami; Yildiz, Cilem; Ozturhan, Hakan; Durmaz, Riza

    2006-04-01

    The frequency, antibiotic susceptibility, and genotypic discrimination of Aeromonas strains isolated from municipally treated drinking tap water distribution systems were investigated in this study. We have analyzed 148 tap water samples collected from 8 different locations by bacterial cultivation and arbitrarily primed polymerase chain reaction (AP-PCR). Gram negative, hemolytic, oxidase (+) and catalase (+) bacterial colonies were applied to the study. Identification of bacterial colonies was done by conventional biochemical method and API ID 20E panel (BioMerieux-France). Molecular epidemiological discrimination of the isolates was done by AP-PCR. Aeromonas spp. was detected in 6 of 148 (4%) tap water samples from 8 different locations. Five isolates were identified as Aeromonas hydrophila and one isolate was identified as Vibrio fluvialis by conventional biochemical method. These data were also confirmed by API 20E panel. One of 6 isolates was resistant to gentamicin, 2 of 6 isolates were resistant to trimethoprim/sulfamethoxazole, 4 of 6 isolates were resistant to ampicillin and ampicillin-sulbactam and all of 6 isolates were resistant to cephalothin. All isolates were found to be susceptible to amikacin, aztreonam, ceftazidime, ceftriaxone, ciprofloxacin. All 6 strains of Aeromonas were discriminated by AP-PCR and were determined that all isolates were from different genotypic sources. Although the frequency of the isolates was under the standard limits, the results indicate that hemolytic A. hydrophila are present in municipally treated tap water samples in Mersin City. While all strains were genotypically distinct, all of them were resistant to first generation beta lactam antibiotics tested in this study. PMID:16427154

  8. Genotyping-By-Sequencing (GBS) Detects Genetic Structure and Confirms Behavioral QTL in Tame and Aggressive Foxes (Vulpes vulpes)

    PubMed Central

    Johnson, Jennifer L.; Wittgenstein, Helena; Mitchell, Sharon E.; Hyma, Katie E.; Temnykh, Svetlana V.; Kharlamova, Anastasiya V.; Gulevich, Rimma G.; Vladimirova, Anastasiya V.; Fong, Hiu Wa Flora; Acland, Gregory M.; Trut, Lyudmila N.; Kukekova, Anna V.

    2015-01-01

    The silver fox (Vulpes vulpes) offers a novel model for studying the genetics of social behavior and animal domestication. Selection of foxes, separately, for tame and for aggressive behavior has yielded two strains with markedly different, genetically determined, behavioral phenotypes. Tame strain foxes are eager to establish human contact while foxes from the aggressive strain are aggressive and difficult to handle. These strains have been maintained as separate outbred lines for over 40 generations but their genetic structure has not been previously investigated. We applied a genotyping-by-sequencing (GBS) approach to provide insights into the genetic composition of these fox populations. Sequence analysis of EcoT22I genomic libraries of tame and aggressive foxes identified 48,294 high quality SNPs. Population structure analysis revealed genetic divergence between the two strains and more diversity in the aggressive strain than in the tame one. Significant differences in allele frequency between the strains were identified for 68 SNPs. Three of these SNPs were located on fox chromosome 14 within an interval of a previously identified behavioral QTL, further supporting the importance of this region for behavior. The GBS SNP data confirmed that significant genetic diversity has been preserved in both fox populations despite many years of selective breeding. Analysis of SNP allele frequencies in the two populations identified several regions of genetic divergence between the tame and aggressive foxes, some of which may represent targets of selection for behavior. The GBS protocol used in this study significantly expanded genomic resources for the fox, and can be adapted for SNP discovery and genotyping in other canid species. PMID:26061395

  9. Detection of the SHV genotype polymorphism of the extended-spectrum β-lactamase-producing Gram-negative bacterium

    PubMed Central

    LI, JUNLONG; JI, XIAOLI; DENG, XIAOHUI; ZHOU, YINGFENG; NI, XIAOQING; LIU, XIAOKANG

    2015-01-01

    The prevalence of extended-spectrum β-lactamases (ESBLs) is due to the extensive usage of the extended-spectrum cephalosporins and leads to huge financial loss worldwide, whilst presenting a challenge to the clinical treatment. The aim of the present study was to delineate the frequency of ESBL occurrence in Enterobacteriaceae and confirm the SHV genotype. A random collection of 153 Escherichia coli isolates (E. coli) and 70 Klebsiella pneumoniae isolates were tested. The amplification products obtained by polymerase chain reaction were sequenced. Isolates with novel mutations were transformed to E. coli DH5 α. The minimum inhibitory concentration (MIC) was obtained by a microdilution method. The relevance ratio of ESBL was 67.7% and the proportion of the SHV β-lactamase gene (blaSHV) was 18.5%. A new genotype of β-lactamase was demonstrated and submitted to GenBank. A total of 12 mutational sites were found in 28 ESBL-producing isolates, including four nonsense mutations. Sensitive-rates of 28 ESBL-producing isolates to imipenem were 100%, and resistant-rates to penicillin, amoxicillin and oxacillin were 100%. The MIC of DH5 α-F8 to penicillin, oxacillin, cefoxitin, cefotaxime, cefepime, cefoperazone/sulbactam, imipenem and netilmicin was 512, 512, 2, 0.03, 0.06, 4, 0.015 and 32 respectively. In conclusion, ESBL and SHV-28 is the most prevalent bla. Imipenem is the most effective antibiotic to ESBL, and the 4th-generation cephalosporins and β-lactamase inhibitor compound are also effective. ESBL is mediated by plasmids and able to spread among different Enterobacteriaceae. In conclusion, new mutations of the blaSHV gene exist from at least 2010. PMID:26075080

  10. Development of a Multiplex PCR Test with Automated Genotyping Targeting E7 for Detection of Six High-Risk Human Papillomaviruses

    PubMed Central

    de Assis, Angela Maria

    2015-01-01

    Cervical cancer is caused by high-risk human papillomaviruses (HPV) and viral detection tests aid in the diagnosis of precursor lesions. In the present study, a molecular test for detection of high-risk HPV DNA, called E7-HPV, was standardized and assessed in samples from women with pre-cancerous lesions. The development of the E7-HPV test for detection and genotyping of six high-risk HPV (types 16, 18, 31, 33, 45 and 52), consisted of evaluating primer quality and adjusting the multiplex PCR conditions. Primer design was based on the E7 region of each HPV, and the fluorochrome 6-FAM was added to PCR primers. Viral detection was performed by capillary electrophoresis in automated sequencer in samples obtained from 60 women (55 with ASC-H/HSIL cytology) from August to September 2013. A non-inferiority analysis was conducted with the cobas HPV test as a reference and following international guidelines for the development of new tests. The two tests had a high concordance rate in HPV16 detection (kappa=0.972), with only one discordant case (cervical intraepithelial neoplasia grade 3, negaive with cobas and positive for HPV16 by E7-HPV) and complete agreement in HPV18 detection. When comparing detection of all high-risk HPV, three cases were positive with cobas but negative with E7-HPV, and another three cases were negative with cobas but positive with E7-HPV (HPV16, 31 and 52). When we evaluate the cases initially suspected by cytology, the two tests had the same sensitivity in detection CIN2 or worse. In conclusion, the E7-HPV test has satisfactory initial results, and its development can be continued. PMID:26087285

  11. High sensitivity, loop-mediated isothermal amplification combined with colorimetric gold-nanoparticle probes for visual detection of high risk human papillomavirus genotypes 16 and 18.

    PubMed

    Kumvongpin, Ratchanida; Jearanaikool, Patcharee; Wilailuckana, Chotechana; Sae-Ung, Nattaya; Prasongdee, Prinya; Daduang, Sakda; Wongsena, Metee; Boonsiri, Patcharee; Kiatpathomchai, Wansika; Swangvaree, Sukumarn Sanersak; Sandee, Alisa; Daduang, Jureerut

    2016-08-01

    High-risk human papillomavirus (HR-HPV) causes cervical cancer. HPV16 and HPV18 are the most prevalent strains of the virus reported in women worldwide. Loop-mediated isothermal amplification (LAMP) is an alternative method for DNA detection under isothermal conditions. However, it results in a turbid amplified product which is not easily detected by the naked eye. This study aimed to develop an improved technique by using gold nanoparticles (AuNPs) attached to a single-stranded DNA probe for the detection of HPV16 and HPV18. Detection of the LAMP product by AuNP color change was compared with detection by visual turbidity. The optimal conditions for this new LAMP-AuNP assay were an incubation time of 20min and a temperature of 65°C. After LAMP amplification was complete, its products were hybridized with the AuNP probe for 5min and then detected by the addition of magnesium salt. The color changed from red to blue as a result of aggregation of the AuNP probe under high ionic strength conditions produced by the addition of the salt. The sensitivity of the LAMP-AuNP assay was greater than the LAMP turbidity assay by up to 10-fold for both HPV genotypes. The LAMP-AuNP assay showed higher sensitivity and ease of visualization than did the LAMP turbidity for the detection of HPV16 and HPV18. Additionally, AuNP-HPV16 and AuNP-HPV18 probes were stable for over 1year. The combination of LAMP and the AuNP-probe colorimetric assay offers a simple, rapid and highly sensitive alternative diagnostic tool for the detection of HPV16 and HPV18 in district hospitals or field studies. PMID:27086727

  12. National Tay-Sachs and Allied Diseases Association, Inc.

    ERIC Educational Resources Information Center

    Exceptional Parent, 1977

    1977-01-01

    Reviewed are the history and organization, purpose and programs, and public services of the National Tay-Sachs and Allied Diseases Association, an organization geared toward eradicating Tay-Sachs disease (a hereditary disorder affecting primarily Jewish infants which generally leads to deterioration and death by the child's fifth year). (SBH)

  13. The National Tay Sachs and Allied Diseases Association.

    ERIC Educational Resources Information Center

    Zeitlin, Paula

    1986-01-01

    The National Tay-Sachs and Allied Diseases Association is involved in education, research, and prevention of Tay-Sachs, an inherited metabolic disorder which destroys the central nervous system, and over 30 related disorders. The group features a parent peer group network and a support group for carrier couples. (CL)

  14. Multicenter Noninferiority Evaluation of Hain GenoType MTBDRplus Version 2 and Nipro NTM+MDRTB Line Probe Assays for Detection of Rifampin and Isoniazid Resistance.

    PubMed

    Nathavitharana, Ruvandhi R; Hillemann, Doris; Schumacher, Samuel G; Schlueter, Birte; Ismail, Nazir; Omar, Shaheed Vally; Sikhondze, Welile; Havumaki, Joshua; Valli, Eloise; Boehme, Catharina; Denkinger, Claudia M

    2016-06-01

    Less than 30% of multidrug-resistant tuberculosis (MDR-TB) patients are currently diagnosed, due to laboratory constraints. Molecular diagnostics enable rapid and simplified diagnosis. Newer-version line probe assays have not been evaluated against the WHO-endorsed Hain GenoType MTBDRplus (referred to as Hain version 1 [V1]) for the rapid detection of rifampin (RIF) and isoniazid (INH) resistance. A two-phase noninferiority study was conducted in two supranational reference laboratories to allow head-to-head comparisons of two new tests, Hain Genotype MTBDRplus version 2 (referred to as Hain version 2 [V2]) and Nipro NTM+MDRTB detection kit 2 (referred to as Nipro), to Hain V1. In phase 1, the results for 379 test strains were compared to a composite reference standard that used phenotypic drug susceptibility testing (DST) and targeted sequencing. In phase 2, the results for 644 sputum samples were compared to a phenotypic DST reference standard alone. Using a challenging set of strains in phase 1, the values for sensitivity and specificity for Hain V1, Hain V2, and Nipro, respectively, were 90.3%/98.5%, 90.3%/98.5%, and 92.0%/98.5% for RIF resistance detection and 89.1%/99.4%, 89.1%/99.4%, and 89.6%/100.0% for INH resistance detection. Testing of sputa in phase 2 yielded values for sensitivity and specificity of 97.1%/97.1%, 98.2%/97.8%, and 96.5%/97.5% for RIF and 94.4%/96.4%, 95.4%/98.8%, and 94.9%/97.6% for INH. Overall, the rates of indeterminate results were low, but there was a higher rate of indeterminate results with Nipro than with Hain V1 and V2 in samples with low smear grades. Noninferiority of Hain V2 and Nipro to Hain V1 was demonstrated for RIF and INH resistance detection in isolates and sputum specimens. These results serve as evidence for WHO policy recommendations on the use of line probe assays, including the Hain V2 and Nipro assays, for MDR-TB detection. PMID:27076658

  15. Multicenter Noninferiority Evaluation of Hain GenoType MTBDRplus Version 2 and Nipro NTM+MDRTB Line Probe Assays for Detection of Rifampin and Isoniazid Resistance

    PubMed Central

    Hillemann, Doris; Schumacher, Samuel G.; Schlueter, Birte; Ismail, Nazir; Omar, Shaheed Vally; Sikhondze, Welile; Havumaki, Joshua; Valli, Eloise; Boehme, Catharina

    2016-01-01

    Less than 30% of multidrug-resistant tuberculosis (MDR-TB) patients are currently diagnosed, due to laboratory constraints. Molecular diagnostics enable rapid and simplified diagnosis. Newer-version line probe assays have not been evaluated against the WHO-endorsed Hain GenoType MTBDRplus (referred to as Hain version 1 [V1]) for the rapid detection of rifampin (RIF) and isoniazid (INH) resistance. A two-phase noninferiority study was conducted in two supranational reference laboratories to allow head-to-head comparisons of two new tests, Hain Genotype MTBDRplus version 2 (referred to as Hain version 2 [V2]) and Nipro NTM+MDRTB detection kit 2 (referred to as Nipro), to Hain V1. In phase 1, the results for 379 test strains were compared to a composite reference standard that used phenotypic drug susceptibility testing (DST) and targeted sequencing. In phase 2, the results for 644 sputum samples were compared to a phenotypic DST reference standard alone. Using a challenging set of strains in phase 1, the values for sensitivity and specificity for Hain V1, Hain V2, and Nipro, respectively, were 90.3%/98.5%, 90.3%/98.5%, and 92.0%/98.5% for RIF resistance detection and 89.1%/99.4%, 89.1%/99.4%, and 89.6%/100.0% for INH resistance detection. Testing of sputa in phase 2 yielded values for sensitivity and specificity of 97.1%/97.1%, 98.2%/97.8%, and 96.5%/97.5% for RIF and 94.4%/96.4%, 95.4%/98.8%, and 94.9%/97.6% for INH. Overall, the rates of indeterminate results were low, but there was a higher rate of indeterminate results with Nipro than with Hain V1 and V2 in samples with low smear grades. Noninferiority of Hain V2 and Nipro to Hain V1 was demonstrated for RIF and INH resistance detection in isolates and sputum specimens. These results serve as evidence for WHO policy recommendations on the use of line probe assays, including the Hain V2 and Nipro assays, for MDR-TB detection. PMID:27076658

  16. Comparison of Xpert MTB/RIF Assay and GenoType MTBDRplus DNA Probes for Detection of Mutations Associated with Rifampicin Resistance in Mycobacterium tuberculosis

    PubMed Central

    Rahman, Arfatur; Sahrin, Mahfuza; Afrin, Sadia; Earley, Keith; Ahmed, Shahriar; Rahman, S. M. Mazidur; Banu, Sayera

    2016-01-01

    Background GeneXpert MTB/RIF (Xpert) and Genotype MTBDRplus (DRplus) are two World Health Organization (WHO) endorsed probe based molecular drug susceptibility testing (DST) methods for rapid diagnosis of drug resistant tuberculosis. Both methods target the same 81 bp Rifampicin Resistance Determining Region (RRDR) of bacterial RNA polymerase β subunit (rpoB) for detection of Rifampicin (RIF) resistance associated mutations using DNA probes. So there is a correspondence of the probes of each other and expected similarity of probe binding. Methods We analyzed 92 sputum specimens by Xpert, DRplus and LJ proportion method (LJ-DST). We compared molecular DSTs with gold standard LJ-DST. We wanted to see the agreement level of two molecular methods for detection of RIF resistance associated mutations. The 81bp RRDR region of rpoB gene of discrepant cases between the two molecular methods was sequenced by Sanger sequencing. Results The agreement of Xpert and DRplus with LJ-DST for detection of RIF susceptibility was found to be 93.5% and 92.4%, respectively. We also found 92.4% overall agreement of two molecular methods for the detection of RIF susceptibility. A total of 84 out of 92 samples (91.3%) had agreement on the molecular locus of RRDR mutation by DRplus and Xpert. Sanger sequencing of 81bp RRDR revealed that Xpert probes detected seven of eight discrepant cases correctly and DRplus was erroneous in all the eight cases. Conclusion Although the overall concordance with LJ-DST was similar for both Xpert and DRplus assay, Xpert demonstrated more accuracy in the detection of RIF susceptibility for discrepant isolates compared with DRplus. This observation would be helpful for the improvement of probe based detection of drug resistance associated mutations especially rpoB mutation in M. tuberculosis. PMID:27054344

  17. Primary Screening for Cervical Cancer Based on High-Risk Human Papillomavirus (HPV) Detection and HPV 16 and HPV 18 Genotyping, in Comparison to Cytology

    PubMed Central

    Constantinidis, Theocharis; Constantinidis, Theodoros C.

    2015-01-01

    Objectives The objective of the present study is to assess the performance of a high-risk human papillomavirus (HR-HPV) DNA test with individual HPV-16/HPV-18 genotyping as a method for primary cervical cancer screening compared with liquid-based cytology (LBC) in a population of Greek women taking part in routine cervical cancer screening. Methods The study, conducted by the “HEllenic Real life Multicentric cErvical Screening” (HERMES) study group, involved the recruitment of 4,009 women, aged 25–55, who took part in routine cervical screening at nine Gynecology Departments in Greece. At first visit cervical specimens were collected for LBC and HPV testing using the Roche Cobas 4800 system. Women found positive for either cytology or HPV were referred for colposcopy, whereas women negative for both tests will be retested after three years. The study is ongoing and the results of the first screening round are reported herein. Results Valid results for cytology and HPV testing were obtained for 3,993 women. The overall prevalence of HR-HPV was 12.7%, of HPV-16 2.7% and of HPV-18 1.4%. Of those referred for colposcopy, cervical intraepithelial neoplasia grade 2 or worse (CIN2+) was detected in 41 women (1.07%). At the threshold of CIN2+, cytology [atypical squamous cells of undetermined significance (ASC-US) or worse] and HPV testing showed a sensitivity of 53.7% and 100% respectively, without change between age groups. Cytology and HPV testing showed specificity of 96.8% and 90.3% respectively, which was increased in older women (≥30) in comparison to younger ones (25–29). Genotyping for HPV16/18 had similar accuracy to cytology for the detection of CIN2+ (sensitivity: 58.5%; specificity 97.5%) as well as for triage to colposcopy (sensitivity: 58.5% vs 53.7% for cytology). Conclusion HPV testing has much better sensitivity than cytology to identify high-grade cervical lesions with slightly lower specificity. HPV testing with individual HPV-16/HPV-18

  18. Inflammatory bowel disease associations with HLA Class II genes

    SciTech Connect

    Castro, R.; Yang, H.; Targan, S.

    1994-09-01

    A PCR-SSOP assay has been used to analyze HLA-Class II DRB1 and DQB1 alleles in 378 Caucasians from a population in Southern California. The data has been analyzed separately for the Ashkenasi Jews and non-Jewish patients (n=286) and controls (n=92). Two common clinical forms of inflammatory bowel disease (IBD) have been studied: ulcerative colitis (UC) and Crohn`s disease (CD). In CD, we observed a susceptible effect with the rare DR1 allele - DRB*0103 [O.R.=4.56; 95% CI (0.96, 42.97); p=0.03]; a trend for an increase in DRB1*0103 was also observed in UC patients. A susceptible effect with DRB1*1502 [O.R.=5.20; 95% CI (1.10, 48.99); p=0.02] was observed in non-Jewish UC patients. This susceptible effect was restricted to UC ANCA-positive (antineutrophil cytoplasmic antibodies) patients. In addition, a significant association with DRB1*1101-DQB1*0301 [O.R.=9.46; 95% CI (1.30, 413.87); p=0.01] was seen with UC among non-Jewish patients: this haplotype was increased with CD among non-Jewish patients. Two protective haplotypes were detected among CD non-Jewish patients: DRB1*1301-DQB1*0603 [O.R.=0.34; 95% CI (0.09, 1.09); p=0.04], and DRB*0404-DQB1*0302 [O.R.=<0.08; 95% CI (0.0, 0.84); p=0.01]. When the same data were analyzed at the serology level, we observed a positive association in UC with DR2 [O.R.6.77; 95% CI (2.47, 22.95); p=2 x 10{sup -4}], and a positive association in CD with DR1 [O.R.=2.63; 95% CI (1.14, 6.62); p=0.01] consistent with previous reports. Thus, some IBD disease associations appear to be common to both UC and CD, while some are unique to one disease.

  19. A Nucleic Acid Biosensor for Detection of Hepatitis C Virus Genotype 1a Using Poly(L-Glutamic Acid)-Modified Electrode.

    PubMed

    Donmez, Soner; Arslan, Fatma; Arslan, Halit

    2015-07-01

    An electrochemical nucleic acid biosensor based on label-free DNA detection method was prepared for the first time by using electropolymerized poly(L-glutamic acid)-modified pencil graphite electrode (PGA/PGE) for detection of hepatitis C virus genotype 1a (HCV1a). Inosine-substituted 20-mer probes related to the HCV1a were immobilized onto PGA/PGE surface by covalent linking with the formation of amide bonds. Square wave voltammetry (SWV) was used to monitor the oxidation signal of guanine in the hybridization events, which gave an oxidation peak at +1.05 V. An increase in the oxidation signal of guanine was showed by hybridization of the probe with the complementary DNA. Noncomplementary oligonucleotides were also used to investigate the selectivity of the biosensor. The proposed nucleic acid biosensor was linear in the range of 50 nM to 1.0 μM, exhibiting a limit of detection of 40.6 nM. Finally, single-stranded synthetic PCR product analogues of HCV1a were performed in optimal condition. This PGA-modified nucleic acid sensor is cost-effective and disposable, and besides, it has superior electrocatalytic effect on the oxidation of guanine. PMID:25947619

  20. Hybrid microarray based on double biomolecular markers of DNA and carbohydrate for simultaneous genotypic and phenotypic detection of cholera toxin-producing Vibrio cholerae.

    PubMed

    Shin, Hwa Hui; Seo, Jeong Hyun; Kim, Chang Sup; Hwang, Byeong Hee; Cha, Hyung Joon

    2016-05-15

    Life-threatening diarrheal cholera is usually caused by water or food contaminated with cholera toxin-producing Vibrio cholerae. For the prevention and surveillance of cholera, it is crucial to rapidly and precisely detect and identify the etiological causes, such as V. cholerae and/or its toxin. In the present work, we propose the use of a hybrid double biomolecular marker (DBM) microarray containing 16S rRNA-based DNA capture probe to genotypically identify V. cholerae and GM1 pentasaccharide capture probe to phenotypically detect cholera toxin. We employed a simple sample preparation method to directly obtain genomic DNA and secreted cholera toxin as target materials from bacterial cells. By utilizing the constructed DBM microarray and prepared samples, V. cholerae and cholera toxin were detected successfully, selectively, and simultaneously; the DBM microarray was able to analyze the pathogenicity of the identified V. cholerae regardless of whether the bacteria produces toxin. Therefore, our proposed DBM microarray is a new effective platform for identifying bacteria and analyzing bacterial pathogenicity simultaneously. PMID:26735874

  1. Detection of hepatitis B virus genotypic resistance mutations by coamplification at lower denaturation temperature-PCR coupled with sanger sequencing.

    PubMed

    Liu, Can; Lin, Jinpiao; Chen, Huijuan; Shang, Hongyan; Jiang, Ling; Chen, Jing; Ye, Yang; Yang, Bin; Ou, Qishui

    2014-08-01

    Mutations in the reverse transcriptase (rt) region of the DNA polymerase gene are the primary cause of hepatitis B virus (HBV) drug resistance. In this study, we established a novel method that couples coamplification at lower denaturation temperature (COLD)-PCR and Sanger sequencing, and we applied it to the detection of known and unknown HBV mutations. Primers were designed based on the common mutations in the HBV rt sequence at positions 180 to 215. The critical denaturation temperature (Tc) was established as a denaturing temperature for both fast and full COLD-PCR procedures. For single mutations, when a melting temperature (Tm)-reducing mutation occurred (e.g., C-G → T-A), the sensitivities of fast and full COLD-PCR for mutant detection were 1% and 2%, respectively; when the mutation caused no change in Tm (e.g., C-G → G-C) or raised Tm (e.g., T-A → C-G), only full COLD-PCR improved the sensitivity for mutant detection (2%). For combination mutations, the sensitivities of both full and fast COLD-PCR were increased to 0.5%. The limits of detection for fast and full COLD-PCR were 50 IU/ml and 100 IU/ml, respectively. In 30 chronic hepatitis B (CHB) cases, no mutations were detected by conventional PCR, whereas 18 mutations were successfully detected by COLD-PCR, including low-prevalence mutations (<10%), as confirmed by ultradeep pyrosequencing. In conclusion, COLD-PCR provides a highly sensitive, simple, inexpensive, and practical tool for significantly improving amplification efficacy and detecting low-level mutations in clinical CHB cases. PMID:24899029

  2. Genotype-based association models of complex diseases to detect gene-gene and gene-environment interactions

    PubMed Central

    Fan, Ruzong; Manga, Prashiela

    2015-01-01

    A central problem in genetic epidemiology is to identify and rank genetic markers involved in a disease. Complex diseases, such as cancer, hypertension, diabetes, are thought to be caused by an interaction of a panel of genetic factors, that can be identified by markers, which modulate environmental factors. Moreover, the effect of each genetic marker may be small. Hence, the association signal may be missed unless a large sample is considered, or a priori biomedical data are used. Recent advances generated a vast variety of a priori information, including linkage maps and information about gene regulatory dependence assembled into curated pathway databases. We propose a genotype-based approach that takes into account linkage disequilibrium (LD) information between genetic markers that are in moderate LD while modeling gene-gene and gene-environment interactions. A major advantage of our method is that the observed genetic information enters a model directly thus eliminating the need to estimate haplotype-phase. Our approach results in an algorithm that is inexpensive computationally and does not suffer from bias induced by haplotype-phase ambiguity. We investigated our model in a series of simulation experiments and demonstrated that the proposed approach results in estimates that are nearly unbiased and have small variability. We applied our method to the analysis of data from a melanoma case-control study and investigated interaction between a set of pigmentation genes and environmental factors defined by age and gender. Furthermore, an application of our method is demonstrated using a study of Alcohol Dependence. PMID:26191336

  3. Integrative analysis reveals disease-associated genes and biomarkers for prostate cancer progression

    PubMed Central

    2014-01-01

    Background Prostate cancer is one of the most common complex diseases with high leading cause of death in men. Identifications of prostate cancer associated genes and biomarkers are thus essential as they can gain insights into the mechanisms underlying disease progression and advancing for early diagnosis and developing effective therapies. Methods In this study, we presented an integrative analysis of gene expression profiling and protein interaction network at a systematic level to reveal candidate disease-associated genes and biomarkers for prostate cancer progression. At first, we reconstructed the human prostate cancer protein-protein interaction network (HPC-PPIN) and the network was then integrated with the prostate cancer gene expression data to identify modules related to different phases in prostate cancer. At last, the candidate module biomarkers were validated by its predictive ability of prostate cancer progression. Results Different phases-specific modules were identified for prostate cancer. Among these modules, transcription Androgen Receptor (AR) nuclear signaling and Epidermal Growth Factor Receptor (EGFR) signalling pathway were shown to be the pathway targets for prostate cancer progression. The identified candidate disease-associated genes showed better predictive ability of prostate cancer progression than those of published biomarkers. In context of functional enrichment analysis, interestingly candidate disease-associated genes were enriched in the nucleus and different functions were encoded for potential transcription factors, for examples key players as AR, Myc, ESR1 and hidden player as Sp1 which was considered as a potential novel biomarker for prostate cancer. Conclusions The successful results on prostate cancer samples demonstrated that the integrative analysis is powerful and useful approach to detect candidate disease-associate genes and modules which can be used as the potential biomarkers for prostate cancer progression. The

  4. Translation of disease associated gene signatures across tissues.

    PubMed

    Kasim, Adetayo; Shkedy, Ziv; Lin, Dan; Van Sanden, Suzy; Abrahantes, Josè Cortiñas; Göhlmann, Hinrich W H; Bijnens, Luc; Yekutieli, Dani; Camilleri, Michael; Aerssens, Jeroen; Talloen, Willem

    2015-01-01

    It has recently been shown that disease associated gene signatures can be identified by profiling tissue other than the disease related tissue. In this paper, we investigate gene signatures for Irritable Bowel Syndrome (IBS) using gene expression profiling of both disease related tissue (colon) and surrogate tissue (rectum). Gene specific joint ANOVA models were used to investigate differentially expressed genes between the IBS patients and the healthy controls taken into account both intra and inter tissue dependencies among expression levels of the same gene. Classification algorithms in combination with feature selection methods were used to investigate the predictive power of gene expression levels from the surrogate and the target tissues. We conclude based on the analyses that expression profiles of the colon and the rectum tissue could result in better predictive accuracy if the disease associated genes are known. PMID:26333264

  5. Detection and Genotyping of Arcobacter and Campylobacter Isolates from Retail Chicken Samples by Use of DNA Oligonucleotide Arrays

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To explore the use of DNA microarrays for pathogen detection in food, we have produced DNA oligonucleotide arrays to identify the presence of Arcobacter and Campylobacter in retail chicken. Probes were selected that target housekeeping and virulence-associated genes in both Arcobacter butzleri and ...

  6. Report of six kidney disease-associated Castleman's disease cases.

    PubMed

    Sui, Yanxia; Zhao, Dongli

    2015-12-01

    Castleman's disease (CD) is an uncommon, benign, non-neoplastic, lymphoproliferative disease of uncertain etiology. Here, we report 6 cases of kidney disease-associated CD in China. All patients exhibited multicentric CD (MCD) with involvement of the mediastinum, neck, bilateral axillary, and bilateral inguinal regions. Clinical manifestations included fever, fatigue, edema, and swollen lymph nodes. Laboratory examinations of all 6 patients found proteinuria or renal insufficiency. Two of the patients were diagnosed with hyaline vascular type MCD, and 4 patients were diagnosed with plasma cell type CD. The case 1 and case 4 patients had mesangial proliferative glomerulonephritis, case 3 had type I membranoproliferative glomerulonephritis, case 2 and case 5 had interstitial nephritis, and case 6 had AA type amyloidosis nephropathy. Three patients were treated with prednisone plus cyclophosphamide, 1 patient received COP therapy (cyclophosphamide, vincristine and prednisone), and 2 patients received COP therapy supplemented by small doses of radiation therapy delivered to local lymph nodes. In all cases, the clinical manifestations of MCD, including fever, fatigue, edema, swollen lymph nodes, and proteinuria, were alleviated or abolished by treatment. One patient responded to treatment with complete MCD remission, and another 4 patients survived. However, 1 patient died due to renal failure. In conclusion, common diagnosis and treatment techniques are suitable for kidney disease-associated CD. However, treatment efficacy might be difficult to predict, and some cases may have poor prognosis with this treatment strategy. Therefore, additional studies investigating kidney disease-associated CD and treatment outcomes in larger patient populations are needed. PMID:26445003

  7. Characterization of Disease-Associated Mutations in Human Transmembrane Proteins

    PubMed Central

    Molnár, János; Szakács, Gergely; Tusnády, Gábor E.

    2016-01-01

    Transmembrane protein coding genes are commonly associated with human diseases. We characterized disease causing mutations and natural polymorphisms in transmembrane proteins by mapping missense genetic variations from the UniProt database on the transmembrane protein topology listed in the Human Transmembrane Proteome database. We found characteristic differences in the spectrum of amino acid changes within transmembrane regions: in the case of disease associated mutations the non-polar to non-polar and non-polar to charged amino acid changes are equally frequent. In contrast, in the case of natural polymorphisms non-polar to charged amino acid changes are rare while non-polar to non-polar changes are common. The majority of disease associated mutations result in glycine to arginine and leucine to proline substitutions. Mutations to positively charged amino acids are more common in the center of the lipid bilayer, where they cause more severe structural and functional anomalies. Our analysis contributes to the better understanding of the effect of disease associated mutations in transmembrane proteins, which can help prioritize genetic variations in personal genomic investigations. PMID:26986070

  8. Inferring drug-disease associations based on known protein complexes

    PubMed Central

    2015-01-01

    Inferring drug-disease associations is critical in unveiling disease mechanisms, as well as discovering novel functions of available drugs, or drug repositioning. Previous work is primarily based on drug-gene-disease relationship, which throws away many important information since genes execute their functions through interacting others. To overcome this issue, we propose a novel methodology that discover the drug-disease association based on protein complexes. Firstly, the integrated heterogeneous network consisting of drugs, protein complexes, and disease are constructed, where we assign weights to the drug-disease association by using probability. Then, from the tripartite network, we get the indirect weighted relationships between drugs and diseases. The larger the weight, the higher the reliability of the correlation. We apply our method to mental disorders and hypertension, and validate the result by using comparative toxicogenomics database. Our ranked results can be directly reinforced by existing biomedical literature, suggesting that our proposed method obtains higher specificity and sensitivity. The proposed method offers new insight into drug-disease discovery. Our method is publicly available at http://1.complexdrug.sinaapp.com/Drug_Complex_Disease/Data_Download.html. PMID:26044949

  9. Implications of Differential Age Distribution of Disease-Associated Meningococcal Lineages for Vaccine Development

    PubMed Central

    Trotter, Caroline L.; Ramsay, Mary E.; Chandra, Manosree; Jolley, Keith A.; van der Ende, Arie; Carion, Françoise; Berthelsen, Lene; Hoffmann, Steen; Harðardóttir, Hjördís; Vazquez, Julio A.; Murphy, Karen; Toropainen, Maija; Caniça, Manuela; Ferreira, Eugenia; Diggle, Mathew; Edwards, Giles F.; Taha, Muhamed-Kheir; Stefanelli, Paola; Kriz, Paula; Gray, Steve J.; Fox, Andrew J.; Jacobsson, Susanne; Claus, Heike; Vogel, Ulrich; Tzanakaki, Georgina; Heuberger, Sigrid; Caugant, Dominique A.; Frosch, Matthias; Maiden, Martin C. J.

    2014-01-01

    New vaccines targeting meningococci expressing serogroup B polysaccharide have been developed, with some being licensed in Europe. Coverage depends on the distribution of disease-associated genotypes, which may vary by age. It is well established that a small number of hyperinvasive lineages account for most disease, and these lineages are associated with particular antigens, including vaccine candidates. A collection of 4,048 representative meningococcal disease isolates from 18 European countries, collected over a 3-year period, were characterized by multilocus sequence typing (MLST). Age data were available for 3,147 isolates. The proportions of hyperinvasive lineages, identified as particular clonal complexes (ccs) by MLST, differed among age groups. Subjects <1 year of age experienced lower risk of sequence type 11 (ST-11) cc, ST-32 cc, and ST-269 cc disease and higher risk of disease due to unassigned STs, 1- to 4-year-olds experienced lower risk of ST-11 cc and ST-32 cc disease, 5- to 14-year-olds were less likely to experience ST-11 cc and ST-269 cc disease, and ≥25-year-olds were more likely to experience disease due to less common ccs and unassigned STs. Younger and older subjects were vulnerable to a more diverse set of genotypes, indicating the more clonal nature of genotypes affecting adolescents and young adults. Knowledge of temporal and spatial diversity and the dynamics of meningococcal populations is essential for disease control by vaccines, as coverage is lineage specific. The nonrandom age distribution of hyperinvasive lineages has consequences for the design and implementation of vaccines, as different variants, or perhaps targets, may be required for different age groups. PMID:24695776

  10. Emergence of telaprevir-resistant variants detected by ultra-deep sequencing after triple therapy in patients infected with HCV genotype 1.

    PubMed

    Akuta, Norio; Suzuki, Fumitaka; Seko, Yuya; Kawamura, Yusuke; Sezaki, Hitomi; Suzuki, Yoshiyuki; Hosaka, Tetsuya; Kobayashi, Masahiro; Hara, Tasuku; Kobayashi, Mariko; Saitoh, Satoshi; Arase, Yasuji; Ikeda, Kenji; Kumada, Hiromitsu

    2013-06-01

    Using ultra-deep sequencing technology, the present was designed to investigate whether the emergence of telaprevir-resistant variants (amino acid substitutions of aa36, aa54, aa155, aa156, and aa170 positions in HCV NS3 region) after commencement of triple therapy of telaprevir/peginterferon (PEG-IFN)/ribavirin could be predicted at baseline in previous non-responders to dual therapy. Fourteen patients infected with HCV genotype 1 who did not respond to previous PEG-IFN/ribavirin, received a 24-week regimen of triple therapy, and were evaluated for appearance of telaprevir-resistant variants (amino acid substitutions of more than 0.2% among the total coverage) by ultra-deep sequencing. The sustained virological response rate was 28.6% (4 of 14 patients), which was significantly higher in patients with Arg70 (substitution at core aa70) and partial response (type of previous response to PEG-IFN/ribavirin) than in other patients. Telaprevir-resistant variants at baseline were detected in 7.1% (1 of 14 patients) by direct sequencing and in 21.4% (3 of 14 patients) by ultra-deep sequencing. The appearance of telaprevir-resistant variants was examined by ultra-deep sequencing in 10 who did not show sustained virological responders. De novo variants emerged at re-elevation of viral load, regardless of variant frequencies at baseline (one patient with very high frequency variants [T54S: 99.9%], two patients with very low frequency variants [V36A: 0.2%; and V170A: 0.4%], and seven patients of undetectable variants). It is concluded that it is difficult to predict at baseline the emergence of telaprevir-resistant variants after commencement of triple therapy in prior non-responders of HCV genotype 1, even with the use of ultra-deep sequencing. PMID:23588728

  11. Genotypic Detection of rpoB and katG Gene Mutations Associated with Rifampicin and Isoniazid Resistance in Mycobacterium Tuberculosis Isolates: A Local Scenario (Kelantan)

    PubMed Central

    Ismail, Nurul-Ain; Ismail, Mohd Fazli; Noor, Siti Suraiya MD; Camalxaman, Siti Nazrina

    2016-01-01

    Background Drug resistant tuberculosis (DR-TB) remains a public health issue that is of major concern on a global scale. The characterisation of clinical isolates may provide key information regarding the underlying mechanisms of drug resistance, and helps to augment therapeutic options. This study aims to evaluate the frequency of gene mutations associated with Rifampicin (RIF) and Isoniazid (INH) resistance among nine clinical isolates. Methods A total of nine drug resistant Mycobacterium tuberculosis clinical isolates were screened for genetic mutations in rpoB and katusing polymerase chain reaction (PCR) amplification and DNA sequencing. Genotypic analysis was performed to detect the mutations in the sequence of the target genes. Results Our findings reveal that 80% of the isolates possess mutations at codon 119 (His119Tyr) and 135 (Arg135Trp and Ser135Leu) within the rpoB gene; and 70% possess mutations in the katG gene at codon 238 with amino acid change (Leu238Arg). Conclusion Findings from this study provide an overview of the current situation of RIF and INH resistance in a hospital Universiti Sains Malaysia (HUSM) located in Kelantan, Malaysia, which could facilitate molecular-based detection methods of drug-resistant strains. Further information regarding the molecular mechanisms involved in resistance in RR-/MDR-TB should be addressed in the near future. PMID:27540322

  12. Distribution and phenotypic and genotypic detection of a metallo-β-lactamase, CphA, among bacteraemic Aeromonas isolates.

    PubMed

    Wu, Chi-Jung; Chen, Po-Lin; Wu, Jiunn-Jong; Yan, Jing-Jou; Lee, Chin-Chi; Lee, Hsin-Chun; Lee, Nan-Yao; Chang, Chia-Ming; Lin, Yu-Tzu; Chiu, Yen-Cheng; Ko, Wen-Chien

    2012-05-01

    The objectives of the study were to investigate the distribution of cphA-related genes (cphA) encoding a CphA metallo-β-lactamase (MBL) among 51 consecutive Aeromonas blood isolates and to compare different phenotypic methods for detecting CphA. The presence of cphA was detected by PCR. Four phenotypic methods, the imipenem-EDTA combined disc test, imipenem-EDTA MBL Etest, agar dilution test and modified Hodge test (MHT), were used to detect imipenem susceptibility and MBL production. The results showed that 35 (69%) blood isolates had cphA. All (100%) of 16 Aeromonas aquariorum isolates and 12 Aeromonas veronii isolates, and 4 (80%) of 5 Aeromonas hydrophila isolates, carried cphA, but none of 15 Aeromonas caviae isolates did. With the standard inocula, irrespective of the presence or absence of cphA, all but one (50, 98%) isolates were susceptible to imipenem tested by disc diffusion, Etest and agar dilution (10(4) c.f.u. spot inocula), and did not exhibit MBL production by the imipenem-EDTA combined disc test and MBL Etest. By the agar dilution test using large inocula (10(7) c.f.u.), 34 (97%) of 35 cphA(+) isolates had imipenem MICs of ≥16 µg ml(-1), higher than the susceptible breakpoint (4 µg ml(-1)), and demonstrated positive results for the MHT, while one cphA(+) and all 17 cphA(-) isolates had imipenem MICs of ≤4 µg ml(-1). In conclusion, the distribution of cphA among aeromonads is species-specific, found in A. aquariorum, A. veronii and A. hydrophila, and the MHT may be a phenotypic screening test for CphA production. PMID:22322339

  13. Genotyping methods.

    PubMed

    Tümmler, Burkhard

    2014-01-01

    Genotyping allows for the identification of bacterial isolates to the strain level and provides basic information about the evolutionary biology, population biology, taxonomy, ecology, and genetics of bacteria. Depending on the underlying question and available resources, Pseudomonas aeruginosa strains may be typed by anonymous fingerprinting techniques or electronically portable sequence-based typing methods such as multiple locus variable number tandem repeat (VNTR) analysis (MLVA), multilocus sequence typing, or oligonucleotide microarray. Macrorestriction fragment pattern analysis is a genotyping method that is globally applicable to all bacteria and hence has been and still is the reference method for strain typing in bacteriology. Agarose-embedded chromosomal DNA is cleaved with a rare-cutting restriction endonuclease and the generated 20-70 fragments are then separated by pulsed-field gel electrophoresis. The chapter provides a detailed step-by-step manual for SpeI genome fingerprinting of Pseudomonas chromosomes that has been optimized for SpeI fragment pattern analysis of P. aeruginosa. PMID:24818895

  14. PCR based detection of HPV 16 and 18 genotypes in normal oral mucosa of tobacco users and non-users.

    PubMed

    Pattanshetty, S; Kotrashetti, V S; Nayak, R; Bhat, K; Somannavar, P; Babji, D

    2014-08-01

    There is increasing evidence of a causal association between human papillomavirus (HPV) and oral squamous cell carcinoma (OSCC). Several studies have shown that HPV is associated with increased risk of oral cancer independent of exposure to tobacco and alcohol. The association is valid for HPVs 16 and 18, which generally are considered high risk types, because they have been detected in oral dysplastic lesions and cancers. We determined the baseline prevalence of HPVs 16 and 18 in normal oral mucosa of individuals with and without tobacco habit. PCR was used for DNA collected by oral smears to detect HPV 16/18 DNA in normal oral mucosa of 60 healthy individuals who were assigned to two groups of 30 subjects each. One group had a tobacco habit, the other did not. The tobacco user group comprised individuals who were tobacco chewers only. Sixty-five percent of individuals were positive for HPV 16/18 DNA, but HPV 16/18 positivity was less in individuals with tobacco habit than in those without tobacco habit. No significant association was found between the presence of HPVs and gender, age or duration of chewing habit, or between groups with and without a tobacco habit. We propose that HPVs16 and 18 commonly are present in normal oral mucosa and emphasize the importance of distinguishing clinical, subclinical and latent HPV infections when investigating HPVs and OSCC. PMID:24588599

  15. Disease associated with exposure to certain herbicide agents: peripheral neuropathy.

    PubMed

    2013-09-01

    The Department of Veterans Affairs (VA) adopts as a final rule its proposal to amend its adjudication regulations by clarifying and expanding the terminology regarding presumptive service connection for acute and subacute peripheral neuropathy associated with exposure to certain herbicide agents. This amendment implements a decision by the Secretary of Veterans Affairs based on findings from the National Academy of Sciences (NAS) Institute of Medicine report, Veterans and Agent Orange: Update 2010. It also amends VA's regulation governing retroactive awards for certain diseases associated with herbicide exposure as required by court orders in the class action litigation of Nehmer v. U.S. Department of Veterans Affairs. PMID:24040683

  16. Detection of the Klebsiella pneumoniae carbapenemase (KPC) in K. pneumoniae Isolated from the Clinical Samples by the Phenotypic and Genotypic Methods

    PubMed Central

    Bina, Masoume; Pournajaf, Abazar; Mirkalantari, Shiva; Talebi, Malihe; Irajian, Gholamreza

    2015-01-01

    Background and Objective: The production of carbapenemases especially Klebsiella pneumoniae carbapenemase (KPC) is the most important mechanism of enzymatic resistance in isolated Enterobacteriaceae such as K. pneumoniae . The purpose of this study was detected of the carbapenemase producer K. pneumoniae strains with phenotypic and genotypic methods. Method: Out of 800 strains, 270 K. pneumoniae strains (33.7%), were obtained. Antibiotic susceptibility test was performed by disk diffusion method in accordance with CLSI guidelines. Carbapenem resistant strains were identified by the Modified Hodge Test based on CLSI instruction and PCR for surveying the presence of bla -KPC gene. Results: A total 270 K. pneumoniae strains were collected. Antibiotic susceptibility test results showed the highest and lowest resistance was related to piperacillin (60.6%) and carbapenems (14.6%) respectively. 80.5% (33 of 41) isolates were positive by MHT, but all of them (100%) were negative for amplification of the bla -KPC gene in the PCR method. Conclusion: The MHT was an appropriate method for approving carbapenemase production. Moreover, a laboratory could accept the carbapenemase production with PCR method for the bla-KPC gene, which has the additional profit of validating which KPC is present. PMID:26351485

  17. Correlation of agar dilution and VITEK2 system for detection of resistance to macrolides, lincosamides and pristinamycin among Staphylococcus aureus and Staphylococcus epidermidis: association with genotypes.

    PubMed

    Bémer, P; Juvin, M-E; Corvec, S; Ros, A; Drugeon, H

    2005-08-01

    The performance of the VITEK2 system was evaluated against the agar dilution reference procedure for testing susceptibility of Staphylococcus aureus and Staphylococcus epidermidis to macrolides, lincosamides and streptogramins (MLS). Eighty clinical isolates were selected according to their resistance phenotype and genotype. Results for erythromycin and clindamycin showed 100% agreement; results for lincomycin showed agreement of 78%, with one very major error and 17 minor errors; and results for pristinamycin showed agreement of 46%, with one major error and 43 minor errors. Most isolates resistant to lincomycin and streptogramin A (L SgAr phenotype) were falsely susceptible to lincomycin, and intermediately-resistant or resistant to pristinamycin, with the VITEK2 system. No resistance gene was detected. Most (80%) isolates resistant constitutively to MLS (MLS(r)BC phenotype) were falsely intermediately-resistant to pristinamycin with the VITEK2 system. The erm(A) gene was more common than erm(C) in MLS(r)BC strains. Resistance to pristinamycin alone (SgA SgB PTr phenotype), or associated with either lincomycin resistance (L SgA SgB PTr phenotype) or constitutive MLS(B) resistance (MLS(BC) SgA PTr phenotype), was well-characterised without discordant results. Resistance to pristinamycin was always associated with resistance to streptogramin A, encoded by the vga(A), vga(B), vgb(A) and vat(A) genes in association with the erm(A) or erm(C) genes. PMID:16008619

  18. Role of a GenoType MTBDRplus line probe assay in early detection of multidrug-resistant tuberculosis at a Brazilian reference center.

    PubMed

    Feliciano, C S; Nascimento, M M P; Anselmo, L M P; Pocente, R H C; Bellissimo-Rodrigues, F; Bollela, V R

    2015-08-01

    Resistance to Mycobacterium tuberculosis is a reality worldwide, and its diagnosis continues to be difficult and time consuming. To face this challenge, the World Health Organization has recommended the use of rapid molecular tests. We evaluated the routine use (once a week) of a line probe assay (Genotype MTBDRplus) for early diagnosis of resistance and for assessment of the main related risk factors over 2 years. A total of 170 samples were tested: 15 (8.8%) were resistant, and multidrug resistance was detected in 10 (5.9%). The sensitivity profile took 3 weeks (2 weeks for culture and 1 week for rapid testing). Previous treatment for tuberculosis and the persistence of positive acid-fast smears after 4 months of supervised treatment were the major risk factors observed. The use of molecular tests enabled early diagnosis of drug-resistant bacilli and led to appropriate treatment of the disease. This information has the potential to interrupt the transmission chain of resistant M. tuberculosis. PMID:26132094

  19. HLA disease association and protection in HIV infection among African Americans and Caucasians.

    PubMed

    Cruse, J M; Brackin, M N; Lewis, R E; Meeks, W; Nolan, R; Brackin, B

    1991-01-01

    In a previous investigation, we demonstrated an increased progression of overt AIDS in the African American population compared to the Caucasian population as reflected by the significantly lower absolute number of CD4+ lymphocytes detected in the African American population in an earlier study. The present study elucidates some of the possible genetic factors which may contribute to disease association or protection against HIV infection. The HLA phenotypes expressed as A, B, C, DR and DQw antigens were revealed by the Amos-modified typing procedure. NIH scoring was utilized to designate positive cells taking up trypan blue. A test of proportion equivalent to the chi 2 approximation was used to compare the disease population (n = 62; 38 African Americans, 24 Caucasians) to race-matched normal heterosexual local controls (323 African Americans, 412 Caucasians). Significant p values were corrected for the number of HLA antigens tested. HLA markers associated with possible protection from infection for African Americans were Cw4 and DRw6, whereas Caucasians expressed none. Disease association markers present in the African American population were A31, B35, Cw6, Cw7, DR5, DR6, DRw11, DRw12, DQw6 and DQw7, whereas in the Caucasian population A28, Aw66, Aw48, Bw65, Bw70, Cw7, DRw10, DRw12, DQw6 and DQw7 were demonstrated. The highest phenotypic frequency for a disease association marker in the study was for HLA-DR5 (62.9%) in the HIV-infected African American population without Kaposi's sarcoma compared to a frequency of 28.9% for the regional control group (p = 0.0012). We conclude that genetic factors do have a role in HIV infection since only 50-60% of those exposed to the AIDS virus will become infected. PMID:1910527

  20. Rotavirus Genotypes in Belarus, 2008–2012

    PubMed Central

    Semeiko, Galina; Yermalovich, Marina; Poliakova, Nadezhda; Mijatovic-Rustempasic, Slavica; Kerin, Tara; Wasley, Annemarie; Videbaek, Dovile; Gentsch, Jon R.; Bowen, Michael D.; Samoilovich, Elena

    2015-01-01

    This study describes group A rotavirus (RVA) genotype prevalence in Belarus from 2008–2012. In 2008, data from 3 sites in Belarus (Brest, Mogilev, Minsk) indicated that G4P[8] was the predominant genotype. Data from Minsk (2008–2012) showed that G4P[8] was the predominant RVA genotype in all years except in 2011 when G3P[8] was most frequently detected. Other RVA genotypes common in Europe (G1P[8], G2P[4]) were detected each year of the study. This study reveals the dominance of genotype G4P[8] in Belarus and helps to establish the baseline genotype prevalence prior to RVA vaccine introduction in the country. PMID:25218086

  1. Next generation sequencing based identification of disease-associated mutations in Swiss patients with retinal dystrophies

    PubMed Central

    Tiwari, Amit; Bahr, Angela; Bähr, Luzy; Fleischhauer, Johannes; Zinkernagel, Martin S.; Winkler, Niklas; Barthelmes, Daniel; Berger, Lieselotte; Gerth-Kahlert, Christina; Neidhardt, John; Berger, Wolfgang

    2016-01-01

    Inherited monogenic diseases of the retina and vitreous affect approximately 1 in 2000 individuals. They are characterized by tremendous genetic heterogeneity and clinical variability involving mutations in approximately 250 genes and more than 20 different clinical phenotypes. Clinical manifestations of retinal dystrophies (RDs) range from mild retinal dysfunctions to severe congenital forms of blindness. A detailed clinical diagnosis and the identification of causative mutations are crucial for genetic counseling of affected patients and their families, for understanding genotype-phenotype correlations and developing therapeutic approaches. Using whole exome sequencing (WES) we have established a reliable and efficient high-throughput analysis pipeline to identify disease-causing mutations. Our data indicate that this approach enables us to genetically diagnose approximately 64% of the patients (n = 58) with variant(s) in known disease-associated genes. We report 20 novel and 26 recurrent variants in genes associated with RDs. We also identified a novel phenotype for mutations in C2orf71 and provide functional evidence for exon skipping due to a splice-site variant identified in FLVCR1. In conclusion, WES can rapidly identify variants in various families affected with different forms of RDs. Our study also expands the clinical and allelic spectrum of genes associated with RDs in the Swiss population. PMID:27353947

  2. Revealing disease-associated pathways by network integration of untargeted metabolomics.

    PubMed

    Pirhaji, Leila; Milani, Pamela; Leidl, Mathias; Curran, Timothy; Avila-Pacheco, Julian; Clish, Clary B; White, Forest M; Saghatelian, Alan; Fraenkel, Ernest

    2016-09-01

    Uncovering the molecular context of dysregulated metabolites is crucial to understand pathogenic pathways. However, their system-level analysis has been limited owing to challenges in global metabolite identification. Most metabolite features detected by untargeted metabolomics carried out by liquid-chromatography-mass spectrometry cannot be uniquely identified without additional, time-consuming experiments. We report a network-based approach, prize-collecting Steiner forest algorithm for integrative analysis of untargeted metabolomics (PIUMet), that infers molecular pathways and components via integrative analysis of metabolite features, without requiring their identification. We demonstrated PIUMet by analyzing changes in metabolism of sphingolipids, fatty acids and steroids in a Huntington's disease model. Additionally, PIUMet enabled us to elucidate putative identities of altered metabolite features in diseased cells, and infer experimentally undetected, disease-associated metabolites and dysregulated proteins. Finally, we established PIUMet's ability for integrative analysis of untargeted metabolomics data with proteomics data, demonstrating that this approach elicits disease-associated metabolites and proteins that cannot be inferred by individual analysis of these data. PMID:27479327

  3. Genotypic Identification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In comparison with traditional, phenotype-based procedures for detection and identification of foodborne pathogen Listeria monocytogenes, molecular techniques are superior in terms of sensitivity, specificity and speed. This chapter provides a comprehensive review on the use of molecular methods for...

  4. Inductive matrix completion for predicting gene–disease associations

    PubMed Central

    Natarajan, Nagarajan; Dhillon, Inderjit S.

    2014-01-01

    Motivation: Most existing methods for predicting causal disease genes rely on specific type of evidence, and are therefore limited in terms of applicability. More often than not, the type of evidence available for diseases varies—for example, we may know linked genes, keywords associated with the disease obtained by mining text, or co-occurrence of disease symptoms in patients. Similarly, the type of evidence available for genes varies—for example, specific microarray probes convey information only for certain sets of genes. In this article, we apply a novel matrix-completion method called Inductive Matrix Completion to the problem of predicting gene-disease associations; it combines multiple types of evidence (features) for diseases and genes to learn latent factors that explain the observed gene–disease associations. We construct features from different biological sources such as microarray expression data and disease-related textual data. A crucial advantage of the method is that it is inductive; it can be applied to diseases not seen at training time, unlike traditional matrix-completion approaches and network-based inference methods that are transductive. Results: Comparison with state-of-the-art methods on diseases from the Online Mendelian Inheritance in Man (OMIM) database shows that the proposed approach is substantially better—it has close to one-in-four chance of recovering a true association in the top 100 predictions, compared to the recently proposed Catapult method (second best) that has <15% chance. We demonstrate that the inductive method is particularly effective for a query disease with no previously known gene associations, and for predicting novel genes, i.e. genes that are previously not linked to diseases. Thus the method is capable of predicting novel genes even for well-characterized diseases. We also validate the novelty of predictions by evaluating the method on recently reported OMIM associations and on associations recently

  5. Evaluation of two commercial kits for the detection of genotypic drug resistance on a panel of HIV type 1 subtypes A through J.

    PubMed

    Fontaine, E; Riva, C; Peeters, M; Schmit, J C; Delaporte, E; Van Laethem, K; Van Vaerenbergh, K; Snoeck, J; Van Wijngaerden, E; De Clercq, E; Van Ranst, M; Vandamme, A M

    2001-11-01

    We compared the two commercially available sequencing kits for HIV-1 drug resistance testing, the ViroSeq Genotyping System (Applied Biosystems, Foster City, CA, U.S.A.) and the TRUGENE HIV-1 Genotyping Kit (Visible Genetics, Inc., Toronto, Ontario, Canada), with our in-house genotyping system. Fifteen viral isolates from African patients (6 treated and 9 untreated) covering a panel of HIV-1 subtypes A through J and 7 plasma samples from Belgian and African patients (2 treated and 5 untreated) were tested. All the samples could be amplified and sequenced by the three systems; however, for all systems, alternative amplification/sequencing primers had to be used for some samples belonging to subtype B as well as to other subtypes. The consensus sequence was partially derived from only one strand for the in-house system and for the ViroSeq Genotyping System. The TRUGENE HIV-1 Genotyping Kit scored the highest number of ambiguities, followed by the ViroSeq Genotyping System and the in-house system. For 11 samples, these differences in reporting mixtures affected 14 resistance-related positions, which altered the interpretation toward protease inhibitors for 2 samples when using version 1.2 RetroGram software (Virology Networks, Utrecht, The Netherlands). All three systems were able to sequence diluted samples with a viral load down to 10 3 or 10 4 RNA copies/ml. Our data therefore suggest that the performance of amplification and sequencing primers must be improved to allow fast and reliable resistance testing for all HIV-1 subtypes. PMID:11694832

  6. Protein-Peptide Arrays for Detection of Specific Anti-Hepatitis D Virus (HDV) Genotype 1, 6, and 8 Antibodies among HDV-Infected Patients by Surface Plasmon Resonance Imaging

    PubMed Central

    Villiers, Marie-Bernadette; Cortay, Jean-Claude; Cortès, Sandra; Bloquel, Bénédicte; Brichler, Ségolène; Brakha, Carine; Kay, Alan; Falah, Nisrine; Zoulim, Fabien; Marquette, Christophe

    2015-01-01

    Liver diseases linked to hepatitis B-hepatitis D virus co- or superinfections are more severe than those during hepatitis B virus (HBV) monoinfection. The diagnosis of hepatitis D virus (HDV) infection therefore remains crucial in monitoring patients but is often overlooked. To integrate HDV markers into high-throughput viral hepatitis diagnostics, we studied the binding of anti-HDV antibodies (Abs) using surface plasmon resonance imaging (SPRi). We focused on the ubiquitous HDV genotype 1 (HDV1) and the more uncommon African-HDV6 and HDV8 genotypes to define an array with recombinant proteins or peptides. Full-length and truncated small hepatitis D antigen (S-HDAg) recombinant proteins of HDV genotype 1 (HDV1) and 11 HDV peptides of HDV1, 6, and 8, representing various portions of the delta antigen were grafted onto biochips, allowing SPRi measurements to be made. Sixteen to 17 serum samples from patients infected with different HDV genotypes were injected onto protein and peptide chips. In all, Abs against HDV proteins and/or peptides were detected in 16 out of 17 infected patients (94.12%), although the amplitude of the SPR signal varied. The amino-terminal part of the protein was poorly immunogenic, while epitope 65-80, exposed on the viral ribonucleoprotein, may be immunodominant, as 9 patient samples led to a specific SPR signal on peptide 65 type 1 (65#1), independently of the infecting genotype. In this pilot study, we confirmed that HDV infection screening based on the reactivity of patient Abs against carefully chosen HDV peptides and/or proteins can be included in a syndrome-based viral hepatitis diagnostic assay. The preliminary results indicated that SPRi studying direct physical HDAg–anti-HDV Ab interactions was more convenient using linear peptide epitopes than full-length S-HDAg proteins, due to the regeneration process, and may represent an innovative approach for a hepatitis syndrome–viral etiology-exploring array. PMID:25631795

  7. Prevalence of HCV genotypes in district Mardan

    PubMed Central

    2013-01-01

    Background Approximately 170 million people are infected with Hepatitis C virus (HCV) worldwide. The prevalence of chronic HCV infections in Pakistan is about 5%, with most individuals being infected with HCV genotype 3a. Data on HCV genotypes distribution across various districts of the country are scarce. One example is district Mardan from where such data is available only from 17 individuals. Accordingly, the present study aimed at determining HCV genotypes distribution among 177 HCV RNA positive individuals from district Mardan. Findings Serum samples (n = 215) from patients suspected of hepatitis C were collected and processed for Nested PCR based detection and subsequent genotyping. Gender-wise and age-wise differences in HCV prevalence and HCV genotypes distribution were determined by χ2 test. Out of the total 215 serum samples, 177 were found to be positive for HCV RNA. The genotype 3a was the most predominant genotype among HCV RNA positive samples with a prevalence of 90.3%, followed by genotype 1a (5.6%), mixed genotypes (2.8%), genotype 3b (0.6%) and genotype 4 (0.6%). The HCV prevalence was higher in young individuals than old people and was indicative of reduced survival rate beyond 40 years. Conclusion HCV genotype 3a is the most predominant genotype in district Mardan. The state of the art preventive and therapeutic strategies should be implemented to control the spread of HCV infections. Further temporal studies involving different geographical areas of Pakistan, are required to improve the control measures for HCV infection. PMID:23514695

  8. Diseases Associated with Defective Responses to DNA Damage

    PubMed Central

    O’Driscoll, Mark

    2012-01-01

    Within the last decade, multiple novel congenital human disorders have been described with genetic defects in known and/or novel components of several well-known DNA repair and damage response pathways. Examples include disorders of impaired nucleotide excision repair, DNA double-strand and single-strand break repair, as well as compromised DNA damage-induced signal transduction including phosphorylation and ubiquitination. These conditions further reinforce the importance of multiple genome stability pathways for health and development in humans. Furthermore, these conditions inform our knowledge of the biology of the mechanics of genome stability and in some cases provide potential routes to help exploit these pathways therapeutically. Here, I will review a selection of these exciting findings from the perspective of the disorders themselves, describing how they were identified, how genotype informs phenotype, and how these defects contribute to our growing understanding of genome stability pathways. PMID:23209155

  9. Biomarkers in connective tissue disease-associated interstitial lung disease.

    PubMed

    Bonella, Francesco; Costabel, Ulrich

    2014-04-01

    This article reviews major biomarkers in serum and bronchoalveolar lavage fluid (BALF) with respect to their diagnostic and prognostic value in connective tissue disease-associated interstitial lung disease (CTD-ILD). In some CTD such as systemic sclerosis (SSc), the incidence of ILD is up to two-third of patients, and currently ILD represents the leading cause of death in SSc. Because of the extremely variable incidence and outcome of ILD in CTD, progress in the discovery and validation of biomarkers for diagnosis, prognosis, patients' subtyping, response to treatment, or as surrogate endpoints in clinical trials is extremely important. In contrast to idiopathic interstitial pneumonias, autoantibodies play a crucial role as biomarkers in CTD-ILD because their presence is strictly linked to the pathogenesis and tissue damage. Patterns of autoantibodies, for instance, anticitrullinated peptide antibodies in rheumatoid arthritis or aminoacyl-tRNA synthetases (ARS) in polymyositis/dermatomyositis, have been found to correlate with the presence and occasionally with the course of ILD in CTD. Besides autoantibodies, an increase in serum or BALF of a biomarker of pulmonary origin may be able to predict or reflect the development of fibrosis, the impairment of lung function, and ideally also the prognosis. Promising biomarkers are lung epithelium-derived proteins such as KL-6 (Krebs von den Lungen-6), SP-D (surfactant protein-D), SP-A (surfactant protein-A), YKL-40 (chitinase-3-like protein 1 [CHI3L1] or cytokines such as CCL18 [chemokine (C-C) motif ligand 18]). In the future, genetic/epigenetic markers, such as human leukocyte antigen (HLA) haplotypes, single nucleotide polymorphisms, and micro-RNA, may help to identify subtypes of patients with different needs of management and treatment strategies. PMID:24668534

  10. Brain Expression Genome-Wide Association Study (eGWAS) Identifies Human Disease-Associated Variants

    PubMed Central

    Crook, Julia; Pankratz, V. Shane; Carrasquillo, Minerva M.; Rowley, Christopher N.; Nair, Asha A.; Middha, Sumit; Maharjan, Sooraj; Nguyen, Thuy; Ma, Li; Malphrus, Kimberly G.; Palusak, Ryan; Lincoln, Sarah; Bisceglio, Gina; Georgescu, Constantin; Kouri, Naomi; Kolbert, Christopher P.; Jen, Jin; Haines, Jonathan L.; Mayeux, Richard; Pericak-Vance, Margaret A.; Farrer, Lindsay A.; Schellenberg, Gerard D.; Petersen, Ronald C.; Graff-Radford, Neill R.; Dickson, Dennis W.; Younkin, Steven G.; Ertekin-Taner, Nilüfer

    2012-01-01

    Genetic variants that modify brain gene expression may also influence risk for human diseases. We measured expression levels of 24,526 transcripts in brain samples from the cerebellum and temporal cortex of autopsied subjects with Alzheimer's disease (AD, cerebellar n = 197, temporal cortex n = 202) and with other brain pathologies (non–AD, cerebellar n = 177, temporal cortex n = 197). We conducted an expression genome-wide association study (eGWAS) using 213,528 cisSNPs within ±100 kb of the tested transcripts. We identified 2,980 cerebellar cisSNP/transcript level associations (2,596 unique cisSNPs) significant in both ADs and non–ADs (q<0.05, p = 7.70×10−5–1.67×10−82). Of these, 2,089 were also significant in the temporal cortex (p = 1.85×10−5–1.70×10−141). The top cerebellar cisSNPs had 2.4-fold enrichment for human disease-associated variants (p<10−6). We identified novel cisSNP/transcript associations for human disease-associated variants, including progressive supranuclear palsy SLCO1A2/rs11568563, Parkinson's disease (PD) MMRN1/rs6532197, Paget's disease OPTN/rs1561570; and we confirmed others, including PD MAPT/rs242557, systemic lupus erythematosus and ulcerative colitis IRF5/rs4728142, and type 1 diabetes mellitus RPS26/rs1701704. In our eGWAS, there was 2.9–3.3 fold enrichment (p<10−6) of significant cisSNPs with suggestive AD–risk association (p<10−3) in the Alzheimer's Disease Genetics Consortium GWAS. These results demonstrate the significant contributions of genetic factors to human brain gene expression, which are reliably detected across different brain regions and pathologies. The significant enrichment of brain cisSNPs among disease-associated variants advocates gene expression changes as a mechanism for many central nervous system (CNS) and non–CNS diseases. Combined assessment of expression and disease GWAS may provide complementary information in discovery of human disease variants with

  11. Brain expression genome-wide association study (eGWAS) identifies human disease-associated variants.

    PubMed

    Zou, Fanggeng; Chai, High Seng; Younkin, Curtis S; Allen, Mariet; Crook, Julia; Pankratz, V Shane; Carrasquillo, Minerva M; Rowley, Christopher N; Nair, Asha A; Middha, Sumit; Maharjan, Sooraj; Nguyen, Thuy; Ma, Li; Malphrus, Kimberly G; Palusak, Ryan; Lincoln, Sarah; Bisceglio, Gina; Georgescu, Constantin; Kouri, Naomi; Kolbert, Christopher P; Jen, Jin; Haines, Jonathan L; Mayeux, Richard; Pericak-Vance, Margaret A; Farrer, Lindsay A; Schellenberg, Gerard D; Petersen, Ronald C; Graff-Radford, Neill R; Dickson, Dennis W; Younkin, Steven G; Ertekin-Taner, Nilüfer

    2012-01-01

    Genetic variants that modify brain gene expression may also influence risk for human diseases. We measured expression levels of 24,526 transcripts in brain samples from the cerebellum and temporal cortex of autopsied subjects with Alzheimer's disease (AD, cerebellar n=197, temporal cortex n=202) and with other brain pathologies (non-AD, cerebellar n=177, temporal cortex n=197). We conducted an expression genome-wide association study (eGWAS) using 213,528 cisSNPs within ± 100 kb of the tested transcripts. We identified 2,980 cerebellar cisSNP/transcript level associations (2,596 unique cisSNPs) significant in both ADs and non-ADs (q<0.05, p=7.70 × 10(-5)-1.67 × 10(-82)). Of these, 2,089 were also significant in the temporal cortex (p=1.85 × 10(-5)-1.70 × 10(-141)). The top cerebellar cisSNPs had 2.4-fold enrichment for human disease-associated variants (p<10(-6)). We identified novel cisSNP/transcript associations for human disease-associated variants, including progressive supranuclear palsy SLCO1A2/rs11568563, Parkinson's disease (PD) MMRN1/rs6532197, Paget's disease OPTN/rs1561570; and we confirmed others, including PD MAPT/rs242557, systemic lupus erythematosus and ulcerative colitis IRF5/rs4728142, and type 1 diabetes mellitus RPS26/rs1701704. In our eGWAS, there was 2.9-3.3 fold enrichment (p<10(-6)) of significant cisSNPs with suggestive AD-risk association (p<10(-3)) in the Alzheimer's Disease Genetics Consortium GWAS. These results demonstrate the significant contributions of genetic factors to human brain gene expression, which are reliably detected across different brain regions and pathologies. The significant enrichment of brain cisSNPs among disease-associated variants advocates gene expression changes as a mechanism for many central nervous system (CNS) and non-CNS diseases. Combined assessment of expression and disease GWAS may provide complementary information in discovery of human disease variants with functional implications. Our findings

  12. Inferring haplotypes from genotypes on a pedigree with mutations, genotyping errors and missing alleles.

    PubMed

    Wang, Wei-Bung; Jiang, Tao

    2011-04-01

    Inferring the haplotypes of the members of a pedigree from their genotypes has been extensively studied. However, most studies do not consider genotyping errors and de novo mutations. In this paper, we study how to infer haplotypes from genotype data that may contain genotyping errors, de novo mutations, and missing alleles. We assume that there are no recombinants in the genotype data, which is usually true for tightly linked markers. We introduce a combinatorial optimization problem, called haplotype configuration with mutations and errors (HCME), which calls for haplotype configurations consistent with the given genotypes that incur no recombinants and require the minimum number of mutations and errors. HCME is NP-hard. To solve the problem, we propose a heuristic algorithm, the core of which is an integer linear program (ILP) using the system of linear equations over Galois field GF(2). Our algorithm can detect and locate genotyping errors that cannot be detected by simply checking the Mendelian law of inheritance. The algorithm also offers error correction in genotypes/haplotypes rather than just detecting inconsistencies and deleting the involved loci. Our experimental results show that the algorithm can infer haplotypes with a very high accuracy and recover 65%-94% of genotyping errors depending on the pedigree topology. PMID:21523936

  13. Population based allele frequencies of disease associated polymorphisms in the Personalized Medicine Research Project

    PubMed Central

    2010-01-01

    Background There is a lack of knowledge regarding the frequency of disease associated polymorphisms in populations and population attributable risk for many populations remains unknown. Factors that could affect the association of the allele with disease, either positively or negatively, such as race, ethnicity, and gender, may not be possible to determine without population based allele frequencies. Here we used a panel of 51 polymorphisms previously associated with at least one disease and determined the allele frequencies within the entire Personalized Medicine Research Project population based cohort. We compared these allele frequencies to those in dbSNP and other data sources stratified by race. Differences in allele frequencies between self reported race, region of origin, and sex were determined. Results There were 19544 individuals who self reported a single racial category, 19027 or (97.4%) self reported white Caucasian, and 11205 (57.3%) individuals were female. Of the 11,208 (57%) individuals with an identifiable region of origin 8337 or (74.4%) were German. 41 polymorphisms were significantly different between self reported race at the 0.05 level. Stratification of our Caucasian population by self reported region of origin revealed 19 polymorphisms that were significantly different (p = 0.05) between individuals of different origins. Further stratification of the population by gender revealed few significant differences in allele frequencies between the genders. Conclusions This represents one of the largest population based allele frequency studies to date. Stratification by self reported race and region of origin revealed wide differences in allele frequencies not only by race but also by region of origin within a single racial group. We report allele frequencies for our Asian/Hmong and American Indian populations; these two minority groups are not typically selected for population allele frequency detection. Population wide allele frequencies are

  14. Simultaneous Genotyping and Quantification of Hepatitis B Virus for Genotypes B and C by Real-Time PCR Assay▿

    PubMed Central

    Zhao, Yao; Zhang, Xiu-Yu; Guo, Jin-Jun; Zeng, Ai-Zhong; Hu, Jie-Li; Huang, Wen-Xiang; Shan, You-Lan; Huang, Ai-Long

    2010-01-01

    Hepatitis B virus (HBV) is an important cause of human chronic liver diseases and is a major public health problem. Viral load and HBV genotype play critical roles in determining clinical outcomes and response to antiviral treatment in hepatitis B patients. Viral genotype detection and quantification assays are currently in use with different levels of effectiveness. In this study, the performance of a real-time genotyping and quantitative PCR (GQ-PCR)-based assay was evaluated. Through the use of genotype-specific primers and probes, this assay provides simultaneous identification and quantification of genotypes B and C in a single reaction. Our GQ-PCR correctly identified all predefined genotypes B and C, and no cross-reaction between genotypes B and C were observed. The GQ-PCR identified more cases of HBV infections with mixed genotypes B and C than direct sequencing did. Samples from 127 HBV-infected Chinese patients were genotyped with GQ-PCR, revealing 56.7% HBV as genotype B, 13.4% as genotype C, and 29.8% as mixed genotypes B and C. This assay provides a reliable, efficient, and cost-effective means for quantification of the B and C genotypes of HBV in single or mixed infections. This assay is suitable for sequential monitoring of viral load levels and for determining the relationship between the genotype viral load and stage of disease in Asians. PMID:20720032

  15. One-step multiplex real-time RT-PCR assay for detecting and genotyping wild-type group A rotavirus strains and vaccine strains (Rotarix® and RotaTeq®) in stool samples.

    PubMed

    Gautam, Rashi; Mijatovic-Rustempasic, Slavica; Esona, Mathew D; Tam, Ka Ian; Quaye, Osbourne; Bowen, Michael D

    2016-01-01

    Background. Group A rotavirus (RVA) infection is the major cause of acute gastroenteritis (AGE) in young children worldwide. Introduction of two live-attenuated rotavirus vaccines, RotaTeq® and Rotarix®, has dramatically reduced RVA associated AGE and mortality in developed as well as in many developing countries. High-throughput methods are needed to genotype rotavirus wild-type strains and to identify vaccine strains in stool samples. Quantitative RT-PCR assays (qRT-PCR) offer several advantages including increased sensitivity, higher throughput, and faster turnaround time. Methods. In this study, a one-step multiplex qRT-PCR assay was developed to detect and genotype wild-type strains and vaccine (Rotarix® and RotaTeq®) rotavirus strains along with an internal processing control (Xeno or MS2 RNA). Real-time RT-PCR assays were designed for VP7 (G1, G2, G3, G4, G9, G12) and VP4 (P[4], P[6] and P[8]) genotypes. The multiplex qRT-PCR assay also included previously published NSP3 qRT-PCR for rotavirus detection and Rotarix® NSP2 and RotaTeq® VP6 qRT-PCRs for detection of Rotarix® and RotaTeq® vaccine strains respectively. The multiplex qRT-PCR assay was validated using 853 sequence confirmed stool samples and 24 lab cultured strains of different rotavirus genotypes. By using thermostable rTth polymerase enzyme, dsRNA denaturation, reverse transcription (RT) and amplification (PCR) steps were performed in single tube by uninterrupted thermocycling profile to reduce chances of sample cross contamination and for rapid generation of results. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments. Results. The VP7 qRT-PCRs exhibited 98.8-100% sensitivity, 99.7-100% specificity, 85-95% efficiency and a limit of detection of 4-60 copies per singleplex reaction. The VP7 qRT-PCRs exhibited 81-92% efficiency and limit of detection of 150-600 copies in multiplex reactions. The VP4 qRT-PCRs exhibited 98

  16. One-step multiplex real-time RT-PCR assay for detecting and genotyping wild-type group A rotavirus strains and vaccine strains (Rotarix® and RotaTeq®) in stool samples

    PubMed Central

    Mijatovic-Rustempasic, Slavica; Esona, Mathew D.; Tam, Ka Ian; Quaye, Osbourne; Bowen, Michael D.

    2016-01-01

    Background. Group A rotavirus (RVA) infection is the major cause of acute gastroenteritis (AGE) in young children worldwide. Introduction of two live-attenuated rotavirus vaccines, RotaTeq® and Rotarix®, has dramatically reduced RVA associated AGE and mortality in developed as well as in many developing countries. High-throughput methods are needed to genotype rotavirus wild-type strains and to identify vaccine strains in stool samples. Quantitative RT-PCR assays (qRT-PCR) offer several advantages including increased sensitivity, higher throughput, and faster turnaround time. Methods. In this study, a one-step multiplex qRT-PCR assay was developed to detect and genotype wild-type strains and vaccine (Rotarix® and RotaTeq®) rotavirus strains along with an internal processing control (Xeno or MS2 RNA). Real-time RT-PCR assays were designed for VP7 (G1, G2, G3, G4, G9, G12) and VP4 (P[4], P[6] and P[8]) genotypes. The multiplex qRT-PCR assay also included previously published NSP3 qRT-PCR for rotavirus detection and Rotarix® NSP2 and RotaTeq® VP6 qRT-PCRs for detection of Rotarix® and RotaTeq® vaccine strains respectively. The multiplex qRT-PCR assay was validated using 853 sequence confirmed stool samples and 24 lab cultured strains of different rotavirus genotypes. By using thermostable rTth polymerase enzyme, dsRNA denaturation, reverse transcription (RT) and amplification (PCR) steps were performed in single tube by uninterrupted thermocycling profile to reduce chances of sample cross contamination and for rapid generation of results. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments. Results. The VP7 qRT-PCRs exhibited 98.8–100% sensitivity, 99.7–100% specificity, 85–95% efficiency and a limit of detection of 4–60 copies per singleplex reaction. The VP7 qRT-PCRs exhibited 81–92% efficiency and limit of detection of 150–600 copies in multiplex reactions. The VP4 qRT-PCRs exhibited 98.8

  17. Transmission and persistence of Ceratonova shasta genotypes in Chinook salmon.

    PubMed

    Hurst, Charlene N; Wong, Peter; Hallett, Sascha L; Ray, R Adam; Bartholomew, Jerri L

    2014-12-01

    Ceratonova shasta is a myxozoan parasite of salmon and trout transmitted by waterborne actinospores. Based on DNA sequence data and host specificity, 4 distinct parasite genotypes are recognized. Genotypes I and II are common in the lower reaches of the Klamath River, Oregon-California, but only infection by genotype I causes mortality in Chinook salmon. We conducted sentinel fish exposures and determined genotype composition in river water during exposure, and in fish gills, intestine, and tank water post-exposure to determine whether: (1) transmission of parasites having different genotypes is host-specific and (2) all transmitted genotypes persist in the host through to release as waterborne stages. Initial parasite transmission to the fish host appears indiscriminant, since we detected both genotypes I and II in 83.6% of the fish gills sampled. However, only genotype I was detected in fish that succumbed to infection, while both genotypes persisted in fish that survived. Persistence was likely dependent on exposure dose, initial infection type (mixed or single) and infection outcome (mortality or survival). The transmission of both genotypes to a majority of Chinook salmon and the persistence of multiple genotypes raises questions about how infection with mixed genotypes could result in within-host interactions that affect disease severity. PMID:24945751

  18. Geographic differences and the role of cagA gene in gastroduodenal diseases associated with Helicobacter pylori infection.

    PubMed

    Valmaseda Pérez, T; Gisbert, J P; Pajares García, J M

    2001-07-01

    Helicobacter pylori (H. pylori) is the major causal agent of gastritis, peptic ulcer and gastric cancer. Several bacterium genes seem to be involved in the pathogenicity mechanism. One of them, the cagA gene, has been extensively studied and characterized. In this article we have carried out a study of characteristics and genetic variability of cagA gene in different geographic areas of the world. At the same time, we have summarized several studies that evaluate possible relation of cagA with gastroduodenal diseases associated by H. pylori infection. In our study we found that the presence of the cagA gene has been confirmed in more than 60% H. pylori strains distributed throughout the world. The prevalence of cagA genotype is of 65.4% in gastritis patients, 84.2% in patients with peptic ulcer and 86.5% in those with gastric cancer. It shows a high genetic variability of cagA associated with gastroduodenal diseases that could serve as a virulence marker in H. pylori infected subjects. However, the high prevalence of H. pylori cagA positive strains in some geographic areas does not confirm the strong association between cagA and virulence of strains as described in other countries. Nowadays, cagA gene is considered as a marker for the presence of cag pathogenicity island (cag-PAI) in H. pylori genoma. This region contains several genes that has been involved with the production of cytokines that results in an increased inflammation of host gastric mucosa, but its function is unknown. Probably, others bacterium factors, such as susceptibility host and environmental cofactors could influence in the risk of developing different gastroduodenal diseases associated with H. pylori infection. PMID:11685943

  19. Construction of High Density Sweet Cherry (Prunus avium L.) Linkage Maps Using Microsatellite Markers and SNPs Detected by Genotyping-by-Sequencing (GBS).

    PubMed

    Guajardo, Verónica; Solís, Simón; Sagredo, Boris; Gainza, Felipe; Muñoz, Carlos; Gasic, Ksenija; Hinrichsen, Patricio

    2015-01-01

    Linkage maps are valuable tools in genetic and genomic studies. For sweet cherry, linkage maps have been constructed using mainly microsatellite markers (SSRs) and, recently, using single nucleotide polymorphism markers (SNPs) from a cherry 6K SNP array. Genotyping-by-sequencing (GBS), a new methodology based on high-throughput sequencing, holds great promise for identification of high number of SNPs and construction of high density linkage maps. In this study, GBS was used to identify SNPs from an intra-specific sweet cherry cross. A total of 8,476 high quality SNPs were selected for mapping. The physical position for each SNP was determined using the peach genome, Peach v1.0, as reference, and a homogeneous distribution of markers along the eight peach scaffolds was obtained. On average, 65.6% of the SNPs were present in genic regions and 49.8% were located in exonic regions. In addition to the SNPs, a group of SSRs was also used for construction of linkage maps. Parental and consensus high density maps were constructed by genotyping 166 siblings from a 'Rainier' x 'Rivedel' (Ra x Ri) cross. Using Ra x Ri population, 462, 489 and 985 markers were mapped into eight linkage groups in 'Rainier', 'Rivedel' and the Ra x Ri map, respectively, with 80% of mapped SNPs located in genic regions. Obtained maps spanned 549.5, 582.6 and 731.3 cM for 'Rainier', 'Rivedel' and consensus maps, respectively, with an average distance of 1.2 cM between adjacent markers for both 'Rainier' and 'Rivedel' maps and of 0.7 cM for Ra x Ri map. High synteny and co-linearity was observed between obtained maps and with Peach v1.0. These new high density linkage maps provide valuable information on the sweet cherry genome, and serve as the basis for identification of QTLs and genes relevant for the breeding of the species. PMID:26011256

  20. Inflammatory bowel disease associated neoplasia: A surgeon’s perspective

    PubMed Central

    Althumairi, Azah A; Lazarev, Mark G; Gearhart, Susan L

    2016-01-01

    Inflammatory bowel disease (IBD) is associated with increased risk of colorectal cancer (CRC). The risk is known to increase with longer duration of the disease, family history of CRC, and history of primary sclerosing cholangitis. The diagnosis of the neoplastic changes associated with IBD is difficult owing to the heterogeneous endoscopic appearance and inter-observer variability of the pathological diagnosis. Screening and surveillance guidelines have been established which aim for early detection of neoplasia. Several surgical options are available for the treatment of IBD-associated neoplasia. Patients’ morbidities, risk factors for CRC, degree and the extent of neoplasia must be considered in choosing the surgical treatment. A multidisciplinary team including the surgeon, gastroenterologist, pathologist, and the patient who has a clear understanding of the nature of their disease is needed to optimize outcomes. PMID:26811640

  1. Connective tissue disease-associated pulmonary arterial hypertension

    PubMed Central

    Howard, Luke S.

    2015-01-01

    Although rare in its idiopathic form, pulmonary arterial hypertension (PAH) is not uncommon in association with various associated medical conditions, most notably connective tissue disease (CTD). In particular, it develops in approximately 10% of patients with systemic sclerosis and so these patients are increasingly screened to enable early detection. The response of patients with systemic sclerosis to PAH-specific therapy appears to be worse than in other forms of PAH. Survival in systemic sclerosis-associated PAH is inferior to that observed in idiopathic PAH. Potential reasons for this include differences in age, the nature of the underlying pulmonary vasculopathy and the ability of the right ventricle to cope with increased afterload between patients with systemic sclerosis-associated PAH and idiopathic PAH, while coexisting cardiac and pulmonary disease is common in systemic sclerosis-associated PAH. Other forms of connective tissue-associated PAH have been less well studied, however PAH associated with systemic lupus erythematosus (SLE) has a better prognosis than systemic sclerosis-associated PAH and likely responds to immunosuppression. PMID:25705389

  2. Outbreak of Legionnaires' disease associated with a supermarket mist machine.

    PubMed

    Barrabeig, I; Rovira, A; Garcia, M; Oliva, J M; Vilamala, A; Ferrer, M D; Sabrià, M; Domínguez, A

    2010-12-01

    An outbreak of Legionnaires' disease affected 12 customers of a supermarket in a town in Catalonia, Spain, between August and November 2006. An epidemiological and environmental investigation was undertaken. Preliminary investigation showed that all patients had visited the same supermarket in this town where a mist machine was found in the fish section. Water samples were collected from the machine and from the supermarket's water distribution system when high-risk samples were excluded. Environmental samples from the mist machine and clinical samples from two patients tested positive for L. pneumophila serogroup 1 and had the same molecular pattern. The PFGE pattern detected in the clinical and mist-machine isolates had never previously been identified in Catalonia prior to the outbreak and has not been identified since. Four days after turning off the machine, new cases ceased appearing. Molecular study supports the hypothesis that the mist machine from the fish section of the supermarket was the source of infection. We believe it is essential to include exposure to mist machines in any legionellosis epidemiological survey. PMID:20392306

  3. Sex and Genotype Differences in Odor Detection in the 3×Tg-AD and 5XFAD Mouse Models of Alzheimer's Disease at 6 Months of Age.

    PubMed

    Roddick, Kyle M; Roberts, Amelia D; Schellinck, Heather M; Brown, Richard E

    2016-06-01

    Deficits in odor identification and detection are early symptoms of Alzheimer's disease (AD). Two transgenic mouse models of AD, the 5XFAD and the 3×Tg-AD mice and their wildtype controls, were assessed for olfactory detection with decreasing concentrations of ethyl acetate in a go no-go operant olfactometer task at 6 months of age. For both the 5XFAD and their B6SJLF1 wildtype littermates, females made fewer errors in detecting the ethyl acetate than males on all but the lowest odor concentrations. Female 5XFAD mice performed slightly better than their female wildtype littermates on the higher odor concentrations, though not at the lowest concentration. The 3×Tg-AD females showed decreased olfactory detection compared with their wildtype B6129S1 controls, whereas there was no difference in the males. Therefore, although the 5XFAD mice showed no olfactory detection deficits, female 3×Tg-AD mice had impaired olfactory detection at low odor concentrations but males did not. This difference in odor detection should be considered in studies of olfactory learning and memory, as differences in performance may be due to sensory rather than cognitive factors, though detection seems unimpaired at high odor concentrations. PMID:26969629

  4. Metabolic bone disease associated with total parenteral nutrition.

    PubMed

    Klein, G L; Coburn, J W

    1984-01-01

    Patients receiving long-term treatment with total parenteral nutrition often develop bony abnormalities characterized by patchy osteomalacia and low bone turnover. The patients present evidence of physiologic hypoparathyroidism, although low levels of iPTH cannot entirely explain the osteomalacia. Abnormally low serum levels of 1,25(OH)2-vitamin D have been demonstrated, but the significance of these reduced levels in the pathogenesis of the bone lesions is not defined. Aluminum has been detected in large quantities in the plasma, urine, and bone of some patients treated with TPN, and there is mounting evidence that aluminum may be associated with skeletal pathology, particularly osteomalacia. There is, however, no clear documentation that aluminum accumulation produces the skeletal lesions observed, although it could be a contributing factor. There has been the unusual empiric observation that the removal of vitamin D2 from the infusate is associated with a decrease in the quantity of unmineralized osteoid in TPN patients. A possible role of vitamin D2 in producing osteomalacia is not easy to understand since normal serum levels of 25(OH)-D2, the circulating form of vitamin D2, have been reported. The long-term consequences of intravenous nutritional support for many aspects of metabolism remain unknown. Administration into the systemic circulation of predetermined quantities of calcium and phosphorus via a route that bypasses their passage across the intestinal mucosa, the portal system and the liver may have adverse consequences. It is possible that bypassing homeostatic mechanisms may affect bone formation and metabolism or lead to alterations in vitamin D sterols. Alternatively, a deficiency of an essential trace metal or the accumulation of a toxic trace substance could be responsible for the bony abnormalities. Much remains to be clarified concerning calcium homeostasis and bone disease during total parenteral nutrition. Among various possible factors, it

  5. Genetic analysis of G12P[8] rotaviruses detected in the largest U.S. G12 genotype outbreak on record

    PubMed Central

    Mijatovic-Rustempasic, Slavica; Teel, Elizabeth N.; Kerin, Tara K.; Hull, Jennifer; Roy, Sunando; Weinberg, Geoffrey A.; Payne, Daniel C.; Parashar, Umesh D.; Gentsch, Jon R.; Bowen, Michael D.

    2015-01-01

    In 2006–07, 77 cases of gastroenteritis in Rochester, NY, USA were associated with rotavirus genotype G12P[8]. Sequence analysis identified a high degree of genetic relatedness among the VP7 and VP4 genes of the Rochester G12P[8] strains and between these strains and currently circulating human G12P[8] strains. Out of 77 samples, two and seven unique nucleotide sequences were identified for VP7 and VP4 genes, respectively. Rochester strain VP7 genes were found to occupy the G12-III lineage and VP4 genes clustered within the P[8]-3 lineage. Six strains contained non-synonymous nucleotide substitutions that produced amino acid changes at 6 sites in the VP8* region of the VP4 gene. Two sites (amino acids 242 and 246) were located in or near a described trypsin cleavage site. Selection analyses identified one positively selected VP7 site (107) and strong purifying selection at 58 sites within the VP7 gene as well as 2 of the 6 variant sites (79 and 218) in VP4. PMID:24270016

  6. Comparing the performance of FAM19A4 methylation analysis, cytology and HPV16/18 genotyping for the detection of cervical (pre)cancer in high-risk HPV-positive women of a gynecologic outpatient population (COMETH study).

    PubMed

    Luttmer, Roosmarijn; De Strooper, Lise M A; Berkhof, Johannes; Snijders, Peter J F; Dijkstra, Maaike G; Uijterwaal, Margot H; Steenbergen, Renske D M; van Kemenade, Folkert J; Rozendaal, Lawrence; Helmerhorst, Theo J M; Verheijen, Rene H M; Ter Harmsel, W Abraham; Van Baal, W Marchien; Graziosi, Peppino G C M; Quint, Wim G V; Heideman, Daniëlle A M; Meijer, Chris J L M

    2016-02-15

    Recently, DNA methylation analysis of FAM19A4 in cervical scrapes has been shown to adequately detect high-grade cervical intraepithelial neoplasia and cervical cancer (≥ CIN3) in high-risk HPV (hrHPV)-positive women. Here, we compared the clinical performance of FAM19A4 methylation analysis to cytology and HPV16/18 genotyping, separately and in combination, for ≥ CIN3 detection in hrHPV-positive women participating in a prospective observational multi-center cohort study. The study population comprised hrHPV-positive women aged 18-66 years, visiting a gynecological outpatient clinic. From these women, cervical scrapes and colposcopy-directed biopsies (for histological confirmation) were obtained. Cervical scrapes were analyzed for FAM19A4 gene promoter methylation, cytology and HPV16/18 genotyping. Methylation analysis was performed by quantitative methylation-specific PCR (qMSP). Sensitivities and specificities for ≥ CIN3 were compared between tests. Stratified analyses were performed for variables that potentially influence marker performance. Of all 508 hrHPV-positive women, the sensitivities for ≥ CIN3 of cytology, FAM19A4 methylation analysis, and cytology combined with HPV16/18 genotyping were 85.6, 75.6 and 92.2%, respectively, with corresponding specificities of 49.8, 71.1 and 29.4%, respectively. Both sensitivity and specificity of FAM19A4 methylation analysis were associated with age (p ≤ 0.001 each). In women ≥ 30 years (n = 287), ≥ CIN3 sensitivity of FAM19A4 methylation analysis was 88.3% (95%CI: 80.2-96.5) which was noninferior to that of cytology [85.5% (95%CI: 76.0-94.0)], at a significantly higher specificity [62.1% (95%CI: 55.8-68.4) compared to 47.6% (95%CI: 41.1-54.1)]. In conclusion, among hrHPV-positive women from an outpatient population aged ≥ 30 years, methylation analysis of FAM19A4 is an attractive marker for the identification of women with ≥ CIN3. PMID:26317579

  7. Predicting lncRNA-disease associations and constructing lncRNA functional similarity network based on the information of miRNA

    PubMed Central

    Chen, Xing

    2015-01-01

    Accumulating experimental studies have indicated that lncRNAs play important roles in various critical biological process and their alterations and dysregulations have been associated with many important complex diseases. Developing effective computational models to predict potential disease-lncRNA association could benefit not only the understanding of disease mechanism at lncRNA level, but also the detection of disease biomarkers for disease diagnosis, treatment, prognosis and prevention. However, known experimentally confirmed disease-lncRNA associations are still very limited. In this study, a novel model of HyperGeometric distribution for LncRNA-Disease Association inference (HGLDA) was developed to predict lncRNA-disease associations by integrating miRNA-disease associations and lncRNA-miRNA interactions. Although HGLDA didn’t rely on any known disease-lncRNA associations, it still obtained an AUC of 0.7621 in the leave-one-out cross validation. Furthermore, 19 predicted associations for breast cancer, lung cancer, and colorectal cancer were verified by biological experimental studies. Furthermore, the model of LncRNA Functional Similarity Calculation based on the information of MiRNA (LFSCM) was developed to calculate lncRNA functional similarity on a large scale by integrating disease semantic similarity, miRNA-disease associations, and miRNA-lncRNA interactions. It is anticipated that HGLDA and LFSCM could be effective biological tools for biomedical research. PMID:26278472

  8. Diagnostic Performance of the New Version (v2.0) of GenoType MTBDRsl Assay for Detection of Resistance to Fluoroquinolones and Second-Line Injectable Drugs: a Multicenter Study.

    PubMed

    Tagliani, Elisa; Cabibbe, Andrea M; Miotto, Paolo; Borroni, Emanuele; Toro, Juan Carlos; Mansjö, Mikael; Hoffner, Sven; Hillemann, Doris; Zalutskaya, Aksana; Skrahina, Alena; Cirillo, Daniela M

    2015-09-01

    Resistance to fluoroquinolones (FLQ) and second-line injectable drugs (SLID) is steadily increasing, especially in eastern European countries, posing a serious threat to effective tuberculosis (TB) infection control and adequate patient management. The availability of rapid molecular tests for the detection of extensively drug-resistant TB (XDR-TB) is critical in areas with high rates of multidrug-resistant TB (MDR-TB) and XDR-TB and limited conventional drug susceptibility testing (DST) capacity. We conducted a multicenter study to evaluate the performance of the new version (v2.0) of the Genotype MTBDRsl assay compared to phenotypic DST and sequencing on a panel of 228 Mycobacterium tuberculosis isolates and 231 smear-positive clinical specimens. The inclusion of probes for the detection of mutations in the eis promoter region in the MTBDRsl v2.0 test resulted in a higher sensitivity for detection of kanamycin resistance for both direct and indirect testing (96% and 95.4%, respectively) than that seen with the original version of the assay, whereas the test sensitivities for detection of FLQ resistance remained unchanged (93% and 83.6% for direct and indirect testing, respectively). Moreover, MTBDRsl v2.0 showed better performance characteristics than v1.0 for the detection of XDR-TB, with high specificity and sensitivities of 81.8% and 80.4% for direct and indirect testing, respectively. MTBDRsl v2.0 thus represents a reliable test for the rapid detection of resistance to second-line drugs and a useful screening tool to guide the initiation of appropriate MDR-TB treatment. PMID:26179309

  9. Mitochondrial proteome: toward the detection and profiling of disease associated alterations.

    PubMed

    Herrmann, Paul C; Herrmann, E Clifford

    2012-01-01

    Existing at the heart of cellular energy metabolism, the mitochondrion is uniquely positioned to have a major impact on human disease processes. Examples of mitochondrial impact on human pathology abound and include etiologies ranging from inborn errors of metabolism to the site of activity of a variety of toxic compounds. In this review, the unique aspects of the mechanisms related to the mitochondrial proteome are discussed along with an overview of the literature related to mitochondrial proteomic exploration. The review includes discussion of potential areas for exploration and advantages of applying proteomic techniques to the study of mitochondria. PMID:22081351

  10. Evidence for two koi herpesvirus (KHV) genotypes in South Korea.

    PubMed

    Kim, Hyoung Jun; Kwon, Se Ryun

    2013-06-13

    The geographic distribution of koi herpesvirus (KHV) has recently been analyzed by polymerase chain reaction (PCR, based on the alleles of 3 domains) and sequence analysis using 3 regions of KHV genomic DNA (SphI-5, 9/5, and the thymidine kinase gene). In this study, samples from 6 carp showing symptoms of KHV infection in 2008 were examined for the presence of KHV by using PCR and cell culture isolation methods. KHV was detected in 2 (Pyeongtaek and Buan) of the samples. Sequence analysis revealed that the genotype of the KHV PT-08 isolate was Asia genotype variant 1 (A1), and the genotype of the KHV BA-08 isolate was European genotype variant 4 (E4). In addition, PCR patterns and sequence analysis based on the alleles of 3 domains of an alternate KHV classification system confirmed that the genotype of the KHV PT-08 isolate was CyHV3-J, and the genotype of the KHV BA-08 isolate was CyHV3-third genotype. To our knowledge, this is the first study to demonstrate the presence of 2 genotypes of KHV (genotype A1/CyHV3-J; genotype E4/CyHV3-third genotype) in South Korea. PMID:23759557

  11. Evaluation of the GenoType Mycobacteria Direct assay for the simultaneous detection of the Mycobacterium tuberculosis complex and four atypical mycobacterial species in smear-positive respiratory specimens.

    PubMed

    Seagar, A-Louise; Prendergast, Carmel; Emmanuel, F Xavier; Rayner, Alan; Thomson, Susan; Laurenson, Ian F

    2008-05-01

    A novel, commercially available reverse hybridization assay [GenoType Mycobacteria Direct (GTMD), version 2.0; Hain Lifescience] was evaluated for the direct detection of five clinically relevant mycobacterial species [Mycobacterium tuberculosis complex (MTBC), Mycobacterium avium, Mycobacterium malmoense, Mycobacterium kansasii and Mycobacterium intracellulare] from 54 smear-positive respiratory specimens and the findings were compared with culture results. Three approaches were used for specimen preparation using either whole or 'split' sample volumes and N-acetyl-l-cysteine/3 % NaOH or 4 % NaOH as decontamination chemicals. Forty-three out of 52 samples in which RNA amplification was successful gave GTMD results that concurred with the identification of the cultured isolate. All cases of MTBC were detected. Twenty-two samples contained M. tuberculosis complex, seven had M. kansasii, four had M. malmoense, seven contained atypical mycobacteria other than those detectable using the GTMD assay and three specimens contained no viable mycobacteria. The assay is easy to use and can be completed in one working day. Results interpretation is facilitated by the inclusion of an internal amplification control with each sample to allow identification of specimens containing amplification inhibitors. A positive GTMD result will quickly identify patients with MTBC infection or provide specific identification of four other atypical mycobacteria from the same specimen. This allows more rapid drug susceptibility testing, treatment, and public health and infection control decisions. PMID:18436594

  12. No more time to stay 'single' in the detection of Anisakis pegreffii, A. simplex (s. s.) and hybridization events between them: a multi-marker nuclear genotyping approach.

    PubMed

    Mattiucci, S; Acerra, V; Paoletti, M; Cipriani, P; Levsen, A; Webb, S C; Canestrelli, D; Nascetti, G

    2016-07-01

    A multi-marker nuclear genotyping approach was performed on larval and adult specimens of Anisakis spp. (N = 689) collected from fish and cetaceans in allopatric and sympatric areas of the two species Anisakis pegreffii and Anisakis simplex (s. s.), in order to: (1) identify specimens belonging to the parental taxa by using nuclear markers (allozymes loci) and sequence analysis of a new diagnostic nuclear DNA locus (i.e. partial sequence of the EF1 α-1 nDNA region) and (2) recognize hybrid categories. According to the Bayesian clustering algorithms, based on those markers, most of the individuals (N = 678) were identified as the parental species [i.e. A. pegreffii or A. simplex (s. s.)], whereas a smaller portion (N = 11) were recognized as F1 hybrids. Discordant results were obtained when using the polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLPs) of the internal transcribed spacer (ITS) ribosomal DNA (rDNA) on the same specimens, which indicated the occurrence of a large number of 'hybrids' both in sympatry and allopatry. These findings raise the question of possible misidentification of specimens belonging to the two parental Anisakis and their hybrid categories derived from the application of that single marker (i.e. PCR-RFLPs analysis of the ITS of rDNA). Finally, Bayesian clustering, using allozymes and EF1 α-1 nDNA markers, has demonstrated that hybridization between A. pegreffii and A. simplex (s. s.) is a contemporary phenomenon in sympatric areas, while no introgressive hybridization takes place between the two species. PMID:27046418

  13. HIV Genotypic Resistance Testing

    MedlinePlus

    ... be limited. Home Visit Global Sites Search Help? HIV Antiretroviral Drug Resistance Testing, Genotypic Share this page: Was this page helpful? Also known as: Anti-retroviral Drug Resistance Testing; ARV Resistance Testing Formal name: ...

  14. HCV genotypes in Morocco.

    PubMed

    Benani, A; El-Turk, J; Benjelloun, S; Sekkat, S; Nadifi, S; Hda, N; Benslimane, A

    1997-08-01

    To determine the hepatitis C virus (HCV) genotypes circulating in Morocco, virus isolates from 105 chronically infected and 19 hemodialysis patients were examined using the line probe assay. Genotypes 1 and 2 only were found among Moroccan patients. Subtypes 1b (47.6%) and 2a/2c (37.1%) were the most common, whereas subtype 1a (2.8%) was less common. Among the hemodialysis patients, only genotype 1 was found with a prevalence of 68.4% for subtype 1b and 15.8% for the subtype 1a. It was also shown that the HCV genotypes distribution varies with age in both studied populations. Subtype 1b was most prevalent among older patients, whereas subtype 2a/2c was mainly found among younger ones. Although Morocco belongs to the African continent, the circulating HCV strains are similar to those observed in some American and European countries. PMID:9260687

  15. APOE Genotyping, Cardiovascular Disease

    MedlinePlus

    ... Risk Assessment ; HDL Cholesterol ; LDL Cholesterol ; Lipid Profile ; Triglycerides Were you looking instead for APOE genotyping ordered ... the skin called xanthomas, a high level of triglycerides in the blood, and atherosclerosis that develops at ...

  16. Prediction of MicroRNA-Disease Associations Based on Social Network Analysis Methods.

    PubMed

    Zou, Quan; Li, Jinjin; Hong, Qingqi; Lin, Ziyu; Wu, Yun; Shi, Hua; Ju, Ying

    2015-01-01

    MicroRNAs constitute an important class of noncoding, single-stranded, ~22 nucleotide long RNA molecules encoded by endogenous genes. They play an important role in regulating gene transcription and the regulation of normal development. MicroRNAs can be associated with disease; however, only a few microRNA-disease associations have been confirmed by traditional experimental approaches. We introduce two methods to predict microRNA-disease association. The first method, KATZ, focuses on integrating the social network analysis method with machine learning and is based on networks derived from known microRNA-disease associations, disease-disease associations, and microRNA-microRNA associations. The other method, CATAPULT, is a supervised machine learning method. We applied the two methods to 242 known microRNA-disease associations and evaluated their performance using leave-one-out cross-validation and 3-fold cross-validation. Experiments proved that our methods outperformed the state-of-the-art methods. PMID:26273645

  17. Genotypic and Phenotypic Detection of AmpC β-lactamases in Enterobacter spp. Isolated from a Teaching Hospital in Malaysia

    PubMed Central

    Mohd Khari, Fatin Izzati; Karunakaran, Rina; Rosli, Roshalina; Tee Tay, Sun

    2016-01-01

    Objectives The objective of this study was to determine the occurrence of chromosomal and plasmid-mediated β-lactamases (AmpC) genes in a collection of Malaysian isolates of Enterobacter species. Several phenotypic tests for detection of AmpC production of Enterobacter spp. were evaluated and the agreements between tests were determined. Methods Antimicrobial susceptibility profiles for 117 Enterobacter clinical isolates obtained from the Medical Microbiology Diagnostic Laboratory, University Malaya Medical Centre, Malaysia, from November 2012—February 2014 were determined in accordance to CLSI guidelines. AmpC genes were detected using a multiplex PCR assay targeting the MIR/ACT gene (closely related to chromosomal EBC family gene) and other plasmid-mediated genes, including DHA, MOX, CMY, ACC, and FOX. The AmpC β-lactamase production of the isolates was assessed using cefoxitin disk screening test, D69C AmpC detection set, cefoxitin-cloxacillin double disk synergy test (CC-DDS) and AmpC induction test. Results Among the Enterobacter isolates in this study, 39.3% were resistant to cefotaxime and ceftriaxone and 23.9% were resistant to ceftazidime. Ten (8.5%) of the isolates were resistant to cefepime, and one isolate was resistant to meropenem. Chromosomal EBC family gene was amplified from 36 (47.4%) E. cloacae and three (25%) E. asburiae. A novel blaDHA type plasmid-mediated AmpC gene was identified for the first time from an E. cloacae isolate. AmpC β-lactamase production was detected in 99 (89.2%) of 111 potential AmpC β-lactamase producers (positive in cefoxitin disk screening) using D69C AmpC detection set. The detection rates were lower with CC-DDS (80.2%) and AmpC induction tests (50.5%). There was low agreement between the D69C AmpC detection set and the other two phenotypic tests. Of the 40 isolates with AmpC genes detected in this study, 87.5%, 77.5% and 50.0% of these isolates were positive by the D69C AmpC detection set, CC-DDS and Amp

  18. Disease-associated mutation in SRSF2 misregulates splicing by altering RNA-binding affinities

    PubMed Central

    Zhang, Jian; Lieu, Yen K.; Ali, Abdullah M.; Penson, Alex; Reggio, Kathryn S.; Rabadan, Raul; Raza, Azra; Mukherjee, Siddhartha; Manley, James L.

    2015-01-01

    Serine/arginine-rich splicing factor 2 (SRSF2) is an RNA-binding protein that plays important roles in splicing of mRNA precursors. SRSF2 mutations are frequently found in patients with myelodysplastic syndromes and certain leukemias, but how these mutations affect SRSF2 function has only begun to be examined. We used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease to introduce the P95H mutation to SRSF2 in K562 leukemia cells, generating an isogenic model so that splicing alterations can be attributed solely to mutant SRSF2. We found that SRSF2 (P95H) misregulates 548 splicing events (<1% of total). Of these events, 374 involved the inclusion of cassette exons, and the inclusion was either increased (206) or decreased (168). We detected a specific motif (UCCA/UG) enriched in the more-included exons and a distinct motif (UGGA/UG) in the more-excluded exons. RNA gel shift assays showed that a mutant SRSF2 derivative bound more tightly than its wild-type counterpart to RNA sites containing UCCAG but bound less tightly to UGGAG sites. Thus in most cases the pattern of exon inclusion or exclusion correlated with stronger or weaker RNA binding, respectively. We further show that the P95H mutation does not affect other functions of SRSF2, i.e., protein–protein interactions with key splicing factors. Our results thus demonstrate that the P95H mutation positively or negatively alters the binding affinity of SRSF2 for cognate RNA sites in target transcripts, leading to misregulation of exon inclusion. Our findings shed light on the mechanism of the disease-associated SRSF2 mutation in splicing regulation and also reveal a group of misspliced mRNA isoforms for potential therapeutic targeting. PMID:26261309

  19. Leukocyte Ig-Like Receptors – A Model for MHC Class I Disease Associations

    PubMed Central

    Hudson, Laura Emily; Allen, Rachel Louise

    2016-01-01

    MHC class I (MHC-I) polymorphisms are associated with the outcome of some viral infections and autoimmune diseases. MHC-I proteins present antigenic peptides and are recognized by receptors on natural killer cells and cytotoxic T lymphocytes, thus enabling the immune system to detect self-antigens and eliminate targets lacking self or expressing foreign antigens. Recognition of MHC-I, however, extends beyond receptors on cytotoxic leukocytes. Members of the leukocyte Ig-like receptor (LILR) family are expressed on monocytic cells and can recognize both classical and non-classical MHC-I alleles. Despite their relatively broad specificity when compared to the T cell receptor or killer Ig-like receptors, variations in the strength of LILR binding between different MHC-I alleles have recently been shown to correlate with control of HIV infection. We suggest that LILR recognition may mediate MHC-I disease association in a manner that does not depend on a binary discrimination of self/non-self by cytotoxic cells. Instead, the effects of LILR activity following engagement by MHC-I may represent a “degrees of self” model, whereby strength of binding to different alleles determines the degree of influence exerted by these receptors on immune cell functions. LILRs are expressed by myelomonocytic cells and lymphocytes, extending their influence across antigen-presenting cell subsets including dendritic cells, macrophages, and B cells. They have been identified as important players in the response to infection, inflammatory diseases, and cancer, with recent literature to indicate that MHC-I recognition by these receptors and consequent allelic effects could extend an influence beyond the immune system. PMID:27504110

  20. Optimization and application of a custom microarray for the detection and genotyping of E. coli O157:H7 in fresh meat samples

    Technology Transfer Automated Retrieval System (TEKTRAN)

    DNA microarrays are promising high-throughput tools for multiple pathogen detection. Currently, the performance and cost of this platform has limited its broad application in identifying microbial contaminants in foods. In this study, an optimized custom DNA microarray with flexibility in design and...

  1. Simultaneous detection of changes in protein expression and oxidative modification as function of age and APOE genotype in human APOE-targeted replacement mice

    EPA Science Inventory

    Background The purpose of this study was to improve the current method for detecting differentially-oxidized (carbonyl-modified) proteins by 2D-DIGE, while at the same time determining changes in total protein expression. Protein oxidation is a widely accepted model of aging and...

  2. Detection of Novel Rotavirus Strain by Vaccine Postlicensure Surveillance

    PubMed Central

    Teel, Elizabeth N.; Mijatovic-Rustempasic, Slavica; Payne, Daniel C.; Roy, Sunando; Foytich, Kimberly; Parashar, Umesh D.; Gentsch, Jon R.; Bowen, Michael D.

    2013-01-01

    Surveillance for rotavirus-associated diarrhea after implementation of rotavirus vaccination can assess vaccine effectiveness and identify disease-associated genotypes. During active vaccine postlicensure surveillance in the United States, we found a novel rotavirus genotype, G14P[24], in a stool sample from a child who had diarrhea. Unusual rotavirus strains may become more prevalent after vaccine implementation. PMID:23876297

  3. Methicillin-Resistant Staphylococcus aureus (MRSA) Detection: Comparison of Two Molecular Methods (IDI-MRSA PCR Assay and GenoType MRSA Direct PCR Assay) with Three Selective MRSA Agars (MRSA ID, MRSASelect, and CHROMagar MRSA) for Use with Infection-Control Swabs▿

    PubMed Central

    van Hal, S. J.; Stark, D.; Lockwood, B.; Marriott, D.; Harkness, J.

    2007-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is an increasing problem. Rapid detection of MRSA-colonized patients has the potential to limit spread of the organism. We evaluated the sensitivities and specificities of MRSA detection by two molecular methods (IDI-MRSA PCR assay and GenoType MRSA Direct PCR assay) and three selective MRSA agars (MRSA ID, MRSASelect, and CHROMagar MRSA), using 205 (101 nasal, 52 groin, and 52 axillary samples) samples from consecutive known MRSA-infected and/or -colonized patients. All detection methods had higher MRSA detection rates for nasal swabs than for axillary and groin swabs. Detection of MRSA by IDI-MRSA was the most sensitive method, independent of the site (94% for nasal samples, 80% for nonnasal samples, and 90% overall). The sensitivities of the GenoType MRSA Direct assay and the MRSA ID, MRSASelect, and CHROMagar MRSA agars with nasal swabs were 70%, 72%, 68%, and 75%, respectively. All detection methods had high specificities (95 to 99%), independent of the swab site. Extended incubation for a further 24 h with selective MRSA agars increased the detection of MRSA, with a corresponding decline in specificity secondary to a significant increase in false-positive results. There was a noticeable difference in test performance of the GenoType MRSA Direct assay in detection of MRSA (28/38 samples [74%]) compared with detection of nonmultiresistant MRSA (17/31 samples [55%]) (susceptible to two or more non-β-lactam antibiotics). This was not observed with selective MRSA agar plates or IDI-MRSA. Although it is more expensive, in addition to rapid turnaround times of 2 to 4 h, IDI-MRSA offers greater detection of MRSA colonization, independent of the swab site, than do conventional selective agars and GenoType MRSA Direct. PMID:17537949

  4. The glutathione-S-transferase Mu 1 null genotype modulates ozone-induced airway inflammation in humans*

    EPA Science Inventory

    Background: The Glutathione-S-Transferase Mu 1 null genotype has been reported to be a risk factor for acute respiratory disease associated with increases in ambient air ozone. Ozone is known to cause an immediate decrease in lung function and increased airway inflammation. Howev...

  5. DiMeX: A Text Mining System for Mutation-Disease Association Extraction.

    PubMed

    Mahmood, A S M Ashique; Wu, Tsung-Jung; Mazumder, Raja; Vijay-Shanker, K

    2016-01-01

    The number of published articles describing associations between mutations and diseases is increasing at a fast pace. There is a pressing need to gather such mutation-disease associations into public knowledge bases, but manual curation slows down the growth of such databases. We have addressed this problem by developing a text-mining system (DiMeX) to extract mutation to disease associations from publication abstracts. DiMeX consists of a series of natural language processing modules that preprocess input text and apply syntactic and semantic patterns to extract mutation-disease associations. DiMeX achieves high precision and recall with F-scores of 0.88, 0.91 and 0.89 when evaluated on three different datasets for mutation-disease associations. DiMeX includes a separate component that extracts mutation mentions in text and associates them with genes. This component has been also evaluated on different datasets and shown to achieve state-of-the-art performance. The results indicate that our system outperforms the existing mutation-disease association tools, addressing the low precision problems suffered by most approaches. DiMeX was applied on a large set of abstracts from Medline to extract mutation-disease associations, as well as other relevant information including patient/cohort size and population data. The results are stored in a database that can be queried and downloaded at http://biotm.cis.udel.edu/dimex/. We conclude that this high-throughput text-mining approach has the potential to significantly assist researchers and curators to enrich mutation databases. PMID:27073839

  6. DiMeX: A Text Mining System for Mutation-Disease Association Extraction

    PubMed Central

    Mahmood, A. S. M. Ashique; Wu, Tsung-Jung; Mazumder, Raja; Vijay-Shanker, K.

    2016-01-01

    The number of published articles describing associations between mutations and diseases is increasing at a fast pace. There is a pressing need to gather such mutation-disease associations into public knowledge bases, but manual curation slows down the growth of such databases. We have addressed this problem by developing a text-mining system (DiMeX) to extract mutation to disease associations from publication abstracts. DiMeX consists of a series of natural language processing modules that preprocess input text and apply syntactic and semantic patterns to extract mutation-disease associations. DiMeX achieves high precision and recall with F-scores of 0.88, 0.91 and 0.89 when evaluated on three different datasets for mutation-disease associations. DiMeX includes a separate component that extracts mutation mentions in text and associates them with genes. This component has been also evaluated on different datasets and shown to achieve state-of-the-art performance. The results indicate that our system outperforms the existing mutation-disease association tools, addressing the low precision problems suffered by most approaches. DiMeX was applied on a large set of abstracts from Medline to extract mutation-disease associations, as well as other relevant information including patient/cohort size and population data. The results are stored in a database that can be queried and downloaded at http://biotm.cis.udel.edu/dimex/. We conclude that this high-throughput text-mining approach has the potential to significantly assist researchers and curators to enrich mutation databases. PMID:27073839

  7. A one-step duplex rRT-PCR assay for the simultaneous detection of duck hepatitis A virus genotypes 1 and 3.

    PubMed

    Hu, Qin; Zhu, Dekang; Ma, Guangpeng; Cheng, Anchun; Wang, Mingshu; Chen, Shun; Jia, Renyong; Liu, Mafeng; Sun, Kunfeng; Yang, Qiao; Wu, Ying; Chen, Xiaoyue

    2016-10-01

    Duck hepatitis A virus (DHAV) is a highly infectious pathogen that causes significant bleeding lesions in the viscera of ducklings less than 3 weeks old. There are three serotypes of DHAV: serotype 1 (DHAV-1), serotype 2 (DHAV-2) and serotype 3 (DHAV-3). These serotypes have no cross-antigenicity with each other. To establish an rRT-PCR assay for the rapid detection of a mixed infection of DHAV-1 and DHAV-3, two pairs of primers and a pair of matching TaqMan probes were designed based on conserved regions of DHAV-1 VP0 and DHAV-3 VP3. Finally, we established a one-step duplex rRT-PCR assay with high specificity and sensitivity for the simultaneous detection of DHAV-1 and DHAV-3. This method showed no cross-antigenicity with the other pathogens tested, including duck plague virus, Muscovy duck parvovirus, Riemerella anatipestifer, and pathogenic E. coli from ducks. Sensitivity tests identified the minimum detection limits of this method as 98 (DHAV-1) and 10 (DHAV-3) copies/reaction. To validate the method, thirty-eight clinical samples and thirty artificially infected samples collected from dead duck embryos were studied. Thirty-seven samples were positive for DHAV-1, seventeen samples were positive for DHAV-3, and fourteen samples were positive for a mixed infection using the duplex rRT-PCR method. The method established in this study is specific, sensitive, convenient and timesaving and is a powerful tool for detecting DHAV-1, DHAV-3, and their mixed infection and for conducting surveys of pandemic virus strains. PMID:27435338

  8. Genotyping of Coxiella burnetii from domestic ruminants in northern Spain

    PubMed Central

    2012-01-01

    Background Information on the genotypic diversity of Coxiella burnetii isolates from infected domestic ruminants in Spain is limited. The aim of this study was to identify the C. burnetii genotypes infecting livestock in Northern Spain and compare them to other European genotypes. A commercial real-time PCR targeting the IS1111a insertion element was used to detect the presence of C. burnetii DNA in domestic ruminants from Spain. Genotypes were determined by a 6-loci Multiple Locus Variable number tandem repeat analysis (MLVA) panel and Multispacer Sequence Typing (MST). Results A total of 45 samples from 4 goat herds (placentas, N = 4), 12 dairy cattle herds (vaginal mucus, individual milk, bulk tank milk, aerosols, N = 20) and 5 sheep flocks (placenta, vaginal swabs, faeces, air samples, dust, N = 21) were included in the study. Samples from goats and sheep were obtained from herds which had suffered abortions suspected to be caused by C. burnetii, whereas cattle samples were obtained from animals with reproductive problems compatible with C. burnetii infection, or consisted of bulk tank milk (BTM) samples from a Q fever surveillance programme. C. burnetii genotypes identified in ruminants from Spain were compared to those detected in other countries. Three MLVA genotypes were found in 4 goat farms, 7 MLVA genotypes were identified in 12 cattle herds and 4 MLVA genotypes were identified in 5 sheep flocks. Clustering of the MLVA genotypes using the minimum spanning tree method showed a high degree of genetic similarity between most MLVA genotypes. Overall 11 different MLVA genotypes were obtained corresponding to 4 different MST genotypes: MST genotype 13, identified in goat, sheep and cattle from Spain; MST genotype 18, only identified in goats; and, MST genotypes 8 and 20, identified in small ruminants and cattle, respectively. All these genotypes had been previously identified in animal and human clinical samples from several European countries, but

  9. Detection of Cryptosporidium sp. in two new seal species, Phoca vitulina and Cystophora cristata, and a novel Cryptosporidium genotype in a third seal species, Pagophilus groenlandicus, from the Gulf of Maine.

    PubMed

    Bass, A L; Wallace, C C; Yund, P O; Ford, T E

    2012-04-01

    Data on the geographic distribution and host specificity of Cryptosporidium spp. are critical for developing an understanding of likely transmission patterns in nature. During a molecular-based survey of fecal samples from 293 terrestrial and aquatic animals in Maine, USA, we detected Cryptosporidium sp. in 11 harbor seals (Phoca vitulina), 1 hooded seal (Cystophora cristata), and 1 harp seal (Pagophilus groenlandicus). None of the terrestrial or freshwater mammal fecal samples or bird samples tested positive for Cryptosporidium sp. However, the sequencing results of the small subunit (ssu) rRNA gene indicate that the seals were infected with an undescribed species of Cryptosporidium , previously isolated only from ringed seals (Phoca hispida) in northern Quebec, Canada. In addition, the Cryptosporidium sp. detected in the harp seal is significantly different from the previously observed Cryptosporidium sp. in other seals. We confirmed the genetic distinctiveness of this Cryptosporidium genotype and the identity of the other Cryptosporidium sp. seal ssu rRNA sequences by using data from the 70-kDa heat shock protein gene. Based on phylogenetic reconstructions of both genes, it seems that either Cryptosporidium canis or C. felis are sister species to the seal associated Cryptosporidium spp. Our findings extend the range of " Cryptosporidium sp. seal" well south of the 55th parallel, add other species to the list of seals affected by Cryptosporidium sp., and highlight the presence of unrecognized population and potentially species level variation in Cryptosporidium. PMID:22017467

  10. Molecular Assay and Genotyping of Hepatitis C Virus among Infected Egyptian and Saudi Arabian Patients

    PubMed Central

    Farag, Mohamed MS; Sofy, Ahmed R; Mousa, Adel A; Ahmed, Mohamed A; Alganzory, Mohamed R

    2015-01-01

    Hepatitis C virus (HCV) infection is a major health problem recognized globally. HCV is a common cause of liver fibrosis that may lead to liver cirrhosis or hepatocellular carcinoma. The aim of this study was to estimate the prevalence of HCV infection and genotyping among Egyptian and Saudi Arabian chronic patients using different molecular techniques. HCV RNA viral load was assessed by real-time polymerase chain reaction (RT-PCR) technology. For HCV genotyping, RT-PCR hybridization fluorescence-based method and reverse hybridization line probe assay (INNO-LiPA) were used. A total of 40 anti-HCV-positive patients with chronic hepatitis C were examined for HCV RNA, genotyping, and different laboratory investigations. In the present study, HCV genotypes 4, mixed 4.1b, and 1 were detected in patients of both countries, while genotype 2 was only detected in Saudi Arabian patients. Genotyping methods for HCV showed no difference in the classification at the genotype level. With regard to HCV subtypes, INNO-LiPA assay was a reliable test in HCV genotyping for the detection of major genotypes and subtypes, while RT-PCR-based assay was a good test at the genotype level only. HCV genotype 4 was found to be the predominant genotype among Egyptian and Saudi Arabian chronic patients. In conclusion, data analysis for detecting and genotyping HCV was an important factor for understanding the epidemiology and treatment strategies of HCV among Egyptian and Saudi Arabian chronic patients. PMID:26512201

  11. Using PCR-based detection and genotyping to trace Streptococcus salivarius meningitis outbreak strain to oral flora of radiology physician assistant.

    PubMed

    Srinivasan, Velusamy; Gertz, Robert E; Shewmaker, Patricia L; Patrick, Sarah; Chitnis, Amit S; O'Connell, Heather; Benowitz, Isaac; Patel, Priti; Guh, Alice Y; Noble-Wang, Judith; Turabelidze, George; Beall, Bernard

    2012-01-01

    We recently investigated three cases of bacterial meningitis that were reported from a midwestern radiology clinic where facemasks were not worn during spinal injection of contrast agent during myelography procedures. Using pulsed field gel electrophoresis we linked a case strain of S. salivarius to an oral specimen of a radiology physician assistant (RPA). We also used a real-time PCR assay to detect S. salivarius DNA within a culture-negative cerebrospinal fluid (CSF) specimen. Here we extend this investigation through using a nested PCR/sequencing strategy to link the culture-negative CSF specimen to the case strain. We also provide validation of the real-time PCR assay used, demonstrating that it is not solely specific for Streptococcus salivarius, but is also highly sensitive for detection of the closely related oral species Streptococcus vestibularis. Through using multilocus sequence typing and 16S rDNA sequencing we further strengthen the link between the CSF case isolate and the RPA carriage isolate. We also demonstrate that the newly characterized strains from this study are distinct from previously characterized S. salivarius strains associated with carriage and meningitis. PMID:22384169

  12. Using PCR-Based Detection and Genotyping to Trace Streptococcus salivarius Meningitis Outbreak Strain to Oral Flora of Radiology Physician Assistant

    PubMed Central

    Srinivasan, Velusamy; Gertz Jr., Robert E.; Shewmaker, Patricia L.; Patrick, Sarah; Chitnis, Amit S.; O'Connell, Heather; Benowitz, Isaac; Patel, Priti; Guh, Alice Y.; Noble-Wang, Judith; Turabelidze, George; Beall, Bernard

    2012-01-01

    We recently investigated three cases of bacterial meningitis that were reported from a midwestern radiology clinic where facemasks were not worn during spinal injection of contrast agent during myelography procedures. Using pulsed field gel electrophoresis we linked a case strain of S. salivarius to an oral specimen of a radiology physician assistant (RPA). We also used a real-time PCR assay to detect S. salivarius DNA within a culture-negative cerebrospinal fluid (CSF) specimen. Here we extend this investigation through using a nested PCR/sequencing strategy to link the culture-negative CSF specimen to the case strain. We also provide validation of the real-time PCR assay used, demonstrating that it is not solely specific for Streptococcus salivarius, but is also highly sensitive for detection of the closely related oral species Streptococcus vestibularis. Through using multilocus sequence typing and 16S rDNA sequencing we further strengthen the link between the CSF case isolate and the RPA carriage isolate. We also demonstrate that the newly characterized strains from this study are distinct from previously characterized S. salivarius strains associated with carriage and meningitis. PMID:22384169

  13. Staphylococcus aureus genotype B and other genotypes isolated from cow milk in European countries.

    PubMed

    Cosandey, A; Boss, R; Luini, M; Artursson, K; Bardiau, M; Breitenwieser, F; Hehenberger, E; Lam, Th; Mansfeld, M; Michel, A; Mösslacher, G; Naskova, J; Nelson, S; Podpečan, O; Raemy, A; Ryan, E; Salat, O; Zangerl, P; Steiner, A; Graber, H U

    2016-01-01

    Staphylococcus aureus is globally one of the most important pathogens causing contagious mastitis in cattle. Previous studies, however, have demonstrated in Swiss cows that Staph. aureus isolated from bovine intramammary infection is genetically heterogeneous, with Staph. aureus genotype B (GTB) and GTC being the most prominent genotypes. In addition, Staph. aureus GTB was found to be contagious, whereas Staph. aureus GTC and all the remaining genotypes were involved in individual cow disease. The aim of this study was to subtype strains of Staph. aureus isolated from bovine mastitic milk and bulk tank milk to obtain a unified view of the presence of bovine staphylococcal subtypes in 12 European countries. A total of 456 strains of Staph. aureus were subjected to different typing methods: ribosomal spacer PCR, detection of enterotoxin genes, and detection of gene polymorphisms (lukE, coa). Major genotypes with their variants were combined into genotypic clusters (CL). This study revealed 5 major CL representing 76% of all strains and comprised CLB, CLC, CLF, CLI, and CLR. The clusters were characterized by the same genetic properties as the Swiss isolates, demonstrating high clonality of bovine Staph. aureus. Interestingly, CLB was situated in central Europe whereas the other CL were widely disseminated. The remaining 24% of the strains comprised 41 genotypes and variants, some of which (GTAM, GTBG) were restricted to certain countries; many others, however, were observed only once. PMID:26585469

  14. Improved ancestry estimation for both genotyping and sequencing data using projection procrustes analysis and genotype imputation.

    PubMed

    Wang, Chaolong; Zhan, Xiaowei; Liang, Liming; Abecasis, Gonçalo R; Lin, Xihong

    2015-06-01

    Accurate estimation of individual ancestry is important in genetic association studies, especially when a large number of samples are collected from multiple sources. However, existing approaches developed for genome-wide SNP data do not work well with modest amounts of genetic data, such as in targeted sequencing or exome chip genotyping experiments. We propose a statistical framework to estimate individual ancestry in a principal component ancestry map generated by a reference set of individuals. This framework extends and improves upon our previous method for estimating ancestry using low-coverage sequence reads (LASER 1.0) to analyze either genotyping or sequencing data. In particular, we introduce a projection Procrustes analysis approach that uses high-dimensional principal components to estimate ancestry in a low-dimensional reference space. Using extensive simulations and empirical data examples, we show that our new method (LASER 2.0), combined with genotype imputation on the reference individuals, can substantially outperform LASER 1.0 in estimating fine-scale genetic ancestry. Specifically, LASER 2.0 can accurately estimate fine-scale ancestry within Europe using either exome chip genotypes or targeted sequencing data with off-target coverage as low as 0.05×. Under the framework of LASER 2.0, we can estimate individual ancestry in a shared reference space for samples assayed at different loci or by different techniques. Therefore, our ancestry estimation method will accelerate discovery in disease association studies not only by helping model ancestry within individual studies but also by facilitating combined analysis of genetic data from multiple sources. PMID:26027497

  15. Improved Ancestry Estimation for both Genotyping and Sequencing Data using Projection Procrustes Analysis and Genotype Imputation

    PubMed Central

    Wang, Chaolong; Zhan, Xiaowei; Liang, Liming; Abecasis, Gonçalo R.; Lin, Xihong

    2015-01-01

    Accurate estimation of individual ancestry is important in genetic association studies, especially when a large number of samples are collected from multiple sources. However, existing approaches developed for genome-wide SNP data do not work well with modest amounts of genetic data, such as in targeted sequencing or exome chip genotyping experiments. We propose a statistical framework to estimate individual ancestry in a principal component ancestry map generated by a reference set of individuals. This framework extends and improves upon our previous method for estimating ancestry using low-coverage sequence reads (LASER 1.0) to analyze either genotyping or sequencing data. In particular, we introduce a projection Procrustes analysis approach that uses high-dimensional principal components to estimate ancestry in a low-dimensional reference space. Using extensive simulations and empirical data examples, we show that our new method (LASER 2.0), combined with genotype imputation on the reference individuals, can substantially outperform LASER 1.0 in estimating fine-scale genetic ancestry. Specifically, LASER 2.0 can accurately estimate fine-scale ancestry within Europe using either exome chip genotypes or targeted sequencing data with off-target coverage as low as 0.05×. Under the framework of LASER 2.0, we can estimate individual ancestry in a shared reference space for samples assayed at different loci or by different techniques. Therefore, our ancestry estimation method will accelerate discovery in disease association studies not only by helping model ancestry within individual studies but also by facilitating combined analysis of genetic data from multiple sources. PMID:26027497

  16. Comparison of Two Widely Used Human Papillomavirus Detection and Genotyping Methods, GP5+/6+-Based PCR Followed by Reverse Line Blot Hybridization and Multiplex Type-Specific E7-Based PCR.

    PubMed

    Clifford, Gary M; Vaccarella, Salvatore; Franceschi, Silvia; Tenet, Vanessa; Umulisa, M Chantal; Tshomo, Ugyen; Dondog, Bolormaa; Vorsters, Alex; Tommasino, Massimo; Heideman, Daniëlle A M; Snijders, Peter J F; Gheit, Tarik

    2016-08-01

    GP5+/6+-based PCR followed by reverse line blot hybridization (GP5+/6+RLB) and multiplex type-specific PCR (E7-MPG) are two human papillomavirus (HPV) genotyping methodologies widely applied in epidemiological research. We investigated their relative analytical performance in 4,662 samples derived from five studies in Bhutan, Rwanda, and Mongolia coordinated by the International Agency for Research on Cancer (IARC). A total of 630 samples were positive by E7-MPG only (13.5%), 24 were positive by GP5+/6+RLB only (0.5%), and 1,014 were positive (21.8%) by both methods. Ratios of HPV type-specific positivity of the two tests (E7-MPG:GP5+/6+RLB ratio) were calculated among 1,668 samples that were HPV positive by one or both tests. E7-MPG:GP5+/6+RLB ratios were >1 for all types and highly reproducible across populations and sample types. E7-MPG:GP5+/6+RLB ratios were highest for HPV53 (7.5) and HPV68 (7.1). HPV16 (1.6) and HPV18 (1.7) had lower than average E7-MPG:GP5+/6+RLB ratios. Among E7-MPG positive infections, median mean fluorescence intensity (MFI; a semiquantitative measure of viral load) tended to be higher among samples positive for the same virus type by GP5+/6+RLB than for those negative for the same type by GP5+/6+RLB. Exceptions, however, included HPV53, -59, and -82, for which the chances of being undetected by GP5+/6+RLB appeared to be MFI independent. Furthermore, the probability of detecting an additional type by E7-MPG was higher when another type was already detected by GP5+/6+RLB, suggesting the existence of masking effects due to competition for GP5+/6+ PCR primers. In conclusion, this analysis is not an evaluation of clinical performance but may inform choices for HPV genotyping methods in epidemiological studies, when the relative merits and dangers of sensitivity versus specificity for individual types should be considered, as well as the potential to unmask nonvaccine types following HPV vaccination. PMID:27225411

  17. Insight into Neutral and Disease-Associated Human Genetic Variants through Interpretable Predictors

    PubMed Central

    van den Berg, Bastiaan A.; Reinders, Marcel J. T.; de Ridder, Dick; de Beer, Tjaart A. P.

    2015-01-01

    A variety of methods that predict human nonsynonymous single nucleotide polymorphisms (SNPs) to be neutral or disease-associated have been developed over the last decade. These methods are used for pinpointing disease-associated variants in the many variants obtained with next-generation sequencing technologies. The high performances of current sequence-based predictors indicate that sequence data contains valuable information about a variant being neutral or disease-associated. However, most predictors do not readily disclose this information, and so it remains unclear what sequence properties are most important. Here, we show how we can obtain insight into sequence characteristics of variants and their surroundings by interpreting predictors. We used an extensive range of features derived from the variant itself, its surrounding sequence, sequence conservation, and sequence annotation, and employed linear support vector machine classifiers to enable extracting feature importance from trained predictors. Our approach is useful for providing additional information about what features are most important for the predictions made. Furthermore, for large sets of known variants, it can provide insight into the mechanisms responsible for variants being disease-associated. PMID:25826299

  18. Insight into neutral and disease-associated human genetic variants through interpretable predictors.

    PubMed

    van den Berg, Bastiaan A; Reinders, Marcel J T; de Ridder, Dick; de Beer, Tjaart A P

    2015-01-01

    A variety of methods that predict human nonsynonymous single nucleotide polymorphisms (SNPs) to be neutral or disease-associated have been developed over the last decade. These methods are used for pinpointing disease-associated variants in the many variants obtained with next-generation sequencing technologies. The high performances of current sequence-based predictors indicate that sequence data contains valuable information about a variant being neutral or disease-associated. However, most predictors do not readily disclose this information, and so it remains unclear what sequence properties are most important. Here, we show how we can obtain insight into sequence characteristics of variants and their surroundings by interpreting predictors. We used an extensive range of features derived from the variant itself, its surrounding sequence, sequence conservation, and sequence annotation, and employed linear support vector machine classifiers to enable extracting feature importance from trained predictors. Our approach is useful for providing additional information about what features are most important for the predictions made. Furthermore, for large sets of known variants, it can provide insight into the mechanisms responsible for variants being disease-associated. PMID:25826299

  19. WBSMDA: Within and Between Score for MiRNA-Disease Association prediction.

    PubMed

    Chen, Xing; Yan, Chenggang Clarence; Zhang, Xu; You, Zhu-Hong; Deng, Lixi; Liu, Ying; Zhang, Yongdong; Dai, Qionghai

    2016-01-01

    Increasing evidences have indicated that microRNAs (miRNAs) are functionally associated with the development and progression of various complex human diseases. However, the roles of miRNAs in multiple biological processes or various diseases and their underlying molecular mechanisms still have not been fully understood yet. Predicting potential miRNA-disease associations by integrating various heterogeneous biological datasets is of great significance to the biomedical research. Computational methods could obtain potential miRNA-disease associations in a short time, which significantly reduce the experimental time and cost. Considering the limitations in previous computational methods, we developed the model of Within and Between Score for MiRNA-Disease Association prediction (WBSMDA) to predict potential miRNAs associated with various complex diseases. WBSMDA could be applied to the diseases without any known related miRNAs. The AUC of 0.8031 based on Leave-one-out cross validation has demonstrated its reliable performance. WBSMDA was further applied to Colon Neoplasms, Prostate Neoplasms, and Lymphoma for the identification of their potential related miRNAs. As a result, 90%, 84%, and 80% of predicted miRNA-disease pairs in the top 50 prediction list for these three diseases have been confirmed by recent experimental literatures, respectively. It is anticipated that WBSMDA would be a useful resource for potential miRNA-disease association identification. PMID:26880032

  20. Performance of the New Version (v2.0) of the GenoType MTBDRsl Test for Detection of Resistance to Second-Line Drugs in Multidrug-Resistant Mycobacterium tuberculosis Complex Strains.

    PubMed

    Brossier, Florence; Guindo, David; Pham, Anne; Reibel, Florence; Sougakoff, Wladimir; Veziris, Nicolas; Aubry, Alexandra

    2016-06-01

    Detecting resistance to fluoroquinolones (FQ) and second-line injectable drugs (amikacin [AMK], kanamycin [KAN], and capreomycin [CAP]) is crucial given the worldwide increase in the incidence of extensively drug-resistant tuberculosis (XDR-TB). A new version of the GenoType MTBDRsl test (v2.0) has been developed to improve the detection of resistance to FQ (involving gyrA and gyrB mutations) and to second-line injectable drugs (involving rrs and eis promoter mutations) in Mycobacterium tuberculosis A collection of 127 multidrug-resistant (MDR) M. tuberculosis complex strains was tested using the first (v1) and second (v2.0) versions of the MTBDRsl test, as well as DNA sequencing. The specificities in resistance detection of v1 and v2.0 were similar throughout, whereas the levels of sensitivity of v2.0 were superior for FQ (94.8% versus 89.6%) and KAN (90.5% versus 59.5%) but similar for AMK (91.3%) and CAP (83.0%). The sensitivity and specificity of v2.0 were superior to those of v1 for the detection of pre-XDR strains (83.3% versus 75.0% and 88.6% versus 67.1%, respectively), whereas the sensitivity of v2.0 was superior to that of v1 only for the detection of XDR strains (83.0% versus 49.1%). In conclusion, MTBDRsl v2.0 is superior to MTBDRsl v1 and efficiently detects the most common mutations involved in resistance to FQ and aminoglycosides/CAP. However, due to mutations not recognized by v2.0 or to the presence of resistance mechanisms not yet characterized (particularly mechanisms related to monoresistance to aminoglycosides or CAP), the results for wild-type strains obtained with MTBDRsl v2.0 should be confirmed by further DNA sequencing and phenotypic drug susceptibility testing. PMID:27053671

  1. Integration of Multiple Genomic and Phenotype Data to Infer Novel miRNA-Disease Associations.

    PubMed

    Shi, Hongbo; Zhang, Guangde; Zhou, Meng; Cheng, Liang; Yang, Haixiu; Wang, Jing; Sun, Jie; Wang, Zhenzhen

    2016-01-01

    MicroRNAs (miRNAs) play an important role in the development and progression of human diseases. The identification of disease-associated miRNAs will be helpful for understanding the molecular mechanisms of diseases at the post-transcriptional level. Based on different types of genomic data sources, computational methods for miRNA-disease association prediction have been proposed. However, individual source of genomic data tends to be incomplete and noisy; therefore, the integration of various types of genomic data for inferring reliable miRNA-disease associations is urgently needed. In this study, we present a computational framework, CHNmiRD, for identifying miRNA-disease associations by integrating multiple genomic and phenotype data, including protein-protein interaction data, gene ontology data, experimentally verified miRNA-target relationships, disease phenotype information and known miRNA-disease connections. The performance of CHNmiRD was evaluated by experimentally verified miRNA-disease associations, which achieved an area under the ROC curve (AUC) of 0.834 for 5-fold cross-validation. In particular, CHNmiRD displayed excellent performance for diseases without any known related miRNAs. The results of case studies for three human diseases (glioblastoma, myocardial infarction and type 1 diabetes) showed that all of the top 10 ranked miRNAs having no known associations with these three diseases in existing miRNA-disease databases were directly or indirectly confirmed by our latest literature mining. All these results demonstrated the reliability and efficiency of CHNmiRD, and it is anticipated that CHNmiRD will serve as a powerful bioinformatics method for mining novel disease-related miRNAs and providing a new perspective into molecular mechanisms underlying human diseases at the post-transcriptional level. CHNmiRD is freely available at http://www.bio-bigdata.com/CHNmiRD. PMID:26849207

  2. Evaluation of Genotypic and Phenotypic Methods for Detection of Methicillin Resistant Staphylococcus aureus in a Tertiary Care Hospital of Eastern Odisha

    PubMed Central

    Panda, Rakesh Kumar; Mallick, Bandana; Chayani, Nirupama

    2016-01-01

    Introduction Methicillin resistant Staphylococcus aureus has emerged as an important pathogen in nosocomial and community acquired infections. Accurate and rapid identification of MRSA in clinical specimens is essential for timely decision of effective antimicrobial chemotherapy. Aim The present study was conducted to compare efficacy of four conventional phenotypic methods, with mec- A based polymerase chain reaction (PCR) for MRSA identification. Materials and Methods Methicillin resistance was determined in 200 S.aureus isolates by oxacillin disc diffusion, cefoxitin disc diffusion, Oxacillin Resistance Screening Agar and E-test. The results were compared with mec-A based PCR. Results Among 200 S.aureus isolates 62 (31%) were positive for mec-A gene by PCR. Cefoxitin disc diffusion, Oxacillin Resistance Screening Agar and E-test showed 100% specificity. Oxacillin disc diffusion had lowest sensitivity (82.5%) and specificity (98.5%) among all. The conventional methods take more time than PCR for diagnosing MRSA. Linezolid, Vancomycin & Dalfopristin were the highly sensitive drugs against MRSA isolates. Conclusion Cefoxitin disc diffusion, is rapid, simple and cheaper, hence can be used routinely as an alternative to PCR for detection of MRSA in resource constraint laboratories. PMID:27042463

  3. Axiom turkey genotyping array

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Axiom®Turkey Genotyping Array interrogates 643,845 probesets on the array, covering 643,845 SNPs. The array development was led by Dr. Julie Long of the USDA-ARS Beltsville Agricultural Research Center under a public-private partnership with Hendrix Genetics, Aviagen, and Affymetrix. The Turk...

  4. Effects of Photo and Genotype-Based Misidentification Error on Estimates of Survival, Detection and State Transition using Multistate Survival Models

    PubMed Central

    Winiarski, Kristopher J.; McGarigal, Kevin

    2016-01-01

    We simulated multistate capture histories (CHs) by varying state survival (ϕ), detection (p) and transition (ψ), number of total capture occasions and releases per capture occasion and then modified these scenarios to mimic false rejection error (FRE), a common misidentification error, resulting from the failure to match samples of the same individual. We then fit a multistate model and estimated accuracy, bias and precision of state-specific ϕ, p and ψ to better understand the effects of FRE on different simulation scenarios. As expected, ϕ, and p, decreased in accuracy with FRE, with lower accuracy when CHs were simulated under a shorter-term study and a lower number of releases per capture occasion (lower sample size). Accuracy of ψ estimates were robust to FRE except in those CH scenarios simulated using low sample size. The effect of FRE on bias was not consistent among parameters and differed by CH scenario. As expected, ϕ was negatively biased with increased FRE (except for the low ϕ low p CH scenario simulated with a low sample size), but we found that the magnitude of bias differed by scenario (high p CH scenarios were more negatively biased). State transition was relatively unbiased, except for the low p CH scenarios simulated with a low sample size, which were positively biased with FRE, and high p CH scenarios simulated with a low sample size. The effect of FRE on precision was not consistent among parameters and differed by scenario and sample size. Precision of ϕ decreased with FRE and was lowest with the low ϕ low p CH scenarios. Precision of p estimates also decreased with FRE under all scenarios, except the low ϕ high p CH scenarios. However, precision of ψ increased with FRE, except for those CH scenarios simulated with a low sample size. Our results demonstrate how FRE leads to loss of accuracy in parameter estimates in a multistate model with the exception of ψ when estimated using an adequate sample size. PMID:26751208

  5. Toxoplasma gondii B1 Gene Detection in Feces of Stray Cats around Seoul, Korea and Genotype Analysis of Two Laboratory-Passaged Isolates

    PubMed Central

    Jung, Bong-Kwang; Lee, Sang-Eun; Lim, Hyemi; Cho, Jaeeun; Kim, Deok-Gyu; Song, Hyemi; Kim, Min-Jae; Shin, Eun-Hee; Chai, Jong-Yil

    2015-01-01

    The increasing prevalence of Toxoplasma gondii infection in the human population in the Republic of Korea (= Korea) is due to various reasons such as an increase in meat consumption. However, the importance of cats in transmitting T. gondii infection through oocysts to humans has seldom been assessed. A total of 300 fecal samples of stray cats captured around Seoul from June to August 2013 were examined for T. gondii B1 gene (indicating the presence of oocysts) using nested-PCR. Fourteen (4.7%) of 300 cats examined were positive for B1 gene. Female cats (7.5%) showed a higher prevalence than male cats (1.4%). Cats younger than 3 months (5.5%) showed a higher prevalence than cats (1.5%) older than 3 months. For laboratory passage of the positive samples, the fecal suspension (0.2 ml) of B1 gene positive cats was orally inoculated into experimental mice. Brain tissues of the mice were obtained after 40 days and examined for the presence of tissue cysts. Two isolates were successfully passaged (designated KNIH-1 and KNIH-2) and were molecularly analyzed using the SAG5D and SAG5E gene sequences. The SAG5D and SAG5E gene sequences showed high homologies with the ME49 strain (less virulent strain). The results indicated the importance of stray cats in transmitting T. gondii to humans in Korea, as revealed by detection of B1 gene in fecal samples. T. gondii isolates from cats were successfully passaged in the laboratory for the first time in Korea. PMID:26174818

  6. Comparison of AMPLICOR and Hybrid Capture II assays for high risk HPV detection in normal and abnormal liquid-based cytology: use of INNO-LiPA Genotyping assay to screen the discordant results.

    PubMed

    Mo, L Z; Monnier-Benoit, S; Kantelip, B; Petitjean, A; Riethmuller, D; Prétet, J L; Mougin, C

    2008-02-01

    The study was aimed to evaluate the feasibility of detecting human papillomavirus (HPV) in women with normal or abnormal cervical smears using the Roche Amplicor MWP HPV Test. We compared by AMPLICOR Test and Hybrid Capture II (HCII) Test, the prevalence of HR-HPV in 470 cervical samples including 55 samples with WNL cytology, 208 ASC-US, 193 LGSIL and 14 HGSIL. Samples with discordant results were retested with INNO-LiPA Genotyping HPV Test v2. The HR-HPV positivity in WNL cytology samples was similar (21.8%) by AMPLICOR and HCII. In ASC-US, the HPV positivity was 42.3% by both tests. In LGSIL, HPV positivity was 66.3% and 66.8% by AMPLICOR and HCII, respectively. In HGSIL, 92.8% of samples were positive by AMPLICOR and 85.7% by HCII. The agreement of both tests was 96.2% with a Kappa value of 0.92. Eighteen cases were discordant: 9 HCII positive/AMPLICOR negative and 9 HCII negative/AMPLICOR positive. The INNO-LiPA test revealed HPV positivity in every case. Interestingly, all HCII+/AMPLICOR- samples were found to harbour HPV53. As for the HCII-/AMPLICOR+ samples, 8 demonstrated a multiple infection with HR 16- and/or 18- and/or 56-phylogenetically related HPV types. Moreover, two of these samples were co-infected with HPV6 and two other with HPV54. By using consensus HR-HPV as our reference HPV positivity, the sensitivity (96.6%) and specificity (100%) of AMPLICOR was similar to that of HCII Test. The AMPLICOR HPV Test is sensitive, specific, feasible and appropriate for routine HPV detection. PMID:18036888

  7. Hepatic and Splenic Acoustic Radiation Force Impulse Shear Wave Velocity Elastography in Children with Liver Disease Associated with Cystic Fibrosis

    PubMed Central

    Cañas, Teresa; Maciá, Araceli; Muñoz-Codoceo, Rosa Ana; Fontanilla, Teresa; González-Rios, Patricia; Miralles, María; Gómez-Mardones, Gloria

    2015-01-01

    Background. Liver disease associated with cystic fibrosis (CFLD) is the second cause of mortality in these patients. The diagnosis is difficult because none of the available tests are specific enough. Noninvasive elastographic techniques have been proven to be useful to diagnose hepatic fibrosis. Acoustic radiation force impulse (ARFI) imaging is an elastography imaging system. The purpose of the work was to study the utility of liver and spleen ARFI Imaging in the detection of CFLD. Method. 72 patients with cystic fibrosis (CF) were studied and received ARFI imaging in the liver and in the spleen. SWV values were compared with the values of 60 healthy controls. Results. Comparing the SWV values of CFLD with the control healthy group, values in the right lobe were higher in patients with CFLD. We found a SWV RHL cut-off value to detect CFLD of 1.27 m/s with a sensitivity of 56.5% and a specificity of 90.5%. CF patients were found to have higher SWC spleen values than the control group. Conclusions. ARFI shear wave elastography in the right hepatic lobe is a noninvasive technique useful to detect CFLD in our sample of patients. Splenic SWV values are higher in CF patients, without any clinical consequence. PMID:26609528

  8. Mass genotyping by sequencing technology

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Large scale genotyping of a moderate number of loci is cost prohibitive with current chip-based technologies. We demonstrate the ability to use next generation sequencing technologies to genotype many DNA samples for a moderate number of loci – a mass genotyping by sequencing technology (MGST). Ou...

  9. SNP genotyping by heteroduplex analysis.

    PubMed

    Paniego, Norma; Fusari, Corina; Lia, Verónica; Puebla, Andrea

    2015-01-01

    Heteroduplex-based genotyping methods have proven to be technologically effective and economically efficient for low- to medium-range throughput single-nucleotide polymorphism (SNP) determination. In this chapter we describe two protocols that were successfully applied for SNP detection and haplotype analysis of candidate genes in association studies. The protocols involve (1) enzymatic mismatch cleavage with endonuclease CEL1 from celery, associated with fragment separation using capillary electrophoresis (CEL1 cleavage), and (2) differential retention of the homo/heteroduplex DNA molecules under partial denaturing conditions on ion pair reversed-phase liquid chromatography (dHPLC). Both methods are complementary since dHPLC is more versatile than CEL1 cleavage for identifying multiple SNP per target region, and the latter is easily optimized for sequences with fewer SNPs or small insertion/deletion polymorphisms. Besides, CEL1 cleavage is a powerful method to localize the position of the mutation when fragment resolution is done using capillary electrophoresis. PMID:25373754

  10. [Combined physiobalneotherapy of vibration disease associated with dyscirculatory encephalopathy in the miners].

    PubMed

    Borzunova, Iu M; Fedorov, A A

    2012-01-01

    The present study included 161 patients with vibration disease associated with dyscirculatory encephalopathy. It was designed to estimate the degree of cognitive dysfunction and the efficacy of its treatment by physiobalneotherapeutic techniiques including low-frequency pulsed currents in combination with general artificial sodium chloride and iodine bromide baths. In addition, the influence of combined therapy on the clinical course of the disease, results of neuropsychological tests, and cerebral hemodynamics was evaluated. PMID:23210356

  11. Crohn's disease associated with Sweet's syndrome and Sjögren's syndrome treated with infliximab.

    PubMed

    Foster, Erina N; Nguyen, Khanh K; Sheikh, Rafiq A; Prindiville, Thomas P

    2005-06-01

    The association of Crohn's disease (CD) and Sweet's syndrome is rare and the presence of Sjögren's syndrome in Crohn's disease is even rarer, with only three reports found in the literature. We describe two cases of Crohn's disease associated with Sweet's syndrome, one of which is the first case of CD and Sweet's concomitantly associated with Sjogren's syndrome. Both cases responded rapidly to Infliximab therapy with complete resolution of the skin lesions. PMID:16050146

  12. Using VAAST to Identify Disease-Associated Variants in Next-Generation Sequencing Data

    PubMed Central

    Kennedy, Brett; Kronenberg, Zev; Hu, Hao; Moore, Barry; Flygare, Steven; Reese, Martin G.; Jorde, Lynn B.; Yandell, Mark; Huff, Chad

    2014-01-01

    The VAAST pipeline is specifically designed to identify disease-associated alleles in next-generation sequencing data. In the protocols presented in this paper, we outline the best practices for variant prioritization using VAAST. Examples and test data are provided for case-control, small pedigree, and large pedigree analyses. These protocols will teach users the fundamentals of VAAST, VAAST 2.0, and pVAAST analyses. PMID:24763993

  13. Mercury in Hair Is Inversely Related to Disease Associated Damage in Systemic Lupus Erythematosus

    PubMed Central

    Crowe, William; Doherty, Leanne; Watson, Gene; Armstrong, David; Ball, Elisabeth; Magee, Pamela; Allsopp, Philip; Bell, Aubrey; Strain, J. J.; McSorley, Emeir

    2015-01-01

    Systemic lupus erythematosus (SLE) is an autoimmune inflammatory disease, and environmental factors are proposed to exacerbate existing symptoms. One such environmental factor is mercury. The aim of this study was to investigate the relationship between exposure to mercury (Hg) and disease activity and disease associated damage in Total Hg concentrations in hair and urine were measured in 52 SLE patients. Dental amalgams were quantified. Disease activity was assessed using three indexes including the British Isles Lupus Assessment Group Index (BILAG). Disease associated damage was measured using the Systemic Lupus International Collaborating Clinics/American College of Rheumatology SLICC/ACR Damage Index. Pearson’s correlation identified a significant negative correlation between hair Hg and BILAG (r = −0.323, p = 0.029) and SLICC/ACR (r = −0.377, p = 0.038). Multiple regression analysis identified hair Hg as a significant predictor of disease associated damage as determined by SLICC/ACR (β = −0.366, 95% confidence interval (CI): −1.769, −0.155 p = 0.019). Urinary Hg was not related to disease activity or damage. Fish consumption is the primary route of MeHg exposure in humans and the inverse association of hair Hg with disease activity observed here might be explained by the anti-inflammatory effects of n-3 long chain polyunsaturated fatty acids also found in fish. PMID:26703710

  14. Cell type-selective disease-association of genes under high regulatory load.

    PubMed

    Galhardo, Mafalda; Berninger, Philipp; Nguyen, Thanh-Phuong; Sauter, Thomas; Sinkkonen, Lasse

    2015-10-15

    We previously showed that disease-linked metabolic genes are often under combinatorial regulation. Using the genome-wide ChIP-Seq binding profiles for 93 transcription factors in nine different cell lines, we show that genes under high regulatory load are significantly enriched for disease-association across cell types. We find that transcription factor load correlates with the enhancer load of the genes and thereby allows the identification of genes under high regulatory load by epigenomic mapping of active enhancers. Identification of the high enhancer load genes across 139 samples from 96 different cell and tissue types reveals a consistent enrichment for disease-associated genes in a cell type-selective manner. The underlying genes are not limited to super-enhancer genes and show several types of disease-association evidence beyond genetic variation (such as biomarkers). Interestingly, the high regulatory load genes are involved in more KEGG pathways than expected by chance, exhibit increased betweenness centrality in the interaction network of liver disease genes, and carry longer 3' UTRs with more microRNA (miRNA) binding sites than genes on average, suggesting a role as hubs integrating signals within regulatory networks. In summary, epigenetic mapping of active enhancers presents a promising and unbiased approach for identification of novel disease genes in a cell type-selective manner. PMID:26338775

  15. A putative disease-associated haplotype within the SCN1A gene in Dravet syndrome.

    PubMed

    Fendri-Kriaa, Nourhène; Boujilbene, Salma; Kammoun, Fatma; Mkaouar-Rebai, Emna; Ben Mahmoud, Afif; Hsairi, Ines; Rebai, Ahmed; Triki, Chahnez; Fakhfakh, Faiza

    2011-05-20

    Dravet syndrome (DS), previously known as severe myoclonic epilepsy of infancy, is one of the most severe forms of childhood epilepsy. DS is caused by a mutation in the neuronal voltage-gated sodium-channel alpha-subunit gene (SCN1A). However, 25-30% of patients with DS are negative for the SCN1A mutation screening, suggesting that other molecular mechanisms may account for these disorders. Recently, the first case of DS caused by a mutation in the neuronal voltage-gated sodium-channel beta-subunit gene (SCN1B) was also reported. In this report we aim to make the molecular analysis of the SCN1A and SCN1B genes in two Tunisian patients affected with DS. The SCN1A and SCN1B genes were tested for mutations by direct sequencing. No mutation was revealed in the SCN1A and SCN1B genes by sequencing analyses. On the other hand, 11 known single nucleotide polymorphisms were identified in the SCN1A gene and composed a putative disease-associated haplotype in patients with DS phenotype. One of the two patients with putative disease-associated haplotype in SCN1A had also one known single nucleotide polymorphism in the SCN1B gene. The sequencing analyses of the SCN1A gene revealed the presence of a putative disease-associated haplotype in two patients affected with Dravet syndrome. PMID:21531204

  16. Using Disease-Associated Coding Sequence Variation to Investigate Functional Compensation by Human Paralogous Proteins

    PubMed Central

    Miura, Sayaka; Tate, Stephanie; Kumar, Sudhir

    2015-01-01

    Gene duplication enables the functional diversification in species. It is thought that duplicated genes may be able to compensate if the function of one of the gene copies is disrupted. This possibility is extensively debated with some studies reporting proteome-wide compensation, whereas others suggest functional compensation among only recent gene duplicates or no compensation at all. We report results from a systematic molecular evolutionary analysis to test the predictions of the functional compensation hypothesis. We contrasted the density of Mendelian disease-associated single nucleotide variants (dSNVs) in proteins with no discernable paralogs (singletons) with the dSNV density in proteins found in multigene families. Under the functional compensation hypothesis, we expected to find greater numbers of dSNVs in singletons due to the lack of any compensating partners. Our analyses produced an opposite pattern; paralogs have over 35% higher dSNV density than singletons. We found that these patterns are concordant with similar differences in the rates of amino acid evolution (ie, functional constraints), as the proteins with paralogs have evolved 33% slower than singletons. Our evolutionary constraint explanation is robust to differences in family sizes, ages (young vs. old duplicates), and degrees of amino acid sequence similarities among paralogs. Therefore, disease-associated human variation does not exhibit significant signals of functional compensation among paralogous proteins, but rather an evolutionary constraint hypothesis provides a better explanation for the observed patterns of disease-associated and neutral polymorphisms in the human genome. PMID:26604664

  17. Lombardia GENS: a collaborative registry for monogenic diseases associated with stroke

    PubMed Central

    Bersano, Anna; Baron, Pierluigi; Lanfranconi, Silvia; Trobia, Nadia; Sterzi, Roberto; Motto, Cristina; Comi, Giancarlo; Sessa, Maria; Martinelli-Boneschi, Filippo; Micieli, Giuseppe; Ferrarese, Carlo; Santoro, Patrizia; Parati, Eugenio; Boncoraglio, Giorgio; Padovani, Alessandro; Pezzini, Alessandro; Candelise, Livia

    2012-01-01

    Summary The Italian region of Lombardy, with its existing stroke centers and high-technology laboratories, provides a favorable context for studying monogenic diseases associated with stroke. The Lombardia GENS project was set up to create a regional network for the diagnosis of six monogenic diseases associated with stroke: CADASIL, Fabry disease, MELAS, familial and sporadic hemiplegic migraine, hereditary cerebral amyloid angiopathy and Marfan syndrome. The network comprises 36 stroke centers and seven high-technology laboratories, performing molecular analysis. In this context, all stroke/TIA patients fulfilling clinical criteria for monogenic diseases are currently being included in an ongoing study. Demographic, clinical and family data and diagnostic criteria are collected using standardized forms. On the basis of stroke incidence in Lombardy and the reported prevalence of the diseases considered, we expect, during the course of the study, to collect datasets and DNA samples from more than 200 stroke patients suspected of having monogenic diseases. This will allow evaluation of the regional burden and better phenotype characterization of monogenic diseases associated with stroke. PMID:23158583

  18. Cell type-selective disease-association of genes under high regulatory load

    PubMed Central

    Galhardo, Mafalda; Berninger, Philipp; Nguyen, Thanh-Phuong; Sauter, Thomas; Sinkkonen, Lasse

    2015-01-01

    We previously showed that disease-linked metabolic genes are often under combinatorial regulation. Using the genome-wide ChIP-Seq binding profiles for 93 transcription factors in nine different cell lines, we show that genes under high regulatory load are significantly enriched for disease-association across cell types. We find that transcription factor load correlates with the enhancer load of the genes and thereby allows the identification of genes under high regulatory load by epigenomic mapping of active enhancers. Identification of the high enhancer load genes across 139 samples from 96 different cell and tissue types reveals a consistent enrichment for disease-associated genes in a cell type-selective manner. The underlying genes are not limited to super-enhancer genes and show several types of disease-association evidence beyond genetic variation (such as biomarkers). Interestingly, the high regulatory load genes are involved in more KEGG pathways than expected by chance, exhibit increased betweenness centrality in the interaction network of liver disease genes, and carry longer 3′ UTRs with more microRNA (miRNA) binding sites than genes on average, suggesting a role as hubs integrating signals within regulatory networks. In summary, epigenetic mapping of active enhancers presents a promising and unbiased approach for identification of novel disease genes in a cell type-selective manner. PMID:26338775

  19. Flavonoid content and antioxidant capacity of spinach genotypes determined by high-performance liquid chromatography/mass spectrometry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Flavonoids in different spinach genotypes were separated, identified, and quantified by a high-performance liquid chromatographic method with photodiode array and mass spectrometric detection. The antioxidant capacities of the genotypes were also measured using two antioxidant assays - oxygen radica...

  20. Genotyping-by-Sequencing in Plants

    PubMed Central

    Deschamps, Stéphane; Llaca, Victor; May, Gregory D.

    2012-01-01

    The advent of next-generation DNA sequencing (NGS) technologies has led to the development of rapid genome-wide Single Nucleotide Polymorphism (SNP) detection applications in various plant species. Recent improvements in sequencing throughput combined with an overall decrease in costs per gigabase of sequence is allowing NGS to be applied to not only the evaluation of small subsets of parental inbred lines, but also the mapping and characterization of traits of interest in much larger populations. Such an approach, where sequences are used simultaneously to detect and score SNPs, therefore bypassing the entire marker assay development stage, is known as genotyping-by-sequencing (GBS). This review will summarize the current state of GBS in plants and the promises it holds as a genome-wide genotyping application. PMID:24832503

  1. Source tracking identifies deer and geese as vectors of human-infectious Cryptosporidium genotypes in an urban/suburban watershed.

    PubMed

    Jellison, Kristen L; Lynch, Amy E; Ziemann, Joseph M

    2009-06-15

    This study identified Cryptosporidium genotypes in the Wissahickon watershed from May 2005 to April 2008. We analyzed 129 samples from Wissahickon Creek, 83 effluent samples from wastewater treatment plants (WWTPs), and 240 fecal droppings. Genotyping was based on the hypervariable region of the 18S rRNA gene. Oocysts were detected year-round, independent of wet weather events, in 22% of Wissahickon Creek samples, 5% of WWTP effluents, and 7% of fecal samples. Of the genotypes detected, 67% were human-infectious: 30% C. hominis or C. hominis-like, 12% C. parvum, 14% cervine genotype, 9% skunk genotype, and 1% chipmunk I genotype. Similar genotype profiles were detected in Wissahickon Creek each year, and human-infectious genotypes were detected year-round. Unusual genotypes detected in a deer (a C. hominis-like genotype) and geese (C. hominis-like genotypes, C. parvum, and muskrat genotype I) show that these animals are vectors of human-infectious genotypes in this watershed. Results suggest that deer, geese, and WWTPs are appropriate targets for source water protection in the Wissahickon watershed. PMID:19603633

  2. Analysis of hepatitis B virus genotype changes in patients with chronic hepatitis B infection on tenofovir therapy.

    PubMed

    Chauhan, Ranjit; Singh, Avishek K; Rooge, Sheetalnath; Varshney, Aditi; Kumar, Manoj; Sarin, Shiv K

    2016-08-01

    Antiviral therapy for chronic hepatitis B (CHB) is often required for prolonged periods. We investigated the instance of one HBV genotype switching to another during tenofovir therapy. Of the 67 patients, genotype A was present in 6 (8.9%), D in 43 (65.6%), C in 1 (1.5%), and mixed in 17 (23.8%) patients. Genotype changes were detected in 51 (76.1%) patients on therapy during a follow-up of 192 (range 52-312) weeks. Inter-genotype changes were seen in 17 (33.3%) and intra-genotype in 28 (55%) and both inter- and intra-genotype in 6 of 51 (11.7%) patients. The distribution of genotypes in patients achieving complete virological response was genotype D, 32/43 (74.4%); genotype A, 6/6 (100%); and mixed genotypes, 13/17 (76.47%). The cumulative time of genotype switch among genotype A was 12 months (range 6-18), in genotype D, 12 months (range 6-48), and mixed genotype, 18 months (range 6-24). The type of inter-genotype switch most frequently detected among genotype A1 was from A1 to D1 5/6 (83.3%), followed by mixed to genotype D3 7/13 (54%) and among intra-genotype changes, from D1 to D3 in 14/20 (70%). Pretreatment HBV genotype was the only factor predicting inter-genotype switches with genotype A or mixed genotypes more likely to undergo inter-genotype switches as compared to genotype D patients (OR 66.6 [13.6-327.0, P < 0.001]). Compared to genotype D, genotype A, and mixed genotypes are more inclined to switch while on tenofovir therapy. Genotypes tend to switch and select to a particular type possibly due to constant antiviral drug pressure. J. Med. Virol. 88:1364-1375, 2016. © 2016 Wiley Periodicals, Inc. PMID:26858138

  3. Enrichment Analysis Identifies Functional MicroRNA-Disease Associations in Humans

    PubMed Central

    Yuan, Dandan; Cui, Xiaomeng; Wang, Yang; Zhao, Yilei; Li, Huiying; Hu, Suangjiu; Chu, Xiaodan; Li, Yan; Li, Qiang; Liu, Qian; Zhu, Wenliang

    2015-01-01

    Substantial evidence has shown that microRNAs (miRNAs) may be causally linked to the occurrence and progression of human diseases. Herein, we conducted an enrichment analysis to identify potential functional miRNA-disease associations (MDAs) in humans by integrating currently known biological data: miRNA-target interactions (MTIs), protein-protein interactions, and gene-disease associations. Two contributing factors to functional miRNA-disease associations were quantitatively considered: the direct effects of miRNA that target disease-related genes, and indirect effects triggered by protein-protein interactions. Ninety-nine miRNAs were scanned for possible functional association with 2223 MeSH-defined human diseases. Each miRNA was experimentally validated to target ≥ 10 mRNA genes. Putative MDAs were identified when at least one MTI was confidently validated for a disease. Overall, 19648 putative MDAs were found, of which 10.0% was experimentally validated. Further results suggest that filtering for miRNAs that target a greater number of disease-related genes (n ≥ 8) can significantly enrich for true MDAs from the set of putative associations (enrichment rate = 60.7%, adjusted hypergeometric p = 2.41×10−91). Considering the indirect effects of miRNAs further elevated the enrichment rate to 72.6%. By using this method, a novel MDA between miR-24 and ovarian cancer was found. Compared with scramble miRNA overexpression of miR-24 was validated to remarkably induce ovarian cancer cells apoptosis. Our study provides novel insight into factors contributing to functional MDAs by integrating large quantities of previously generated biological data, and establishes a feasible method to identify plausible associations with high confidence. PMID:26296081

  4. Enrichment Analysis Identifies Functional MicroRNA-Disease Associations in Humans.

    PubMed

    Yuan, Dandan; Cui, Xiaomeng; Wang, Yang; Zhao, Yilei; Li, Huiying; Hu, Suangjiu; Chu, Xiaodan; Li, Yan; Li, Qiang; Liu, Qian; Zhu, Wenliang

    2015-01-01

    Substantial evidence has shown that microRNAs (miRNAs) may be causally linked to the occurrence and progression of human diseases. Herein, we conducted an enrichment analysis to identify potential functional miRNA-disease associations (MDAs) in humans by integrating currently known biological data: miRNA-target interactions (MTIs), protein-protein interactions, and gene-disease associations. Two contributing factors to functional miRNA-disease associations were quantitatively considered: the direct effects of miRNA that target disease-related genes, and indirect effects triggered by protein-protein interactions. Ninety-nine miRNAs were scanned for possible functional association with 2223 MeSH-defined human diseases. Each miRNA was experimentally validated to target ≥ 10 mRNA genes. Putative MDAs were identified when at least one MTI was confidently validated for a disease. Overall, 19648 putative MDAs were found, of which 10.0% was experimentally validated. Further results suggest that filtering for miRNAs that target a greater number of disease-related genes (n ≥ 8) can significantly enrich for true MDAs from the set of putative associations (enrichment rate = 60.7%, adjusted hypergeometric p = 2.41×10-91). Considering the indirect effects of miRNAs further elevated the enrichment rate to 72.6%. By using this method, a novel MDA between miR-24 and ovarian cancer was found. Compared with scramble miRNA overexpression of miR-24 was validated to remarkably induce ovarian cancer cells apoptosis. Our study provides novel insight into factors contributing to functional MDAs by integrating large quantities of previously generated biological data, and establishes a feasible method to identify plausible associations with high confidence. PMID:26296081

  5. [Hirschsprung's disease associated with intestinal malrotation in an adult and a review of literature].

    PubMed

    Ko, S; Fujii, H; Yamamoto, K; Sado, S; Yamamoto, M; Nakano, H

    1991-04-01

    A 19-year-old woman was admitted to our clinic because of abdominal distention and severe constipation from infancy. Barium enema revealed a grossly dilated descending colon in the right of the abdomen. At laparotomy, with diagnosis of Hirschsprung's disease associated with non-obstructing intestinal malrotation (non-rotation type), division of the adhesion between caecum and duodenum, and modified Duhamel's procedure were performed. The association of Hirschsprung's disease and intestinal malrotation is rare, and to date only 20 infantile case have been reported. The present case would be the first adult case. PMID:1870577

  6. Genotyping Sleep Disorders Patients

    PubMed Central

    Shadan, Farhad F.; Dawson, Arthur; Cronin, John W.; Jamil, Shazia M.; Grizas, Alexandra P.; Koziol, James A.; Kline, Lawrence E.

    2010-01-01

    Objective The genetic susceptibility factors underlying sleep disorders might help us predict prognoses and responses to treatment. Several candidate polymorphisms for sleep disorders have been proposed, but there has as yet inadequate replication or validation that the candidates may be useful in the clinical setting. Methods To assess the validity of several candidate associations, we obtained saliva deoxyribonucleic acid (DNA) samples and clinical information from 360 consenting research participants who were undergoing clinical polysomnograms. Ten single nucleotide polymorphisms (SNPs) were genotyped. These were thought to be related to depression, circadian sleep disorders, sleep apnea, restless legs syndrome (RLS), excessive sleepiness, or to slow waves in sleep. Results With multivariate generalized linear models, the association of TEF rs738499 with depressive symptoms was confirmed. Equivocal statistical evidence of association of rs1801260 (the C3111T SNP in the CLOCK gene) with morningness/eveningness and an association of Apolipoprotein E (APOE) rs429358 with the Epworth Sleepiness Scale (ESS) were obtained, but these associations were not strong enough to be of clinical value by themselves. Predicted association of SNPs with sleep apnea, RLS, and slow wave sleep were not confirmed. Conclusion The SNPs tested would not, by themselves, be of use for clinical genotyping in a sleep clinic. PMID:20396431

  7. Single nucleotide polymorphism genotyping using BeadChip microarrays.

    PubMed

    Lambert, Gilliam; Tsinajinnie, Darwin; Duggan, David

    2013-07-01

    The genotyping of single nucleotide polymorphisms (SNPs) has successfully contributed to the study of complex diseases more than any other technology to date. Genome-wide association studies (GWAS) using 10,000s to >1,000,000 SNPs have identified 1000s of statistically significant SNPs pertaining to 17 different human disease and trait categories. Post-GWAS fine-mapping studies using 10,000s to 100,000s SNPs on a microarray have narrowed the region of interest for many of these GWAS findings; in addition, independent signals within the original GWAS region have been identified. Focused content, SNP-based microarrays such as the human exome, for example, have too been used successfully to identify novel disease associations. Success has come to studies where 100s to 10,000s (mostly) to >100,000 samples were genotyped. For the time being, SNP-based microarrays remain cost-effective especially when studying large numbers of samples compared to other "genotyping" technologies including next generation sequencing. In this unit, protocols for manual (LIMS-free), semi-manual, and automated processing of BeadChip microarrays are presented. Lower throughput studies will find value in the manual and semi-manual protocols, while all types of studies--low-, medium-, and high-throughput--will find value in the semi-manual and automated protocols. PMID:23853082

  8. [Chronic liver disease associated with cystic fibrosis in the Hospital Alberto Sabogal Sologuren, Lima, Peru: report of a case].

    PubMed

    Ormeño J, Alexis; García D, Cesar; Sumire U, Julia; Asato H, Carmen

    2013-01-01

    Cystic fibrosis (CF) is the most frequent recessive genetic disorder in the caucasian population and is produced by the alteration of electrolyte and water transport in the epithelial cell membrane. Liver disease is a frequent complication towards the end of the first decade of life, being weird its onset, except in patients with a history of meconium ileus. The characteristic liver injury in CF is focal biliary cirrhosis, but fatty infiltration can also be found. The diagnosis is made considering the clinical, laboratory and imaging results having in consideration that the normal liver function tests do not rule out the disease. Ultrasound is the most widely used and can detect the presence of steatosis, stones, fibrosis, cirrhosis, portal hypertension or abnormalities of the biliary tree. There is an also available technique such as computed tomography or magnetic resonance imaging, which allows a morphological study. Important aspects in the treatment are nutritional management, administration of soluble vitamins and the use of ursodeoxycholic acid (UDCA). In cases of advanced cirrhosis, transplantation, isolated or combined with the lung, is an option to consider, with acceptable survival rates. We report the case of an 11 year old patient with a diagnosis of chronic liver disease associated with cystic fibrosis. PMID:24108378

  9. Semi-supervised spectral clustering with application to detect population stratification

    PubMed Central

    Liu, Binghui; Shen, Xiaotong; Pan, Wei

    2013-01-01

    In genetic association studies, unaccounted population stratification can cause spurious associations in a discovery process of identifying disease-associated genetic markers. In such a situation, prior information is often available for some subjects' population identities. To leverage the additional information, we propose a semi-supervised clustering approach for detecting population stratification. This approach maintains the advantages of spectral clustering, while is integrated with the additional identity information, leading to sharper clustering performance. To demonstrate utility of our approach, we analyze a whole-genome sequencing dataset from the 1000 Genomes Project, consisting of the genotypes of 607 individuals sampled from three continental groups involving 10 subpopulations. This is compared against a semi-supervised spectral clustering method, in addition to a spectral clustering method, with the known subpopulation information by the Rand index and an adjusted Rand (ARand) index. The numerical results suggest that the proposed method outperforms its competitors in detecting population stratification. PMID:24298278

  10. Comparison of Genotypes I and III in Japanese Encephalitis Virus Reveals Distinct Differences in Their Genetic and Host Diversity

    PubMed Central

    Han, Na; Adams, James; Chen, Ping; Guo, Zhen-yang; Zhong, Xiang-fu; Fang, Wei; Li, Na; Wen, Lei; Tao, Xiao-yan; Yuan, Zhi-ming

    2014-01-01

    ABSTRACT Japanese encephalitis (JE) is an arthropod-borne disease associated with the majority of viral encephalitis cases in the Asia-Pacific region. The causative agent, Japanese encephalitis virus (JEV), has been phylogenetically divided into five genotypes. Recent surveillance data indicate that genotype I (GI) is gradually replacing genotype III (GIII) as the dominant genotype. To investigate the mechanism behind the genotype shift and the potential consequences in terms of vaccine efficacy, human cases, and virus dissemination, we collected (i) all full-length and partial JEV molecular sequences and (ii) associated genotype and host information comprising a data set of 873 sequences. We then examined differences between the two genotypes at the genetic and epidemiological level by investigating amino acid mutations, positive selection, and host range. We found that although GI is dominant, it has fewer sites predicted to be under positive selection, a narrower host range, and significantly fewer human isolates. For the E protein, the sites under positive selection define a haplotype set for each genotype that shows striking differences in their composition and diversity, with GIII showing significantly more variety than GI. Our results suggest that GI has displaced GIII by achieving a replication cycle that is more efficient but is also more restricted in its host range. IMPORTANCE Japanese encephalitis is an arthropod-borne disease associated with the majority of viral encephalitis cases in the Asia-Pacific region. The causative agent, Japanese encephalitis virus (JEV), has been divided into five genotypes based on sequence similarity. Recent data indicate that genotype I (GI) is gradually replacing genotype III (GIII) as the dominant genotype. Understanding the reasons behind this shift and the potential consequences in terms of vaccine efficacy, human cases, and virus dissemination is important for controlling the spread of the virus and reducing human

  11. Semi-supervised learning for potential human microRNA-disease associations inference.

    PubMed

    Chen, Xing; Yan, Gui-Ying

    2014-01-01

    MicroRNAs play critical role in the development and progression of various diseases. Predicting potential miRNA-disease associations from vast amount of biological data is an important problem in the biomedical research. Considering the limitations in previous methods, we developed Regularized Least Squares for MiRNA-Disease Association (RLSMDA) to uncover the relationship between diseases and miRNAs. RLSMDA can work for diseases without known related miRNAs. Furthermore, it is a semi-supervised (does not need negative samples) and global method (prioritize associations for all the diseases simultaneously). Based on leave-one-out cross validation, reliable AUC have demonstrated the reliable performance of RLSMDA. We also applied RLSMDA to Hepatocellular cancer and Lung cancer and implemented global prediction for all the diseases simultaneously. As a result, 80% (Hepatocellular cancer) and 84% (Lung cancer) of top 50 predicted miRNAs and 75% of top 20 potential associations based on global prediction have been confirmed by biological experiments. We also applied RLSMDA to diseases without known related miRNAs in golden standard dataset. As a result, in the top 3 potential related miRNA list predicted by RLSMDA for 32 diseases, 34 disease-miRNA associations were successfully confirmed by experiments. It is anticipated that RLSMDA would be a useful bioinformatics resource for biomedical researches. PMID:24975600

  12. Prediction of miRNA-disease associations with a vector space model

    PubMed Central

    Pasquier, Claude; Gardès, Julien

    2016-01-01

    MicroRNAs play critical roles in many physiological processes. Their dysregulations are also closely related to the development and progression of various human diseases, including cancer. Therefore, identifying new microRNAs that are associated with diseases contributes to a better understanding of pathogenicity mechanisms. MicroRNAs also represent a tremendous opportunity in biotechnology for early diagnosis. To date, several in silico methods have been developed to address the issue of microRNA-disease association prediction. However, these methods have various limitations. In this study, we investigate the hypothesis that information attached to miRNAs and diseases can be revealed by distributional semantics. Our basic approach is to represent distributional information on miRNAs and diseases in a high-dimensional vector space and to define associations between miRNAs and diseases in terms of their vector similarity. Cross validations performed on a dataset of known miRNA-disease associations demonstrate the excellent performance of our method. Moreover, the case study focused on breast cancer confirms the ability of our method to discover new disease-miRNA associations and to identify putative false associations reported in databases. PMID:27246786

  13. Prediction of miRNA-disease associations with a vector space model.

    PubMed

    Pasquier, Claude; Gardès, Julien

    2016-01-01

    MicroRNAs play critical roles in many physiological processes. Their dysregulations are also closely related to the development and progression of various human diseases, including cancer. Therefore, identifying new microRNAs that are associated with diseases contributes to a better understanding of pathogenicity mechanisms. MicroRNAs also represent a tremendous opportunity in biotechnology for early diagnosis. To date, several in silico methods have been developed to address the issue of microRNA-disease association prediction. However, these methods have various limitations. In this study, we investigate the hypothesis that information attached to miRNAs and diseases can be revealed by distributional semantics. Our basic approach is to represent distributional information on miRNAs and diseases in a high-dimensional vector space and to define associations between miRNAs and diseases in terms of their vector similarity. Cross validations performed on a dataset of known miRNA-disease associations demonstrate the excellent performance of our method. Moreover, the case study focused on breast cancer confirms the ability of our method to discover new disease-miRNA associations and to identify putative false associations reported in databases. PMID:27246786

  14. Semi-supervised learning for potential human microRNA-disease associations inference

    PubMed Central

    Chen, Xing; Yan, Gui-Ying

    2014-01-01

    MicroRNAs play critical role in the development and progression of various diseases. Predicting potential miRNA-disease associations from vast amount of biological data is an important problem in the biomedical research. Considering the limitations in previous methods, we developed Regularized Least Squares for MiRNA-Disease Association (RLSMDA) to uncover the relationship between diseases and miRNAs. RLSMDA can work for diseases without known related miRNAs. Furthermore, it is a semi-supervised (does not need negative samples) and global method (prioritize associations for all the diseases simultaneously). Based on leave-one-out cross validation, reliable AUC have demonstrated the reliable performance of RLSMDA. We also applied RLSMDA to Hepatocellular cancer and Lung cancer and implemented global prediction for all the diseases simultaneously. As a result, 80% (Hepatocellular cancer) and 84% (Lung cancer) of top 50 predicted miRNAs and 75% of top 20 potential associations based on global prediction have been confirmed by biological experiments. We also applied RLSMDA to diseases without known related miRNAs in golden standard dataset. As a result, in the top 3 potential related miRNA list predicted by RLSMDA for 32 diseases, 34 disease-miRNA associations were successfully confirmed by experiments. It is anticipated that RLSMDA would be a useful bioinformatics resource for biomedical researches. PMID:24975600

  15. Hajdu-Cheney Syndrome, a Disease Associated with NOTCH2 Mutations.

    PubMed

    Canalis, Ernesto; Zanotti, Stefano

    2016-08-01

    Notch plays an important function in skeletal homeostasis, osteoblastogenesis, and osteoclastogenesis. Hajdu-Cheney syndrome (HCS) is a rare disease associated with mutations in NOTCH2 leading to the translation of a truncated NOTCH2 stable protein. As a consequence, a gain-of-NOTCH2 function is manifested. HCS is inherited as an autosomal dominant disease although sporadic cases exist. HCS is characterized by craniofacial developmental defects, including platybasia and wormian bones, osteoporosis with fractures, and acro-osteolysis. Subjects may suffer severe neurological complications, and HCS presents with cardiovascular defects and polycystic kidneys. An experimental mouse model harboring a HCSNotch2 mutation exhibits osteopenia secondary to enhanced bone resorption suggesting this as a possible mechanism for the skeletal disease. If the same mechanisms were operational in humans, anti-resorptive therapy could correct the bone loss, but not necessarily the acro-osteolysis. In conclusion, HCS is a devastating disease associated with a gain-of-NOTCH2 function resulting in diverse clinical manifestations. PMID:27241678

  16. A review of outbreaks of foodborne disease associated with passenger ships: evidence for risk management.

    PubMed Central

    Rooney, Roisin M.; Cramer, Elaine H.; Mantha, Stacey; Nichols, Gordon; Bartram, Jamie K.; Farber, Jeffrey M.; Benembarek, Peter K.

    2004-01-01

    OBJECTIVE: Foodborne disease outbreaks on ships are of concern because of their potentially serious health consequences for passengers and crew and high costs to the industry. The authors conducted a review of outbreaks of foodborne diseases associated with passenger ships in the framework of a World Health Organization project on setting guidelines for ship sanitation. METHODS: The authors reviewed data on 50 outbreaks of foodborne disease associated with passenger ships. For each outbreak, data on pathogens/toxins, type of ship, factors contributing to outbreaks, mortality and morbidity, and food vehicles were collected. RESULTS: The findings of this review show that the majority of reported outbreaks were associated with cruise ships and that almost 10,000 people were affected. Salmonella spp were most frequently associated with outbreaks. Foodborne outbreaks due to enterotoxigenic E. coli spp, Shigella spp, noroviruses (formally called Norwalk-like viruses), Vibrio spp, Staphylococcus aureus, Clostridium perfringens, Cyclospora sp, and Trichinella sp also occurred on ships. Factors associated with the outbreaks reviewed include inadequate temperature control, infected food handlers, contaminated raw ingredients, cross-contamination, inadequate heat treatment, and onshore excursions. Seafood was the most common food vehicle implicated in outbreaks. CONCLUSIONS: Many ship-associated outbreaks could have been prevented if measures had been taken to ensure adequate temperature control, avoidance of cross-contamination, reliable food sources, adequate heat treatment, and exclusion of infected food handlers from work. PMID:15219800

  17. Kinase inhibitor profiling reveals unexpected opportunities to inhibit disease-associated mutant kinases

    PubMed Central

    Duong-Ly, Krisna C.; Devarajan, Karthik; Liang, Shuguang; Horiuchi, Kurumi Y.; Wang, Yuren; Ma, Haiching; Peterson, Jeffrey R.

    2016-01-01

    Summary Small-molecule kinase inhibitors have typically been designed to inhibit wild-type kinases rather than the mutant forms that frequently arise in diseases such as cancer. Mutations can have serious clinical implications by increasing kinase catalytic activity or conferring therapeutic resistance. To identify opportunities to repurpose inhibitors against disease-associated mutant kinases, we conducted a large-scale functional screen of 183 known kinase inhibitors against 76 recombinant, mutant kinases. The results revealed lead compounds with activity against clinically important mutant kinases including ALK, LRRK2, RET, and EGFR as well as unexpected opportunities for repurposing FDA-approved kinase inhibitors as leads for additional indications. Furthermore, using T674I PDGFRα as an example, we show how single-dose screening data can provide predictive structure-activity data to guide subsequent inhibitor optimization. This study provides a resource for the development of inhibitors against numerous disease-associated mutant kinases and illustrates the potential of unbiased profiling as an approach to compound-centric inhibitor development. PMID:26776524

  18. Computational screening of disease-associated mutations in OCA2 gene.

    PubMed

    Kamaraj, Balu; Purohit, Rituraj

    2014-01-01

    Oculocutaneous albinism type 2 (OCA2), caused by mutations of OCA2 gene, is an autosomal recessive disorder characterized by reduced biosynthesis of melanin pigment in the skin, hair, and eyes. The OCA2 gene encodes instructions for making a protein called the P protein. This protein plays a crucial role in melanosome biogenesis, and controls the eumelanin content in melanocytes in part via the processing and trafficking of tyrosinase which is the rate-limiting enzyme in melanin synthesis. In this study we analyzed the pathogenic effect of 95 non-synonymous single nucleotide polymorphisms reported in OCA2 gene using computational methods. We found R305W mutation as most deleterious and disease associated using SIFT, PolyPhen, PANTHER, PhD-SNP, Pmut, and MutPred tools. To understand the atomic arrangement in 3D space, the native and mutant (R305W) structures were modeled. Molecular dynamics simulation was conducted to observe the structural significance of computationally prioritized disease-associated mutation (R305W). Root-mean-square deviation, root-mean-square fluctuation, radius of gyration, solvent accessibility surface area, hydrogen bond (NH bond), trace of covariance matrix, eigenvector projection analysis, and density analysis results showed prominent loss of stability and rise in mutant flexibility values in 3D space. This study presents a well designed computational methodology to examine the albinism-associated SNPs. PMID:23824587

  19. LincSNP: a database of linking disease-associated SNPs to human large intergenic non-coding RNAs

    PubMed Central

    2014-01-01

    Background Genome-wide association studies (GWAS) have successfully identified a large number of single nucleotide polymorphisms (SNPs) that are associated with a wide range of human diseases. However, many of these disease-associated SNPs are located in non-coding regions and have remained largely unexplained. Recent findings indicate that disease-associated SNPs in human large intergenic non-coding RNA (lincRNA) may lead to susceptibility to diseases through their effects on lincRNA expression. There is, therefore, a need to specifically record these SNPs and annotate them as potential candidates for disease. Description We have built LincSNP, an integrated database, to identify and annotate disease-associated SNPs in human lincRNAs. The current release of LincSNP contains approximately 140,000 disease-associated SNPs (or linkage disequilibrium SNPs), which can be mapped to around 5,000 human lincRNAs, together with their comprehensive functional annotations. The database also contains annotated, experimentally supported SNP-lincRNA-disease associations and disease-associated lincRNAs. It provides flexible search options for data extraction and searches can be performed by disease/phenotype name, SNP ID, lincRNA name and chromosome region. In addition, we provide users with a link to download all the data from LincSNP and have developed a web interface for the submission of novel identified SNP-lincRNA-disease associations. Conclusions The LincSNP database aims to integrate disease-associated SNPs and human lincRNAs, which will be an important resource for the investigation of the functions and mechanisms of lincRNAs in human disease. The database is available at http://bioinfo.hrbmu.edu.cn/LincSNP. PMID:24885522

  20. Liquid Biopsies: Genotyping Circulating Tumor DNA

    PubMed Central

    Diaz, Luis A.; Bardelli, Alberto

    2016-01-01

    Genotyping tumor tissue in search of somatic genetic alterations for actionable information has become routine practice in clinical oncology. Although these sequence alterations are highly informative, sampling tumor tissue has significant inherent limitations; tumor tissue is a single snapshot in time, is subject to selection bias resulting from tumor heterogeneity, and can be difficult to obtain. Cell-free fragments of DNA are shed into the bloodstream by cells undergoing apoptosis or necrosis, and the load of circulating cell-free DNA (cfDNA) correlates with tumor staging and prognosis. Moreover, recent advances in the sensitivity and accuracy of DNA analysis have allowed for genotyping of cfDNA for somatic genomic alterations found in tumors. The ability to detect and quantify tumor mutations has proven effective in tracking tumor dynamics in real time as well as serving as a liquid biopsy that can be used for a variety of clinical and investigational applications not previously possible. PMID:24449238

  1. Role of cytochrome P450 genotype in the steps toward personalized drug therapy

    PubMed Central

    Cavallari, Larisa H; Jeong, Hyunyoung; Bress, Adam

    2011-01-01

    Genetic polymorphism for cytochrome 450 (P450) enzymes leads to interindividual variability in the plasma concentrations of many drugs. In some cases, P450 genotype results in decreased enzyme activity and an increased risk for adverse drug effects. For example, individuals with the CYP2D6 loss-of-function genotype are at increased risk for ventricular arrhythmia if treated with usual does of thioridazine. In other cases, P450 genotype may influence the dose of a drug required to achieve a desired effect. This is the case with warfarin, with lower doses often necessary in carriers of a variant CYP2C9*2 or *3 allele to avoid supratherapeutic anticoagulation. When a prodrug, such as clopidogrel or codeine, must undergo hepatic biotransformation to its active form, a loss-of-function P450 genotype leads to reduced concentrations of the active drug and decreased drug efficacy. In contrast, patients with multiple CYP2D6 gene copies are at risk for opioid-related toxicity if treated with usual doses of codeine-containing analgesics. At least 25 drugs contain information in their US Food and Drug Administration-approved labeling regarding P450 genotype. The CYP2C9, CYP2C19, and CYP2D6 genes are the P450 genes most often cited. To date, integration of P450 genetic information into clinical decision making is limited. However, some institutions are beginning to embrace routine P450 genotyping to assist in the treatment of their patients. Genotyping for P450 variants may carry less risk for discrimination compared with genotyping for disease-associated variants. As such, P450 genotyping is likely to lead the way in the clinical implementation of pharmacogenomics. This review discusses variability in the CYP2C9, CYP2C19, and CYP2D6 genes and the implications of this for drug efficacy and safety. PMID:23226058

  2. HBV Genotypic Variability in Cuba

    PubMed Central

    Loureiro, Carmen L.; Aguilar, Julio C.; Aguiar, Jorge; Muzio, Verena; Pentón, Eduardo; Garcia, Daymir; Guillen, Gerardo; Pujol, Flor H.

    2015-01-01

    The genetic diversity of HBV in human population is often a reflection of its genetic admixture. The aim of this study was to explore the genotypic diversity of HBV in Cuba. The S genomic region of Cuban HBV isolates was sequenced and for selected isolates the complete genome or precore-core sequence was analyzed. The most frequent genotype was A (167/250, 67%), mainly A2 (149, 60%) but also A1 and one A4. A total of 77 isolates were classified as genotype D (31%), with co-circulation of several subgenotypes (56 D4, 2 D1, 5 D2, 7 D3/6 and 7 D7). Three isolates belonged to genotype E, two to H and one to B3. Complete genome sequence analysis of selected isolates confirmed the phylogenetic analysis performed with the S region. Mutations or polymorphisms in precore region were more common among genotype D compared to genotype A isolates. The HBV genotypic distribution in this Caribbean island correlates with the Y lineage genetic background of the population, where a European and African origin prevails. HBV genotypes E, B3 and H isolates might represent more recent introductions. PMID:25742179

  3. Porphyromonas gingivalis Fim-A genotype distribution among Colombians

    PubMed Central

    Jaramillo, Adriana; Parra, Beatriz; Botero, Javier Enrique; Contreras, Adolfo

    2015-01-01

    Introduction: Porphyromonas gingivalis is associated with periodontitis and exhibit a wide array of virulence factors, including fimbriae which is encoded by the FimA gene representing six known genotypes. Objetive: To identify FimA genotypes of P. gingivalis in subjects from Cali-Colombia, including the co-infection with Aggregatibacter actinomycetemcomitans, Treponema denticola, and Tannerella forsythia. Methods: Subgingival samples were collected from 151 people exhibiting diverse periodontal condition. The occurrence of P. gingivalis, FimA genotypes and other bacteria was determined by PCR. Results: P. gingivalis was positive in 85 patients. Genotype FimA II was more prevalent without reach significant differences among study groups (54.3%), FimA IV was also prevalent in gingivitis (13.0%). A high correlation (p= 0.000) was found among P. gingivalis, T. denticola, and T. forsythia co-infection. The FimA II genotype correlated with concomitant detection of T. denticola and T. forsythia. Conclusions: Porphyromonas gingivalis was high even in the healthy group at the study population. A trend toward a greater frequency of FimA II genotype in patients with moderate and severe periodontitis was determined. The FimA II genotype was also associated with increased pocket depth, greater loss of attachment level, and patients co-infected with T. denticola and T. forsythia. PMID:26600627

  4. Complete genotyping of unusual species A rotavirus G12P[11] and G10P[14] isolates and evidence of frequent in vivo reassortment among the rotaviruses detected in children with diarrhea in Kolkata, India, during 2014.

    PubMed

    Mandal, Paulami; Mullick, Satarupa; Nayak, Mukti Kant; Mukherjee, Anupam; Ganguly, Nupur; Niyogi, Prabal; Panda, Samiran; Chawla-Sarkar, Mamta

    2016-10-01

    Species A rotaviruses (RVA) are the most important cause of acute gastroenteritis in the young of humans and many animal species globally. G1P[8], G2P[4], G3P[8], G4P[8], G9P[6/8] and G12P[6/8] are the predominantly isolated genotypes throughout the world including India. Unusual genotypes from different host species such as G5, G6, G8, G10 and G11 have also been reported in humans with low frequency. In the present study, among >650 RVA positive stool samples collected from children with diarrhea in Kolkata, India, during 2014, two isolates each of the genotype G12P[11] and G10P[14] were obtained and their genomes completely sequenced. The full genotype constellations were G12-P[11]-I1-R1-C1-M2-A1-N1-T2-E1-H1 and G12-P[11]-I1-R1-C1-M1-A5-N1-T1-E1-H1 for G12P[11] viruses, suggesting several reassortments between Wa- and DS-1-like human RVA strains, including possible reassortment of a simian NSP1 gene. The G10P[14] viruses (G10-P[14]-I2-R2-C2-M2-A11-N2-T6-E2-H3) were found to contain multiple genes closely related to RVAs of artiodactyl origin, highlighting the role of inter-host species transmissions of RVAs. From the G/P constellation of all RVA isolates, it could be concluded that approximately one quarter had likely arisen from reassortment events in vivo among RVAs of 'usual' genotypes. PMID:27447463

  5. Disease-associated PrP in the enteric nervous system of scrapie-affected Suffolk sheep.

    PubMed

    Heggebø, Ragna; González, Lorenzo; Press, Charles McL; Gunnes, Gjermund; Espenes, Arild; Jeffrey, Martin

    2003-05-01

    Disease-associated prion protein (PrP(d)) in the enteric nervous system (ENS) of 20- to 24-month-old Suffolk sheep in the late subclinical and early clinical phase of scrapie was studied. Sites in the alimentary tract extending from the forestomachs and abomasum to the colon from scrapie-affected sheep (PrP(ARQ/ARQ)) and scrapie-resistant sheep (PrP(ARR/ARQ) and PrP(ARR/ARR)) were examined. PrP(d) was found only in scrapie-affected sheep and was most prominent in the ENS when abundant deposits of PrP(d) were also present in adjacent lymphoid nodules. Immunolabelling with the nerve fibre markers PgP 9.5 and neuron-specific enolase and the satellite cell marker glial fibrillary acidic protein revealed the extensive ganglionated networks of the myenteric and submucosal plexi. Fewer nerve fibres were present in the lamina propria, T-cell dominated interfollicular areas and dome regions of Peyer's patches. A substantial network of nerve fibres was detected in many lymphoid nodules of both the scrapie-affected and scrapie-resistant sheep. Nerve fibres were also detected within the capsule of lymphoid nodules. Electron microscopy revealed the presence of nerves in the lymphoid nodules, showing a close association with follicular dendritic cells, lymphocytes and tingible body macrophages. In demonstrating that lymphoid nodules in the Peyer's patches of scrapie-affected sheep possess a substantial network of nerve fibres, the present study shows that nodules provide close contact between nerve fibres and cell populations known to contain abundant PrP(d), including follicular dendritic cells and tingible body macrophages, and that gut-associated lymphoid nodules in sheep may represent an important site for neuroinvasion. PMID:12692300

  6. Representativeness of Tuberculosis Genotyping Surveillance in the United States, 2009-2010.

    PubMed

    Shak, Emma B; France, Anne Marie; Cowan, Lauren; Starks, Angela M; Grant, Juliana

    2015-01-01

    Genotyping of Mycobacterium tuberculosis isolates contributes to tuberculosis (TB) control through detection of possible outbreaks. However, 20% of U.S. cases do not have an isolate for testing, and 10% of cases with isolates do not have a genotype reported. TB outbreaks in populations with incomplete genotyping data might be missed by genotyping-based outbreak detection. Therefore, we assessed the representativeness of TB genotyping data by comparing characteristics of cases reported during January 1, 2009-December 31, 2010, that had a genotype result with those cases that did not. Of 22,476 cases, 14,922 (66%) had a genotype result. Cases without genotype results were more likely to be patients <19 years of age, with unknown HIV status, of female sex, U.S.-born, and with no recent history of homelessness or substance abuse. Although cases with a genotype result are largely representative of all reported U.S. TB cases, outbreak detection methods that rely solely on genotyping data may underestimate TB transmission among certain groups. PMID:26556930

  7. Disease-associated variants in different categories of disease located in distinct regulatory elements

    PubMed Central

    2015-01-01

    Background The invention of high throughput sequencing technologies has led to the discoveries of hundreds of thousands of genetic variants associated with thousands of human diseases. Many of these genetic variants are located outside the protein coding regions, and as such, it is challenging to interpret the function of these genetic variants by traditional genetic approaches. Recent genome-wide functional genomics studies, such as FANTOM5 and ENCODE have uncovered a large number of regulatory elements across hundreds of different tissues or cell lines in the human genome. These findings provide an opportunity to study the interaction between regulatory elements and disease-associated genetic variants. Identifying these diseased-related regulatory elements will shed light on understanding the mechanisms of how these variants regulate gene expression and ultimately result in disease formation and progression. Results In this study, we curated and categorized 27,558 Mendelian disease variants, 20,964 complex disease variants, 5,809 cancer predisposing germline variants, and 43,364 recurrent cancer somatic mutations. Compared against nine different types of regulatory regions from FANTOM5 and ENCODE projects, we found that different types of disease variants show distinctive propensity for particular regulatory elements. Mendelian disease variants and recurrent cancer somatic mutations are 22-fold and 10- fold significantly enriched in promoter regions respectively (q<0.001), compared with allele-frequency-matched genomic background. Separate from these two categories, cancer predisposing germline variants are 27-fold enriched in histone modification regions (q<0.001), 10-fold enriched in chromatin physical interaction regions (q<0.001), and 6-fold enriched in transcription promoters (q<0.001). Furthermore, Mendelian disease variants and recurrent cancer somatic mutations share very similar distribution across types of functional effects. We further found that

  8. Assessing inflammatory bowel disease-associated antibodies in Caucasian and First Nations cohorts

    PubMed Central

    Bernstein, Charles N; El-Gabalawy, Hani; Sargent, Michael; Landers, Carol J; Rawsthorne, Patricia; Elias, Brenda; Targan, Stephan R

    2011-01-01

    BACKGROUND: First Nation populations in Canada have a very low incidence of inflammatory bowel disease (IBD). Based on typical infections in this population, it is plausible that the First Nations react differently to microbial antigens with a different antibody response pattern, which may shed some light as to why they experience a low rate of IBD. OBJECTIVE: To compare the positivity rates of antibodies known to be associated with IBD in Canadian First Nations compared with a Canadian Caucasian population. METHODS: Subjects with Crohn’s disease, ulcerative colitis (UC), rheumatoid arthritis (RA) (as an immune disease control) and healthy controls without a personal or family history of chronic immune diseases, were enrolled in a cohort study aimed to determine differences between First Nations and Caucasians with IBD or RA. Serum from a random sample of these subjects (n=50 for each of First Nations with RA, First Nations controls, Caucasians with RA, Caucasians with Crohn’s disease, Caucasians with UC and Caucasians controls, and as many First Nations with either Crohn’s disease or UC as could be enrolled) was analyzed in the laboratory for the following antibodies: perinuclear antineutrophil cytoplasmic antibody (pANCA), and four Crohn’s disease-associated antibodies including anti-Saccharomyces cerevisiae, the outer membrane porin C of Escherichia coli, I2 – a fragment of bacterial DNA associated with Pseudomonas fluorescens, and the bacterial flagellin CBir-1. The rates of positive antibody responses and mean titres among positive results were compared. RESULTS: For pANCA, First Nations had a positivity rate of 55% in those with UC, 32% in healthy controls and 48% in those with RA. The pANCA positivity rate was 32% among Caucasians with RA. The rates of the Crohn’s disease-associated antibodies for the First Nations and Caucasians were comparable. Among First Nations, up to one in four healthy controls were positive for any one of the Crohn

  9. Hepatitis E virus genotype 3 in wild rats, United States.

    PubMed

    Lack, Justin B; Volk, Kylie; Van Den Bussche, Ronald A

    2012-08-01

    The role of rodents in the epidemiology of zoonotic hepatitis E virus (HEV) infection has been a subject of considerable debate. Seroprevalence studies suggest widespread HEV infection in commensal Rattus spp. rats, but experimental transmission has been largely unsuccessful and recovery of zoonotic genotype 3 HEV RNA from wild Rattus spp. rats has never been confirmed. We surveyed R. rattus and R. norvegicus rats from across the United States and several international populations by using a hemi-nested reverse transcription PCR approach. We isolated HEV RNA in liver tissues from 35 of 446 rats examined. All but 1 of these isolates was relegated to the zoonotic HEV genotype 3, and the remaining sequence represented the recently discovered rat genotype from the United States and Germany. HEV-positive rats were detected in urban and remote localities. Genetic analyses suggest all HEV genotype 3 isolates obtained from wild Rattus spp. rats were closely related. PMID:22840202

  10. Hepatitis E Virus Genotype 3 in Wild Rats, United States

    PubMed Central

    Volk, Kylie; Van Den Bussche, Ronald A.

    2012-01-01

    The role of rodents in the epidemiology of zoonotic hepatitis E virus (HEV) infection has been a subject of considerable debate. Seroprevalence studies suggest widespread HEV infection in commensal Rattus spp. rats, but experimental transmission has been largely unsuccessful and recovery of zoonotic genotype 3 HEV RNA from wild Rattus spp. rats has never been confirmed. We surveyed R. rattus and R. norvegicus rats from across the United States and several international populations by using a hemi-nested reverse transcription PCR approach. We isolated HEV RNA in liver tissues from 35 of 446 rats examined. All but 1 of these isolates was relegated to the zoonotic HEV genotype 3, and the remaining sequence represented the recently discovered rat genotype from the United States and Germany. HEV-positive rats were detected in urban and remote localities. Genetic analyses suggest all HEV genotype 3 isolates obtained from wild Rattus spp. rats were closely related. PMID:22840202

  11. Determination of self-incompatibility groups of sweet cherry genotypes from Turkey.

    PubMed

    Ipek, A; Gulen, H; Akcay, M E; Ipek, M; Ergin, S; Eris, A

    2011-01-01

    Determination of S-allele combinations of sweet cherry genotypes and cultivars has importance for both growers and breeders. We determined S-allele combinations of 40 local Turkish sweet cherry genotypes using a PCR-based method. Ten different S-alleles were detected. Although the most common S-allele was S3, as also found in Western genotypes and cultivars, there were some differences in the frequencies of some S-alleles between Turkish and Western sweet cherry genotypes. According to their S-allele compositions, 30 local Turkish sweet cherry genotypes were assigned to 10 previously identified incompatibility groups. For the remaining genotypes, whose S-allele combinations did not fit to any previous incompatibility groups, three more incompatibility groups, XLII, XLIII and XLIV, were proposed. Results obtained from this study will help both sweet cherry growers and breeders to better manage these local Turkish sweet cherry genotypes in their orchards. PMID:21341217

  12. Complete genome sequence of Colocasia bobone disease-associated virus, a putative cytorhabdovirus infecting taro.

    PubMed

    Higgins, Colleen M; Bejerman, Nicolas; Li, Ming; James, Anthony P; Dietzgen, Ralf G; Pearson, Michael N; Revill, Peter A; Harding, Robert M

    2016-03-01

    We report the first genome sequence of a Colocasia bobone disease-associated virus (CBDaV) derived from bobone-affected taro [Colocasia esculenta L. Schott] from Solomon Islands. The negative-strand RNA genome is 12,193 nt long, with six major open reading frames (ORFs) with the arrangement 3'-N-P-P3-M-G-L-5'. Typical of all rhabdoviruses, the 3' leader and 5' trailer sequences show complementarity to each other. Phylogenetic analysis indicated that CBDaV is a member of the genus Cytorhabdovirus, supporting previous reports of virus particles within the cytoplasm of bobone-infected taro cells. The availability of the CBDaV genome sequence now makes it possible to assess the role of this virus in bobone, and possibly alomae disease of taro and confirm that this sequence is that of Colocasia bobone disease virus (CBDV). PMID:26687584

  13. Identification of four novel potentially Parkinson's disease associated LRRK2 variations among Iranian patients.

    PubMed

    Shojaee, Seyedmehdi; Fazlali, Zeinab; Ghazavi, Farzaneh; Banihosseini, Setareh Sadat; Kazemi, Mohammad Hossein; Parsa, Khosro; Sadeghi, Homa; Sina, Farzad; Shahidi, Gholam-Ali; Ronaghi, Mostafa; Elahi, Elahe

    2009-12-25

    The results of mutation screening of 24 exons of LRRK2 in 60 Iranian Parkinson's Disease patients are presented. The Iranian cohort represents a novel population and was notably young (average age at onset of disease: 36.0 years). Fifty sequence variations were found, seventeen of which are novel. Variations considered possibly associated with disease were screened in available family members, 145 additional patients and 220 control individuals. It was surmised that four novel sequence variations (IVS49+178A>G, p.R1725Q, p.Q1823K, and p.D2175H) may be associated with PD status, albeit they may be very rare non-disease associated variations. The four variations were all observed in the heterozygous state in early onset cases. If one or more of the variations do indeed contribute to disease status, their penetrance is expected to be low. PMID:19800393

  14. An Update on the Streptococcus bovis Group: Classification, Identification, and Disease Associations.

    PubMed

    Dekker, John P; Lau, Anna F

    2016-07-01

    The Streptococcus bovis group has undergone significant taxonomic changes over the past 2 decades with the advent of new identification methods with higher discriminatory power. Although the current classification system is not yet embraced by all researchers in the field and debate remains over the performance of molecular techniques for identification to the species level within the group, important disease associations for several members of the group have been clarified. Here, we provide a brief overview of the history of the S. bovis group, an outline of the currently accepted classification scheme, a review of associated clinical syndromes, and a summary of the performance and diagnostic accuracy of currently available identification methods. PMID:26912760

  15. Tight junctions in inflammatory bowel diseases and inflammatory bowel disease associated colorectal cancer

    PubMed Central

    Landy, Jonathan; Ronde, Emma; English, Nick; Clark, Sue K; Hart, Ailsa L; Knight, Stella C; Ciclitira, Paul J; Al-Hassi, Hafid Omar

    2016-01-01

    Inflammatory bowel diseases are characterised by inflammation that compromises the integrity of the epithelial barrier. The intestinal epithelium is not only a static barrier but has evolved complex mechanisms to control and regulate bacterial interactions with the mucosal surface. Apical tight junction proteins are critical in the maintenance of epithelial barrier function and control of paracellular permeability. The characterisation of alterations in tight junction proteins as key players in epithelial barrier function in inflammatory bowel diseases is rapidly enhancing our understanding of critical mechanisms in disease pathogenesis as well as novel therapeutic opportunities. Here we give an overview of recent literature focusing on the role of tight junction proteins, in particular claudins, in inflammatory bowel diseases and inflammatory bowel disease associated colorectal cancer. PMID:27003989

  16. Early-onset Parkinson's Disease Associated with Chromosome 22q11.2 Deletion Syndrome.

    PubMed

    Oki, Mitsuaki; Hori, Shin-ichiro; Asayama, Shinya; Wate, Reika; Kaneko, Satoshi; Kusaka, Hirofumi

    2016-01-01

    We herein report the case of a 43-year-old man with a 4-year history of resting tremor and akinesia. His resting tremor and rigidity were more prominent on the left side. He also presented retropulsion. His symptoms responded to the administration of levodopa. The patient also had a cleft lip and palate, cavum vergae, and hypoparathyroidism. A chromosome analysis disclosed a hemizygous deletion in 22q11.2, and he was diagnosed with early-onset Parkinson's disease associated with 22q11.2 deletion syndrome. However, the patient lacked autonomic nerve dysfunction, and his cardiac uptake of (123)I-metaiodobenzylguanidine was normal, indicating an underlying pathological mechanism that differed to that of sporadic Parkinson's disease. PMID:26831029

  17. Diffuse Parenchymal Diseases Associated With Aluminum Use and Primary Aluminum Production

    PubMed Central

    2014-01-01

    Aluminum use and primary aluminum production results in the generation of various particles, fumes, gases, and airborne materials with the potential for inducing a wide range of lung pathology. Nevertheless, the presence of diffuse parenchymal or interstitial lung disease related to these processes remains controversial. The relatively uncommon occurrence of interstitial lung diseases in aluminum-exposed workers—despite the extensive industrial use of aluminum—the potential for concurrent exposure to other fibrogenic fibers, and the previous use of inhaled aluminum powder for the prevention of silicosis without apparent adverse respiratory effects are some of the reasons for this continuing controversy. Specific aluminum-induced parenchymal diseases described in the literature, including existing evidence of interstitial lung diseases, associated with primary aluminum production are reviewed. PMID:24806728

  18. Disease-associated mutations of TDP-43 promote turnover of the protein through the proteasomal pathway.

    PubMed

    Araki, Wataru; Minegishi, Seiji; Motoki, Kazumi; Kume, Hideaki; Hohjoh, Hirohiko; Araki, Yumiko M; Tamaoka, Akira

    2014-12-01

    TAR DNA-binding protein (TDP-43) is a major component of most ubiquitin-positive neuronal and glial inclusions of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). A number of missense mutations in the TARDBP gene have been identified in patients with familial and sporadic ALS, as well as familial FTLD with ALS. In the diseased states, TDP-43 proteins exhibit characteristic alterations, including truncation, abnormal phosphorylation, and altered subcellular distribution. However, the mechanisms by which TDP-43 mutations induce neurodegeneration remain unclear at present. In the current study, we analyzed protein turnover and subcellular distribution of wild-type TDP-43 and two disease-associated mutants (G298S and A382T) in human neuroblastoma SH-SY5Y cells stably expressing TDP-43 with a C-terminal tag. Cycloheximide chase experiments revealed more rapid turnover of TDP-43 mutant proteins than their wild-type counterpart. The decrease in the TDP-43 level after cycloheximide treatment was partially recovered upon co-treatment with the proteasome inhibitor, epoxomicin, but not the lysosomotropic agent, chloroquine, suggesting involvement of the proteasomal pathway in TDP-43 degradation. Analysis of the subcellular distribution of TDP-43 revealed predominant localization in the nuclear fraction, whereas the relative level in the cytoplasm remained unaltered in cells expressing either mutant protein, compared with wild-type protein. Our results suggest that higher turnover of disease-associated mutant TDP-43 proteins through the ubiquitin proteasome system is pathogenetically relevant and highlight the significance of proteolysis in the pathogenetic mechanism of TDP-43 proteinopathy. PMID:24477737

  19. LncDisease: a sequence based bioinformatics tool for predicting lncRNA-disease associations

    PubMed Central

    Wang, Junyi; Ma, Ruixia; Ma, Wei; Chen, Ji; Yang, Jichun; Xi, Yaguang; Cui, Qinghua

    2016-01-01

    LncRNAs represent a large class of noncoding RNA molecules that have important functions and play key roles in a variety of human diseases. There is an urgent need to develop bioinformatics tools as to gain insight into lncRNAs. This study developed a sequence-based bioinformatics method, LncDisease, to predict the lncRNA-disease associations based on the crosstalk between lncRNAs and miRNAs. Using LncDisease, we predicted the lncRNAs associated with breast cancer and hypertension. The breast-cancer-associated lncRNAs were studied in two breast tumor cell lines, MCF-7 and MDA-MB-231. The qRT-PCR results showed that 11 (91.7%) of the 12 predicted lncRNAs could be validated in both breast cancer cell lines. The hypertension-associated lncRNAs were further evaluated in human vascular smooth muscle cells (VSMCs) stimulated with angiotensin II (Ang II). The qRT-PCR results showed that 3 (75.0%) of the 4 predicted lncRNAs could be validated in Ang II-treated human VSMCs. In addition, we predicted 6 diseases associated with the lncRNA GAS5 and validated 4 (66.7%) of them by literature mining. These results greatly support the specificity and efficacy of LncDisease in the study of lncRNAs in human diseases. The LncDisease software is freely available on the Software Page: http://www.cuilab.cn/. PMID:26887819

  20. LncDisease: a sequence based bioinformatics tool for predicting lncRNA-disease associations.

    PubMed

    Wang, Junyi; Ma, Ruixia; Ma, Wei; Chen, Ji; Yang, Jichun; Xi, Yaguang; Cui, Qinghua

    2016-05-19

    LncRNAs represent a large class of noncoding RNA molecules that have important functions and play key roles in a variety of human diseases. There is an urgent need to develop bioinformatics tools as to gain insight into lncRNAs. This study developed a sequence-based bioinformatics method, LncDisease, to predict the lncRNA-disease associations based on the crosstalk between lncRNAs and miRNAs. Using LncDisease, we predicted the lncRNAs associated with breast cancer and hypertension. The breast-cancer-associated lncRNAs were studied in two breast tumor cell lines, MCF-7 and MDA-MB-231. The qRT-PCR results showed that 11 (91.7%) of the 12 predicted lncRNAs could be validated in both breast cancer cell lines. The hypertension-associated lncRNAs were further evaluated in human vascular smooth muscle cells (VSMCs) stimulated with angiotensin II (Ang II). The qRT-PCR results showed that 3 (75.0%) of the 4 predicted lncRNAs could be validated in Ang II-treated human VSMCs. In addition, we predicted 6 diseases associated with the lncRNA GAS5 and validated 4 (66.7%) of them by literature mining. These results greatly support the specificity and efficacy of LncDisease in the study of lncRNAs in human diseases. The LncDisease software is freely available on the Software Page: http://www.cuilab.cn/. PMID:26887819

  1. A review of outbreaks of waterborne disease associated with ships: evidence for risk management.

    PubMed Central

    Rooney, Roisin M.; Bartram, Jamie K.; Cramer, Elaine H.; Mantha, Stacey; Nichols, Gordon; Suraj, Rohini; Todd, Ewen C. D.

    2004-01-01

    OBJECTIVE: The organization of water supply to and on ships differs considerably from that of water supply on land. Risks of contamination can arise from source water at the port or during loading, storage, or distribution on the ship. The purpose of this article is to review documented outbreaks of waterborne diseases associated with passenger, cargo, fishing, and naval ships to identify contributing factors so that similar outbreaks can be prevented in the future. METHODS: The authors reviewed 21 reported outbreaks of waterborne diseases associated with ships. For each outbreak, data on pathogens/toxins, type of ship, factors contributing to outbreaks, mortality and morbidity, and remedial action are presented. RESULTS: The findings of this review show that the majority of reported outbreaks were associated with passenger ships and that more than 6,400 people were affected. Waterborne outbreaks due to Enterotoxigenic Escherichia coli, noroviruses, Salmonella spp, Shigella sp, Cryptosporidium sp, and Giardia lamblia occurred on ships. Enterotoxigenic E. coli was the pathogen most frequently associated with outbreaks. One outbreak of chemical water poisoning also occurred on a ship. Risk factors included contaminated port water, inadequate treatment, improper loading techniques, poor design and maintenance of storage tanks, ingress of contamination during repair and maintenance, cross-connections, back siphonage, and insufficient residual disinfectant. CONCLUSIONS: Waterborne disease outbreaks on ships can be prevented. The factors contributing to outbreaks emphasize the need for hygienic handling of water along the supply chain from source to consumption. A comprehensive approach to water safety on ships is essential. This may be achieved by the adoption of Water Safety Plans that cover design, construction, operation, and routine inspection and maintenance. PMID:15219801

  2. An Integrated Data Driven Approach to Drug Repositioning Using Gene-Disease Associations

    PubMed Central

    Mullen, Joseph; Cockell, Simon J.; Woollard, Peter; Wipat, Anil

    2016-01-01

    Drug development is both increasing in cost whilst decreasing in productivity. There is a general acceptance that the current paradigm of R&D needs to change. One alternative approach is drug repositioning. With target-based approaches utilised heavily in the field of drug discovery, it becomes increasingly necessary to have a systematic method to rank gene-disease associations. Although methods already exist to collect, integrate and score these associations, they are often not a reliable reflection of expert knowledge. Furthermore, the amount of data available in all areas covered by bioinformatics is increasing dramatically year on year. It thus makes sense to move away from more generalised hypothesis driven approaches to research to one that allows data to generate their own hypothesis. We introduce an integrated, data driven approach to drug repositioning. We first apply a Bayesian statistics approach to rank 309,885 gene-disease associations using existing knowledge. Ranked associations are then integrated with other biological data to produce a semantically-rich drug discovery network. Using this network, we show how our approach identifies diseases of the central nervous system (CNS) to be an area of interest. CNS disorders are identified due to the low numbers of such disorders that currently have marketed treatments, in comparison to other therapeutic areas. We then systematically mine our network for semantic subgraphs that allow us to infer drug-disease relations that are not captured in the network. We identify and rank 275,934 drug-disease has_indication associations after filtering those that are more likely to be side effects, whilst commenting on the top ranked associations in more detail. The dataset has been created in Neo4j and is available for download at https://bitbucket.org/ncl-intbio/genediseaserepositioning along with a Java implementation of the searching algorithm. PMID:27196054

  3. An Integrated Data Driven Approach to Drug Repositioning Using Gene-Disease Associations.

    PubMed

    Mullen, Joseph; Cockell, Simon J; Woollard, Peter; Wipat, Anil

    2016-01-01

    Drug development is both increasing in cost whilst decreasing in productivity. There is a general acceptance that the current paradigm of R&D needs to change. One alternative approach is drug repositioning. With target-based approaches utilised heavily in the field of drug discovery, it becomes increasingly necessary to have a systematic method to rank gene-disease associations. Although methods already exist to collect, integrate and score these associations, they are often not a reliable reflection of expert knowledge. Furthermore, the amount of data available in all areas covered by bioinformatics is increasing dramatically year on year. It thus makes sense to move away from more generalised hypothesis driven approaches to research to one that allows data to generate their own hypothesis. We introduce an integrated, data driven approach to drug repositioning. We first apply a Bayesian statistics approach to rank 309,885 gene-disease associations using existing knowledge. Ranked associations are then integrated with other biological data to produce a semantically-rich drug discovery network. Using this network, we show how our approach identifies diseases of the central nervous system (CNS) to be an area of interest. CNS disorders are identified due to the low numbers of such disorders that currently have marketed treatments, in comparison to other therapeutic areas. We then systematically mine our network for semantic subgraphs that allow us to infer drug-disease relations that are not captured in the network. We identify and rank 275,934 drug-disease has_indication associations after filtering those that are more likely to be side effects, whilst commenting on the top ranked associations in more detail. The dataset has been created in Neo4j and is available for download at https://bitbucket.org/ncl-intbio/genediseaserepositioning along with a Java implementation of the searching algorithm. PMID:27196054

  4. Sensitive detection of chromatin-altering polymorphisms reveals autoimmune disease mechanisms.

    PubMed

    del Rosario, Ricardo Cruz-Herrera; Poschmann, Jeremie; Rouam, Sigrid Laure; Png, Eileen; Khor, Chiea Chuen; Hibberd, Martin Lloyd; Prabhakar, Shyam

    2015-05-01

    Most disease associations detected by genome-wide association studies (GWAS) lie outside coding genes, but very few have been mapped to causal regulatory variants. Here, we present a method for detecting regulatory quantitative trait loci (QTLs) that does not require genotyping or whole-genome sequencing. The method combines deep, long-read chromatin immunoprecipitation-sequencing (ChIP-seq) with a statistical test that simultaneously scores peak height correlation and allelic imbalance: the genotype-independent signal correlation and imbalance (G-SCI) test. We performed histone acetylation ChIP-seq on 57 human lymphoblastoid cell lines and used the resulting reads to call 500,066 single-nucleotide polymorphisms de novo within regulatory elements. The G-SCI test annotated 8,764 of these as histone acetylation QTLs (haQTLs)—an order of magnitude larger than the set of candidates detected by expression QTL analysis. Lymphoblastoid haQTLs were highly predictive of autoimmune disease mechanisms. Thus, our method facilitates large-scale regulatory variant detection in any moderately sized cohort for which functional profiling data can be generated, thereby simplifying identification of causal variants within GWAS loci. PMID:25799442

  5. Genotype Specification Language.

    PubMed

    Wilson, Erin H; Sagawa, Shiori; Weis, James W; Schubert, Max G; Bissell, Michael; Hawthorne, Brian; Reeves, Christopher D; Dean, Jed; Platt, Darren

    2016-06-17

    We describe here the Genotype Specification Language (GSL), a language that facilitates the rapid design of large and complex DNA constructs used to engineer genomes. The GSL compiler implements a high-level language based on traditional genetic notation, as well as a set of low-level DNA manipulation primitives. The language allows facile incorporation of parts from a library of cloned DNA constructs and from the "natural" library of parts in fully sequenced and annotated genomes. GSL was designed to engage genetic engineers in their native language while providing a framework for higher level abstract tooling. To this end we define four language levels, Level 0 (literal DNA sequence) through Level 3, with increasing abstraction of part selection and construction paths. GSL targets an intermediate language based on DNA slices that translates efficiently into a wide range of final output formats, such as FASTA and GenBank, and includes formats that specify instructions and materials such as oligonucleotide primers to allow the physical construction of the GSL designs by individual strain engineers or an automated DNA assembly core facility. PMID:26886161

  6. Echinococcus granulosus genotypes in Iran

    PubMed Central

    Sharafi, Seyedeh Maryam; Rostami-Nejad, Mohammad; Moazeni, Mohammad; Yousefi, Morteza; Saneie, Behnam; Hosseini-Safa, Ahmad

    2014-01-01

    Hydatidosis, caused by Echinococcus granulosus is one of the most important zoonotic diseases, throughout most parts of the world. Hydatidosis is endemic in Iran and responsible for approximately 1% of admission to surgical wards. There are extensive genetic variations within E. granulosus and 10 different genotypes (G1–G10) within this parasite have been reported. Identification of strains is important for improvement of control and prevention of the disease. No new review article presented the situation of Echinococcus granulosus genotypes in Iran in the recent years; therefore in this paper we reviewed the different studies regarding Echinococcus granulosus genotypes in Iran. PMID:24834298

  7. Identification of new sub-genotypes of virulent Newcastle disease virus with potential panzootic features.

    PubMed

    Miller, Patti J; Haddas, Ruth; Simanov, Luba; Lublin, Avishay; Rehmani, Shafqat Fatima; Wajid, Abdul; Bibi, Tasra; Khan, Taseer Ahmad; Yaqub, Tahir; Setiyaningsih, Surachmi; Afonso, Claudio L

    2015-01-01

    Virulent Newcastle disease virus (NDV) isolates from new sub-genotypes within genotype VII are rapidly spreading through Asia and the Middle East causing outbreaks of Newcastle disease (ND) characterized by significant illness and mortality in poultry, suggesting the existence of a fifth panzootic. These viruses, which belong to the new sub-genotypes VIIh and VIIi, have epizootic characteristics and do not appear to have originated directly from other genotype VII NDV isolates that are currently circulating elsewhere, but are related to the present and past Indonesian NDV viruses isolated from wild birds since the 80s. Viruses from sub-genotype VIIh were isolated in Indonesia (2009-2010), Malaysia (2011), China (2011), and Cambodia (2011-2012) and are closely related to the Indonesian NDV isolated in 2007, APMV1/Chicken/Karangasem, Indonesia (Bali-01)/2007. Since 2011 and during 2012 highly related NDV isolates from sub-genotype VIIi have been isolated from poultry production facilities and occasionally from pet birds, throughout Indonesia, Pakistan and Israel. In Pakistan, the viruses of sub-genotype VIIi have replaced NDV isolates of genotype XIII, which were commonly isolated in 2009-2011, and they have become the predominant sub-genotype causing ND outbreaks since 2012. In a similar fashion, the numbers of viruses of sub-genotype VIIi isolated in Israel increased in 2012, and isolates from this sub-genotype are now found more frequently than viruses from the previously predominant sub-genotypes VIId and VIIb, from 2009 to 2012. All NDV isolates of sub-genotype VIIi are approximately 99% identical to each other and are more closely related to Indonesian viruses isolated from 1983 through 1990 than to those of genotype VII, still circulating in the region. Similarly, in addition to the Pakistani NDV isolates of the original genotype XIII (now called sub-genotype XIIIa), there is an additional sub-genotype (XIIIb) that was initially detected in India and Iran

  8. Impact of Human Enterovirus 71 Genotypes in Meningoencephalitis in Iran

    PubMed Central

    Rahimi, Pooneh; Roohandeh, Akram; Sohrabi, Amir; Mostafavi, Ehsan; Bahram Ali, Golnaz

    2015-01-01

    Background: Since the importance of poliovirus has diminished, as a result of its elimination in the majority of countries, non-polioviruses are emerging as causative agents of severe central nervous system (CNS) involvement. Outbreaks of enterovirus 71 (EV71)-associated CNS infections have recently been reported in Asia, Australia, and Europe. Objectives: This is the first study on genotyping of EV71 in children with meningoencephalitis to be carried out in Iran, and it was conducted in order to obtain an improved understanding of the disease burden of this virus, particularly with regard to CNS involvement. Patients and Methods: Viral RNA was extracted from 170 cerebrospinal fluid samples obtained from children aged under 8 years with a primary diagnosis of aseptic meningitis. Specific EV71 PCR was conducted to identify the genotype of the detected EV71 viruses. Results: Human enteroviruses (HEVs) were detected in 89 patients (52.3%). EV71 infection was detected in 19 (21.3%) of the 89 EV71-positive patients, and the C genotype was identified in 15 isolates. Conclusions: The C genotype should be considered as the prevalent EV71 circulating genotype in Iran, particularly in cases of aseptic meningitis. PMID:26865943

  9. Dynamics of Theileria orientalis genotype population in cattle in a year-round grazing system.

    PubMed

    Masatani, Tatsunori; Yoshihara, Shunpei; Matsubara, Atsuko; Gotoh, Takafumi; Takahashi, Hideyuki; Tanaka, Tetsuya; Andoh, Masako; Endo, Yasuyuki; Matsuo, Tomohide

    2016-03-01

    Theirelia orientalis is a tick-borne haemoprotozoan parasite, and infection with this parasite is one of the most important diseases for grazing cattle. Co-infection of cattle with different genotypes of T. orientalis often occurs. In this study, we investigated the temporal dynamics of genotypes in cattle in a year-round grazing system in Japan. Genotype-specific PCR assays to determine major piroplasm surface protein (MPSP) genotypes (types 1 to 5) of T. orientalis were performed by using time-course blood samples collected from grazing cattle and ticks in a pasture. All 20 cattle investigated in this study were infected with T. orientalis. By using genotype-specific PCR, we detected the combination of genotypes of T. orientalis (types 1 to 5) from each cattle. These multiple genotypes of T. orientalis were also confirmed in ticks. Notably, each genotype of T. orientalis in cattle was temporally detected from cattle and more variable genotypes were found in summer. The observed temporal dynamics of the MPSP genotypes of T. orientalis in cattle could be explained by host immunity against the parasites or genetic recombination of parasite in ticks. PMID:27078669

  10. Hepatitis B Virus Genotypes and Precore and Core Mutants in Brazilian Patients

    PubMed Central

    Sitnik, Roberta; Pinho, João Renato Rebello; Bertolini, Dennis Armando; Bernardini, Antonio Plinio; da Silva, Luiz Caetano; Carrilho, Flair José

    2004-01-01

    A method for genotyping hepatitis B virus by partial HBsAg gene sequencing with primers common to all known genotypes was developed. Mutations related to anti-HBs resistance are also detected with this method. Samples from 103 Brazilian patients were analyzed. Precore and core region of these viruses were also sequenced in 101 patients. Genotypes A, B, C, D, and F were found with frequencies of 49.5, 2.9, 13.6, 24.3, and 9.7%, respectively. Genotypes B and C were found only in Asian patients, whereas genotypes A, D, and F were more common in patients without an Asian background. Precore mutants were found in 32 (31.7%) of 101 patients, with a higher frequency in those infected with genotype D (22 of 25 [88.0%]). Analysis of nucleotide 1858 showed presence of thymine in all patients with genotypes B, C, and D and in a few patients with genotypes A (10.0%) and F (30.0%), who showed more frequently the presence of cytosine. This nucleotide was closely related to the presence of precore mutants. Mutations in the basal core promoter were found in 64 of 101 (63.4%) samples. These mutations were more frequent in patients infected with genotype F (90.0%) and less frequent in patients infected with genotype B (33.3%). Deletions in this region were found in two genotype C-infected patients. PMID:15184419

  11. Whole Genome Re-Sequencing and Characterization of Powdery Mildew Disease-Associated Allelic Variation in Melon

    PubMed Central

    Natarajan, Sathishkumar; Kim, Hoy-Taek; Thamilarasan, Senthil Kumar; Veerappan, Karpagam; Park, Jong-In; Nou, Ill-Sup

    2016-01-01

    Powdery mildew is one of the most common fungal diseases in the world. This disease frequently affects melon (Cucumis melo L.) and other Cucurbitaceous family crops in both open field and greenhouse cultivation. One of the goals of genomics is to identify the polymorphic loci responsible for variation in phenotypic traits. In this study, powdery mildew disease assessment scores were calculated for four melon accessions, ‘SCNU1154’, ‘Edisto47’, ‘MR-1’, and ‘PMR5’. To investigate the genetic variation of these accessions, whole genome re-sequencing using the Illumina HiSeq 2000 platform was performed. A total of 754,759,704 quality-filtered reads were generated, with an average of 82.64% coverage relative to the reference genome. Comparisons of the sequences for the melon accessions revealed around 7.4 million single nucleotide polymorphisms (SNPs), 1.9 million InDels, and 182,398 putative structural variations (SVs). Functional enrichment analysis of detected variations classified them into biological process, cellular component and molecular function categories. Further, a disease-associated QTL map was constructed for 390 SNPs and 45 InDels identified as related to defense-response genes. Among them 112 SNPs and 12 InDels were observed in powdery mildew responsive chromosomes. Accordingly, this whole genome re-sequencing study identified SNPs and InDels associated with defense genes that will serve as candidate polymorphisms in the search for sources of resistance against powdery mildew disease and could accelerate marker-assisted breeding in melon. PMID:27311063

  12. Whole Genome Re-Sequencing and Characterization of Powdery Mildew Disease-Associated Allelic Variation in Melon.

    PubMed

    Natarajan, Sathishkumar; Kim, Hoy-Taek; Thamilarasan, Senthil Kumar; Veerappan, Karpagam; Park, Jong-In; Nou, Ill-Sup

    2016-01-01

    Powdery mildew is one of the most common fungal diseases in the world. This disease frequently affects melon (Cucumis melo L.) and other Cucurbitaceous family crops in both open field and greenhouse cultivation. One of the goals of genomics is to identify the polymorphic loci responsible for variation in phenotypic traits. In this study, powdery mildew disease assessment scores were calculated for four melon accessions, 'SCNU1154', 'Edisto47', 'MR-1', and 'PMR5'. To investigate the genetic variation of these accessions, whole genome re-sequencing using the Illumina HiSeq 2000 platform was performed. A total of 754,759,704 quality-filtered reads were generated, with an average of 82.64% coverage relative to the reference genome. Comparisons of the sequences for the melon accessions revealed around 7.4 million single nucleotide polymorphisms (SNPs), 1.9 million InDels, and 182,398 putative structural variations (SVs). Functional enrichment analysis of detected variations classified them into biological process, cellular component and molecular function categories. Further, a disease-associated QTL map was constructed for 390 SNPs and 45 InDels identified as related to defense-response genes. Among them 112 SNPs and 12 InDels were observed in powdery mildew responsive chromosomes. Accordingly, this whole genome re-sequencing study identified SNPs and InDels associated with defense genes that will serve as candidate polymorphisms in the search for sources of resistance against powdery mildew disease and could accelerate marker-assisted breeding in melon. PMID:27311063

  13. In utero transmission and tissue distribution of chronic wasting disease-associated prions in free-ranging Rocky Mountain elk.

    PubMed

    Selariu, Anca; Powers, Jenny G; Nalls, Amy; Brandhuber, Monica; Mayfield, Amber; Fullaway, Stephenie; Wyckoff, Christy A; Goldmann, Wilfred; Zabel, Mark M; Wild, Margaret A; Hoover, Edward A; Mathiason, Candace K

    2015-11-01

    The presence of disease-associated prions in tissues and bodily fluids of chronic wasting disease (CWD)-infected cervids has received much investigation, yet little is known about mother-to-offspring transmission of CWD. Our previous work demonstrated that mother-to-offspring transmission is efficient in an experimental setting. To address the question of relevance in a naturally exposed free-ranging population, we assessed maternal and fetal tissues derived from 19 elk dam-calf pairs collected from free-ranging Rocky Mountain elk from north-central Colorado, a known CWD endemic region. Conventional immunohistochemistry identified three of 19 CWD-positive dams, whereas a more sensitive assay [serial protein misfolding cyclic amplification (sPMCA)] detected CWD prion seeding activity (PrPCWD) in 15 of 19 dams. PrPCWD distribution in tissues was widespread, and included the central nervous system (CNS), lymphoreticular system, and reproductive, secretory, excretory and adipose tissues. Interestingly, five of 15 sPMCA-positive dams showed no evidence of PrPCWD in either CNS or lymphoreticular system, sites typically assessed in diagnosing CWD. Analysis of fetal tissues harvested from the 15 sPMCA-positive dams revealed PrPCWD in 80 % of fetuses (12 of 15), regardless of gestational stage. These findings demonstrated that PrPCWD is more abundant in peripheral tissues of CWD-exposed elk than current diagnostic methods suggest, and that transmission of prions from mother to offspring may contribute to the efficient transmission of CWD in naturally exposed cervid populations. PMID:26358706

  14. Correlated genotypes in friendship networks

    PubMed Central

    Fowler, James H.; Settle, Jaime E.; Christakis, Nicholas A.

    2011-01-01

    It is well known that humans tend to associate with other humans who have similar characteristics, but it is unclear whether this tendency has consequences for the distribution of genotypes in a population. Although geneticists have shown that populations tend to stratify genetically, this process results from geographic sorting or assortative mating, and it is unknown whether genotypes may be correlated as a consequence of nonreproductive associations or other processes. Here, we study six available genotypes from the National Longitudinal Study of Adolescent Health to test for genetic similarity between friends. Maps of the friendship networks show clustering of genotypes and, after we apply strict controls for population stratification, the results show that one genotype is positively correlated (homophily) and one genotype is negatively correlated (heterophily). A replication study in an independent sample from the Framingham Heart Study verifies that DRD2 exhibits significant homophily and that CYP2A6 exhibits significant heterophily. These unique results show that homophily and heterophily obtain on a genetic (indeed, an allelic) level, which has implications for the study of population genetics and social behavior. In particular, the results suggest that association tests should include friends’ genes and that theories of evolution should take into account the fact that humans might, in some sense, be metagenomic with respect to the humans around them. PMID:21245293

  15. Genotypes and phenotypes of Shiga toxin-producing Escherichia coli (STEC) in Abeokuta, Southwestern Nigeria

    PubMed Central

    Olowe, Olugbenga Adekunle; Aboderin, Bukola W; Idris, Olayinka O; Mabayoje, Victor O; Opaleye, Oluyinka O; Adekunle, O Catherine; Olowe, Rita Ayanbolade; Akinduti, Paul Akinniyi; Ojurongbe, Olusola

    2014-01-01

    Purpose To characterize the prevalence of hemolytic Shiga toxin-producing Escherichia coli (STEC) with a multidrug-resistant pattern in different age groups in Abeokuta, Nigeria. Methods Nonrepetitive E. coli isolates were collected from 202 subjects with or without evidence of diarrhea. Each isolate was biochemically identified and antimicrobial susceptibility testing was performed using the disk diffusion method. A sorbitol fermentation test of all the E. coli isolates was done and the minimum inhibitory concentration of suspected STEC was measured by the standard broth microdilution method to determine antibiotic resistance. The genotypes of stx1, stx2, and hlyA were determined by polymerase chain reaction assay. Results The majority of subjects were aged ≥40 years (41.6%) and were female (61.9%). Of the 202 subjects, 86.1% had STEC isolates (P<0.05). A high rate of STEC isolates resistant to amoxicillin (90.6%), cefotaxime (77.7%), and cefuroxime (75.7%) was observed. Resistance to amoxicillin, gentamicin, and cefotaxime was demonstrated with a minimum inhibitory concentration >16 μg/mL in 13.9%, 11.4%, and 10.4% of the isolates, respectively. The prevalence of stx1, stx2, and hlyA was 13.9%, 6.9%, and 2.0%, respectively; 5.5% of stx1 were in the 0–10-year-old age group, 3.5% of stx2 were aged ≥40 and above, and 1.0% of the hlyA isolates were in the 0–10-year-old age group. Conclusion The prevalence of virulent STEC is a public health concern. The use of polymerase chain reaction assay should aid quick detection of this virulent serotype and help curb the severe epidemic of human diseases associated with STEC infections. PMID:25342913

  16. A zoonotic genotype of Enterocytozoon bieneusi in horses.

    PubMed

    Santín, Mónica; Vecino, Jesús A Cortés; Fayer, Ronald

    2010-02-01

    This is the first report of Enterocytozoon bieneusi in an equid species. Feces from 195 horses from 4 locations in Colombia were examined for E. bieneusi by polymerase chain reaction. Of these, 21 horses (10.8%) were found positive for E. bieneusi . The prevalence of E. bieneusi in horses <1 yr of age was significantly higher (23.7%) than in horses >1 yr of age (2.5%). No significant differences in prevalence were observed between male (13.7%) and female horses (9%). Sequencing of the internal transcribed spacer region of the SSUrRNA locus identified 3 genotypes. Two genotypes appear to be unique to horses and were named Horse 1 and Horse 2. A third genotype, identified as genotype D, was detected in 4 horses. This genotype, previously reported to infect humans, beaver, cattle, dogs, falcons, foxes, macaques, muskrats, pigs, and raccoons, is the most ubiquitous of the E. bieneusi zoonotic genotypes. Our findings indicate that E. bieneusi from horses can be a potential source of infection for humans. PMID:19799490

  17. Polymerase chain reaction-based genotype classification among human Blastocystis hominis populations isolated from different countries.

    PubMed

    Yoshikawa, Hisao; Wu, Zhiliang; Kimata, Isao; Iseki, Motohiro; Ali, Ibne Karim M D; Hossain, Momammad B; Zaman, Viqar; Haque, Rashidul; Takahashi, Yuzo

    2004-01-01

    Since the genotype of human Blastocystis hominis isolates is highly polymorphic, PCR-based genotype classification using known sequenced-tagged site (STS) primers would allow the identification or classification of different genotypes. Five populations of human B. hominis isolates obtained from Japan, Pakistan, Bangladesh, Germany, and Thailand were subjected to genotype analysis by using seven kinds of STS primers. Ninety-nine out of 102 isolates were identified as one of the known genotypes, while one isolate from Thailand showed two distinct genotypes and two isolates from Japan were negative with all the STS primers. The most dominant genotype among four populations, except for all four isolates from Thailand, was subtype 3 and it varied from 41.7% to 92.3%. The second most common genotype among four populations was either subtype 1 (7.7-25.0%) or subtype 4 (10.0-22.9%). Subtype 2, subtype 5, and/or subtype 7 were only rarely detected among the isolates from Japan and Germany, while subtype 6 was not detected. The phylogenetic position of the two isolates which were negative with all STS primers, was inferred from the small subunit rRNA (SSU rRNA) genes with the known sequence data of 20 Blastocystis isolates. Since the two isolates were positioned in an additional clade in the phylogenetic tree, this suggested they were a new genotype. These results demonstrated that PCR-based genotype classification is a powerful tool with which to analyse genotypes of Blastocystis isolates obtained from clinical samples. In addition, two groups of the isolates from 15 symptomatic and 11 asymptomatic patients in Bangladesh were compared with the PCR-based subtype classification. Since both groups were only classified into two distinct genotypes of subtype 1 or subtype 3 and no statistically significant difference was observed between the two groups, in this study it could not be shown that the specific genotype correlated with the pathogenic potential of B. hominis. PMID

  18. Newcastle disease: genotypic variation and gross pathology

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There are currently 18 genotypes described for class II Newcastle disease viruses and one genotype for class I. While class I viruses are globally distributed in wild birds, the class II genotypes are found more selectively in different countries. For instance the NDV isolates in genotype V are mo...

  19. Analytical performance evaluation of Anyplex II HPV28 and Euroarray HPV for genotyping of cervical samples.

    PubMed

    Latsuzbaia, Ardashel; Tapp, Jessica; Nguyen, Trung; Fischer, Marc; Arbyn, Marc; Weyers, Steven; Mossong, Joël

    2016-07-01

    Analytically accurate human papillomavirus (HPV) genotyping methods are required to assess the impact of HPV vaccination. The aim of this study was to evaluate the analytical performance of Anyplex II HPV28 (Seegene, Korea) and Euroarray HPV (Euroimmun, Germany) genotyping kits, for conducting a future HPV vaccine efficacy monitoring study in Luxembourg. A total number of 150 cervical swabs were collected from women with mean age 31.4 years. Agreements for detecting any HPV between Aptima/Anyplex (88.0%) and Aptima/Euroarray (90.7%) were similar. Agreement of Anyplex/EuroArray with Aptima was higher for Genotypes 16, 18 or 45 than for the other 11 HPVs. The average number of HPV genotypes detected per sample was similar with 2.6 and 2.5, for Anyplex and EuroArray, respectively. In conclusion, Anyplex and Euroarray showed high agreement in general and in particular for detecting genotypes contained in HPV vaccines. PMID:27156793

  20. Reevaluation of Phosphorylation Sites in the Parkinson Disease-associated Leucine-rich Repeat Kinase 2*

    PubMed Central

    Li, Xiaojie; Moore, Darren J.; Xiong, Yulan; Dawson, Ted M.; Dawson, Valina L.

    2010-01-01

    Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been identified as an important cause of late-onset, autosomal dominant familial Parkinson disease and contribute to sporadic Parkinson disease. LRRK2 is a large complex protein with multiple functional domains, including a Roc-GTPase, protein kinase, and multiple protein-protein interaction domains. Previous studies have suggested an important role for kinase activity in LRRK2-induced neuronal toxicity and inclusion body formation. Disease-associated mutations in LRRK2 also tend to increase kinase activity. Thus, enhanced kinase activity may therefore underlie LRRK2-linked disease. Similar to the closely related mixed-lineage kinases, LRRK2 can undergo autophosphorylation in vitro. Three putative autophosphorylation sites (Thr-2031, Ser-2032, and Thr-2035) have been identified within the activation segment of the LRRK2 kinase domain based on sequence homology to mixed-lineage kinases. Phosphorylation at one or more of these sites is critical for the kinase activity of LRRK2. Sensitive phopho-specific antibodies to each of these three sites have been developed and validated by ELISA, dot-blot, and Western blot analysis. Using these antibodies, we have found that all three putative sites are phosphorylated in LRRK2, and Ser-2032 and Thr-2035 are the two important sites that regulate LRRK2 kinase activity. PMID:20595391

  1. Discovering Disease Associations by Integrating Electronic Clinical Data and Medical Literature

    PubMed Central

    Holmes, Antony B.; Hawson, Alexander; Liu, Feng; Friedman, Carol; Khiabanian, Hossein; Rabadan, Raul

    2011-01-01

    Electronic health record (EHR) systems offer an exceptional opportunity for studying many diseases and their associated medical conditions within a population. The increasing number of clinical record entries that have become available electronically provides access to rich, large sets of patients' longitudinal medical information. By integrating and comparing relations found in the EHRs with those already reported in the literature, we are able to verify existing and to identify rare or novel associations. Of particular interest is the identification of rare disease co-morbidities, where the small numbers of diagnosed patients make robust statistical analysis difficult. Here, we introduce ADAMS, an Application for Discovering Disease Associations using Multiple Sources, which contains various statistical and language processing operations. We apply ADAMS to the New York-Presbyterian Hospital's EHR to combine the information from the relational diagnosis tables and textual discharge summaries with those from PubMed and Wikipedia in order to investigate the co-morbidities of the rare diseases Kaposi sarcoma, toxoplasmosis, and Kawasaki disease. In addition to finding well-known characteristics of diseases, ADAMS can identify rare or previously unreported associations. In particular, we report a statistically significant association between Kawasaki disease and diagnosis of autistic disorder. PMID:21731656

  2. Factors Regulating Immunoglobulin Production by Normal and Disease-Associated Plasma Cells

    PubMed Central

    Jackson, David A.; Elsawa, Sherine F.

    2015-01-01

    Immunoglobulins are molecules produced by activated B cells and plasma cells in response to exposure to antigens. Upon antigen exposure, these molecules are secreted allowing the immune system to recognize and effectively respond to a myriad of pathogens. Immunoglobulin or antibody secreting cells are the mature form of B lymphocytes, which during their development undergo gene rearrangements and selection in the bone marrow ultimately leading to the generation of B cells, each expressing a single antigen-specific receptor/immunoglobulin molecule. Each individual immunoglobulin molecule has an affinity for a unique motif, or epitope, found on a given antigen. When presented with an antigen, activated B cells differentiate into either plasma cells (which secrete large amounts of antibody that is specific for the inducing antigen), or memory B cells (which are long-lived and elicit a stronger and faster response if the host is re-exposed to the same antigen). The secreted form of immunoglobulin, when bound to an antigen, serves as an effector molecule that directs other cells of the immune system to facilitate the neutralization of soluble antigen or the eradication of the antigen-expressing pathogen. This review will focus on the regulation of secreted immunoglobulin by long-lived normal or disease-associated plasma cells. Specifically, the focus will be on signaling and transcriptional events that regulate the development and homeostasis of long-lived immunoglobulin secreting plasma cells. PMID:25615546

  3. Diseases associated with pronounced eosinophilia: a study of 105 dogs in Sweden.

    PubMed

    Lilliehöök, I; Gunnarsson, L; Zakrisson, G; Tvedten, H

    2000-06-01

    Records of 105 dogs with pronounced eosinophilia (>2.2 x 10(9) eosinophils/litre) were evaluated in a retrospective study to determine diseases associated with the abnormality in dogs in Sweden. Inflammatory disease in organs with large epithelial surfaces, such as the gut, lungs or skin, was found in 36 per cent of the dogs. A further one-quarter of the 105 cases were placed in the 'miscellaneous' category, which comprised various diseases found at low frequency. The most well defined diagnosis was pulmonary infiltrates with eosinophils in 12 per cent of the dogs. A further 11 per cent had parasitic disease caused by either sarcoptic mange or nasal mite. No atopic dog was found and rottweilers were over-represented in most disease groups. Pronounced eosinophilia, in many cases transient, seems to be associated with a variety of disorders in dogs. In the present study, rottweilers appeared to be more prone to a high eosinophil response than other breeds. PMID:10879402

  4. Treatment strategies in the left main coronary artery disease associated with acute coronary syndromes.

    PubMed

    Karabulut, Ahmet; Cakmak, Mahmut

    2015-10-01

    Significant left main coronary artery (LMCA) stenosis is not rare and reported 3 to 10% of patients undergoing coronary angiography. Unprotected LMCA intervention is a still clinical challenge and surgery is still going to be a traditional management method in many cardiac centers. With a presentation of drug eluting stent (DES), extensive use of IVUS and skilled operators, number of such interventions increased rapidly which lead to change in recommendation in the guidelines regarding LMCA procedures in the stable angina (Class 2a recommendation for ostial and shaft lesion and class 2b recommendation for distal bifurcation lesion). However, there was not clear consensus about the management of unprotected LMCA lesion associated with acute myocardial infarction (MI) with a LMCA culprit lesion itself or distinct culprit lesion of other major coronary arteries. Surgery could be preferred as an obligatory management strategy even in the high risk patients. With this review, we aimed to demonstrate treatment strategies of LMCA disease associated with acute coronary syndrome, particularly acute myocardial infarction (MI). In addition, we presented a short case series with LMCA lesion and ST elevated acute MI in which culprit lesion placed either in the left anterior descending artery or circumflex artery. We reviewed the current medical literature and propose simple algorithm for management. PMID:26557745

  5. Recombination affects accumulation of damaging and disease-associated mutations in human populations.

    PubMed

    Hussin, Julie G; Hodgkinson, Alan; Idaghdour, Youssef; Grenier, Jean-Christophe; Goulet, Jean-Philippe; Gbeha, Elias; Hip-Ki, Elodie; Awadalla, Philip

    2015-04-01

    Many decades of theory have demonstrated that, in non-recombining systems, slightly deleterious mutations accumulate non-reversibly, potentially driving the extinction of many asexual species. Non-recombining chromosomes in sexual organisms are thought to have degenerated in a similar fashion; however, it is not clear the extent to which damaging mutations accumulate along chromosomes with highly variable rates of crossing over. Using high-coverage sequencing data from over 1,400 individuals in the 1000 Genomes and CARTaGENE projects, we show that recombination rate modulates the distribution of putatively deleterious variants across the entire human genome. Exons in regions of low recombination are significantly enriched for deleterious and disease-associated variants, a signature varying in strength across worldwide human populations with different demographic histories. Regions with low recombination rates are enriched for highly conserved genes with essential cellular functions and show an excess of mutations with demonstrated effects on health, a phenomenon likely affecting disease susceptibility in humans. PMID:25685891

  6. The Neuropsychiatric Disease-Associated Gene cacna1c Mediates Survival of Young Hippocampal Neurons.

    PubMed

    Lee, Anni S; De Jesús-Cortés, Héctor; Kabir, Zeeba D; Knobbe, Whitney; Orr, Madeline; Burgdorf, Caitlin; Huntington, Paula; McDaniel, Latisha; Britt, Jeremiah K; Hoffmann, Franz; Brat, Daniel J; Rajadhyaksha, Anjali M; Pieper, Andrew A

    2016-01-01

    Genetic variations in CACNA1C, which encodes the Cav1.2 subunit of L-type calcium channels (LTCCs), are associated with multiple forms of neuropsychiatric disease that manifest high anxiety in patients. In parallel, mice harboring forebrain-specific conditional knockout of cacna1c (forebrain-Cav1.2 cKO) display unusually high anxiety-like behavior. LTCCs in general, including the Cav1.3 subunit, have been shown to mediate differentiation of neural precursor cells (NPCs). However, it has not previously been determined whether Cav1.2 affects postnatal hippocampal neurogenesis in vivo. Here, we show that forebrain-Cav1.2 cKO mice exhibit enhanced cell death of young hippocampal neurons, with no change in NPC proliferation, hippocampal size, dentate gyrus thickness, or corticosterone levels compared with wild-type littermates. These mice also exhibit deficits in brain levels of brain-derived neurotrophic factor (BDNF), and Cre recombinase-mediated knockdown of adult hippocampal Cav1.2 recapitulates the deficit in young hippocampal neurons survival. Treatment of forebrain-Cav1.2 cKO mice with the neuroprotective agent P7C3-A20 restored the net magnitude of postnatal hippocampal neurogenesis to wild-type levels without ameliorating their deficit in BDNF expression. The role of Cav1.2 in young hippocampal neurons survival may provide new approaches for understanding and treating neuropsychiatric disease associated with aberrations in CACNA1C. Visual Abstract. PMID:27066530

  7. A Computational Framework to Infer Human Disease-Associated Long Noncoding RNAs

    PubMed Central

    Liu, Ming-Xi; Chen, Xing; Chen, Geng; Cui, Qing-Hua; Yan, Gui-Ying

    2014-01-01

    As a major class of noncoding RNAs, long noncoding RNAs (lncRNAs) have been implicated in various critical biological processes. Accumulating researches have linked dysregulations and mutations of lncRNAs to a variety of human disorders and diseases. However, to date, only a few human lncRNAs have been associated with diseases. Therefore, it is very important to develop a computational method to globally predict potential associated diseases for human lncRNAs. In this paper, we developed a computational framework to accomplish this by combining human lncRNA expression profiles, gene expression profiles, and human disease-associated gene data. Applying this framework to available human long intergenic noncoding RNAs (lincRNAs) expression data, we showed that the framework has reliable accuracy. As a result, for non-tissue-specific lincRNAs, the AUC of our algorithm is 0.7645, and the prediction accuracy is about 89%. This study will be helpful for identifying novel lncRNAs for human diseases, which will help in understanding the roles of lncRNAs in human diseases and facilitate treatment. The corresponding codes for our method and the predicted results are all available at http://asdcd.amss.ac.cn/MingXiLiu/lncRNA-disease.html. PMID:24392133

  8. Diseases associated with spontaneous feline leukemia virus (FeLV) infection in cats.

    PubMed

    Reinacher, M

    1989-05-01

    More than 2000 cats sent for necropsy in order to provide a diagnosis were investigated immunohistologically using paraffin sections for the presence of a persistent infection with feline leukemia virus (FeLV). The spectrum of neoplastic and non-neoplastic diseases associated significantly with FeLV infection was determined statistically. Three-quarters of the cats with persistent FeLV infections died of non-neoplastic diseases and about 23% died of tumors, nearly exclusively those of the leukemia/lymphoma disease complex. A strong association with liver degeneration, icterus and a FeLV-associated enteritis was found in addition to the known association with non-neoplastic diseases and conditions such as anemia, bacterial secondary infections and respiratory tract inflammations due to the immunosuppressive effect of FeLV, hemorrhages and feline infectious peritonitis. Surprisingly, diseases and conditions like feline infectious panleukopenia, enteritis (of other types than FeLV-associated enteritis and feline infectious panleukopenia), glomerulonephritis, uremia and hemorrhagic cystitis were not associated with persistent FeLV infection. Another unexpected finding was that most pathogenic infectious agents demonstrated in the cats were not FeLV-associated either. Thus, immunosuppression due to FeLV infection seems to make the animals susceptible to certain pathogenic infectious agents, but not to the majority. PMID:2549696

  9. The Neuropsychiatric Disease-Associated Gene cacna1c Mediates Survival of Young Hippocampal Neurons123

    PubMed Central

    Lee, Anni S.; Kabir, Zeeba D.; Knobbe, Whitney; Orr, Madeline; Burgdorf, Caitlin; Huntington, Paula; McDaniel, Latisha; Britt, Jeremiah K.; Hoffmann, Franz; Brat, Daniel J.; Rajadhyaksha, Anjali M.

    2016-01-01

    Genetic variations in CACNA1C, which encodes the Cav1.2 subunit of L-type calcium channels (LTCCs), are associated with multiple forms of neuropsychiatric disease that manifest high anxiety in patients. In parallel, mice harboring forebrain-specific conditional knockout of cacna1c (forebrain-Cav1.2 cKO) display unusually high anxiety-like behavior. LTCCs in general, including the Cav1.3 subunit, have been shown to mediate differentiation of neural precursor cells (NPCs). However, it has not previously been determined whether Cav1.2 affects postnatal hippocampal neurogenesis in vivo. Here, we show that forebrain-Cav1.2 cKO mice exhibit enhanced cell death of young hippocampal neurons, with no change in NPC proliferation, hippocampal size, dentate gyrus thickness, or corticosterone levels compared with wild-type littermates. These mice also exhibit deficits in brain levels of brain-derived neurotrophic factor (BDNF), and Cre recombinase-mediated knockdown of adult hippocampal Cav1.2 recapitulates the deficit in young hippocampal neurons survival. Treatment of forebrain-Cav1.2 cKO mice with the neuroprotective agent P7C3-A20 restored the net magnitude of postnatal hippocampal neurogenesis to wild-type levels without ameliorating their deficit in BDNF expression. The role of Cav1.2 in young hippocampal neurons survival may provide new approaches for understanding and treating neuropsychiatric disease associated with aberrations in CACNA1C. Visual Abstract PMID:27066530

  10. DDA: A Novel Network-Based Scoring Method to Identify Disease–Disease Associations

    PubMed Central

    Suratanee, Apichat; Plaimas, Kitiporn

    2015-01-01

    Categorizing human diseases provides higher efficiency and accuracy for disease diagnosis, prognosis, and treatment. Disease–disease association (DDA) is a precious information that indicates the large-scale structure of complex relationships of diseases. However, the number of known and reliable associations is very small. Therefore, identification of DDAs is a challenging task in systems biology and medicine. Here, we developed a novel network-based scoring algorithm called DDA to identify the relationships between diseases in a large-scale study. Our method is developed based on a random walk prioritization in a protein–protein interaction network. This approach considers not only whether two diseases directly share associated genes but also the statistical relationships between two different diseases using known disease-related genes. Predicted associations were validated by known DDAs from a database and literature supports. The method yielded a good performance with an area under the curve of 71% and outperformed other standard association indices. Furthermore, novel DDAs and relationships among diseases from the clusters analysis were reported. This method is efficient to identify disease–disease relationships on an interaction network and can also be generalized to other association studies to further enhance knowledge in medical studies. PMID:26673408

  11. Coral transplantation triggers shift in microbiome and promotion of coral disease associated potential pathogens

    PubMed Central

    Casey, Jordan M.; Connolly, Sean R.; Ainsworth, Tracy D.

    2015-01-01

    By cultivating turf algae and aggressively defending their territories, territorial damselfishes in the genus Stegastes play a major role in shaping coral-algal dynamics on coral reefs. The epilithic algal matrix (EAM) inside Stegastes’ territories is known to harbor high abundances of potential coral disease pathogens. To determine the impact of territorial grazers on coral microbial assemblages, we established a coral transplant inside and outside of Stegastes’ territories. Over the course of one year, the percent mortality of transplanted corals was monitored and coral samples were collected for microbial analysis. As compared to outside damselfish territories, Stegastes were associated with a higher rate of mortality of transplanted corals. However, 16S rDNA sequencing revealed that territorial grazers do not differentially impact the microbial assemblage of corals exposed to the EAM. Regardless of Stegastes presence or absence, coral transplantation resulted in a shift in the coral-associated microbial community and an increase in coral disease associated potential pathogens. Further, transplanted corals that suffer low to high mortality undergo a microbial transition from a microbiome similar to that of healthy corals to that resembling the EAM. These findings demonstrate that coral transplantation significantly impacts coral microbial communities, and transplantation may increase susceptibility to coral disease. PMID:26144865

  12. Ribosome profiling reveals features of normal and disease-associated mitochondrial translation

    NASA Astrophysics Data System (ADS)

    Rooijers, Koos; Loayza-Puch, Fabricio; Nijtmans, Leo G.; Agami, Reuven

    2013-12-01

    Mitochondria are essential cellular organelles for generation of energy and their dysfunction may cause diabetes, Parkinson’s disease and multi-systemic failure marked by failure to thrive, gastrointestinal problems, lactic acidosis and early lethality. Disease-associated mitochondrial mutations often affect components of the mitochondrial translation machinery. Here we perform ribosome profiling to measure mitochondrial translation at nucleotide resolution. Using a protocol optimized for the retrieval of mitochondrial ribosome protected fragments (RPFs) we show that the size distribution of wild-type mitochondrial RPFs follows a bimodal distribution peaking at 27 and 33 nucleotides, which is distinct from the 30-nucleotide peak of nuclear RPFs. Their cross-correlation suggests generation of mitochondrial RPFs during ribosome progression. In contrast, RPFs from patient-derived mitochondria mutated in tRNA-Tryptophan are centered on tryptophan codons and reduced downstream, indicating ribosome stalling. Intriguingly, long RPFs are enriched in mutated mitochondria, suggesting they characterize stalled ribosomes. Our findings provide the first model for translation in wild-type and disease-triggering mitochondria.

  13. Multifocal-motor-neuropathy-like disease associated with Infliximab treatment in a patient with Crohn's disease.

    PubMed

    Fernández-Menéndez, S; González Nafría, N; Redondo-Robles, L; Sierra-Ausín, M; García-Santiago, R; Saponaro-González, A

    2015-02-15

    Multifocal motor neuropathy is an immune-mediated disorder characterized by motor-conduction block in nerve-conduction studies. It is recognized that anti-TNF-α therapies are associated with immune-mediated conditions as adverse events. We report a case of multifocal-motor-neuropathy-like disease associated with the use of Infliximab in a patient with Crohn's disease. The diagnosis was based on neurophysiological evaluation and complete screening tests. Clinical and laboratory findings were not compatible with other potential causes. There was a mild response to the IVIg treatment, and once Infliximab treatment was withdrawn, the patient made slow but substantial progress in his motor function, with partial improvement of motor conduction blocks in the last neurophysiological evaluation. We believe there is a causal relationship between anti-TNF-α treatment and the disorder in this patient. There are few well-documented reports of this association. To our knowledge, our case is the first occurring in a patient with Crohn's disease. PMID:25592414

  14. Disease-association analysis of an inflammation-related feedback loop.

    PubMed

    Murakami, Masaaki; Harada, Masaya; Kamimura, Daisuke; Ogura, Hideki; Okuyama, Yuko; Kumai, Noriko; Okuyama, Azusa; Singh, Rajeev; Jiang, Jing-Jing; Atsumi, Toru; Shiraya, Sayaka; Nakatsuji, Yuji; Kinoshita, Makoto; Kohsaka, Hitoshi; Nishida, Makoto; Sakoda, Saburo; Miyasaka, Nobuyuki; Yamauchi-Takihara, Keiko; Yamaguchi-Takihara, Keiko; Hirano, Toshio

    2013-03-28

    The IL-6-triggered positive feedback loop for NFκB signaling (or the IL-6 amplifier/Inflammation amplifier) was originally discovered as a synergistic-activation signal that follows IL-17/IL-6 stimulation in nonimmune cells. Subsequent results from animal models have shown that the amplifier is activated by stimulation of NFκB and STAT3 and induces chemokines and inflammation via an NFκB loop. However, its role in human diseases is unclear. Here, we combined two genome-wide mouse screens with SNP-based disease association studies, revealing 1,700 genes related to the IL-6 amplifier, 202 of which showed 492 indications of association with ailments beyond autoimmune diseases. We followed up on ErbB1 from our list. Blocking ErbB1 signaling suppressed the IL-6 amplifier, whereas the expression of epiregulin, an ErbB1 ligand, was higher in patients with inflammatory diseases. These results indicate that the IL-6 amplifier is indeed associated with human diseases and disorders and that the identified genes may make for potential therapeutic targets. PMID:23434511

  15. Curcumin and genistein: the combined effects on disease-associated CFTR mutants and their clinical implications.

    PubMed

    Sohma, Yoshiro; Yu, Ying-Chun; Hwang, Tzyh-Chang

    2013-01-01

    Genistein and curcumin are major components of Asian foods, soybean and curry turmeric respectively. These compounds have been intensively investigated for their chemical and biological features conferring their anti-cancer activity. Genistein and curcumin have also been investigated for their potentiation effects on disease-associated CFTR mutants such as ΔF508 and G551D. Recently, we investigated the combined effect of genistein and curcumin on G551D-CFTR, which exhibits gating defects without abnormalities in protein synthesis or trafficking using the patch-clamp technique. We found that genistein and curcumin showed additive effects on their potentiation of G551D-CFTR in high concentration range and also, more importantly, showed a significant synergistic effect in their minimum concentration ranges. These results are consistent with the idea that multiple mechanisms are involved in the action of these CFTR potentiators. In this review, we revisit the pharmacology of genistein and curcumin on CFTR and also propose new pharmaceutical implications of combined use of these compounds in the development of drugs for CF pharmacotherapy. PMID:23331029

  16. Curcumin and Genistein: the Combined Effects on Disease-associated CFTR Mutants and their Clinical Implications

    PubMed Central

    Sohma, Yoshiro; Yu, Ying-Chun; Hwang, Tzyh-Chang

    2016-01-01

    Genistein and curcumin are major components of Asian foods, soybean and curry turmeric respectively. These compounds have been intensively investigated for their chemical and biological features conferring their anti-cancer activity. Genistein and curcumin have also been investigated for their potentiation effects on disease-associated CFTR mutants such as ΔF508 and G551D. Recently, we investigated the combined effect of genistein and curcumin on G551D-CFTR, which exhibits gating defects without abnormalities in protein synthesis or trafficking using the patch-clamp technique. We found that genistein and curcumin showed additive effects on their potentiation of G551D-CFTR in high concentration range and also, more importantly, showed a significant synergistic effect in their minimum concentration ranges. These results are consistent with the idea that multiple mechanisms are involved in the action of these CFTR potentiators. In this review, we revisit the pharmacology of genistein and curcumin on CFTR and also propose new pharmaceutical implications of combined use of these compounds in the development of drugs for CF pharmacotherapy. PMID:23331029

  17. Effective diagnosis of genetic disease by computational phenotype analysis of the disease-associated genome

    PubMed Central

    Zemojtel, Tomasz; Köhler, Sebastian; Mackenroth, Luisa; Jäger, Marten; Hecht, Jochen; Krawitz, Peter; Graul-Neumann, Luitgard; Doelken, Sandra; Ehmke, Nadja; Spielmann, Malte; Øien, Nancy Christine; Schweiger, Michal R.; Krüger, Ulrike; Frommer, Götz; Fischer, Björn; Kornak, Uwe; Flöttmann, Ricarda; Ardeshirdavani, Amin; Moreau, Yves; Lewis, Suzanna E.; Haendel, Melissa; Smedley, Damian; Horn, Denise; Mundlos, Stefan; Robinson, Peter N.

    2015-01-01

    Less than half of patients with suspected genetic disease receive a molecular diagnosis. We have therefore integrated next-generation sequencing (NGS), bioinformatics, and clinical data into an effective diagnostic workflow. We used variants in the 2741 established Mendelian disease genes [the disease-associated genome (DAG)] to develop a targeted enrichment DAG panel (7.1 Mb), which achieves a coverage of 20-fold or better for 98% of bases. Furthermore, we established a computational method [Phenotypic Interpretation of eXomes (PhenIX)] that evaluated and ranked variants based on pathogenicity and semantic similarity of patients’ phenotype described by Human Phenotype Ontology (HPO) terms to those of 3991 Mendelian diseases. In computer simulations, ranking genes based on the variant score put the true gene in first place less than 5% of the time; PhenIX placed the correct gene in first place more than 86% of the time. In a retrospective test of PhenIX on 52 patients with previously identified mutations and known diagnoses, the correct gene achieved a mean rank of 2.1. In a prospective study on 40 individuals without a diagnosis, PhenIX analysis enabled a diagnosis in 11 cases (28%, at a mean rank of 2.4). Thus, the NGS of the DAG followed by phenotype-driven bioinformatic analysis allows quick and effective differential diagnostics in medical genetics. PMID:25186178

  18. Coral transplantation triggers shift in microbiome and promotion of coral disease associated potential pathogens.

    PubMed

    Casey, Jordan M; Connolly, Sean R; Ainsworth, Tracy D

    2015-01-01

    By cultivating turf algae and aggressively defending their territories, territorial damselfishes in the genus Stegastes play a major role in shaping coral-algal dynamics on coral reefs. The epilithic algal matrix (EAM) inside Stegastes' territories is known to harbor high abundances of potential coral disease pathogens. To determine the impact of territorial grazers on coral microbial assemblages, we established a coral transplant inside and outside of Stegastes' territories. Over the course of one year, the percent mortality of transplanted corals was monitored and coral samples were collected for microbial analysis. As compared to outside damselfish territories, Stegastes were associated with a higher rate of mortality of transplanted corals. However, 16S rDNA sequencing revealed that territorial grazers do not differentially impact the microbial assemblage of corals exposed to the EAM. Regardless of Stegastes presence or absence, coral transplantation resulted in a shift in the coral-associated microbial community and an increase in coral disease associated potential pathogens. Further, transplanted corals that suffer low to high mortality undergo a microbial transition from a microbiome similar to that of healthy corals to that resembling the EAM. These findings demonstrate that coral transplantation significantly impacts coral microbial communities, and transplantation may increase susceptibility to coral disease. PMID:26144865

  19. Overexpression of caveolin-1 is sufficient to phenocopy the behavior of a disease-associated mutant

    PubMed Central

    Hanson, Caroline A.; Drake, Kimberly R.; Baird, Michelle A.; Han, Bing; Kraft, Lewis J.; Davidson, Michael W.; Kenworthy, Anne K.

    2013-01-01

    Mutations and alterations in caveolin-1 expression levels have been linked to a number of human diseases. How misregulation of caveolin-1 contributes to disease is not fully understood, but has been proposed to involve the intracellular accumulation of mutant forms of the protein. To better understand the molecular basis for trafficking defects that trap caveolin-1 intracellularly, we compared the properties of a GFP-tagged version of caveolin-1 P132L, a mutant form of caveolin-1 previously linked to breast cancer, with wild type caveolin-1. Unexpectedly, wild type caveolin-1-GFP also accumulated intracellularly, leading us to examine the mechanisms underlying the abnormal localization of the wild type and mutant protein in more detail. We show that both the nature of the tag and cellular context impact the subcellular distribution of caveolin-1, demonstrate that even the wild type form of caveolin-1 can function as a dominant negative under some conditions, and identify specific conformation changes associated with incorrectly targeted forms of the protein. In addition, we find intracellular caveolin-1 is phosphorylated on Tyr14, but phosphorylation is not required for mistrafficking of the protein. These findings identify novel properties of mistargeted forms of caveolin-1 and raise the possibility that common trafficking defects underlie diseases associated with overexpression and mutations in caveolin-1. PMID:23469926

  20. Super-enhancers delineate disease-associated regulatory nodes in T cells.

    PubMed

    Vahedi, Golnaz; Kanno, Yuka; Furumoto, Yasuko; Jiang, Kan; Parker, Stephen C J; Erdos, Michael R; Davis, Sean R; Roychoudhuri, Rahul; Restifo, Nicholas P; Gadina, Massimo; Tang, Zhonghui; Ruan, Yijun; Collins, Francis S; Sartorelli, Vittorio; O'Shea, John J

    2015-04-23

    Enhancers regulate spatiotemporal gene expression and impart cell-specific transcriptional outputs that drive cell identity. Super-enhancers (SEs), also known as stretch-enhancers, are a subset of enhancers especially important for genes associated with cell identity and genetic risk of disease. CD4(+) T cells are critical for host defence and autoimmunity. Here we analysed maps of mouse T-cell SEs as a non-biased means of identifying key regulatory nodes involved in cell specification. We found that cytokines and cytokine receptors were the dominant class of genes exhibiting SE architecture in T cells. Nonetheless, the locus encoding Bach2, a key negative regulator of effector differentiation, emerged as the most prominent T-cell SE, revealing a network in which SE-associated genes critical for T-cell biology are repressed by BACH2. Disease-associated single-nucleotide polymorphisms for immune-mediated disorders, including rheumatoid arthritis, were highly enriched for T-cell SEs versus typical enhancers or SEs in other cell lineages. Intriguingly, treatment of T cells with the Janus kinase (JAK) inhibitor tofacitinib disproportionately altered the expression of rheumatoid arthritis risk genes with SE structures. Together, these results indicate that genes with SE architecture in T cells encompass a variety of cytokines and cytokine receptors but are controlled by a 'guardian' transcription factor, itself endowed with an SE. Thus, enumeration of SEs allows the unbiased determination of key regulatory nodes in T cells, which are preferentially modulated by pharmacological intervention. PMID:25686607

  1. Systematic identification of trans-eQTLs as putative drivers of known disease associations

    PubMed Central

    Fairfax, Benjamin P.; Schramm, Katharina; Powell, Joseph E.; Zhernakova, Alexandra; Zhernakova, Daria V; Veldink, Jan H.; Van den Berg, Leonard H.; Karjalainen, Juha; Withoff, Sebo; Uitterlinden, André G.; Hofman, Albert; Rivadeneira, Fernando; Hoen, Peter A C 't; Reinmaa, Eva; Fischer, Krista; Nelis, Mari; Milani, Lili; Melzer, David; Ferrucci, Luigi; Singleton, Andrew B.; Hernandez, Dena G.; Nalls, Michael A.; Homuth, Georg; Nauck, Matthias; Radke, Dörte; Völker, Uwe; Perola, Markus; Salomaa, Veikko; Brody, Jennifer; Suchy-Dicey, Astrid; Gharib, Sina A.; Enquobahrie, Daniel A.; Lumley, Thomas; Montgomery, Grant W.; Makino, Seiko; Prokisch, Holger; Herder, Christian; Roden, Michael; Grallert, Harald; Meitinger, Thomas; Strauch, Konstantin; Li, Yang; Jansen, Ritsert C.; Visscher, Peter M.; Knight, Julian C.

    2014-01-01

    Identifying the downstream effects of disease-associated single nucleotide polymorphisms (SNPs) is challenging: the causal gene is often unknown or it is unclear how the SNP affects the causal gene, making it difficult to design experiments that reveal functional consequences. To help overcome this problem, we performed the largest expression quantitative trait locus (eQTL) meta-analysis so far reported in non-transformed peripheral blood samples of 5,311 individuals, with replication in 2,775 individuals. We identified and replicated trans-eQTLs for 233 SNPs (reflecting 103 independent loci) that were previously associated with complex traits at genome-wide significance. Although we did not study specific patient cohorts, we identified trait-associated SNPs that affect multiple trans-genes that are known to be markedly altered in patients: for example, systemic lupus erythematosus (SLE) SNP rs49170141 altered C1QB and five type 1 interferon response genes, both hallmarks of SLE2-4. Subsequent ChIP-seq data analysis on these trans-genes implicated transcription factor IKZF1 as the causal gene at this locus, with DeepSAGE RNA-sequencing revealing that rs4917014 strongly alters 3’ UTR levels of IKZF1. Variants associated with cholesterol metabolism and type 1 diabetes showed similar phenomena, indicating that large-scale eQTL mapping provides insight into the downstream effects of many trait-associated variants. PMID:24013639

  2. Acquired cystic disease-associated renal cell carcinoma: further characterization of the morphologic and immunopathologic features.

    PubMed

    Ahn, Soomin; Kwon, Ghee Young; Cho, Yong Mee; Jun, Sun-Young; Choi, Chan; Kim, Hyun-Jung; Park, Yong Wook; Park, Weon Seo; Shim, Jung Won

    2013-12-01

    Acquired cystic disease-associated renal cell carcinoma (ACD-RCC) is a subtype of renal cell carcinoma (RCC) with unique morphologic features found exclusively in the background of end-stage renal disease. We analyzed the clinicopathologic features and immumoreactive profiles of 12 cases of ACD-RCC to further characterize this recently recognized entity. Review of histologic slides was performed in conjunction with immunohistochemical staining directed to the contemporary diagnostic antibodies and the putative target therapy-related markers. Histologically, the tumors showed characteristic inter-or intracellular microlumens and eosinophilic tumor cells. Intratumoral hemosiderin deposition and degenerating foamy tumor cells were consistent findings which were not previously described. Immunohistochemically, all the tumors were positive for alpha-methylacyl-CoA-racemase, CD10, pan-cytokeratin, PTEN (phosphatase and tensin homolog deleted on chromosome 10) and c-met, while negative for carbonic anhydrase-9, CD57, CD68, c-kit, pax-2, platelet-derived growth factor receptor (PDGFR)-α or vascular endothelial growth factor receptor (VEGFR)-2. Heterogenous staining was found for CK7 and kidney-specific cadherin. Positive reaction to c-met suggests its utility as a plausible therapeutic target in ACD-RCC. Thus, we present the unique morphologic and immunopathologic features of ACD-RCC, which may be helpful in both diagnostic and therapeutic aspects. PMID:23471757

  3. Dengue virus 3 genotype I in Aedes aegypti mosquitoes and eggs, Brazil, 2005-2006.

    PubMed

    Vilela, Ana P P; Figueiredo, Leandra B; dos Santos, João R; Eiras, Alvaro E; Bonjardim, Cláudio A; Ferreira, Paulo C P; Kroon, Erna G

    2010-06-01

    Dengue virus type 3 genotype I was detected in Brazil during epidemics in 2002-2004. To confirm this finding, we identified this virus genotype in naturally infected field-caught Aedes aegypti mosquitoes and eggs. Results showed usefulness of virus investigations in vectors as a component of active epidemiologic surveillance. PMID:20507754

  4. Dengue Virus 3 Genotype I in Aedes aegypti Mosquitoes and Eggs, Brazil, 2005–2006

    PubMed Central

    Vilela, Ana P.P.; Figueiredo, Leandra B.; dos Santos, João R.; Eiras, Álvaro E.; Bonjardim, Cláudio A.; Ferreira, Paulo C.P.

    2010-01-01

    Dengue virus type 3 genotype I was detected in Brazil during epidemics in 2002–2004. To confirm this finding, we identified this virus genotype in naturally infected field-caught Aedes aegypti mosquitoes and eggs. Results showed usefulness of virus investigations in vectors as a component of active epidemiologic surveillance. PMID:20507754

  5. [Genotyping of Giardia intestinalis isolates in the Thrace Region, Turkey].

    PubMed

    Çiçek, Cemal; Şakru, Nermin

    2015-10-01

    Giardia intestinalis is a common protozoon that infects humans and may cause water and food-borne outbreaks. It is regarded as a major public health problem worldwide and in Turkey as well. Molecular techniques are widely used to determine the epidemiology, genetic populations and taxonomy of G.intestinalis. It has two genotypes including genotype A and genotype B in humans. The purpose of the present study is to implement the molecular analysis and genotyping of the isolates of G.intestinalis obtained from human stool samples. A total of 39 isolates obtained from the stool samples of persons (30 male, 9 female; age range: 1-74 years, median age: 20) who have admitted to Trakya University Medical Research and Practice Health Center and Edirne State Hospital between September 2011- April 2013 were included in the study. The average number of cysts were identified both with native and lugol methods among all microscopically detected samples by screening at least 50 field with x400 magnification. The samples were then analyzed through loop-mediated isothermal amplification method (LAMP) for the presence of elongation factor-1 alpha (EF-1α) gene, and polymerase chain reaction (PCR) method for the presence of beta-giardin (bg) gene regions. In addition, sequence analysis of bg gene was performed. Of 39 samples, 32 (82%) and 19 (48.7%) were found to be positive for G.intestinalis EF-1α and bg genes by LAMP and PCR methods, respectively. Genotyping was implemented in 17 out of 19 samples yielding nine genotype A and eight genotype B strains. The sub-genotypes of these strains were identified as A2 (n= 6), A3 (n= 3), B2 (n= 6), B3 (n= 1) and B4 (n= 1). In eight isolates that could be typed among 21 symptomatic patients, genotype B (n= 5) and in nine isolates that could be typed among 18 asymptomatic patients, genotype A (n= 6) were more frequently observed. There was no significant association between symptomatic or asymptomatic status and genotypic patterns of the cases

  6. New Hepatitis E Virus Genotype in Camels, the Middle East

    PubMed Central

    Lau, Susanna K.P.; Teng, Jade L.L.; Tsang, Alan K. L.; Joseph, Marina; Wong, Emily Y.M.; Tang, Ying; Sivakumar, Saritha; Xie, Jun; Bai, Ru; Wernery, Renate; Wernery, Ulrich; Yuen, Kwok-Yung

    2014-01-01

    In a molecular epidemiology study of hepatitis E virus (HEV) in dromedaries in Dubai, United Arab Emirates, HEV was detected in fecal samples from 3 camels. Complete genome sequencing of 2 strains showed >20% overall nucleotide difference to known HEVs. Comparative genomic and phylogenetic analyses revealed a previously unrecognized HEV genotype. PMID:24856611

  7. Hepatitis E Virus Genotype 4 Outbreak, Italy, 2011

    PubMed Central

    Garbuglia, Anna R.; Scognamiglio, Paola; Petrosillo, Nicola; Mastroianni, Claudio Maria; Sordillo, Pasquale; Gentile, Daniele; La Scala, Patrizia; Girardi, Enrico

    2013-01-01

    During 2011, 5 persons in the area of Lazio, Italy were infected with a monophyletic strain of hepatitis E virus that showed high sequence homology with isolates from swine in China. Detection of this genotype in Italy parallels findings in other countries in Europe, signaling the possible spread of strains new to Western countries. PMID:23260079

  8. Hepatitis E Virus Genotype 3 in Humans and Swine, Bolivia

    PubMed Central

    Cavallo, Annalisa; Gonzales, José Luis; Bonelli, Sara Irene; Valda, Ybar; Pieri, Angela; Segundo, Higinio; Ibañez, Ramón; Mantella, Antonia; Bartalesi, Filippo; Tolari, Francesco; Bartoloni, Alessandro

    2011-01-01

    We determined the seroprevalence of hepatitis E virus (HEV) in persons in 2 rural communities in southeastern Bolivia and the presence of HEV in human and swine fecal samples. HEV seroprevalence was 6.3%, and HEV genotype 3 strains with high sequence homology were detected. PMID:21801630

  9. Evaluation of cotton genotypes for ginning energy and ginning rate

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Reducing ginning energy use through cultivar improvement could reduce ginning and energy cost. The objective of this study was to detect genetic variability for ginning energy and ginning rate. Thirty four conventional and twelve transgenic genotypes were evaluated in 2008 and 2009 for ginning energ...

  10. A Pleiotropy-Informed Bayesian False Discovery Rate Adapted to a Shared Control Design Finds New Disease Associations From GWAS Summary Statistics

    PubMed Central

    Liley, James; Wallace, Chris

    2015-01-01

    Genome-wide association studies (GWAS) have been successful in identifying single nucleotide polymorphisms (SNPs) associated with many traits and diseases. However, at existing sample sizes, these variants explain only part of the estimated heritability. Leverage of GWAS results from related phenotypes may improve detection without the need for larger datasets. The Bayesian conditional false discovery rate (cFDR) constitutes an upper bound on the expected false discovery rate (FDR) across a set of SNPs whose p values for two diseases are both less than two disease-specific thresholds. Calculation of the cFDR requires only summary statistics and have several advantages over traditional GWAS analysis. However, existing methods require distinct control samples between studies. Here, we extend the technique to allow for some or all controls to be shared, increasing applicability. Several different SNP sets can be defined with the same cFDR value, and we show that the expected FDR across the union of these sets may exceed expected FDR in any single set. We describe a procedure to establish an upper bound for the expected FDR among the union of such sets of SNPs. We apply our technique to pairwise analysis of p values from ten autoimmune diseases with variable sharing of controls, enabling discovery of 59 SNP-disease associations which do not reach GWAS significance after genomic control in individual datasets. Most of the SNPs we highlight have previously been confirmed using replication studies or larger GWAS, a useful validation of our technique; we report eight SNP-disease associations across five diseases not previously declared. Our technique extends and strengthens the previous algorithm, and establishes robust limits on the expected FDR. This approach can improve SNP detection in GWAS, and give insight into shared aetiology between phenotypically related conditions. PMID:25658688

  11. Improved detection of disease-associated variation by sex-specific characterization and prediction of genes required for fertility

    PubMed Central

    Ho, Nicholas Rui Yuan; Huang, Ni; Conrad, Donald F.

    2016-01-01

    Despite its great potential, high-throughput functional genomic data is rarely integrated and applied to characterizing the genomic basis of fertility. We obtained and reprocessed over 30 functional genomics datasets from human and mouse germ cells to perform genomewide prediction of genes underlying various reproductive phenotypes in both species. Genes involved in male fertility are easier to predict than their female analogs. Of the multiple genomic data types examined, protein-protein interactions are by far the most informative for gene prediction, followed by gene expression, and then epigenetic marks. As an application of our predictions, we show that CNVs disrupting predicted fertility genes are more strongly associated with gonadal dysfunction in male and female case-control cohorts when compared to all gene-disrupting CNVs (OR=1.64, p< 1.64 × 10−8 versus OR=1.25, p < 4 × 10−6). Using gender-specific fertility gene annotations further increased the observed associations (OR = 2.31, p< 2.2 × 10−16). We provide our gene predictions as a resource with this paper. PMID:26473511

  12. Atypical Toxoplasma gondii genotype in feral cats from the Fernando de Noronha Island, northeastern Brazil.

    PubMed

    Melo, R P B; Almeida, J C; Lima, D C V; Pedrosa, C M; Magalhães, F J R; Alcântara, A M; Barros, L D; Vieira, R F C; Garcia, J L; Mota, R A

    2016-07-15

    Toxoplasma gondii isolates from Brazil have a different phenotypic and genotypic pattern, with predominance of virulent isolates and recombinant genotypes, compared to the North Hemisphere. Considering that a new T. gondii genotype, non-pathogenic to mice, was previously identified from free-range chickens from the Fernando de Noronha Island, Brazil, this study aimed to identify genotypes of this parasite in tissue samples of feral cats (Felis catus) from this Brazilian Island. Anti-T. gondii IgG antibodies were detected in 18/31 (58%) feral cats. Two non-virulent T. gondii isolates were obtained by mouse bioassay. Genotyping was performed by PCR-RFLP using 10 genetic markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, PK1, L358 and Apico) and an atypical strain of T. gondii (ToxoDB #146) was identified. This is the first report of this genotype in feral cats. PMID:27270396

  13. Campylobacter coli pulsed field gel electrophoresis genotypic diversity among sows and piglets in a farrowing barn.

    PubMed

    Hume, Michael E; Droleskey, Robert E; Sheffield, Cynthia L; Harvey, Roger B

    2002-08-01

    Genotypes of Campylobacter coli isolates from feces of three sows and rectal swabs of 17 piglets were examined by pulsed field gel electrophoresis (PFGE). All of the animals originated from a single farrowing barn of a farrow-to-finish swine operation. Five Campylobacter colonies were picked from a single agar plate for each sample after broth enrichment and growth on Campy-Cefex agar. Genotypes were examined by PFGE after genomic DNA digestion with SmaI and SacII restriction endonucleases. Twenty SmaI genotypes and 12 SacII genotypes were detected among 99 Campylobacter coli isolates. There was no pattern of shared genotypes between sows and their respective piglets, nor between littermates. Results indicate that a high number of Campylobacter genotypes may coexist in related pigs from a single housing facility. PMID:12070692

  14. Constructing lncRNA functional similarity network based on lncRNA-disease associations and disease semantic similarity.

    PubMed

    Chen, Xing; Yan, Chenggang Clarence; Luo, Cai; Ji, Wen; Zhang, Yongdong; Dai, Qionghai

    2015-01-01

    Increasing evidence has indicated that plenty of lncRNAs play important roles in many critical biological processes. Developing powerful computational models to construct lncRNA functional similarity network based on heterogeneous biological datasets is one of the most important and popular topics in the fields of both lncRNAs and complex diseases. Functional similarity network construction could benefit the model development for both lncRNA function inference and lncRNA-disease association identification. However, little effort has been attempted to analysis and calculate lncRNA functional similarity on a large scale. In this study, based on the assumption that functionally similar lncRNAs tend to be associated with similar diseases, we developed two novel lncRNA functional similarity calculation models (LNCSIM). LNCSIM was evaluated by introducing similarity scores into the model of Laplacian Regularized Least Squares for LncRNA-Disease Association (LRLSLDA) for lncRNA-disease association prediction. As a result, new predictive models improved the performance of LRLSLDA in the leave-one-out cross validation of various known lncRNA-disease associations datasets. Furthermore, some of the predictive results for colorectal cancer and lung cancer were verified by independent biological experimental studies. It is anticipated that LNCSIM could be a useful and important biological tool for human disease diagnosis, treatment, and prevention. PMID:26061969

  15. Concordance of randomized and nonrandomized studies was unrelated to translational patterns of two nutrient-disease associations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There are several examples in nutrition of discordance between the results of observational studies and randomized controlled trials (RCTs). We hypothesized that this discordance is attributable to differences in the translational paths of nutrient–disease associations. Translational paths can be as...

  16. Constructing lncRNA functional similarity network based on lncRNA-disease associations and disease semantic similarity

    PubMed Central

    Chen, Xing; Clarence Yan, Chenggang; Luo, Cai; Ji, Wen; Zhang, Yongdong; Dai, Qionghai

    2015-01-01

    Increasing evidence has indicated that plenty of lncRNAs play important roles in many critical biological processes. Developing powerful computational models to construct lncRNA functional similarity network based on heterogeneous biological datasets is one of the most important and popular topics in the fields of both lncRNAs and complex diseases. Functional similarity network consturction could benefit the model development for both lncRNA function inference and lncRNA-disease association identification. However, little effort has been attempted to analysis and calculate lncRNA functional similarity on a large scale. In this study, based on the assumption that functionally similar lncRNAs tend to be associated with similar diseases, we developed two novel lncRNA functional similarity calculation models (LNCSIM). LNCSIM was evaluated by introducing similarity scores into the model of Laplacian Regularized Least Squares for LncRNA–Disease Association (LRLSLDA) for lncRNA-disease association prediction. As a result, new predictive models improved the performance of LRLSLDA in the leave-one-out cross validation of various known lncRNA-disease associations datasets. Furthermore, some of the predictive results for colorectal cancer and lung cancer were verified by independent biological experimental studies. It is anticipated that LNCSIM could be a useful and important biological tool for human disease diagnosis, treatment, and prevention. PMID:26061969

  17. Strain-Level Differences in Porphyrin Production and Regulation in Propionibacterium acnes Elucidate Disease Associations.

    PubMed

    Johnson, Tremylla; Kang, Dezhi; Barnard, Emma; Li, Huiying

    2016-01-01

    Propionibacterium acnes is an important skin commensal, but it is also considered a pathogenic factor in several diseases including acne vulgaris, the most common skin disease. While previous studies have revealed P. acnes strain-level differences in health and disease associations, the underlying molecular mechanisms remain unknown. Recently, we demonstrated that vitamin B12 supplementation increases P. acnes production of porphyrins, a group of proinflammatory metabolites important in acne development (D. Kang, B. Shi, M. C. Erfe, N. Craft, and H. Li, Sci. Transl. Med. 7:293ra103, 2015, doi:10.1126/scitranslmed.aab2009). In this study, we compared the porphyrin production and regulation of multiple P. acnes strains. We revealed that acne-associated type IA-2 strains inherently produced significantly higher levels of porphyrins, which were further enhanced by vitamin B12 supplementation. On the other hand, health-associated type II strains produced low levels of porphyrins and did not respond to vitamin B12. Using a small-molecule substrate and inhibitor, we demonstrated that porphyrin biosynthesis was modulated at the metabolic level. We identified a repressor gene (deoR) of porphyrin biosynthesis that was carried in all health-associated type II strains, but not in acne-associated type IA-2 strains. The expression of deoR suggests additional regulation of porphyrin production at the transcriptional level in health-associated strains. Our findings provide one potential molecular mechanism for the different contributions of P. acnes strains to skin health and disease and support the role of vitamin B12 in acne pathogenesis. Our study emphasizes the importance of understanding the role of the commensal microbial community in health and disease at the strain level and suggests potential utility of health-associated P. acnes strains in acne treatment. IMPORTANCE Propionibacterium acnes is a dominant bacterium residing on skin, and it has been thought to play a

  18. Strain-Level Differences in Porphyrin Production and Regulation in Propionibacterium acnes Elucidate Disease Associations

    PubMed Central

    Johnson, Tremylla; Kang, Dezhi; Barnard, Emma

    2016-01-01

    ABSTRACT Propionibacterium acnes is an important skin commensal, but it is also considered a pathogenic factor in several diseases including acne vulgaris, the most common skin disease. While previous studies have revealed P. acnes strain-level differences in health and disease associations, the underlying molecular mechanisms remain unknown. Recently, we demonstrated that vitamin B12 supplementation increases P. acnes production of porphyrins, a group of proinflammatory metabolites important in acne development (D. Kang, B. Shi, M. C. Erfe, N. Craft, and H. Li, Sci. Transl. Med. 7:293ra103, 2015, doi:10.1126/scitranslmed.aab2009). In this study, we compared the porphyrin production and regulation of multiple P. acnes strains. We revealed that acne-associated type IA-2 strains inherently produced significantly higher levels of porphyrins, which were further enhanced by vitamin B12 supplementation. On the other hand, health-associated type II strains produced low levels of porphyrins and did not respond to vitamin B12. Using a small-molecule substrate and inhibitor, we demonstrated that porphyrin biosynthesis was modulated at the metabolic level. We identified a repressor gene (deoR) of porphyrin biosynthesis that was carried in all health-associated type II strains, but not in acne-associated type IA-2 strains. The expression of deoR suggests additional regulation of porphyrin production at the transcriptional level in health-associated strains. Our findings provide one potential molecular mechanism for the different contributions of P. acnes strains to skin health and disease and support the role of vitamin B12 in acne pathogenesis. Our study emphasizes the importance of understanding the role of the commensal microbial community in health and disease at the strain level and suggests potential utility of health-associated P. acnes strains in acne treatment. IMPORTANCE Propionibacterium acnes is a dominant bacterium residing on skin, and it has been thought

  19. Fine mapping of the celiac disease-associated LPP locus reveals a potential functional variant

    PubMed Central

    Almeida, Rodrigo; Ricaño-Ponce, Isis; Kumar, Vinod; Deelen, Patrick; Szperl, Agata; Trynka, Gosia; Gutierrez-Achury, Javier; Kanterakis, Alexandros; Westra, Harm-Jan; Franke, Lude; Swertz, Morris A.; Platteel, Mathieu; Bilbao, Jose Ramon; Barisani, Donatella; Greco, Luigi; Mearin, Luisa; Wolters, Victorien M.; Mulder, Chris; Mazzilli, Maria Cristina; Sood, Ajit; Cukrowska, Bozena; Núñez, Concepción; Pratesi, Riccardo; Withoff, Sebo; Wijmenga, Cisca

    2014-01-01

    Using the Immunochip for genotyping, we identified 39 non-human leukocyte antigen (non-HLA) loci associated to celiac disease (CeD), an immune-mediated disease with a worldwide frequency of ∼1%. The most significant non-HLA signal mapped to the intronic region of 70 kb in the LPP gene. Our aim was to fine map and identify possible functional variants in the LPP locus. We performed a meta-analysis in a cohort of 25 169 individuals from six different populations previously genotyped using Immunochip. Imputation using data from the Genome of the Netherlands and 1000 Genomes projects, followed by meta-analysis, confirmed the strong association signal on the LPP locus (rs2030519, P = 1.79 × 10−49), without any novel associations. The conditional analysis on this top SNP-indicated association to a single common haplotype. By performing haplotype analyses in each population separately, as well as in a combined group of the four populations that reach the significant threshold after correction (P < 0.008), we narrowed down the CeD-associated region from 70 to 2.8 kb (P = 1.35 × 10−44). By intersecting regulatory data from the ENCODE project, we found a functional SNP, rs4686484 (P = 3.12 × 10−49), that maps to several B-cell enhancer elements and a highly conserved region. This SNP was also predicted to change the binding motif of the transcription factors IRF4, IRF11, Nkx2.7 and Nkx2.9, suggesting its role in transcriptional regulation. We later found significantly low levels of LPP mRNA in CeD biopsies compared with controls, thus our results suggest that rs4686484 is the functional variant in this locus, while LPP expression is decreased in CeD. PMID:24334606

  20. Using Hamming Distance as Information for SNP-Sets Clustering and Testing in Disease Association Studies

    PubMed Central

    Wang, Charlotte; Kao, Wen-Hsin; Hsiao, Chuhsing Kate

    2015-01-01

    The availability of high-throughput genomic data has led to several challenges in recent genetic association studies, including the large number of genetic variants that must be considered and the computational complexity in statistical analyses. Tackling these problems with a marker-set study such as SNP-set analysis can be an efficient solution. To construct SNP-sets, we first propose a clustering algorithm, which employs Hamming distance to measure the similarity between strings of SNP genotypes and evaluates whether the given SNPs or SNP-sets should be clustered. A dendrogram can then be constructed based on such distance measure, and the number of clusters can be determined. With the resulting SNP-sets, we next develop an association test HDAT to examine susceptibility to the disease of interest. This proposed test assesses, based on Hamming distance, whether the similarity between a diseased and a normal individual differs from the similarity between two individuals of the same disease status. In our proposed methodology, only genotype information is needed. No inference of haplotypes is required, and SNPs under consideration do not need to locate in nearby regions. The proposed clustering algorithm and association test are illustrated with applications and simulation studies. As compared with other existing methods, the clustering algorithm is faster and better at identifying sets containing SNPs exerting a similar effect. In addition, the simulation studies demonstrated that the proposed test works well for SNP-sets containing a large proportion of neutral SNPs. Furthermore, employing the clustering algorithm before testing a large set of data improves the knowledge in confining the genetic regions for susceptible genetic markers. PMID:26302001

  1. Heterogeneous Network Edge Prediction: A Data Integration Approach to Prioritize Disease-Associated Genes

    PubMed Central

    Himmelstein, Daniel S.; Baranzini, Sergio E.

    2015-01-01

    The first decade of Genome Wide Association Studies (GWAS) has uncovered a wealth of disease-associated variants. Two important derivations will be the translation of this information into a multiscale understanding of pathogenic variants and leveraging existing data to increase the power of existing and future studies through prioritization. We explore edge prediction on heterogeneous networks—graphs with multiple node and edge types—for accomplishing both tasks. First we constructed a network with 18 node types—genes, diseases, tissues, pathophysiologies, and 14 MSigDB (molecular signatures database) collections—and 19 edge types from high-throughput publicly-available resources. From this network composed of 40,343 nodes and 1,608,168 edges, we extracted features that describe the topology between specific genes and diseases. Next, we trained a model from GWAS associations and predicted the probability of association between each protein-coding gene and each of 29 well-studied complex diseases. The model, which achieved 132-fold enrichment in precision at 10% recall, outperformed any individual domain, highlighting the benefit of integrative approaches. We identified pleiotropy, transcriptional signatures of perturbations, pathways, and protein interactions as influential mechanisms explaining pathogenesis. Our method successfully predicted the results (with AUROC = 0.79) from a withheld multiple sclerosis (MS) GWAS despite starting with only 13 previously associated genes. Finally, we combined our network predictions with statistical evidence of association to propose four novel MS genes, three of which (JAK2, REL, RUNX3) validated on the masked GWAS. Furthermore, our predictions provide biological support highlighting REL as the causal gene within its gene-rich locus. Users can browse all predictions online (http://het.io). Heterogeneous network edge prediction effectively prioritized genetic associations and provides a powerful new approach for data

  2. Degradation of the disease-associated prion protein by a serine protease from lichens

    USGS Publications Warehouse

    Johnson, C.J.; Bennett, J.P.; Biro, S.M.; Duque-Velasquez, J.C.; Rodriguez, C.M.; Bessen, R.A.; Rocke, T.E.

    2011-01-01

    The disease-associated prion protein (PrP(TSE)), the probable etiological agent of the transmissible spongiform encephalopathies (TSEs), is resistant to degradation and can persist in the environment. Lichens, mutualistic symbioses containing fungi, algae, bacteria and occasionally cyanobacteria, are ubiquitous in the environment and have evolved unique biological activities allowing their survival in challenging ecological niches. We investigated PrP(TSE) inactivation by lichens and found acetone extracts of three lichen species (Parmelia sulcata, Cladonia rangiferina and Lobaria pulmonaria) have the ability to degrade prion protein (PrP) from TSE-infected hamsters, mice and deer. Immunoblots measuring PrP levels and protein misfolding cyclic amplification indicated at least two logs of reductions in PrP(TSE). Degradative activity was not found in closely related lichen species or in algae or a cyanobacterium that inhabit lichens. Degradation was blocked by Pefabloc SC, a serine protease inhibitor, but not inhibitors of other proteases or enzymes. Additionally, we found that PrP levels in PrP(TSE)-enriched preps or infected brain homogenates are also reduced following exposure to freshly-collected P. sulcata or an aqueous extract of the lichen. Our findings indicate that these lichen extracts efficiently degrade PrP(TSE) and suggest that some lichens could have potential to inactivate TSE infectivity on the landscape or be a source for agents to degrade prions. Further work to clone and characterize the protease, assess its effect on TSE infectivity and determine which organism or organisms present in lichens produce or influence the protease activity is warranted.

  3. Degradation of the disease-associated prion protein by a serine protease from lichens

    USGS Publications Warehouse

    Johnson, C.J.; Bennett, J.P.; Biro, S.M.; Duque-Velasquez, J. C.; Rodriguez, C.M.; Bessen, R.A.; Rocke, T.E.

    2011-01-01

    The disease-associated prion protein (PrPTSE), the probable etiological agent of the transmissible spongiform encephalopathies (TSEs), is resistant to degradation and can persist in the environment. Lichens, mutualistic symbioses containing fungi, algae, bacteria and occasionally cyanobacteria, are ubiquitous in the environment and have evolved unique biological activities allowing their survival in challenging ecological niches. We investigated PrPTSE inactivation by lichens and found acetone extracts of three lichen species (Parmelia sulcata, Cladonia rangiferina and Lobaria pulmonaria) have the ability to degrade prion protein (PrP) from TSE-infected hamsters, mice and deer. Immunoblots measuring PrP levels and protein misfolding cyclic amplification indicated at least two logs of reductions in PrPTSE. Degradative activity was not found in closely related lichen species or in algae or a cyanobacterium that inhabit lichens. Degradation was blocked by Pefabloc SC, a serine protease inhibitor, but not inhibitors of other proteases or enzymes. Additionally, we found that PrP levels in PrPTSE-enriched preps or infected brain homogenates are also reduced following exposure to freshly-collected P. sulcata or an aqueous extract of the lichen. Our findings indicate that these lichen extracts efficiently degrade PrPTSE and suggest that some lichens could have potential to inactivate TSE infectivity on the landscape or be a source for agents to degrade prions. Further work to clone and characterize the protease, assess its effect on TSE infectivity and determine which organism or organisms present in lichens produce or influence the protease activity is warranted.

  4. Degradation of the disease-associated prion protein by a serine protease from lichens.

    USGS Publications Warehouse

    Johnson, C.J.; Bennett, J.P.; Biro, S.M.; Duque-Velasquez, J. C.; Rodriguez, C.M.; Bessen, R.A.; Rocke, T.E.

    2011-01-01

    The disease-associated prion protein (PrPTSE), the probable etiological agent of the transmissible spongiform encephalopathies (TSEs), is resistant to degradation and can persist in the environment. Lichens, mutualistic symbioses containing fungi, algae, bacteria and occasionally cyanobacteria, are ubiquitous in the environment and have evolved unique biological activities allowing their survival in challenging ecological niches. We investigated PrPTSE inactivation by lichens and found acetone extracts of three lichen species (Parmelia sulcata, Cladonia rangiferina and Lobaria pulmonaria) have the ability to degrade prion protein (PrP) from TSE-infected hamsters, mice and deer. Immunoblots measuring PrP levels and protein misfolding cyclic amplification indicated at least two logs of reductions in PrPTSE. Degradative activity was not found in closely related lichen species or in algae or a cyanobacterium that inhabit lichens. Degradation was blocked by Pefabloc SC, a serine protease inhibitor, but not inhibitors of other proteases or enzymes. Additionally, we found that PrP levels in PrPTSE-enriched preps or infected brain homogenates are also reduced following exposure to freshly-collected P. sulcata or an aqueous extract of the lichen. Our findings indicate that these lichen extracts efficiently degrade PrPTSE and suggest that some lichens could have potential to inactivate TSE infectivity on the landscape or be a source for agents to degrade prions. Further work to clone and characterize the protease, assess its effect on TSE infectivity and determine which organism or organisms present in lichens produce or influence the protease activity is warranted.

  5. Disease-associated Mutations in the Prion Protein Impair Laminin-induced Process Outgrowth and Survival*

    PubMed Central

    Machado, Cleiton F.; Beraldo, Flavio H.; Santos, Tiago G.; Bourgeon, Dominique; Landemberger, Michele C.; Roffé, Martin; Martins, Vilma R.

    2012-01-01

    Prions, the agents of transmissible spongiform encephalopathies, require the expression of prion protein (PrPC) to propagate disease. PrPC is converted into an abnormal insoluble form, PrPSc, that gains neurotoxic activity. Conversely, clinical manifestations of prion disease may occur either before or in the absence of PrPSc deposits, but the loss of normal PrPC function contribution for the etiology of these diseases is still debatable. Prion disease-associated mutations in PrPC represent one of the best models to understand the impact of PrPC loss-of-function. PrPC associates with various molecules and, in particular, the interaction of PrPC with laminin (Ln) modulates neuronal plasticity and memory formation. To assess the functional alterations associated with PrPC mutations, wild-type and mutated PrPC proteins were expressed in a neural cell line derived from a PrPC-null mouse. Treatment with the laminin γ1 chain peptide (Ln γ1), which mimics the Ln binding site for PrPC, increased intracellular calcium in cells expressing wild-type PrPC, whereas a significantly lower response was observed in cells expressing mutated PrPC molecules. The Ln γ1 did not promote process outgrowth or protect against staurosporine-induced cell death in cells expressing mutated PrPC molecules in contrast to cells expressing wild-type PrPC. The co-expression of wild-type PrPC with mutated PrPC molecules was able to rescue the Ln protective effects, indicating the lack of negative dominance of PrPC mutated molecules. These results indicate that PrPC mutations impair process outgrowth and survival mediated by Ln γ1 peptide in neural cells, which may contribute to the pathogenesis of genetic prion diseases. PMID:23132868

  6. Disease-Associated miRNA-mRNA Networks in Oral Lichen Planus

    PubMed Central

    Gassling, Volker; Hampe, Jochen; Açil, Yahya; Braesen, Jan Hinrich; Wiltfang, Jörg; Häsler, Robert

    2013-01-01

    Background A large number of pathophysiological mechanisms are regulated by microRNAs (miRNAs), which represent a new class of posttranscriptional regulators of gene expression. To date, little is known about their role in oral lichen planus (OLP), a chronic inflammatory mucocutaneous disease of unknown etiology which is being discussed as a potentially premalignant condition of oropharyngeal cancer. The aim of the present investigation was to assess the pathophysiological impact of miRNAs and to determine regulatory miRNA networks which are directly linked to potentially disease-associated target transcripts in OLP. Methods Native tissue samples were collected from the oral mucosa of seven patients with OLP. The control group was composed of native tissue from elective oral surgery. The mRNA profiling was performed using the Affymetrix Human Gene 1.0 ST Array while miRNA profiling was performed using the microRNA Galaxy Array. Subsequent validation of initial results was carried out using TaqMan real time PCR. Results We identified 24 differentially regulated miRNA and 2,694 regulated transcripts. Linking the miRNAs to their potential targets we found 11 potential miRNA-mRNA pairs, of which several are functionally related to premalignant as well as to inflammatory events. Conclusions Our data shows miRNA associated with transcripts which are regulated when comparing OLP patients with healthy control individuals. This suggests that miRNAs may potentially regulate disease-relevant transcripts, proposing the concept of therapeutic interventions based on miRNAs. PMID:23723971

  7. HDL-S1P: cardiovascular functions, disease-associated alterations, and therapeutic applications

    PubMed Central

    Levkau, Bodo

    2015-01-01

    Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid contained in High-density lipoproteins (HDL) and has drawn considerable attention in the lipoprotein field as numerous studies have demonstrated its contribution to several functions inherent to HDL. Some of them are partly and some entirely due to the S1P contained in HDL (HDL-S1P). Despite the presence of over 1000 different lipids in HDL, S1P stands out as it possesses its own cell surface receptors through which it exercises key physiological functions. Most of the S1P in human plasma is associated with HDL, and the amount of HDL-S1P influences the quality and quantity of HDL-dependent functions. The main binding partner of S1P in HDL is apolipoprotein M but others may also exist particularly under conditions of acute S1P elevations. HDL not only exercise functions through their S1P content but have also an impact on genuine S1P signaling by influencing S1P bioactivity and receptor presentation. HDL-S1P content is altered in human diseases such as atherosclerosis, coronary artery disease, myocardial infarction, renal insufficiency and diabetes mellitus. Low HDL-S1P has also been linked to impaired HDL functions associated with these disorders. Although the pathophysiological and molecular reasons for such disease-associated shifts in HDL-S1P are little understood, there have been successful approaches to circumvent their adverse implications by pharmacologically increasing HDL-S1P as means to improve HDL function. This mini-review will cover the current understanding of the contribution of HDL-S1P to physiological HDL function, its alteration in disease and ways for its restoration to correct HDL dysfunction. PMID:26539121

  8. Scoring multiple features to predict drug disease associations using information fusion and aggregation.

    PubMed

    Moghadam, H; Rahgozar, M; Gharaghani, S

    2016-08-01

    Prediction of drug-disease associations is one of the current fields in drug repositioning that has turned into a challenging topic in pharmaceutical science. Several available computational methods use network-based and machine learning approaches to reposition old drugs for new indications. However, they often ignore features of drugs and diseases as well as the priority and importance of each feature, relation, or interactions between features and the degree of uncertainty. When predicting unknown drug-disease interactions there are diverse data sources and multiple features available that can provide more accurate and reliable results. This information can be collectively mined using data fusion methods and aggregation operators. Therefore, we can use the feature fusion method to make high-level features. We have proposed a computational method named scored mean kernel fusion (SMKF), which uses a new method to score the average aggregation operator called scored mean. To predict novel drug indications, this method systematically combines multiple features related to drugs or diseases at two levels: the drug-drug level and the drug-disease level. The purpose of this study was to investigate the effect of drug and disease features as well as data fusion to predict drug-disease interactions. The method was validated against a well-established drug-disease gold-standard dataset. When compared with the available methods, our proposed method outperformed them and competed well in performance with area under cover (AUC) of 0.91, F-measure of 84.9% and Matthews correlation coefficient of 70.31%. PMID:27455069

  9. Heterogeneous Network Edge Prediction: A Data Integration Approach to Prioritize Disease-Associated Genes.

    PubMed

    Himmelstein, Daniel S; Baranzini, Sergio E

    2015-07-01

    The first decade of Genome Wide Association Studies (GWAS) has uncovered a wealth of disease-associated variants. Two important derivations will be the translation of this information into a multiscale understanding of pathogenic variants and leveraging existing data to increase the power of existing and future studies through prioritization. We explore edge prediction on heterogeneous networks--graphs with multiple node and edge types--for accomplishing both tasks. First we constructed a network with 18 node types--genes, diseases, tissues, pathophysiologies, and 14 MSigDB (molecular signatures database) collections--and 19 edge types from high-throughput publicly-available resources. From this network composed of 40,343 nodes and 1,608,168 edges, we extracted features that describe the topology between specific genes and diseases. Next, we trained a model from GWAS associations and predicted the probability of association between each protein-coding gene and each of 29 well-studied complex diseases. The model, which achieved 132-fold enrichment in precision at 10% recall, outperformed any individual domain, highlighting the benefit of integrative approaches. We identified pleiotropy, transcriptional signatures of perturbations, pathways, and protein interactions as influential mechanisms explaining pathogenesis. Our method successfully predicted the results (with AUROC = 0.79) from a withheld multiple sclerosis (MS) GWAS despite starting with only 13 previously associated genes. Finally, we combined our network predictions with statistical evidence of association to propose four novel MS genes, three of which (JAK2, REL, RUNX3) validated on the masked GWAS. Furthermore, our predictions provide biological support highlighting REL as the causal gene within its gene-rich locus. Users can browse all predictions online (http://het.io). Heterogeneous network edge prediction effectively prioritized genetic associations and provides a powerful new approach for data

  10. Degradation of the Disease-Associated Prion Protein by a Serine Protease from Lichens

    PubMed Central

    Johnson, Christopher J.; Bennett, James P.; Biro, Steven M.; Duque-Velasquez, Juan Camilo; Rodriguez, Cynthia M.; Bessen, Richard A.; Rocke, Tonie E.

    2011-01-01

    The disease-associated prion protein (PrPTSE), the probable etiological agent of the transmissible spongiform encephalopathies (TSEs), is resistant to degradation and can persist in the environment. Lichens, mutualistic symbioses containing fungi, algae, bacteria and occasionally cyanobacteria, are ubiquitous in the environment and have evolved unique biological activities allowing their survival in challenging ecological niches. We investigated PrPTSE inactivation by lichens and found acetone extracts of three lichen species (Parmelia sulcata, Cladonia rangiferina and Lobaria pulmonaria) have the ability to degrade prion protein (PrP) from TSE-infected hamsters, mice and deer. Immunoblots measuring PrP levels and protein misfolding cyclic amplification indicated at least two logs of reductions in PrPTSE. Degradative activity was not found in closely related lichen species or in algae or a cyanobacterium that inhabit lichens. Degradation was blocked by Pefabloc SC, a serine protease inhibitor, but not inhibitors of other proteases or enzymes. Additionally, we found that PrP levels in PrPTSE-enriched preps or infected brain homogenates are also reduced following exposure to freshly-collected P. sulcata or an aqueous extract of the lichen. Our findings indicate that these lichen extracts efficiently degrade PrPTSE and suggest that some lichens could have potential to inactivate TSE infectivity on the landscape or be a source for agents to degrade prions. Further work to clone and characterize the protease, assess its effect on TSE infectivity and determine which organism or organisms present in lichens produce or influence the protease activity is warranted. PMID:21589935

  11. Distribution of hepatitis C virus genotypes among hemodialysis patients in Tehran--a multicenter study.

    PubMed

    Hosseini-Moghaddam, Seyed Mohammadmehdi; Keyvani, Hossein; Kasiri, Hossein; Kazemeyni, Seyed Mohammad; Basiri, Abbas; Aghel, Nazanin; Alavian, Seyed-Moayed

    2006-05-01

    Hepatitis C virus has substantial heterogeneity of genotypes throughout the world. The aim of this study was to determine the frequency of HCV genotypes, risk factors and clinical implications in cases of hemodialysis living in Tehran. A total of 155 patients treated by hemodialysis, who had been identified to be anti-HCV positive at 45 medical centers in Tehran, were enrolled. Genotyping was using restriction fragment length polymorphism (RFLP) on HCV-RNA positive samples. HCV-RNA was detected in 66 (42.6%) patients. Genotyping of HCV-RNA positive serum samples demonstrated that subtypes 3a and 1a were predominant accounting for 30.3 and 28.8%, respectively. The distribution of other HCV genotypes showed genotype 1b, 18.2%; genotype 4, 16.7%; mixed genotypes 1a and 1b, 3%; and genotype 3b, 3%. Genotype 2 was not detected in this study. Statistically significant differences were identified between HCV infected and non-HCV infected patients regarding history of hemodialysis unit changes more than two times (P = 0.01), and history of hemodialysis for more than 20 years (P = 0.02). However, blood transfusion, mean duration of hemodialysis therapy and the history of solid organ transplantation did not differ between these two groups. This study indicates that the dominant HCV genotypes among patients treated by hemodialysis living in Tehran were 3a and 1a, and considering previous reports from the general population, genotype 4 was strongly associated with hemodialysis. The duration of treatment by hemodialysis and, in turn, more hemodialysis unit changes will lead to more frequent HCV infections. PMID:16555284

  12. Biochemical Classification of Disease-associated Mutants of RAS-like Protein Expressed in Many Tissues (RIT1).

    PubMed

    Fang, Zhenhao; Marshall, Christopher B; Yin, Jiani C; Mazhab-Jafari, Mohammad T; Gasmi-Seabrook, Geneviève M C; Smith, Matthew J; Nishikawa, Tadateru; Xu, Yang; Neel, Benjamin G; Ikura, Mitsuhiko

    2016-07-22

    RAS-like protein expressed in many tissues 1 (RIT1) is a disease-associated RAS subfamily small guanosine triphosphatase (GTPase). Recent studies revealed that germ-line and somatic RIT1 mutations can cause Noonan syndrome (NS), and drive proliferation of lung adenocarcinomas, respectively, akin to RAS mutations in these diseases. However, the locations of these RIT1 mutations differ significantly from those found in RAS, and do not affect the three mutational "hot spots" of RAS. Moreover, few studies have characterized the GTPase cycle of RIT1 and its disease-associated mutants. Here we developed a real-time NMR-based GTPase assay for RIT1 and investigated the effect of disease-associated mutations on GTPase cycle. RIT1 exhibits an intrinsic GTP hydrolysis rate similar to that of H-RAS, but its intrinsic nucleotide exchange rate is ∼4-fold faster, likely as a result of divergent residues near the nucleotide binding site. All of the disease-associated mutations investigated increased the GTP-loaded, activated state of RIT1 in vitro, but they could be classified into two groups with different intrinsic GTPase properties. The S35T, A57G, and Y89H mutants exhibited more rapid nucleotide exchange, whereas F82V and T83P impaired GTP hydrolysis. A RAS-binding domain pulldown assay indicated that RIT1 A57G and Y89H were highly activated in HEK293T cells, whereas T83P and F82V exhibited more modest activation. All five mutations are associated with NS, whereas two (A57G and F82V) have also been identified in urinary tract cancers and myeloid malignancies. Characterization of the effects on the GTPase cycle of RIT1 disease-associated mutations should enable better understanding of their role in disease processes. PMID:27226556

  13. ENTPRISE: An Algorithm for Predicting Human Disease-Associated Amino Acid Substitutions from Sequence Entropy and Predicted Protein Structures

    PubMed Central

    Zhou, Hongyi; Gao, Mu; Skolnick, Jeffrey

    2016-01-01

    The advance of next-generation sequencing technologies has made exome sequencing rapid and relatively inexpensive. A major application of exome sequencing is the identification of genetic variations likely to cause Mendelian diseases. This requires processing large amounts of sequence information and therefore computational approaches that can accurately and efficiently identify the subset of disease-associated variations are needed. The accuracy and high false positive rates of existing computational tools leave much room for improvement. Here, we develop a boosted tree regression machine-learning approach to predict human disease-associated amino acid variations by utilizing a comprehensive combination of protein sequence and structure features. On comparing our method, ENTPRISE, to the state-of-the-art methods SIFT, PolyPhen-2, MUTATIONASSESSOR, MUTATIONTASTER, FATHMM, ENTPRISE exhibits significant improvement. In particular, on a testing dataset consisting of only proteins with balanced disease-associated and neutral variations defined as having the ratio of neutral/disease-associated variations between 0.3 and 3, the Mathews Correlation Coefficient by ENTPRISE is 0.493 as compared to 0.432 by PPH2-HumVar, 0.406 by SIFT, 0.403 by MUTATIONASSESSOR, 0.402 by PPH2-HumDiv, 0.305 by MUTATIONTASTER, and 0.181 by FATHMM. ENTPRISE is then applied to nucleic acid binding proteins in the human proteome. Disease-associated predictions are shown to be highly correlated with the number of protein-protein interactions. Both these predictions and the ENTPRISE server are freely available for academic users as a web service at http://cssb.biology.gatech.edu/entprise/. PMID:26982818

  14. Genotype imputation via matrix completion.

    PubMed

    Chi, Eric C; Zhou, Hua; Chen, Gary K; Del Vecchyo, Diego Ortega; Lange, Kenneth

    2013-03-01

    Most current genotype imputation methods are model-based and computationally intensive, taking days to impute one chromosome pair on 1000 people. We describe an efficient genotype imputation method based on matrix completion. Our matrix completion method is implemented in MATLAB and tested on real data from HapMap 3, simulated pedigree data, and simulated low-coverage sequencing data derived from the 1000 Genomes Project. Compared with leading imputation programs, the matrix completion algorithm embodied in our program MENDEL-IMPUTE achieves comparable imputation accuracy while reducing run times significantly. Implementation in a lower-level language such as Fortran or C is apt to further improve computational efficiency. PMID:23233546

  15. Hepatitis C virus genotypes in north eastern Algeria: A retrospective study

    PubMed Central

    Rouabhia, Samir; Sadelaoud, Mourad; Chaabna-Mokrane, Karima; Toumi, Wided; Abenavoli, Ludovico

    2013-01-01

    AIM: To determine the frequency of various hepatitis C virus (HCV) genotypes present in patients from north eastern Algeria. METHODS: This is a retrospective cross-sectional study of 435 HCV infected patients from northeast Algeria, detected in the Sadelaoud laboratory and diagnosed between January 2010 and December 2012. The patients were diagnosed with HCV infection in their local hospitals and referred to be assessed for HCV genotype before the antiviral treatment. Demographic information (sex, age and address), genotype, subtype and viral load were retrieved from the patient medical records. The serum samples were tested by the type-specific genotyping assay. RESULTS: The majority of the patients (82.5%) were from the central part of the examined region (P = 0.002). The mean age of the patients studied was 53.6 ± 11.5 years. HCV genotype 1 was the most frequent (88.7%), followed by genotypes 2 (8.5%), 4 (1.1%), 3 (0.9%) and 5 (0.2%). Genotype 6 was not detected in these patients. Mixed infection across the HCV subtypes was detected in twenty patients (4.6%). The genotype distribution was related to age and region. Genotype 1 was significantly less frequent in the ≥ 60 age group than in the younger age group (OR = 0.2; 95%CI: 0.1-0.5, P < 0.001). Furthermore, genotype 1 was more frequent in the central part of the examined region than elsewhere (P < 0.01). CONCLUSION: The HCV genotype (type 1b was dominant) distribution in Algeria is different from those in other northern countries of Africa. PMID:23898373

  16. [Determination of hepatitis C virus genotypes among hepatitis C patients in Eastern Black Sea Region, Turkey].

    PubMed

    Buruk, Celal Kurtuluş; Bayramoğlu, Gülçin; Reis, Ahu; Kaklıkkaya, Neşe; Tosun, Ilknur; Aydın, Faruk

    2013-10-01

    Hepatitis C virus (HCV), the major cause of transfusion-associated hepatitis, is an important public health problem in the world as well as in Turkey. HCV is grouped as six distinct genotypes and a large number of closely-related subtypes. Genotyping of HCV is an important tool for providing epidemiological data, prediction of prognosis, and optimization of antiviral therapy. This study was carried out to determine the distribution of HCV genotypes in hepatitis C patients residing in different provinces of the Eastern Black Sea Region, Turkey. A total of 304 HCV-RNA positive cases (151 male, 153 female; age range: 11-93 years, mean age: 55.2 ± 13.3 years) who were admitted to the Molecular Microbiology Unit of Department of Medical Microbiology, Karadeniz Technical University Faculty of Medicine, between January 2009 to December 2012, were included in the study. HCV genotypes were detected in plasma samples of the patients by using commercial assays [INNO-LiPA HCV II (Innogenetics, Belgium) or Abbott RealTime HCV Genotype II (Abbott Molecular Inc, USA)]. Due to the ambiguous genotyping results in some samples with these methods, an in-house multiplex polymerase chain reaction (PCR) assay with genotype-specific primers was also used in the study. Similar to the previous reports from Turkey, our results showed that four HCV genotypes (1, 2, 3, and 4) prevailed in the Eastern Black Sea Region and the predominant genotype and subtype were genotype 1 (92.8%) and 1b (87.5%), respectively. Distribution of genotypes were observed to vary according to the province. Prevalences of subtype 1a, genotype 2, 3, and 4 were noted as 5.3%, 1.6%, 4.9%, and 0.7%, respectively. Furthermore, the samples from Giresun, Gumushane and Bayburt provinces, which are relatively less immigrated, had higher genotype 1, and the prevalence rates in the region was affected by the presence of non-citizen residents. This study is the first report on distribution of HCV genotypes in chronic hepatitis

  17. A MEMS-Based Approach to Single Nucleotide Polymorphism Genotyping

    PubMed Central

    Zhu, Jing; Palla, Mirkó; Ronca, Stefano; Warpner, Ronald; Ju, Jingyue; Lin, Qiao

    2014-01-01

    Genotyping of single nucleotide polymorphisms (SNPs) allows diagnosis of human genetic disorders associated with single base mutations. Conventional SNP genotyping methods are capable of providing either accurate or high-throughput detection, but are still labor-, time-, and resource-intensive. Microfluidics has been applied to SNP detection to provide fast, low-cost, and automated alternatives, although these applications are still limited by either accuracy or throughput issues. To address this challenge, we present a MEMS-based SNP genotyping approach that uses solid-phase-based reactions in a single microchamber on a temperature control chip. Polymerase chain reaction (PCR), allele specific single base extension (SBE), and desalting on microbeads are performed in the microchamber, which is coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to analyze the SBE product. Experimental results from genotyping of the SNP on exon 1 of the HBB gene, which causes sickle cell anemia, demonstrate the potential of the device for rapid, accurate, multiplexed and high-throughput detection of SNPs. PMID:24729659

  18. A Microfluidic Device for Multiplex Single-Nucleotide Polymorphism Genotyping

    PubMed Central

    Zhu, Jing; Qiu, Chunmei; Palla, Mirkó; Nguyen, ThaiHuu; Russo, James J.; Ju, Jingyue; Lin, Qiao

    2015-01-01

    Single-nucleotide polymorphisms (SNPs) are the most abundant type of genetic variations; they provide the genetic fingerprint of individuals and are essential for genetic biomarker discoveries. Accurate detection of SNPs is of great significance for disease prevention, diagnosis and prognosis, and for prediction of drug response and clinical outcomes in patients. Nevertheless, conventional SNP genotyping methods are still limited by insufficient accuracy or labor-, time-, and resource-intensive procedures. Microfluidics has been increasingly utilized to improve efficiency; however, the currently available microfluidic genotyping systems still have shortcomings in accuracy, sensitivity, throughput and multiplexing capability. To address these challenges, we developed a multi-step SNP genotyping microfluidic device, which performs single-base extension of SNP specific primers and solid-phase purification of the extension products on a temperature-controlled chip. The products are ready for immediate detection by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), providing identification of the alleles at the target loci. The integrated device enables efficient and automated operation, while maintaining the high accuracy and sensitivity provided by MS. The multiplex genotyping capability was validated by performing rapid, accurate and simultaneous detection of 4 loci on a synthetic template. The microfluidic device has the potential to perform automatic, accurate, quantitative and high-throughput assays covering a broad spectrum of applications in biological and clinical research, drug development and forensics. PMID:26594354

  19. Single Nucleotide Polymorphism Genotyping and Distribution of Coxiella burnetii Strains from Field Samples in Belgium

    PubMed Central

    Dal Pozzo, Fabiana; Renaville, Bénédicte; Martinelle, Ludovic; Renaville, Robert; Thys, Christine; Smeets, François; Kirschvink, Nathalie; Grégoire, Fabien; Delooz, Laurent; Czaplicki, Guy

    2015-01-01

    The genotypic characterization of Coxiella burnetii provides useful information about the strains circulating at the farm, region, or country level and may be used to identify the source of infection for animals and humans. The aim of the present study was to investigate the strains of C. burnetii circulating in caprine and bovine Belgian farms using a single nucleotide polymorphism (SNP) technique. Direct genotyping was applied to different samples (bulk tank milk, individual milk, vaginal swab, fetal product, and air sample). Besides the well-known SNP genotypes, unreported ones were found in bovine and caprine samples, increasing the variability of the strains found in the two species in Belgium. Moreover, multiple genotypes were detected contemporarily in caprine farms at different years of sampling and by using different samples. Interestingly, certain SNP genotypes were detected in both bovine and caprine samples, raising the question of interspecies transmission of the pathogen. PMID:26475104

  20. Transforming microbial genotyping: a robotic pipeline for genotyping bacterial strains.

    PubMed

    O'Farrell, Brian; Haase, Jana K; Velayudhan, Vimalkumar; Murphy, Ronan A; Achtman, Mark

    2012-01-01

    Microbial genotyping increasingly deals with large numbers of samples, and data are commonly evaluated by unstructured approaches, such as spread-sheets. The efficiency, reliability and throughput of genotyping would benefit from the automation of manual manipulations within the context of sophisticated data storage. We developed a medium- throughput genotyping pipeline for MultiLocus Sequence Typing (MLST) of bacterial pathogens. This pipeline was implemented through a combination of four automated liquid handling systems, a Laboratory Information Management System (LIMS) consisting of a variety of dedicated commercial operating systems and programs, including a Sample Management System, plus numerous Python scripts. All tubes and microwell racks were bar-coded and their locations and status were recorded in the LIMS. We also created a hierarchical set of items that could be used to represent bacterial species, their products and experiments. The LIMS allowed reliable, semi-automated, traceable bacterial genotyping from initial single colony isolation and sub-cultivation through DNA extraction and normalization to PCRs, sequencing and MLST sequence trace evaluation. We also describe robotic sequencing to facilitate cherrypicking of sequence dropouts. This pipeline is user-friendly, with a throughput of 96 strains within 10 working days at a total cost of < €25 per strain. Since developing this pipeline, >200,000 items were processed by two to three people. Our sophisticated automated pipeline can be implemented by a small microbiology group without extensive external support, and provides a general framework for semi-automated bacterial genotyping of large numbers of samples at low cost. PMID:23144721

  1. Transforming Microbial Genotyping: A Robotic Pipeline for Genotyping Bacterial Strains

    PubMed Central

    Velayudhan, Vimalkumar; Murphy, Ronan A.; Achtman, Mark

    2012-01-01

    Microbial genotyping increasingly deals with large numbers of samples, and data are commonly evaluated by unstructured approaches, such as spread-sheets. The efficiency, reliability and throughput of genotyping would benefit from the automation of manual manipulations within the context of sophisticated data storage. We developed a medium- throughput genotyping pipeline for MultiLocus Sequence Typing (MLST) of bacterial pathogens. This pipeline was implemented through a combination of four automated liquid handling systems, a Laboratory Information Management System (LIMS) consisting of a variety of dedicated commercial operating systems and programs, including a Sample Management System, plus numerous Python scripts. All tubes and microwell racks were bar-coded and their locations and status were recorded in the LIMS. We also created a hierarchical set of items that could be used to represent bacterial species, their products and experiments. The LIMS allowed reliable, semi-automated, traceable bacterial genotyping from initial single colony isolation and sub-cultivation through DNA extraction and normalization to PCRs, sequencing and MLST sequence trace evaluation. We also describe robotic sequencing to facilitate cherrypicking of sequence dropouts. This pipeline is user-friendly, with a throughput of 96 strains within 10 working days at a total cost of < €25 per strain. Since developing this pipeline, >200,000 items were processed by two to three people. Our sophisticated automated pipeline can be implemented by a small microbiology group without extensive external support, and provides a general framework for semi-automated bacterial genotyping of large numbers of samples at low cost. PMID:23144721

  2. MHC microsatellite diversity and linkage disequilibrium among common HLA-A, HLA-B, DRB1 haplotypes: implications for unrelated donor hematopoietic transplantation and disease association studies.

    PubMed

    Malkki, M; Single, R; Carrington, M; Thomson, G; Petersdorf, E

    2005-08-01

    Twenty-two human major histocompatibility complex (MHC) region microsatellite (Msat) markers were studied for diversity and linkage disequilibrium (LD) with HLA loci in hematopoietic cell transplant recipients and their HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 allele-matched unrelated donors. These Msats showed highly significant LD over much of the MHC region. The Msat diversity of five common Caucasian haplotypes (HLA-A1-B8-DR3, A3-B7-DR15, A2-B44-DR4, A29-B44-DR7, and A2-B7-DR15) was examined using a new measure called 'haplotype specific heterozygosity' (HSH). Each of the five haplotypes had at least one Msat marker with an HSH value of zero indicating that only one Msat allele was observed for the particular HLA haplotype. In addition, the ability of Msats to predict HLA-A-B-DRB1 haplotypes was studied. Over 90% prediction probability of two common haplotypes (HLA-A1-B8-DR3 and HLA-A3-B7-DR15) was achieved with information from three Msats (D6S265/D6S2787/D6S2894 and D6S510/D6S2810/D6S2876, respectively). We demonstrate how the HSH index can be used in the selection of informative Msats for transplantation and disease association studies. Markers with low HSH values can be used to predict specific HLA haplotypes or multilocus genotypes to supplement the screening of HLA-matched donors for transplantation. Markers with high HSH values will be most informative in studies investigating MHC region disease-susceptibility genes where HLA haplotypic effects are known to exist. PMID:16029431

  3. Disease-Associated Prion Protein in Neural and Lymphoid Tissues of Mink (Mustela vison) Inoculated with Transmissible Mink Encephalopathy

    PubMed Central

    Schneider, D. A.; Harrington, R. D.; Zhuang, D.; Yan, H.; Truscott, T. C.; Dassanayake, R. P.; O'Rourke, K. I.

    2012-01-01

    Summary Transmissible spongiform encephalopathies (TSEs) are diagnosed by immunodetection of disease-associated prion protein (PrPd). The distribution of PrPd within the body varies with the time-course of infection and between species, during interspecies transmission, as well as with prion strain. Mink are susceptible to a form of TSE known as transmissible mink encephalopathy (TME), presumed to arise due to consumption of feed contaminated with a single prion strain of ruminant origin. After extended passage of TME isolates in hamsters, two strains emerge, HY and DY, each of which is associated with unique structural isoforms of PrPTME and of which only the HY strain is associated with accumulation of PrPTME in lymphoid tissues. Information on the structural nature and lymphoid accumulation of PrPTME in mink is limited. In this study, 13 mink were challenged by intracerebral inoculation using late passage TME inoculum after which brain and lymphoid tissues were collected at preclinical and clinical time points. The distribution and molecular nature of PrPTME was investigated by techniques including blotting of paraffin wax-embedded tissue and epitope mapping by western blotting. PrPTME was detected readily in the brain and retropharyngeal lymph node during preclinical infection with delayed progression of accumulation within other lymphoid tissues. For comparison, three mink were inoculated by the oral route and examined during clinical disease. Accumulation of PrPTME in these mink was greater and more widespread, including follicles of rectoanal mucosa-associated lymphoid tissue. Western blot analyses revealed that PrPTME accumulating in the brain of mink is structurally most similar to that accumulating in the brain of hamsters infected with the DY strain. Collectively, the results of extended passage in mink are consistent with the presence of only a single strain of TME, the DY strain, capable of inducing accumulation of PrPTME in the lymphoid tissues of

  4. Genotyping Tools for Mycobacterium ulcerans-Drawbacks and Future Prospects

    PubMed Central

    Narh, Charles A; Mosi, Lydia; Quaye, Charles; Tay, Samuel CK; Bonfoh, Bassirou; de Souza, Dziedzom K

    2014-01-01

    Mycobacterium ulcerans infection (Buruli ulcer) is a neglected but treatable skin disease endemic in over 30 countries. M. ulcerans is an environmental mycobacteria with an elusive mode of transmission to humans. Ecological and Molecular epidemiological studies to identify reservoirs and transmission vectors are important for source tracking infections especially during outbreaks and elucidating transmission routes. Research efforts have therefore focused on genotyping strains of the mycobacteria from clinical and environmental samples. This review discusses genotyping tools for differentiating M. ulcerans strains from other environmental and Mycolactone Producing Mycobacteria (MPMs). We highlight tools that have been adapted from related fields and propose ways these could be enhanced to resolve intra-species variation for epidemiological, transmission, evolutionary studies, and detection of emerging drug resistant strains. In the wake of increasing cases of Buruli ulcer, cumulative efforts including improvement in diagnostic methods and fine-tuning of genotyping tools are crucial to complement public health efforts in reducing infections. PMID:24900947

  5. Bridging the Gap between Genotype and Phenotype via Network Approaches

    PubMed Central

    Kim, Yoo-Ah; Przytycka, Teresa M.

    2013-01-01

    In the last few years we have witnessed tremendous progress in detecting associations between genetic variations and complex traits. While genome-wide association studies have been able to discover genomic regions that may influence many common human diseases, these discoveries created an urgent need for methods that extend the knowledge of genotype-phenotype relationships to the level of the molecular mechanisms behind them. To address this emerging need, computational approaches increasingly utilize a pathway-centric perspective. These new methods often utilize known or predicted interactions between genes and/or gene products. In this review, we survey recently developed network based methods that attempt to bridge the genotype-phenotype gap. We note that although these methods help narrow the gap between genotype and phenotype relationships, these approaches alone cannot provide the precise details of underlying mechanisms and current research is still far from closing the gap. PMID:23755063

  6. High prevalences of infection with Giardia intestinalis genotype B among children in urban and rural areas of Argentina.

    PubMed

    Molina, N; Minvielle, M; Grenóvero, S; Salomón, C; Basualdo, J

    2011-06-01

    The protozoan parasite most frequently associated with diarrhoea worldwide is Giardia intestinalis. In 2005, a study was initiated to identify the genotypes of this parasite infecting children in the Argentinian provinces of Buenos Aires, Mendoza and Chaco, and to explore the associations between the genotype detected in a child, the characteristics of the child's household and the child's clinical presentation. Overall, 998 children (504 boys and 494 girls) aged between 2-14 years, with or without symptoms, were enrolled. The G. intestinalis in 94 of the 117 stool samples found positive for the parasite by microscopy were successfully genotyped by PCR. Seventy-seven of the children were found to be infected with genotype B only and 14 with genotype AII only, three children being found to have mixed (AII and B) infections. Only genotype B was detected in children from rural areas (P<0·05) and most Giardia detected in children from households with a piped water supply were also of this genotype (P<0·05). The other household characteristics investigated (quality of building, history of flooding, type of sanitation, level of overcrowding, and presence/absence of pet dogs) had no significant effect on the genotype distribution. Children infected with genotype AII were significantly younger than those infected with genotype B (P<0·05) and there was a significant positive association between infection with genotype B and abdominal pain (P<0·05). Diarrhoea was not, however, found to be significantly associated with genotype-AII or genotype-B infection. This is the first published report on the Giardia genotypes circulating in the provinces of Mendoza and Chaco. The results indicate the importance of asymptomatic children in the transmission of Giardia among the young. PMID:21871166

  7. Toward high-throughput genotyping: dynamic and automatic software for manipulating large-scale genotype data using fluorescently labeled dinucleotide markers.

    PubMed

    Li, J L; Deng, H; Lai, D B; Xu, F; Chen, J; Gao, G; Recker, R R; Deng, H W

    2001-07-01

    To efficiently manipulate large amounts of genotype data generated with fluorescently labeled dinucleotide markers, we developed a Microsoft database management system, named. offers several advantages. First, it accommodates the dynamic nature of the accumulations of genotype data during the genotyping process; some data need to be confirmed or replaced by repeat lab procedures. By using, the raw genotype data can be imported easily and continuously and incorporated into the database during the genotyping process that may continue over an extended period of time in large projects. Second, almost all of the procedures are automatic, including autocomparison of the raw data read by different technicians from the same gel, autoadjustment among the allele fragment-size data from cross-runs or cross-platforms, autobinning of alleles, and autocompilation of genotype data for suitable programs to perform inheritance check in pedigrees. Third, provides functions to track electrophoresis gel files to locate gel or sample sources for any resultant genotype data, which is extremely helpful for double-checking consistency of raw and final data and for directing repeat experiments. In addition, the user-friendly graphic interface of renders processing of large amounts of data much less labor-intensive. Furthermore, has built-in mechanisms to detect some genotyping errors and to assess the quality of genotype data that then are summarized in the statistic reports automatically generated by. The can easily handle >500,000 genotype data entries, a number more than sufficient for typical whole-genome linkage studies. The modules and programs we developed for the can be extended to other database platforms, such as Microsoft SQL server, if the capability to handle still greater quantities of genotype data simultaneously is desired. PMID:11435414

  8. Genotyping of cutaneous melanoma

    PubMed Central

    Glitza, Isabella C.; Davies, Michael A.

    2014-01-01

    Until recently, treatment options for patients with metastatic melanoma were very limited. This landscape has evolved dramatically since the discovery of activating mutations in the BRAF gene in ~45% of cutaneous melanomas. Vemurafenib, dabrafenib, and trametinib have all received regulatory approval for the treatment of metastatic melanoma patients with a BRAFV600 mutation. Based on the necessity to document the presence of a BRAFV600 mutation to prescribe these agents, molecular testing is now the standard of care in this disease. However, the options and rationale for testing are evolving rapidly due to an improved understanding of the molecular drivers and heterogeneity of melanoma. Such testing may identify rational combinatorial approaches to prevent or overcome resistance for the approved BRAF inhibitors. In addition, new clinical strategies have been identified for a number of other molecular changes that are detected in this disease, including somatic changes in NRAS, PTEN, CDKN2A, and c-KIT, among others. This review summarizes the current understanding of the genetic landscape of mutations in melanoma, their associations with clinicopathological features, and their implications for clinical testing and treatment. PMID:25632386

  9. Humoral Responses to Diverse Autoimmune Disease-Associated Antigens in Multiple Sclerosis

    PubMed Central

    Malyavantham, Kishore; Weinstock-Guttman, Bianca; Suresh, Lakshmanan; Zivadinov, Robert; Shanahan, Thomas; Badgett, Darlene; Ramanathan, Murali

    2015-01-01

    To compare frequencies of autoreactive antibody responses to endogenous disease-associated antigens in healthy controls (HC), relapsing and progressive MS and to assess their associations with clinical and MRI measures of MS disease progression. Methods The study analyzed 969 serum samples from 315 HC, 411 relapsing remitting MS (RR-MS), 128 secondary progressive MS (SP-MS), 33 primary progressive MS (PP-MS) and 82 patients with other neurological diseases for autoantibodies against two putative MS antigens CSF114(Glc) and KIR4.1a and KIR4.1b and against 24 key endogenous antigens linked to diseases such as vasculitis, systemic sclerosis, rheumatoid arthritis, Sjogren’s syndrome, systemic lupus erythematosus, polymyositis, scleroderma, polymyositis, dermatomyositis, mixed connective tissue disease and primary biliary cirrhosis. Associations with disability and MRI measures of lesional injury and neurodegeneration were assessed. Results The frequencies of anti-KIR4.1a and anti-KIR4.1b peptide IgG positivity were 9.8% and 11.4% in HC compared to 4.9% and 7.5% in RR-MS, 8.6% for both peptides in SP-MS and 6.1% for both peptides in PP-MS (p = 0.13 for KIR4.1a and p = 0.34 for KIR4.1b), respectively. Antibodies against CSF114(Glc), KIR4.1a and KIR4.1b peptides were not associated with MS compared to HC, or with MS disease progression. HLA DRB1*15:01 positivity and anti-Epstein Barr virus antibodies, which are MS risk factors, were not associated with these putative MS antibodies. Conclusions Antibody responses to KIR4.1a and KIR4.1b peptides are not increased in MS compared to HC nor associated with MS disease progression. The frequencies of the diverse autoreactive antibodies investigated are similar in MS and HC. PMID:26065913

  10. Disease-associated mutations in the prion protein impair laminin-induced process outgrowth and survival.

    PubMed

    Machado, Cleiton F; Beraldo, Flavio H; Santos, Tiago G; Bourgeon, Dominique; Landemberger, Michele C; Roffé, Martin; Martins, Vilma R

    2012-12-21

    Prions, the agents of transmissible spongiform encephalopathies, require the expression of prion protein (PrP(C)) to propagate disease. PrP(C) is converted into an abnormal insoluble form, PrP(Sc), that gains neurotoxic activity. Conversely, clinical manifestations of prion disease may occur either before or in the absence of PrP(Sc) deposits, but the loss of normal PrP(C) function contribution for the etiology of these diseases is still debatable. Prion disease-associated mutations in PrP(C) represent one of the best models to understand the impact of PrP(C) loss-of-function. PrP(C) associates with various molecules and, in particular, the interaction of PrP(C) with laminin (Ln) modulates neuronal plasticity and memory formation. To assess the functional alterations associated with PrP(C) mutations, wild-type and mutated PrP(C) proteins were expressed in a neural cell line derived from a PrP(C)-null mouse. Treatment with the laminin γ1 chain peptide (Ln γ1), which mimics the Ln binding site for PrP(C), increased intracellular calcium in cells expressing wild-type PrP(C), whereas a significantly lower response was observed in cells expressing mutated PrP(C) molecules. The Ln γ1 did not promote process outgrowth or protect against staurosporine-induced cell death in cells expressing mutated PrP(C) molecules in contrast to cells expressing wild-type PrP(C). The co-expression of wild-type PrP(C) with mutated PrP(C) molecules was able to rescue the Ln protective effects, indicating the lack of negative dominance of PrP(C) mutated molecules. These results indicate that PrP(C) mutations impair process outgrowth and survival mediated by Ln γ1 peptide in neural cells, which may contribute to the pathogenesis of genetic prion diseases. PMID:23132868

  11. Potentiation of disease-associated cystic fibrosis transmembrane conductance regulator mutants by hydrolyzable ATP analogs.

    PubMed

    Miki, Haruna; Zhou, Zhen; Li, Min; Hwang, Tzyh-Chang; Bompadre, Silvia G

    2010-06-25

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the ATP-binding cassette transporter superfamily. CFTR is gated by ATP binding and hydrolysis at its two nucleotide-binding domains (NBDs), which dimerize in the presence of ATP to form two ATP-binding pockets (ABP1 and ABP2). Mutations reducing the activity of CFTR result in the genetic disease cystic fibrosis. Two of the most common mutations causing a severe phenotype are G551D and DeltaF508. Previously we found that the ATP analog N(6)-(2-phenylethyl)-ATP (P-ATP) potentiates the activity of G551D by approximately 7-fold. Here we show that 2'-deoxy-ATP (dATP), but not 3'-deoxy-ATP, increases the activity of G551D-CFTR by approximately 8-fold. We custom synthesized N(6)-(2-phenylethyl)-2'-deoxy-ATP (P-dATP), an analog combining the chemical modifications in dATP and P-ATP. This new analog enhances G551D current by 36.2 +/- 5.4-fold suggesting an independent but energetically additive action of these two different chemical modifications. We show that P-dATP binds to ABP1 to potentiate the activity of G551D, and mutations in both sides of ABP1 (W401G and S1347G) decrease its potentiation effect, suggesting that the action of P-dATP takes place at the interface of both NBDs. Interestingly, P-dATP completely rectified the gating abnormality of DeltaF508-CFTR by increasing its activity by 19.5 +/- 3.8-fold through binding to both ABPs. This result highlights the severity of the gating defect associated with DeltaF508, the most prevalent disease-associated mutation. The new analog P-dATP can be not only an invaluable tool to study CFTR gating, but it can also serve as a proof-of-principle that, by combining elements that potentiate the channel activity independently, the increase in chloride transport necessary to reach a therapeutic target is attainable. PMID:20406820

  12. Potentiation of Disease-associated Cystic Fibrosis Transmembrane Conductance Regulator Mutants by Hydrolyzable ATP Analogs*

    PubMed Central

    Miki, Haruna; Zhou, Zhen; Li, Min; Hwang, Tzyh-Chang; Bompadre, Silvia G.

    2010-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the ATP-binding cassette transporter superfamily. CFTR is gated by ATP binding and hydrolysis at its two nucleotide-binding domains (NBDs), which dimerize in the presence of ATP to form two ATP-binding pockets (ABP1 and ABP2). Mutations reducing the activity of CFTR result in the genetic disease cystic fibrosis. Two of the most common mutations causing a severe phenotype are G551D and ΔF508. Previously we found that the ATP analog N6-(2-phenylethyl)-ATP (P-ATP) potentiates the activity of G551D by ∼7-fold. Here we show that 2′-deoxy-ATP (dATP), but not 3′-deoxy-ATP, increases the activity of G551D-CFTR by ∼8-fold. We custom synthesized N6-(2-phenylethyl)-2′-deoxy-ATP (P-dATP), an analog combining the chemical modifications in dATP and P-ATP. This new analog enhances G551D current by 36.2 ± 5.4-fold suggesting an independent but energetically additive action of these two different chemical modifications. We show that P-dATP binds to ABP1 to potentiate the activity of G551D, and mutations in both sides of ABP1 (W401G and S1347G) decrease its potentiation effect, suggesting that the action of P-dATP takes place at the interface of both NBDs. Interestingly, P-dATP completely rectified the gating abnormality of ΔF508-CFTR by increasing its activity by 19.5 ± 3.8-fold through binding to both ABPs. This result highlights the severity of the gating defect associated with ΔF508, the most prevalent disease-associated mutation. The new analog P-dATP can be not only an invaluable tool to study CFTR gating, but it can also serve as a proof-of-principle that, by combining elements that potentiate the channel activity independently, the increase in chloride transport necessary to reach a therapeutic target is attainable. PMID:20406820

  13. Genotype × genotype interactions between the toxic cyanobacterium Microcystis and its grazer, the waterflea Daphnia

    PubMed Central

    Lemaire, Veerle; Brusciotti, Silvia; van Gremberghe, Ineke; Vyverman, Wim; Vanoverbeke, Joost; De Meester, Luc

    2012-01-01

    Toxic algal blooms are an important problem worldwide. The literature on toxic cyanobacteria blooms in inland waters reports widely divergent results on whether zooplankton can control cyanobacteria blooms or cyanobacteria suppress zooplankton by their toxins. Here we test whether this may be due to genotype × genotype interactions, in which interactions between the large-bodied and efficient grazer Daphnia and the widespread cyanobacterium Microcystis are not only dependent on Microcystis strain or Daphnia genotype but are specific to genotype × genotype combinations. We show that genotype × genotype interactions are important in explaining mortality in short-time exposures of Daphnia to Microcystis. These genotype × genotype interactions may result in local coadaptation and a geographic mosaic of coevolution. Genotype × genotype interactions can explain why the literature on zooplankton–cyanobacteria interactions is seemingly inconsistent, and provide hope that zooplankton can contribute to the suppression of cyanobacteria blooms in restoration projects. PMID:25568039

  14. Comparison of INNO-LiPA HPV Genotyping v2 with PCR product subcloning and sequencing for identification of genital human papillomavirus genotypes in African women.

    PubMed

    Didelot-Rousseau, Marie-Noëlle; Courgnaud, Valérie; Nagot, Nicolas; Ouedraogo, Abdoulaye; Konate, Issouf; Mayaud, Philippe; Weiss, Helen; Van de Perre, Philippe; Segondy, Michel

    2006-08-01

    The performance characteristics of the INNO-LiPA Genotyping v2 test for human papillomavirus (HPV) identification were assessed by comparing results with those obtained by PCR product sequencing after subcloning, in genital samples from 20 highly sexually exposed African women. The INNO-LiPA HPV Genotyping v2 test identified more HPV types than subcloning/sequencing (56 versus 37, respectively). Overall, 86.5% (32/37) of the HPV types identified by subcloning/sequencing were identified by the INNO-LiPA HPV Genotyping v2 test, whereas 57.1% (32/56) of the HPV types identified by the INNO-LiPA HPV Genotyping v2 test were identified by subcloning/sequencing. Of the 20 clinical samples tested, 7 had identical types detected under both methods and a further 11 had more types detected under INNO-LiPA HPV Genotyping v2 than subcloning/sequencing. Of the remaining two samples, the same number of types were detected under both methods, but different types were detected. INNO-LiPA HPV Genotyping v2 test appears as a valid method for identifying HPV subtypes in women with multiple HPV infection. PMID:16675035

  15. The Effect of Reference Panels and Software Tools on Genotype Imputation

    PubMed Central

    Nho, Kwangsik; Shen, Li; Kim, Sungeun; Swaminathan, Shanker; Risacher, Shannon L.; Saykin, Andrew J.

    2011-01-01

    Genotype imputation is increasingly employed in genome-wide association studies, particularly for integrative and cross-platform analysis. Several imputation algorithms use reference panels with a larger set of genotyped markers to infer genotypes at ungenotyped marker locations. Our objective was to assess which method and reference panel was more accurate when carrying out imputation. We investigated the influence of choice of two most popular imputation methods, IMPUTE and MACH, on two reference panels from the HapMap and the 1000 Genomes Project. Our results indicated that for the HapMap, MACH consistently yielded more accurate imputation results than IMPUTE, while for the 1000 Genomes Project, IMPUTE performed slightly better. The best imputation results were achieved by IMPUTE with the combined reference panel (HapMap + 1000 Genomes Project). IMPUTE with the combined reference panel is a promising strategy for genotype imputation, which should facilitate fine-mapping for discovery as well as known disease-associated candidate regions. PMID:22195161

  16. A literature search tool for intelligent extraction of disease-associated genes

    PubMed Central

    Jung, Jae-Yoon; DeLuca, Todd F; Nelson, Tristan H; Wall, Dennis P

    2014-01-01

    Objective To extract disorder-associated genes from the scientific literature in PubMed with greater sensitivity for literature-based support than existing methods. Methods We developed a PubMed query to retrieve disorder-related, original research articles. Then we applied a rule-based text-mining algorithm with keyword matching to extract target disorders, genes with significant results, and the type of study described by the article. Results We compared our resulting candidate disorder genes and supporting references with existing databases. We demonstrated that our candidate gene set covers nearly all genes in manually curated databases, and that the references supporting the disorder–gene link are more extensive and accurate than other general purpose gene-to-disorder association databases. Conclusions We implemented a novel publication search tool to find target articles, specifically focused on links between disorders and genotypes. Through comparison against gold-standard manually updated gene–disorder databases and comparison with automated databases of similar functionality we show that our tool can search through the entirety of PubMed to extract the main gene findings for human diseases rapidly and accurately. PMID:23999671

  17. EXCEPTIONAL AGGRESSIVENESS OF CEREBRAL CAVERNOUS MALFORMATION DISEASE ASSOCIATED WITH PDCD10 MUTATIONS

    PubMed Central

    Rebeiz, Tania; Stockton, Rebecca A.; McDonald, David A.; Mikati, Abdul Ghani; Zhang, Lingjiao; Austin, Cecilia; Akers, Amy L.; Gallione, Carol J.; Rorrer, Autumn; Gunel, Murat; Min, Wang; De Souza, Jorge Marcondes; Lee, Connie

    2014-01-01

    Purpose The phenotypic manifestations of cerebral cavernous malformation (CCM) disease caused by rare PDCD10 mutations have not been systematically examined, and a mechanistic link to Rho kinase (ROCK) mediated hyperpermeability, a potential therapeutic target, has not been established. Methods We analyze PDCD10-siRNA treated endothelial cells for stress fibers, ROCK activity and permeability. ROCK activity is assessed in CCM lesions. Brain permeability and CCM lesion burden is quantified, and clinical manifestations are assessed in prospectively enrolled subjects with PDCD10 mutations. Results We determine that PDCD10 protein suppresses endothelial stress fibers, ROCK activity and permeability in vitro. Pdcd10 heterozygous mice have greater lesion burden than other Ccm genotypes. We demonstrate robust ROCK activity in murine and human CCM vasculature, and increased brain vascular permeability in humans with PDCD10 mutation. Clinical phenotype is exceptionally aggressive compared to the more common KRIT1 and CCM2 familial and sporadic CCM, with greater lesion burden and more frequent hemorrhages earlier in life. We first report other phenotypic features including scoliosis, cognitive disability and skin lesions, unrelated to lesion burden or bleeding. Conclusion These findings define a unique CCM disease with exceptional aggressiveness, and they inform preclinical therapeutic testing, clinical counseling and the design of trials. PMID:25122144

  18. Characterization of functional methylomes by next-generation capture sequencing identifies novel disease-associated variants

    PubMed Central

    Allum, Fiona; Shao, Xiaojian; Guénard, Frédéric; Simon, Marie-Michelle; Busche, Stephan; Caron, Maxime; Lambourne, John; Lessard, Julie; Tandre, Karolina; Hedman, Åsa K.; Kwan, Tony; Ge, Bing; Rönnblom, Lars; McCarthy, Mark I.; Deloukas, Panos; Richmond, Todd; Burgess, Daniel; Spector, Timothy D.; Tchernof, André; Marceau, Simon; Lathrop, Mark; Vohl, Marie-Claude; Pastinen, Tomi; Grundberg, Elin; Ahmadi, Kourosh R.; Ainali, Chrysanthi; Barrett, Amy; Bataille, Veronique; Bell, Jordana T.; Buil, Alfonso; Dermitzakis, Emmanouil T.; Dimas, Antigone S.; Durbin, Richard; Glass, Daniel; Hassanali, Neelam; Ingle, Catherine; Knowles, David; Krestyaninova, Maria; Lindgren, Cecilia M.; Lowe, Christopher E.; Meduri, Eshwar; di Meglio, Paola; Min, Josine L.; Montgomery, Stephen B.; Nestle, Frank O.; Nica, Alexandra C.; Nisbet, James; O'Rahilly, Stephen; Parts, Leopold; Potter, Simon; Sandling, Johanna; Sekowska, Magdalena; Shin, So-Youn; Small, Kerrin S.; Soranzo, Nicole; Surdulescu, Gabriela; Travers, Mary E.; Tsaprouni, Loukia; Tsoka, Sophia; Wilk, Alicja; Yang, Tsun-Po; Zondervan, Krina T.

    2015-01-01

    Most genome-wide methylation studies (EWAS) of multifactorial disease traits use targeted arrays or enrichment methodologies preferentially covering CpG-dense regions, to characterize sufficiently large samples. To overcome this limitation, we present here a new customizable, cost-effective approach, methylC-capture sequencing (MCC-Seq), for sequencing functional methylomes, while simultaneously providing genetic variation information. To illustrate MCC-Seq, we use whole-genome bisulfite sequencing on adipose tissue (AT) samples and public databases to design AT-specific panels. We establish its efficiency for high-density interrogation of methylome variability by systematic comparisons with other approaches and demonstrate its applicability by identifying novel methylation variation within enhancers strongly correlated to plasma triglyceride and HDL-cholesterol, including at CD36. Our more comprehensive AT panel assesses tissue methylation and genotypes in parallel at ∼4 and ∼3 M sites, respectively. Our study demonstrates that MCC-Seq provides comparable accuracy to alternative approaches but enables more efficient cataloguing of functional and disease-relevant epigenetic and genetic variants for large-scale EWAS. PMID:26021296

  19. Overlapping Toxoplasma gondii Genotypes Circulating in Domestic Animals and Humans in Southeastern Brazil

    PubMed Central

    Silva, Letícia A.; Andrade, Renata O.; Carneiro, Ana Carolina A. V.; Vitor, Ricardo W. A.

    2014-01-01

    Although several Toxoplasma gondii genotyping studies have been performed in Brazil, studies of isolates from animals in the state of Minas Gerais are rare. The objective of this study was to conduct a genotypic characterization of T. gondii isolates obtained from dogs, free-range chickens, and humans in Minas Gerais and to verify whether the T. gondii genotypes circulating in domestic animals correspond to the genotypes detected in humans. Genetic variability was assessed by restricted fragment length polymorphism at 11 loci (SAG1, 5′+3′SAG2, SAG2 alt, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico). Twelve different genotypes were identified among the 24 isolates studied, including 8 previously identified genotypes and 4 new genotypes. The genetic relationship of the 24 T. gondii isolates, together with the genotypes previously described from 24 human newborns with congenital toxoplasmosis, revealed a high degree of similarity among the genotypes circulating in humans and animals in Minas Gerais. The most common genotypes among these species were BrII, BrIII, ToxoDB #108, and ToxoDB #206. Restricted fragment length polymorphism at the CS3 locus of these 48 isolates showed that the majority of isolates presented alleles I (50%) or II (27%). Isolates harboring allele III at the CS3 locus presented low virulence for mice, whereas those harboring alleles I or II presented higher virulence. These results confirm the utility of marker CS3 for predicting the virulence of Brazilian isolates of T. gondii in mice. No association was found between the allele type and clinical manifestations of human congenital toxoplasmosis. This is the first report of T. gondii genotyping that verifies the overlapping genotypes of T. gondii from humans and animals in the same geographic region of Brazil. Our results suggest that there is a common source of infection to the species studied, most likely oocysts contaminating the environment. PMID:24587295

  20. Pseudo-outbreak of pre-extensively drug-resistant (Pre-XDR) tuberculosis in Kinshasa: collateral damage caused by false detection of fluoroquinolone resistance by GenoType MTBDRsl.

    PubMed

    Kaswa, Michel K; Aloni, Muriel; Nkuku, Léontine; Bakoko, Brian; Lebeke, Rossin; Nzita, Albert; Muyembe, Jean Jacques; de Jong, Bouke C; de Rijk, Pim; Verhaegen, Jan; Boelaert, Marleen; Ieven, Margareta; Van Deun, Armand

    2014-08-01

    Fluoroquinolones are the core drugs for the management of multidrug-resistant tuberculosis (MDR-TB). Molecular drug susceptibility testing methods provide considerable advantages for scaling up programmatic management and surveillance of drug-resistant TB. We describe here the misidentification of fluoroquinolone resistance by the GenoType MTBDRsl (MTBDRsl) (Hain Lifescience GmbH, Nehren, Germany) line probe assay (LPA) encountered during a feasibility and validation study for the introduction of this rapid drug susceptibility test in Kinshasa, Democratic Republic of Congo. The double gyrA mutation 80Ala and 90Gly represented 57% of all fluoroquinolone mutations identified from MDR-TB patient sputum samples, as confirmed by DNA sequencing. This double mutation was previously found to be associated with susceptibility to fluoroquinolones, yet it leads to absent hybridization of a wild-type band in the MTBDRsl and is thus falsely scored as resistance. Our findings suggest that MTBDRsl results must be interpreted with caution when the interpretation is based solely on the absence of a wild-type band without confirmation by visualization of a mutant band. Performance of the MTBDRsl LPA might be improved by replacing the gyrA wild-type probes by additional probes specific for well-documented gyrA mutations that confer clinically relevant resistance. PMID:24871222

  1. A microsampling method for genotyping coral symbionts

    NASA Astrophysics Data System (ADS)

    Kemp, D. W.; Fitt, W. K.; Schmidt, G. W.

    2008-06-01

    Genotypic characterization of Symbiodinium symbionts in hard corals has routinely involved coring, or the removal of branches or a piece of the coral colony. These methods can potentially underestimate the complexity of the Symbiodinium community structure and may produce lesions. This study demonstrates that microscale sampling of individual coral polyps provided sufficient DNA for identifying zooxanthellae clades by RFLP analyses, and subclades through the use of PCR amplification of the ITS-2 region of rDNA and denaturing-gradient gel electrophoresis. Using this technique it was possible to detect distinct ITS-2 types of Symbiodinium from two or three adjacent coral polyps. These methods can be used to intensely sample coral-symbiont population/communities while causing minimal damage. The effectiveness and fine scale capabilities of these methods were demonstrated by sampling and identifying phylotypes of Symbiodinium clades A, B, and C that co-reside within a single Montastraea faveolata colony.

  2. Phthalocyanines as Molecular Scaffolds to Block Disease-Associated Protein Aggregation.

    PubMed

    Valiente-Gabioud, Ariel A; Miotto, Marco C; Chesta, María E; Lombardo, Verónica; Binolfi, Andres; Fernández, Claudio O

    2016-05-17

    amyloidogenic proteins. Analysis of the structure-activity relationship in phthalocyanines revealed that their anti-amyloid activity is highly dependent on the type of metal ion coordinated to the tetrapyrrolic system but is not sensitive to the number of peripheral charged substituents. The tendency of phthalocyanines to oligomerize (self-association) via aromatic-aromatic stacking interactions correlates precisely with their binding capabilities to target proteins and, more importantly, determines their efficiency as anti-amyloid agents. The ability to block different types of disease-associated protein aggregation raises the possibility that these cyclic tetrapyrrole compounds have a common mechanism of action to impair the formation of a variety of pathological aggregates. Because the structural and molecular basis for the anti-amyloid effects of these molecules is starting to emerge, combined efforts from the fields of structural, cellular, and animal biology will result critical for the rational design and discovery of new drugs for the treatment of amyloid related neurological disorders. PMID:27136297

  3. Diseases associated with hidranitis suppurativa: part 2 of a series on hidradenitis.

    PubMed

    Scheinfeld, Noah

    2013-06-01

    diseases is likely underreported. Pyoderma vegetans has been noted in 2 cases of HS and 4 cases of Inflammatory Bowel Disease (IBD) and is likely a clue to the linkage of the pathology of IBD and HS. Pityriasis rubra pilaris, in particular Type VI related to HIV, has a relationship more commonly with acne conglobata, but with HS also. Single case reports of diseases associated with HS include systemic lupus erythematosus, acromegaly, Down syndrome, Bazex-Dupre´-Christol, and prurtis ani, but these might be coincidences. Pyogenic Arthritis, Pyoderma gangrenosum, and Acne (PAPA Syndrome) and Pyoderma gangrenosum, Acne, and Suppurative Hidradenitis (PASH Syndrome) are pyodermic-arthritic syndromes that are associated with HS. Erythema nodosum and granulomatous lobular mastitis have been reported with HS but the significance of these reports is uncertain. Because of scarring, HS can result in lymphedema including scrotal elephantiasis and verrucous lymphedema. HS is sometimes accompanied by obesity, hypertension, and anemia and can be considered a disease in the spectrum of metabolic syndrome, a skin disease with systemic consequences. HS, like other types of chronic inflammation when long standing in the perianal and perineal areas, can result in squamous cell cancer. A variety of drugs can induce HS. These include lithium, sirolimus, cyclosporine, vemurafenib, and oral contraceptives. Inverse psoriasis or psoriasis vulgaris as a side effect of infliximab therapy may be associated with HS. These associations aside, most cases of HS occur in isolation without coincident morbidity. PMID:24011308

  4. Selection of common bean (Phaseolus vulgaris L.) genotypes using a genotype plus genotype x environment interaction biplot.

    PubMed

    Corrêa, A M; Teodoro, P E; Gonçalves, M C; Santos, A; Torres, F E

    2016-01-01

    Recently, the genotype plus genotype x environment interaction (GGE) biplot methodology has been used to investigate genotype x environment interactions in several crop species, but has not been applied to the common bean (Phaseolus vulgaris L.) crop in Brazil. The aim of this study was to identify common bean genotypes that exhibit high grain yield and stability in the State of Mato Grosso do Sul, Brazil. We conducted 12 trials from 2000 to 2006 in the municipalities of Aquidauana and Dourados, and evaluated 13 genotypes in a randomized block design with three replications. Grain yield data were subjected to individual and joint analyses of variance. After analyzing the GE interaction, the adaptability and phenotypic stability of the common bean genotypes were analyzed using GGE biplot methodology. The genotypes EMGOPA-201, Xamego, and Aporé are recommended for growing in Mato Grosso do Sul, because they exhibited high grain yield and phenotypic stability. PMID:27525915

  5. Distribution of HPV genotypes in Shanghai women

    PubMed Central

    Singh, Suman; Zhou, Qian; Yu, Yunyun; Xu, Xianghong; Huang, Xiaojie; Zhao, Junwei; Han, Lingfei; Wang, Kai; Sun, Jing; Li, Fang

    2015-01-01

    Objective: To study the Distribution of HPV genotypes in Shanghai women. Design: Cohort study. Setting: Shanghai First Maternity and Infant Hospital affiliated with Tongji University. Population: Patients those attended in the cervical disease diagnosis and treatment center of Shanghai First Maternity and Infant Hospital between January 2011 and December 2014. Methods: HPV GenoArray test kit (HybriBio Ltd) was used to perform HPV genotyping and was also used in DNA amplification and HybriBio’s proprietary flow-through hybridization technique. Results: In this study, total patients analyzed were 4585. Among 4585 sample the HPV positive patients were 1460 i.e. 31.84% in total. On the basis of pathological report normal were 1358, with inflammation 2441, with low grade lesion were 399, high grade lesion were 353, CIN were 19 and cervical carcinoma were 15. Among normal HPV positive were 215 (15.8%), among inflammation HPV positive were 735 (30.11%). HPV positive in low grade lesion were 353 i.e. 59.77%. In high grade lesion 211 were HPV positive among 272 (68.17%). The percentage of HPV positive was 73.68% i.e. 14 out of 19 patient in cervical carcinoma in situ. 13 patient out of 15 i.e. 86.67% of Cervical carcinoma were HPV positive. Among all percentage of HPV positive was high among cervical carcinoma then cervical carcinoma in situ then high grade lesion in decreasing fashion to low grade lesion and in normal. Highest prevalence i.e. 22.67% is of HPV 52 subtype and HPV 16 has second highest prevalence with 17.67% among HPV positive cases. Sensitivity of TCT detection is 71.6%. Specificity of TCT detection is 79.6%. Sensitivity of HPV-DNA detection is 65.2%. Specificity of HPV-DNA detection is 78.2%. Conclusion: HPV is one of major health concern in shanghai having high prevalence rate in comparison to other part of china and other part of world. This has implications for the future cervical cancer burden and the priority to be given to prevent cervical cancer in

  6. Current software for genotype imputation.

    PubMed

    Ellinghaus, David; Schreiber, Stefan; Franke, Andre; Nothnagel, Michael

    2009-07-01

    Genotype imputation for single nucleotide polymorphisms (SNPs) has been shown to be a powerful means to include genetic markers in exploratory genetic association studies without having to genotype them, and is becoming a standard procedure. A number of different software programs are available. In our experience, user-friendliness is often the deciding factor in the choice of software to solve a particular task. We therefore evaluated the usability of three publicly available imputation programs: BEAGLE, IMPUTE and MACH. We found all three programs to perform well with HapMap reference data, with little effort needed for data preparation and subsequent association analysis. Each of them has different strengths and weaknesses, however, and none is optimal for all situations. PMID:19706367

  7. Comparison of Hybribio GenoArray and Roche Human Papillomavirus (HPV) Linear Array for HPV Genotyping in Anal Swab Samples

    PubMed Central

    Silver, Michelle I.; Brown, Brandon J.; Leng, Chan Yoon; Blas, Magaly M.; Gravitt, Patti E.; Woo, Yin Ling

    2014-01-01

    Human papillomavirus (HPV) is causally associated with anal cancer, as HPV DNA is detected in up to 90% of anal intraepithelial neoplasias and anal cancers. With the gradual increase of anal cancer rates, there is a growing need to establish reliable and clinically relevant methods to detect anal cancer precursors. In resource-limited settings, HPV DNA detection is a potentially relevant tool for anal cancer screening. Here, we evaluated the performance of the Hybribio GenoArray (GA) for genotyping HPV in anal samples, against the reference standard Roche Linear Array (LA). Anal swab samples were obtained from sexually active men who have sex with men. Following DNA extraction, each sample was genotyped using GA and LA. The overall interassay agreement, type-specific, and single and multiple genotype agreements were evaluated by kappa statistics and McNemar's χ2 tests. Using GA and LA, 68% and 76% of samples were HPV DNA positive, respectively. There was substantial interassay agreements for the detection of all HPV genotypes (κ = 0.70, 86% agreement). Although LA was able to detect more genotypes per sample, the interassay agreement was acceptable (κ = 0.53, 63% agreement). GA had poorer specific detection of HPV genotypes 35, 42, and 51 (κ < 0.60). In conclusion, GA and LA showed good interassay agreement for the detection of most HPV genotypes in anal samples. However, the detection of HPV DNA in up to 76% of anal samples warrants further evaluation of its clinical significance. PMID:25502520

  8. Comparison of Hybribio GenoArray and Roche human papillomavirus (HPV) linear array for HPV genotyping in anal swab samples.

    PubMed

    Low, Huey Chi; Silver, Michelle I; Brown, Brandon J; Leng, Chan Yoon; Blas, Magaly M; Gravitt, Patti E; Woo, Yin Ling

    2015-02-01

    Human papillomavirus (HPV) is causally associated with anal cancer, as HPV DNA is detected in up to 90% of anal intraepithelial neoplasias and anal cancers. With the gradual increase of anal cancer rates, there is a growing need to establish reliable and clinically relevant methods to detect anal cancer precursors. In resource-limited settings, HPV DNA detection is a potentially relevant tool for anal cancer screening. Here, we evaluated the performance of the Hybribio GenoArray (GA) for genotyping HPV in anal samples, against the reference standard Roche Linear Array (LA). Anal swab samples were obtained from sexually active men who have sex with men. Following DNA extraction, each sample was genotyped using GA and LA. The overall interassay agreement, type-specific, and single and multiple genotype agreements were evaluated by kappa statistics and McNemar's χ(2) tests. Using GA and LA, 68% and 76% of samples were HPV DNA positive, respectively. There was substantial interassay agreements for the detection of all HPV genotypes (κ = 0.70, 86% agreement). Although LA was able to detect more genotypes per sample, the interassay agreement was acceptable (κ = 0.53, 63% agreement). GA had poorer specific detection of HPV genotypes 35, 42, and 51 (κ < 0.60). In conclusion, GA and LA showed good interassay agreement for the detection of most HPV genotypes in anal samples. However, the detection of HPV DNA in up to 76% of anal samples warrants further evaluation of its clinical significance. PMID:25502520

  9. Genotypic composition and the relationship between genotypic composition and geographical proximity of the cyanobacterium Microcystis aeruginosa in western Japan.

    PubMed

    Ohbayashi, Kako; Hodoki, Yoshikuni; Kobayashi, Yuki; Okuda, Noboru; Nakano, Shin-ichi

    2013-04-01

    Microcystis aeruginosa is one of the bloom-forming harmful algae in freshwater ecosystems. We genetically characterized Microcystis populations during bloom-forming periods in various reservoirs, lakes, and ponds in Japan during 2009. Using phylogenetic analysis, we evaluated the relationship between current genotype expansions and geographic location within western Japan and intraspecific variation. Microcystis aeruginosa colonies were isolated at 15 sites and were analyzed by sequencing the 16S-23S internal transcribed spacer (ITS) region of the ribosomal operon, and the potential to produce toxins was assessed by PCR-based detection of the microcystin synthetase gene mcyG. In total, 171 colonies were separated into 41 genotypes. The highest genotypic composition was detected in the south basin of Lake Biwa and the lowest in Lagoon Iba. Cluster analysis indicated no obvious association between genotypic composition and geographic distance. Thus, clear genetic differentiation accompanied by geographic origins was not found in western Japan. The resulting neighbor-joining tree revealed 3 clusters, 2 of which contained strains that showed both nonamplification and amplification of the mcyG gene. PMID:23586751

  10. A novel method for multiplex genotyping in a single reactor using GTPlex-PyroSeq: genotyping HPV as a prototype.

    PubMed

    Oh, Myungsok; Hoehn, Benjamin Douglass; Moon, Youngho; Oh, Taejeong; Ko, Youngbok; An, Sungwhan

    2012-07-01

    Herein, we describe a novel multiplex genotyping method, GTPlex-PyroSeq. This method consists of two phases: multiplex PCR followed by a single reaction of pyrosequencing. This study demonstrates how GTPlex-PyroSeq can be adapted for the determination of multiple human papillomavirus (HPV) genotypes. A biotinylated consensus primer, GP6+, and 15 high-risk HPV type-specific primers are used for multiplex PCR. Each type-specific primer has a 5'-tag unique ID sequence connected to a pyrosequencing primer binding region. The unique ID sequence is composed of three parts: i) a single nucleotide ID representing a specific genotype; ii) a sign post; and iii) an end mark. This design allows multiple genotype determination under an ID sequence-dependent nucleotide dispensation order during pyrosequencing. Following initial studies using HPV plasmids and cell lines, we evaluated the clinical utility and effectiveness by comparing our assay with direct sequencing and HPV DNA chip analysis of 80 samples from high-risk, HPV-positive patients. We found in single-type infections, 100% concordance with direct sequencing (70 of 80 perfect matches) and 97.5% concordance with HPV DNA chip data (50 of 80 perfect matches). Additionally, our system was superior to direct sequencing in detection of multiple infections (12 of 80), with a limit of detection of 100 copies. The scalability of this multiplex system, with its open-platform design and ability to use various sample types, makes the GTPlex applicable for use in multiple settings. PMID:22575716

  11. Host Specificity and Source of Enterocytozoon bieneusi Genotypes in a Drinking Source Watershed

    PubMed Central

    Guo, Yaqiong; Alderisio, Kerri A.; Yang, Wenli; Cama, Vitaliano; Xiao, Lihua

    2014-01-01

    To assess the host specificity of Enterocytozoon bieneusi and to track the sources of E. bieneusi contamination, we genotyped E. bieneusi in wildlife and stormwater from the watershed of New York City's source water, using ribosomal internal transcribed spacer (ITS)-based PCR and sequence analyses. A total of 255 specimens from 23 species of wild mammals and 67 samples from stormwater were analyzed. Seventy-four (29.0%) of the wildlife specimens and 39 (58.2%) of the stormwater samples from streams were PCR positive. Altogether, 20 E. bieneusi genotypes were found, including 8 known genotypes and 12 new ones. Sixteen and five of the genotypes were seen in animals and stormwater from the watershed, respectively, with WL4 being the most common genotype in both animals (35 samples) and stormwater (23 samples). The 20 E. bieneusi genotypes belonged to five genogroups (groups 1, 3, 4, and 7 and an outlier), with only 23/113 (20.4%) E. bieneusi-positive samples belonging to zoonotic genogroup 1 and 3/20 genotypes ever being detected in humans. The two genogroups previously considered host specific, groups 3 and 4, were both detected in multiple groups of mammals. Thus, with the exception of the type IV, Peru11, and D genotypes, which were detected in only 7, 5, and 2 animals, respectively, most E. bieneusi strains in most wildlife samples and all stormwater samples in the watershed had no known public health significance, as these types have not previously been detected in humans. The role of different species of wild mammals in the contribution of E. bieneusi contamination in stormwater was supported by determinations of host-adapted Cryptosporidium species/genotypes in the same water samples. Data from this study indicate that the host specificity of E. bieneusi group 3 is broader than originally thought, and wildlife is the main source of E. bieneusi in stormwater in the watershed. PMID:24141128

  12. A Visual Dual-Aptamer Logic Gate for Sensitive Discrimination of Prion Diseases-Associated Isoform with Reusable Magnetic Microparticles and Fluorescence Quantum Dots

    PubMed Central

    Xiao, Sai Jin; Hu, Ping Ping; Chen, Li Qiang; Zhen, Shu Jun; Peng, Li; Li, Yuan Fang; Huang, Cheng Zhi

    2013-01-01

    Molecular logic gates, which have attracted increasing research interest and are crucial for the development of molecular-scale computers, simplify the results of measurements and detections, leaving the diagnosis of disease either “yes” or “no”. Prion diseases are a group of fatal neurodegenerative disorders that happen in human and animals. The main problem with a diagnosis of prion diseases is how to sensitively and selectively discriminate and detection of the minute amount of PrPRes in biological samples. Our previous work had demonstrated that dual-aptamer strategy could achieve highly sensitive and selective discrimination and detection of prion protein (cellular prion protein, PrPC, and the diseases associated isoform, PrPRes) in serum and brain. Inspired by the advantages of molecular logic gate, we further conceived a new concept for dual-aptamer logic gate that responds to two chemical input signals (PrPC or PrPRes and Gdn-HCl) and generates a change in fluorescence intensity as the output signal. It was found that PrPRes performs the “OR” logic operation while PrPC performs “XOR” logic operation when they get through the gate consisted of aptamer modified reusable magnetic microparticles (MMPs-Apt1) and quantum dots (QDs-Apt2). The dual-aptamer logic gate simplifies the discrimination results of PrPRes, leaving the detection of PrPRes either “yes” or “no”. The development of OR logic gate based on dual-aptamer strategy and two chemical input signals (PrPRes and Gdn-HCl) is an important step toward the design of prion diseases diagnosis and therapy systems. PMID:23393552

  13. People of the British Isles: preliminary analysis of genotypes and surnames in a UK-control population

    PubMed Central

    Winney, Bruce; Boumertit, Abdelhamid; Day, Tammy; Davison, Dan; Echeta, Chikodi; Evseeva, Irina; Hutnik, Katarzyna; Leslie, Stephen; Nicodemus, Kristin; Royrvik, Ellen C; Tonks, Susan; Yang, Xiaofeng; Cheshire, James; Longley, Paul; Mateos, Pablo; Groom, Alexandra; Relton, Caroline; Bishop, D Tim; Black, Kathryn; Northwood, Emma; Parkinson, Louise; Frayling, Timothy M; Steele, Anna; Sampson, Julian R; King, Turi; Dixon, Ron; Middleton, Derek; Jennings, Barbara; Bowden, Rory; Donnelly, Peter; Bodmer, Walter

    2012-01-01

    There is a great deal of interest in a fine-scale population structure in the UK, both as a signature of historical immigration events and because of the effect population structure may have on disease association studies. Although population structure appears to have a minor impact on the current generation of genome-wide association studies, it is likely to have a significant part in the next generation of studies designed to search for rare variants. A powerful way of detecting such structure is to control and document carefully the provenance of the samples involved. In this study, we describe the collection of a cohort of rural UK samples (The People of the British Isles), aimed at providing a well-characterised UK-control population that can be used as a resource by the research community, as well as providing a fine-scale genetic information on the British population. So far, some 4000 samples have been collected, the majority of which fit the criteria of coming from a rural area and having all four grandparents from approximately the same area. Analysis of the first 3865 samples that have been geocoded indicates that 75% have a mean distance between grandparental places of birth of 37.3 km, and that about 70% of grandparental places of birth can be classed as rural. Preliminary genotyping of 1057 samples demonstrates the value of these samples for investigating a fine-scale population structure within the UK, and shows how this can be enhanced by the use of surnames. PMID:21829225

  14. Family history and apoE genotype interaction in Alzheimer`s disease (AD)

    SciTech Connect

    Jarvik, G.P.; Kukull, W.A.; Goddards, K.

    1994-09-01

    The apoE {epsilon}4 allele is associated with increased risk and decreased age of onset of AD. The {epsilon}4 allele may have opposing effects. We determined that family history of a parent or sib with memory problems (famhx+) modified the effect of apoE genotype in a population-based, case (n=165, 72 famhx+)-control (n=233, 73 famhx+) sample. Logistic regression analyses detected a significant apoE genotype (E) by family history (F) by age (A) interaction (ExFxA, p=0.003) and ExF interaction (p=0.0001) in the prediction of AD. ExFxA remained significant when only {epsilon}4+ genotypes were included (p<0.01). ExFxSex (p=0.04) and ExF (p<0.0001) were significant when only {epsilon}4- genotypes were included. Similary, multiple regression detected significant ExF interaction in the prediction of age of AD onset for {epsilon}4+ genotypes (p=0.04) or {epsilon}4- genotypes (p=0.04). Sex did not interact in the prediction of age of onset. Famhx+ increased risk of AD differentially and reduced age of onset except in {epsilon}2+ genotypes. Family history modifies the apoE genotype influence on risk and onset age of AD, suggesting that non-apoE genetic effects interact with apoE in AD. It is most predictive of risk in those with the {epsilon}2{epsilon}3 genotype. Variation in risk and onset among both {epsilon}4+ and {epsilon}4- genotypes demonstrate that {epsilon}2 and {epsilon}3 mediate {epsilon}4 allele effects in AD.

  15. Predicted redistribution of Ceratomyxa shasta genotypes with salmonid passage in the Deschutes River, Oregon.

    PubMed

    Stinson, Matthew E T; Bartholomew, Jerri L

    2012-12-01

    A series of dams on the Deschutes River, Oregon, act as migration barriers that segregate the river system into upper and lower basins. Proposed fish passage between basins would reunite populations of native potamodromous fish and allow anadromous fish of Deschutes River origin access to the upper basin. We assessed the potential redistribution of host-species-specific genotypes (O, I, II, III) of the myxozoan parasite Ceratomyxa shasta that could occur with fish passage and examined the influence of nonnative fish on genotype composition. To determine the present distribution of the parasite genotypes, we exposed eight salmonid species-three native and five stocked for sport fishing-in present and predicted anadromous salmonid habitats. We monitored fish for infection by C. shasta and sequenced a section of the parasite ribosomal DNA gene from fish and water samples to determine parasite genotype. Genotype O was present in both upper and lower basins and detected only in steelhead Oncorhynchus mykiss. Genotype I was spatially limited to the lower basin, isolated predominantly from Chinook salmon O. tshawytscha, and lethal for this species only. Genotype II was detected in both basins and in multiple species, but only as a minor component of the infection. Genotype III was also present in both basins, had a wide host range, and caused mortality in native steelhead and multiple nonnative species. Atlantic salmon Salmo salar and kokanee O. nerka were the least susceptible to infection by any genotype of C. shasta. Our findings confirmed the host-specific patterns of C. shasta infections and indicated that passage of Chinook salmon would probably spread genotype I into the upper Deschutes River basin, but with little risk to native salmonid populations. PMID:23146111

  16. APOE genotype alters immunoglobulin subtypes in knock-in mice

    PubMed Central

    Zhou, Ye; Zhao, Wenjuan; Al-muhtasib, Nour; Rebeck, G. William

    2016-01-01

    Apolipoprotein E (APOE) alleles are strongly related to the risk of Alzheimer’s disease (AD). APOE genotype also affects inflammatory processes in response to damage. We tested whether APOE genotype affected the levels of specific immunoglobulins in healthy, uninfected APOE knock-in mice. We measured specific immunoglobulins in brain, spleen and plasma. Levels of total IgG in brain and spleen were highest in APOE-ε3 mice, significantly higher than in APOE-ε2 and APOE-ε4 mice; no differences were observed for levels of total IgG plasma. We also measured specific subtypes of IgG. IgG1 was only detectable in plasma, and did not differ by APOE genotype. IgG3 was detectable in plasma and spleen, and also did not differ by APOE genotype. IgG2b showed the same pattern as levels of total IgG by APOE genotype, with the highest levels of IgG2b in brain, spleen, and plasma of APOE-ε3 mice. IgG2a showed an entirely different pattern, with significantly higher levels in spleen and plasma of APOE-ε4 mice compared to APOE-ε2 and APOE-ε3 mice. We also measured IgM and IgA in spleens and plasma of these mice. In spleen, APOE-ε4 mice had the lowest IgA levels and the highest levels of IgM; both being significantly different from APOE-ε2 mice. In total, murine IgG2a and IgM were highest in APOE-ε4 mice, while total IgG and Ig2b were highest in APOE-ε3 mice. These dramatically different distributions of immunoglobulins could allow for human AD risk biomarkers based on specific immunoglobulin subtypes. PMID:25737044

  17. [First case of hepatitis B virus genotype H infection in Turkey].

    PubMed

    Ural, Onur; Sayan, Murat; Akhan, Sıla; Sümer, Sua; Simşek, Funda

    2013-07-01

    Clinical studies reported from Turkey indicate that hepatitis B virus (HBV) genotype D is more prevalent than other genotypes. Epidemiological and clinical information on genotype H infection is currently limited. Genotype H infection is most likely due to its regional (Central and South America) prevalence throughout the world. The aim of this report is to present the first HBV genotype H infection in a chronic hepatitis B patient in Turkey. Laboratory findings of a 42 years old male patient admitted to our hospital revealed HBsAg (+), anti-HBs (-), HBeAg (-), anti-HBe (+), anti-HBc IgM (-), anti-HBc IgG (+), anti-HAV IgG (+), HBV-DNA: 5.689.776 IU/ml and high liver enzymes (ALT: 223 U/L, AST: 121 U/L). History of the patient indicated no risk factor (intravenous drug use, blood transfusion, suspicious sexual contact) related to HBV transmission. Since liver ultrasonography showed multiple hemangiomas, biopsy was performed and histologic activity index was found as 6/18 and fibrosis as 2/6, according to modified Knodell score system. HBV DNA isolated from the serum sample of the patient was amplified by polymerase chain reaction and polymerase gene segment of HBV was directly sequenced. UPGMA method was used for phylogenetic analysis, and the genotype of the virus was identified accordingly. The nucleotide sequence was compared to those from the international DNA data bank (GenBank). The genotyping of the patient revealed that the isolated HBV was genotype H. Treatment with tenofovir disoproxil fumarate was initiated and the patient responded to the treatment. This finding suggested that other HBV genotypes, except the predominant genotype D may also be in circulation in Turkey. In conclusion, detection of epidemiologic and molecular characteristics of HBV genotype H which is related to chronic hepatitis, seems to be necessary in order to better understand its circulation and progression around the world. PMID:23971934

  18. Rapid fluorescent lateral-flow immunoassay for hepatitis B virus genotyping.

    PubMed

    Song, Liu-Wei; Wang, Ying-Bin; Fang, Lin-Lin; Wu, Yong; Yang, Lin; Chen, Jie-Yu; Ge, Sheng-Xiang; Zhang, Jing; Xiong, You-Zheng; Deng, Xiu-Mei; Min, Xiao-Ping; Zhang, Jun; Chen, Pei-Jer; Yuan, Quan; Xia, Ning-Shao

    2015-01-01

    Hepatitis B virus (HBV) genotyping plays an important role in the clinical management of chronic hepatitis B (CHB) patients. However, the current nucleic acid based techniques are expensive, time-consuming, and inconvenient. Here, we developed a novel DNA-independent HBV genotyping tool based on a one-step fluorescent lateral flow immunoassay (LFIA). Epitope-targeting immunization and screening techniques were used to develop HBV genotype specific monoclonal antibodies (mAbs). These mAbs were used to develop a multitest LFIA with a matched scanning luminoscope for HBV genotyping (named the GT-LFIA). The performance of this novel assay was carefully evaluated in well-characterized clinical cohorts. The GT-LFIA, which can specifically differentiate HBV genotypes A, B, C, and D in a pretreatment-free single test, was successfully developed using four genotype specific mAbs. The detection limits of the GT-LFIA for HBV genotypes A, B, C, and D were 2.5-10.0 IU HBV surface antigen/mL, respectively. Among the sera from 456 CHB patients, 439 (96.3%; 95% confidence interval (CI), 94.1-97.8%) were genotype-differentiable by the GT-LFIA and 437 (99.5%; 95% CI, 98.4-99.9%) were consistent with viral genome sequencing. In the 21 patients receiving nucleos(t)ide analogue therapy, for end-of-treatment specimens that were HBV DNA undetectable and were not applicable for DNA-dependent genotyping, the GT-LFIA presented genotyping results that were consistent with those obtained in pretreatment specimens by viral genome sequencing and the GT-LFIA. In conclusion, the novel GT-LFIA is a convenient, fast, and reliable tool for differential HBV genotyping, especially in patients with low or undetectable HBV DNA levels. PMID:25892477

  19. Conformational modulation mediated by polyglutamine expansion in CAG repeat expansion disease-associated proteins.

    PubMed

    Verani, Margherita; Bustamante, Maria; Martufi, Paola; Daldin, Manuel; Cariulo, Cristina; Azzollini, Lucia; Fodale, Valentina; Puglisi, Francesca; Weiss, Andreas; Macdonald, Douglas; Petricca, Lara; Caricasole, Andrea

    2016-09-16

    We have previously reported TR-FRET based immunoassays to detect a conformational change imparted on huntingtin protein by the polyglutamine expansion, which we confirmed using biophysical methodologies. Using these immunoassays, we now report that polyglutamine expansion influences the conformational properties of other polyglutamine disease proteins, exemplified by the androgen receptor (associated with spinal bulbar muscular atrophy) and TATA binding protein (associated with spinocerebellar ataxia 17). Using artificial constructs bearing short or long polyglutamine expansions or a multimerized, unrelated epitope (mimicking the increase in anti-polyglutamine antibody epitopes present in polyglutamine repeats of increasing length) we confirmed that the conformational TR-FRET based immunoassay detects an intrinsic conformational property of polyglutamine repeats. The TR-FRET based conformational immunoassay may represent a rapid, scalable tool to identify modulators of polyglutamine-mediated conformational change in different proteins associated with CAG triplet repeat disorders. PMID:27520369

  20. Comparison of the Digene Hybrid Capture 2 Assay and Roche AMPLICOR and LINEAR ARRAY Human Papillomavirus (HPV) Tests in Detecting High-Risk HPV Genotypes in Specimens from Women with Previous Abnormal Pap Smear Results▿

    PubMed Central

    Stevens, Matthew P.; Garland, Suzanne M.; Rudland, Elice; Tan, Jeffrey; Quinn, Michael A.; Tabrizi, Sepehr N.

    2007-01-01

    The development of cervical cancer is strongly associated with the presence of persistent high-risk (HR) human papillomavirus (HPV) infection. Recently, the commercially manufactured PCR-based Roche AMPLICOR (AMP) and LINEAR ARRAY (LA) HPV tests have become available for HPV detection. However, knowledge of their clinical performance compared to the U.S. Food and Drug Administration-approved Hybrid Capture 2 (HC2) assay is limited. This study evaluated the concordance between the HC2, AMP, and LA tests in detecting HR-HPV among a cohort of 1,679 women with previous abnormal Pap smear results. Overall, 1,393 specimens (81.3%) generated concordant results for HR-HPV presence or absence by the three assays. The concordance levels were substantial between the HC2 and AMP tests (84.4%, κ = 0.6419) and between the HC2 and LA tests (84.0%, κ = 0.6341) and nearly perfect between the AMP and LA tests (97.8%, κ = 0.9441). HR-HPV prevalence, as detected by the AMP or LA tests, was significantly higher among women with cytological or histological high-grade disease (CIN2 or greater) than that detected by HC2 (P < 0.0001). The AMP and LA tests exhibited greater sensitivity, but lower specificity, than HC2 for detecting HR-HPV among this cohort of women with underlying cervical abnormalities, particularly among subjects with histologically proven high-grade disease. Both PCR-based HPV tests may be valuable in the management of care for women with underlying cervical abnormalities, in predicting treatment success, and in studying the clearance or acquisition of new infections. PMID:17494721

  1. Rapid and sensitive genotyping of hepatitis C virus by single-strand conformation polymorphism.

    PubMed

    Lareu, R R; Swanson, N R; Fox, S A

    1997-02-01

    There is an increasing demand for genotyping hepatitis C virus (HCV) isolates due to the rapidly expanding list of distinct HCV genotypes and the mounting evidence of genotype-specific clinical consequences. We describe an SSCP-based assay for determining genotypes in HCV infections. HCV RNA extracted from serum was amplified by a sensitive nested-PCR assay producing a 287 bp fragment of the conserved 5' non-coding region (NCR) and analysed by non-denaturing polyacrylamide gel electrophoresis. Following empirical optimisation of the SSCP assay we identified distinct conformation polymorphisms (characteristic band patterns) corresponding to types 1a, 1b, 2a, 2b, 2c, 3 and 4 found in the Western Australian population. Seventy-three HCV RNA-positive samples were used to evaluate the SSCP genotyping assay for accuracy and efficiency by comparison with the previously established genotyping methods of manual direct sequencing and dideoxy fingerprinting. SSCP genotyping was in concord with control methods while performing more rapidly and at a fraction of the cost. Moreover, SSCP detected two co-infected samples that were not shown by the control methods. The PCR-SSCP assay provides an accurate and rapid method for genotyping of HCV RNA-positive samples at the 5' NCR by type-specific sequence polymorphisms which is applicable to large-scale screening. PMID:9029525

  2. Distribution of HPV genotypes in cervical intraepithelial lesions and cervical cancer in Tanzanian women

    PubMed Central

    2011-01-01

    Background Infection with human papillomavirus (HPV) is associated with uterine cervical intraepithelial neoplasia (CIN) and invasive cancers (ICC). Approximately 80% of ICC cases are diagnosed in under-developed countries. Vaccine development relies on knowledge of HPV genotypes characteristic of LSIL, HSIL and cancer; however, these genotypes remain poorly characterized in many African countries. To contribute to the characterization of HPV genotypes in Northeastern Tanzania, we recruited 215 women from the Reproductive Health Clinic at Kilimanjaro Christian Medical Centre. Cervical scrapes and biopsies were obtained for cytology and HPV DNA detection. Results 79 out of 215 (36.7%) enrolled participants tested positive for HPV DNA, with a large proportion being multiple infections (74%). The prevalence of HPV infection increased with lesion grade (14% in controls, 67% in CIN1 cases and 88% in CIN2-3). Among ICC cases, 89% had detectable HPV. Overall, 31 HPV genotypes were detected; the three most common HPV genotypes among ICC were HPV16, 35 and 45. In addition to these genotypes, co-infection with HPV18, 31, 33, 52, 58, 68 and 82 was found in 91% of ICC. Among women with CIN2-3, HPV53, 58 and 84/83 were the most common. HPV35, 45, 53/58/59 were the most common among CIN1 cases. Conclusions In women with no evidence of cytological abnormalities, the most prevalent genotypes were HPV58 with HPV16, 35, 52, 66 and 73 occurring equally. Although numerical constraints limit inference, findings that 91% of ICC harbor only a small number of HPV genotypes suggests that prevention efforts including vaccine development or adjuvant screening should focus on these genotypes. PMID:22081870

  3. Intraspecies Genotype Variability of the Microsporidian Parasite Encephalitozoon hellem

    PubMed Central

    Haro, María; del Águila, Carmen; Fenoy, Soledad; Henriques-Gil, Nuno

    2003-01-01

    Seven isolates of Encephalitozoon hellem from human immunodeficiency virus-positive patients were genotyped through a series of markers: the internal transcribed spacer (ITS) of ribosomal DNA, the polar tube protein (PTP) gene, and two intergenic spacers (IGS-TH and IGS-HZ) whose polymorphism is newly reported. The genome markers were all analyzed at three levels: PCR amplification followed by polyacrylamide gel electrophoresis, single-strand conformation analysis (SSCA), and DNA sequencing. The polymorphisms detected involve insertions/deletions and point mutations. SSCA can distinguish any pair of sequences, even those differing by a single base pair. The different isolates studied fit into the previously described ITS genotype 1A, except one which seems to be a 2A derivative variant (2D). When PTP and the new markers IGS-TH and IGS-HZ were analyzed, most of the isolates displayed different genotypes, demonstrating that E. hellem has a strong intraspecies variability. A set of markers such as those used here may be very useful in genotyping of clinical samples and in the assessment of epidemiological relationships among E. hellem strains. PMID:12958242

  4. Bayesian inference of recent migration rates using multilocus genotypes.

    PubMed Central

    Wilson, Gregory A; Rannala, Bruce

    2003-01-01

    A new Bayesian method that uses individual multilocus genotypes to estimate rates of recent immigration (over the last several generations) among populations is presented. The method also estimates the posterior probability distributions of individual immigrant ancestries, population allele frequencies, population inbreeding coefficients, and other parameters of potential interest. The method is implemented in a computer program that relies on Markov chain Monte Carlo techniques to carry out the estimation of posterior probabilities. The program can be used with allozyme, microsatellite, RFLP, SNP, and other kinds of genotype data. We relax several assumptions of early methods for detecting recent immigrants, using genotype data; most significantly, we allow genotype frequencies to deviate from Hardy-Weinberg equilibrium proportions within populations. The program is demonstrated by applying it to two recently published microsatellite data sets for populations of the plant species Centaurea corymbosa and the gray wolf species Canis lupus. A computer simulation study suggests that the program can provide highly accurate estimates of migration rates and individual migrant ancestries, given sufficient genetic differentiation among populations and sufficient numbers of marker loci. PMID:12663554

  5. Genotype and ancestry modulate brain's DAT availability in healthy humans

    SciTech Connect

    Shumay, E.; Shumay, E.; Chen, J.; Fowler, J.S.; Volkow, N.D.

    2011-08-01

    The dopamine transporter (DAT) is a principal regulator of dopaminergic neurotransmission and its gene (the SLC6A3) is a strong biological candidate gene for various behavioral- and neurological disorders. Intense investigation of the link between the SLC6A3 polymorphisms and behavioral phenotypes yielded inconsistent and even contradictory results. Reliance on objective brain phenotype measures, for example, those afforded by brain imaging, might critically improve detection of DAT genotype-phenotype association. Here, we tested the relationship between the DAT brain availability and the SLC6A3 genotypes using an aggregate sample of 95 healthy participants of several imaging studies. These studies employed positron emission tomography (PET) with [{sup 11}C] cocaine wherein the DAT availability was estimated as Bmax/Kd; while the genotype values were obtained on two repeat polymorphisms - 3-UTR- and intron 8- VNTRs. The main findings are the following: (1) both polymorphisms analyzed as single genetic markers and in combination (haplotype) modulate DAT density in midbrain; (2) ethnic background and age influence the strength of these associations; and (3) age-related changes in DAT availability differ in the 3-UTR and intron8 - genotype groups.

  6. Automated genotyping of dinucleotide repeat markers

    SciTech Connect

    Perlin, M.W.; Hoffman, E.P. |

    1994-09-01

    The dinucleotide repeats (i.e., microsatellites) such as CA-repeats are a highly polymorphic, highly abundant class of PCR-amplifiable markers that have greatly streamlined genetic mapping experimentation. It is expected that over 30,000 such markers (including tri- and tetranucleotide repeats) will be characterized for routine use in the next few years. Since only size determination, and not sequencing, is required to determine alleles, in principle, dinucleotide repeat genotyping is easily performed on electrophoretic gels, and can be automated using DNA sequencers. Unfortunately, PCR stuttering with these markers generates not one band for each allele, but a pattern of bands. Since closely spaced alleles must be disambiguated by human scoring, this poses a key obstacle to full automation. We have developed methods that overcome this obstacle. Our model is that the observed data is generated by arithmetic superposition (i.e., convolution) of multiple allele patterns. By quantitatively measuring the size of each component band, and exploiting the unique stutter pattern associated with each marker, closely spaced alleles can be deconvolved; this unambiguously reconstructs the {open_quotes}true{close_quotes} allele bands, with stutter artifact removed. We used this approach in a system for automated diagnosis of (X-linked) Duchenne muscular dystrophy; four multiplexed CA-repeats within the dystrophin gene were assayed on a DNA sequencer. Our method accurately detected small variations in gel migration that shifted the allele size estimate. In 167 nonmutated alleles, 89% (149/167) showed no size variation, 9% (15/167) showed 1 bp variation, and 2% (3/167) showed 2 bp variation. We are currently developing a library of dinucleotide repeat patterns; together with our deconvolution methods, this library will enable fully automated genotyping of dinucleotide repeats from sizing data.

  7. Sex influences on the penetrance of HLA shared-epitope genotypes for rheumatoid arthritis

    SciTech Connect

    Meyer, J.M.

    1996-02-01

    The association between rheumatoid arthritis (RA) and HLA DRB1 alleles may arise through linkage disequilibrium with a disease locus or the direct involvement of HLA alleles in RA. In support of the latter possibility, the shared-epitope hypothesis has been postulated, stating that conformationally similar DR{beta} chains encoded by several DRB1 alleles confer disease susceptibility. To examine these alternative hypotheses of marker-disease association and to investigate gender differences in RA susceptibility, we analyzed the distributions of PCR-based DRB1 genotypes of 309 Caucasian RA patients and 283 Caucasian controls. Initially, the marker-association-segregation {chi}{sup 2} method was used to evaluate evidence for linkage disequilibrium and the direct involvement of markers DR4 Dw4, DR4 Dw14, and DR1 in RA susceptibility. Additional shared-epitope models that grouped DRB1 alleles into five classes (*0401, *0404/*0102, *0405/*0408/*0101, *1001, and all others) and postulated relationships between genotypes and RA susceptibility were also fitted to observed genotypic distributions by the method of minimal {chi}{sup 2}. For females, a linkage-disequilibrium model provided a good fit to the data, as did a shared-epitope model with RA most penetrant among individuals with the *0401, *0401 genotype. For males, the best model indicated highest RA penetrance among shared-epitope compound heterozygotes. Clinically, male RA patients had more subcutaneous nodules and greater use of slowly acting antirheumatic drugs, while female RA patients had earlier disease onset. This study therefore suggests that sex-related factors influence the RA penetrance associated with DRB1 shared-epitope genotypes and that DRB1 effects on RA prognosis and pathogenesis should be considered separately for men and women. 67 refs., 7 tabs.

  8. The prevalence and genotype diversity of Human Rotavirus A circulating in Thailand, 2011-2014.

    PubMed

    Chieochansin, Thaweesak; Vutithanachot, Viboonsak; Phumpholsup, Tikumporn; Posuwan, Nawarat; Theamboonlers, Apiradee; Poovorawan, Yong

    2016-01-01

    Human rotavirus A (RVA) is the major infectious virus causing acute watery diarrhea in children, especially those younger than 5 years of age, and is a major public health problem in Thailand. Outbreaks of this virus have been reported worldwide. Besides the common genotypes, unusual genotypes providing evidence of inter-species transmission have also been described. Therefore, the aim of this study was to investigate the prevalence and genotypes of RVA in Thailand. A total of 688 samples were collected from children who were hospitalized with acute diarrhea in Chumphae Hospital in Khon Kaen and Chulalongkorn Hospital in Bangkok. RVA was detected using one-step RT-PCR and the genotypes were evaluated by sequencing. Overall, 204 of the 688 samples (30%) were positive for RVA. Nine genotypes were identified: three common in humans (G1P[8] [53%], G2P[4] [18%], G3P[8] [12%]), one feline-like (G3P[9] [1%]), four porcine-like (G4P[6] [0.5%], G5P[6] [0.5%], G9P[8] [0.5%], G12P[6] [1.5%]), and one bovine-like (G8P[8] [13%]). The variation in virus genotypes and the animal-like genotypes detected in this study suggested that a high diversity of RVA types is circulating in the Thai population. Therefore, continuous molecular epidemiological monitoring of RVA is essential and has implications for the national vaccination program. PMID:26593177

  9. [Genotypic specificity of schizophrenic psychoses].

    PubMed

    Shakhmatova-Pavlova, I V; Gindilis, V M; Rokhlina, M L; Kozlova, I A

    1980-01-01

    A total of 610 probands with a disease manifestation in childhood, middle and old age and their families were examined by the clinico-genealogical method. The results allowed conclusions that (1) there is an undoubted genetic relationship between schizophrenia of childhood, middle and old age; and that (2) among the closest relatives in families of the probands there is no significant (in comparison to the general population) accumulation of non-schizophrenic pathology. The latter indicates a high genotypic specificity of schizophrenia. PMID:7415694

  10. Bulk Genotyping of Biopsies Can Create Spurious Evidence for Hetereogeneity in Mutation Content

    PubMed Central

    Kostadinov, Rumen; Maley, Carlo C.; Kuhner, Mary K.

    2016-01-01

    When multiple samples are taken from the neoplastic tissues of a single patient, it is natural to compare their mutation content. This is often done by bulk genotyping of whole biopsies, but the chance that a mutation will be detected in bulk genotyping depends on its local frequency in the sample. When the underlying mutation count per cell is equal, homogenous biopsies will have more high-frequency mutations, and thus more detectable mutations, than heterogeneous ones. Using simulations, we show that bulk genotyping of data simulated under a neutral model of somatic evolution generates strong spurious evidence for non-neutrality, because the pattern of tissue growth systematically generates differences in biopsy heterogeneity. Any experiment which compares mutation content across bulk-genotyped biopsies may therefore suggest mutation rate or selection intensity variation even when these forces are absent. We discuss computational and experimental approaches for resolving this problem. PMID:27105344

  11. Bulk Genotyping of Biopsies Can Create Spurious Evidence for Hetereogeneity in Mutation Content.

    PubMed

    Kostadinov, Rumen; Maley, Carlo C; Kuhner, Mary K

    2016-04-01

    When multiple samples are taken from the neoplastic tissues of a single patient, it is natural to compare their mutation content. This is often done by bulk genotyping of whole biopsies, but the chance that a mutation will be detected in bulk genotyping depends on its local frequency in the sample. When the underlying mutation count per cell is equal, homogenous biopsies will have more high-frequency mutations, and thus more detectable mutations, than heterogeneous ones. Using simulations, we show that bulk genotyping of data simulated under a neutral model of somatic evolution generates strong spurious evidence for non-neutrality, because the pattern of tissue growth systematically generates differences in biopsy heterogeneity. Any experiment which compares mutation content across bulk-genotyped biopsies may therefore suggest mutation rate or selection intensity variation even when these forces are absent. We discuss computational and experimental approaches for resolving this problem. PMID:27105344

  12. [Application of the PCR-APLP method to determine ABO genotypes in forensic samples].

    PubMed

    Watanabe, G; Umetsu, K; Osawa, M

    2000-08-01

    We carried out ABO genotyping of forensic samples by the amplified product length polymorphism (APLP) technique. We present two novel systems. One is termed as eight primers system, in which eight allele-specific primers are added into a single PCR reaction. Another is termed as six primers system. In both APLP systems, all alleles were clearly detected using DNA purified from forensic samples. In PCR amplification with direct addition of specimen, ABO genotyping was also possible to blood stain, seminal stain, blood, saliva and urine. Furthermore, ABO genotyping worked only to chimpanzee. This PCR-APLP method should be convepffnt and valuable for forensic practice. PMID:11060991

  13. Evidence for increased recombination near the human insulin gene: implication for disease association studies

    SciTech Connect

    Chakravarti, A.; Elbein, S.C.; Permutt, M.A.

    1986-02-01

    Haplotypes for four new restriction site polymorphisms (detected by Rsa I, Taq I, HincII, and Sac I) and a previously identified DNA length polymorphism (5'FP), all at the insulin locus, have been studied in US Blacks, African Blacks, Caucasians, and Pima Indians. Black populations are polymorphic for all five markers, whereas the other groups are polymorphic for Rsa I, Taq I, and 5'FP only. The data suggest that approx. = 1 in 550 base pairs is variant in this region. The polymorphisms, even though located within 20 kilobases, display low levels of nonrandom association. Population genetic analysis suggests that recombination within this 20-kilobase segment occurs 24 times more frequently than expected if crossing-over occurred uniformly throughout the human genome. These findings suggest that population association between DNA polymorphisms and disease susceptibility genes near the insulin gene or structural mutations in the insulin gene will be weak. Thus, population studies would probably require large sample sizes to detect association. However, the low levels of nonrandom association increase the information content of the locus for linkage studies, which is the best alternative for discovering disease susceptibility genes.

  14. Clinical Presentation Resembling Mucosal Disease Associated with 'HoBi'-like Pestivirus in a Field Outbreak.

    PubMed

    Weber, M N; Mósena, A C S; Simões, S V D; Almeida, L L; Pessoa, C R M; Budaszewski, R F; Silva, T R; Ridpath, J F; Riet-Correa, F; Driemeier, D; Canal, C W

    2016-02-01

    The genus Pestivirus of the family Flaviviridae consists of four recognized species: Bovine viral diarrhoea virus 1 (BVDV-1), Bovine viral diarrhoea virus 2 (BVDV-2), Classical swine fever virus (CSFV) and Border disease virus (BDV). Recently, atypical pestiviruses ('HoBi'-like pestiviruses) were identified in batches of contaminated foetal calf serum and in naturally infected cattle with and without clinical symptoms. Here, we describe the first report of a mucosal disease-like clinical presentation (MD) associated with a 'HoBi'-like pestivirus occurring in a cattle herd. The outbreak was investigated using immunohistochemistry, antibody detection, viral isolation and RT-PCR. The sequence and phylogenetic analysis of 5'NCR, N(pro) and E2 regions of the RT-PCR positive samples showed that four different 'HoBi'-like strains were circulating in the herd. The main clinical signs and lesions were observed in the respiratory and digestive systems, but skin lesions and corneal opacity were also observed. MD characteristic lesions and a pestivirus with cytopathic biotype were detected in one calf. The present study is the first report of a MD like presentation associated with natural infection with 'HoBi'-like pestivirus. This report describes the clinical signs and provides a pathologic framework of an outbreak associated with at least two different 'HoBi'-like strains. Based on these observations, it appears that these atypical pestiviruses are most likely underdiagnosed in Brazilian cattle. PMID:24735072

  15. The effects of female sex, viral genotype and IL28B genotype on spontaneous clearance of acute hepatitis C virus infection

    PubMed Central

    Grebely, Jason; Page, Kimberly; Sacks-Davis, Rachel; van der Loeff, Maarten Schim; Rice, Thomas M.; Bruneau, Julie; Morris, Meghan D.; Hajarizadeh, Behzad; Amin, Janaki; Cox, Andrea L.; Kim, Arthur Y.; McGovern, Barbara H.; Schinkel, Janke; George, Jacob; Shoukry, Naglaa H.; Lauer, Georg M.; Maher, Lisa; Lloyd, Andrew R.; Hellard, Margaret; Dore, Gregory J.; Prins, Maria

    2014-01-01

    Although 20–40% of persons with acute HCV infection demonstrate spontaneous clearance, the time-course and factors associated with clearance remain poorly understood. We investigated the time to spontaneous clearance and predictors among participants with acute HCV using Cox proportional hazards analyses. Data for this analysis were drawn from an international collaboration of nine prospective cohorts evaluating outcomes following acute HCV infection. Among 632 participants with acute HCV, 35% were female, 82% were Caucasian, 49% had IL28B CC genotype (rs12979860), 96% had injected drugs ever, 47% were infected with HCV genotype 1 and 5% had HIV co-infection. Twenty-eight percent were HCV antibody negative/RNA positive at the time of acute HCV detection (early acute HCV). During follow-up, spontaneous clearance occurred in 173 of 632 and at one year following infection, 25% (95%CI: 21%, 29%) had cleared virus. Among those with clearance, the median time to clearance was 16.5 weeks (IQR: 10.5, 33.4 weeks), with 34%, 67% and 83% demonstrating clearance at three, six and twelve months. Adjusting for age, factors independently associated with time to spontaneous clearance included female sex [adjusted hazards ratio (AHR) 2.16; 95%CI 1.48, 3.18], IL28B CC genotype (vs. CT/TT, AHR 2.26; 95%CI 1.52, 3.34), and HCV genotype 1 (vs. non-genotype 1, AHR 1.56; 95%CI 1.06, 2.30). The effect of IL28B genotype and HCV genotype on spontaneous clearance was greater among females compared to males. Conclusions Female sex, favorable IL28B genotype and HCV genotype 1 are independent predictors of spontaneous clearance. Further research is required to elucidate the observed sex-based differences in HCV control. PMID:23908124

  16. The location of a disease-associated polymorphism and genomic structure of the human 52-kDa Ro/SSA locus (SSA1)

    SciTech Connect

    Tsugu, H.; Horowitz, R.; Gibson, N.

    1994-12-01

    Sera from approximately 30% of patients with systemic lupus erythematosus (SLE) contain high titers of autoantibodies that bind to the 52-kDa Ro/SSA protein. We previously detected polymorphisms in the 52-kDa Ro/SSA gene (SSA1) with restriction enzymes, one of which is strongly associated with the presence of SLE (P < 0.0005) in African Americans. A higher disease frequency and more severe forms of the disease are commonly noted among these female patients. To determine the location and nature of this polymorphism, we obtained two clones that span 8.5 kb of the 52-kDa Ro/SSA locus including its upstream regulatory region. Six exons were identified, and their nucleotide sequences plus adjacent noncoding regions were determined. No differences were found between these exons and the coding region of one of the reported cDNAs. The disease-associated polymorphic site suggested by a restriction enzyme map and confirmed by DNA amplification and nucleotide sequencing was present upstream of exon 1. This polymorphism may be a genetic marker for a disease-related variation in the coding region for the protein or in the upstream regulatory region of this gene. Although this RFLP is present in Japanese, it is not associated with lupus in this race. 41 refs., 4 figs., 2 tabs.

  17. Disease-associated mutations in the extracytoplasmic loops of cystic fibrosis transmembrane conductance regulator do not impede biosynthetic processing but impair chloride channel stability.

    PubMed

    Hämmerle, M M; Aleksandrov, A A; Riordan, J R

    2001-05-01

    Consistent with its function as a chloride channel regulated entirely from the cytoplasmic side of the plasma membrane, the cystic fibrosis transmembrane conductance regulator (CFTR) glycoprotein exposes little of its mass on the exterior surface of cells. The first and fourth extracytoplasmic loops (ELs) contain approximately 15 and 30 residues, respectively; the other four ELs are extremely short. To examine the influence of missense mutants in ELs detected in patients with cystic fibrosis, we have expressed them in mammalian (baby hamster kidney (BHK21)) cells and assessed their biosynthetic processing and chloride channel activity. In contrast to previous findings that 18 of 30 disease-associated missense mutations in cytoplasmic loops caused retention of the nascent polypeptides in the endoplasmic reticulum, all the EL mutants studied matured and were transported to the cell surface. This pronounced asymmetry is consistent with the notion that endoplasmic reticulum quality control of nascent CFTR is exerted primarily on the cytoplasmic side of the membrane. Although this set of EL mutations has little effect on CFTR maturation, most of them seriously compromise its chloride channel activity. Substitutions at six different positions in EL1 and single positions in EL2 and EL4 all destabilized the open state, some of them severely, indicating that the ELs contribute to the stability of the CFTR ion pore. PMID:11278813

  18. Variation in the hyphal growth rate and the virulence of two genotypes of the crayfish plague organism Aphanomyces astaci.

    PubMed

    Viljamaa-Dirks, S; Heinikainen, S; Virtala, A-M K; Torssonen, H; Pelkonen, S

    2016-06-01

    Crayfish plague, a devastating disease of freshwater crayfish, is caused by an oomycete organism, Aphanomyces astaci. Currently five genotypes of A. astaci are known, but variable features between the strains or genotypes have not been studied extensively. This study analysed 28 isolates of the As genotype and 25 isolates of the Ps1 genotype and reveals that the radial growth rate is significantly (P < 0.001) different between these two genotypes, although highly variable inside the genotype As. Two Ps1 genotype isolates and two As genotype isolates with different radial growth rates were tested in an infection trial. Clear differences were detected in the development of mortality in the test groups. The representatives of the Ps1 genotype caused total mortality within a short time span. The As genotype isolates were much less virulent. The slow-growing As isolate showed higher virulence than the As isolate with a high growth capacity. Although slow growth could be one survival strategy of the pathogen, several other mechanisms are involved in the pathogenicity and warrant further studies. PMID:26332454

  19. MICB Allele Genotyping on Microarrays by Improving the Specificity of Extension Primers

    PubMed Central

    Baek, In-Cheol; Jang, Jung-Pil; Choi, Eun-Jeong; Kim, Tai-Gyu

    2015-01-01

    Major histocompatibility complex (MHC) class I chain-related gene B (MICB) encodes a ligand for activating NKG2D that expressed in natural killer cells, γδ T cells, and αβ CD8+ T cells, which is associated with autoimmune diseases, cancer, and infectious diseases. Here, we have established a system for genotyping MICB alleles using allele-specific primer extension (ASPE) on microarrays. Thirty-six high quality, allele-specific extension primers were evaluated using strict and reliable cut-off values using mean fluorescence intensity (MFI), whereby an MFI >30,000 represented a positive signal and an MFI <10,000 represented a negative signal. Eight allele-specific extension primers were found to be false positives, five of which were improved by adjusting their length, and three of which were optimized by refractory modification. The MICB alleles (*002:01, *003, *005:02/*010, *005:03, *008, *009N, *018, and *024) present in the quality control panel could be exactly defined by 22 allele-specific extension primers. MICB genotypes that were identified by ASPE on microarrays were in full concordance with those identified by PCR-sequence-based typing. In conclusion, we have developed a method for genotyping MICB alleles using ASPE on microarrays; which can be applicable for large-scale single nucleotide polymorphism typing studies of population and disease associations. PMID:26569110

  20. Phenotypic and genotypic detection of virulence factors of Staphylococcus aureus isolated from clinical and subclinical mastitis in cattle and water buffaloes from different farms of Sadat City in Egypt

    PubMed Central

    Elsayed, Mohamed Sabry; Mahmoud El-Bagoury, Abd Elrahman; Dawoud, Mai Abdallah

    2015-01-01

    Aim: To characterize Staphylococcus aureus from clinical and subclinical mastitis and identify virulence factors. Materials and Methods: Two hundred and two milk samples were collected, 143 from mastitic cattle and buffaloes 94 and 49, respectively, and 59 from apparently healthy cattle and buffaloes 35 and 24, respectively. Results: California mastitis test was applied and positive prevalence were 91.48% and 75.51% for cattle and buffalo with clinical mastitis and 37.14% and 45.83% for cattle and buffalo with subclinical mastitis. S. aureus was isolated from clinically mastitic cattle and buffaloes were 39.29% and 50%, respectively. While, from subclinical mastitic cattle and buffaloes were 80% and 72.73%, respectively. Hemolytic activity was assessed for S. aureus isolated from clinically and subclinical mastitic cases with prevalences of 100% and 56.25%, respectively. Thermo nuclease production from clinically and subclinical mastitic cases was 25% and 56.25%, respectively. Simplex polymerase chain reaction (PCR) conducted on S. aureus using 16S rRNA, clumping factor A, Panton valentine leukocidin, coagulase (Coa), alpha-hemolysin and beta-hemolysin those proved existence in 100%, 46.9%, 65.6%, 100%, 34.4%, and 43.75% of the isolates, respectively. While, multiplex PCR is utilized for detection of enterotoxins and proved that 12.5% was positive for enterotoxine Type D. Conclusions: It is concluded that simplex and multiplex PCR assays can be used as rapid and sensitive diagnostic tools to detect the presence of S. aureus and characterize its virulence factors that help in detection of severity of infection, distribution and stating preventive and control strategies. PMID:27047197

  1. Transmission of enteric disease associated with wastewater irrigation: a prospective epidemiological study.

    PubMed Central

    Shuval, H I; Wax, Y; Yekutiel, P; Fattal, B

    1989-01-01

    We conducted a prospective epidemiological study of possible enteric disease transmission by aerosolized pathogens from sprinkler irrigation of partially treated wastewater in 20 kibbutzim (collective agricultural settlements) in Israel between March 1981 and February 1982. Medical data were collected from the patients' files and daily logs of physicians and nurses at each kibbutz clinic (total population 10,231). Episodes of enteric disease were similar in the kibbutzim most exposed to wastewater aerosols (11.6 per 100 person-year) and the kibbutzim not exposed to wastewater in any form (11.0 per 100 person-year). No excess of enteric disease was seen among wastewater contact workers or their families as compared with the unexposed. No negative health effects were detected in this study which involved a large population, including many young children, exposed to treated wastewater aerosols generated at distances of 300-600 m. PMID:2735471

  2. Toward understanding MHC disease associations: partial resequencing of 46 distinct HLA haplotypes.

    PubMed

    Smith, Wade P; Vu, Quyen; Li, Shuying Sue; Hansen, John A; Zhao, Lue Ping; Geraghty, Daniel E

    2006-05-01

    We carried out a resequencing project that examined 552 kb of sequence from each of 46 individual HLA haplotypes representing a diversity of HLA allele types, generating nearly 27 Mb of fully phased genomic sequence. Haplotype blocks were defined extending from telomeric of HLA-F to centromeric of HLA-DP including in total 5186 MHC SNPs. To investigate basic questions about the evolutionary origin of common HLA haplotypes, and to obtain an estimate of rare variation in the MHC, we similarly examined two additional sets of samples. In 19 independent HLA-A1, B8, DR3 chromosomes, the most common HLA haplotype in Northern European Caucasians, variation was found at 11 SNP positions in the 3600-kb region from HLA-A to DR. Partial resequencing of 282 individuals in the gene-dense class III region identified significant variability beyond what could have been detected by linkage to common SNPs. PMID:16434165

  3. Periodontal-disease-associated biofilm: A reservoir for pathogens of medical importance.

    PubMed

    Vieira Colombo, Ana Paula; Magalhães, Clarissa Bichara; Hartenbach, Fátima Aparecida Rocha Resende; Martins do Souto, Renata; Maciel da Silva-Boghossian, Carina

    2016-05-01

    The ecological diversity of the periodontal microenvironment may provide suitable conditions for the colonization of species not usually considered members of the oral microbiota. In this investigation, we aimed to determine the prevalence and levels of pathogenic species of medical relevance in the microbiota of individuals with distinct periodontal clinical status. Subgingival biofilm was obtained from patients with periodontal health (H, n = 81), gingivitis (G, n = 55), generalized aggressive (AgP, n = 36) or chronic periodontitis (CP, n = 98), and analyzed for 39 microbial taxa using a checkerboard DNA-DNA hybridization technique. Microbial differences among groups, as well as associations between clinical and microbiological parameters were sought by non-parametric and univariate correlation tests. Neisseria spp., Peptostreptococus anaerobius, Candida albicans, enterobacteria, Pseudomonas aeruginosa, Eubacterium saphenum, Clostridium difficile and Olsenella uli were detected in high mean prevalence and counts in the subgingival microbiota of the study population. Species that were more related to periodontal inflammation and tissue destruction at the patient and site levels included enterobacteria, C. albicans, Neisseria spp., P. aeruginosa, O. uli, Hafnia alvei, Serratia marcescens and Filifactor alocis (p < 0.05). In contrast, Fusobacterium necrophorum, Lactobacillus acidophilus, Staphylococcus aureus and Streptococcus pneumoniae were associated with periodontal health (p < 0.05). Pathogenic species of medical importance may be detected in high prevalence and levels in the periodontal microbiota. Regardless of their role in periodontal health or disease, the periodontal biofilm may be a source for dissemination and development of systemic infections by these pathogenic microorganisms. PMID:26416306

  4. Molecular analysis of HLA-DQ A alleles in coeliac disease lack of a unique disease-associated sequence.

    PubMed Central

    Mantovani, V; Corazza, G R; Angelini, G; Delfino, L; Frisoni, M; Mirri, P; Valentini, R A; Barboni, P; Gasbarrini, G; Ferrara, G B

    1991-01-01

    Susceptibility to coeliac disease is strongly associated with some HLA class II antigens, encoded by the HLA-D region. Since the HLA-DQ locus seems to be primarily involved, we have analysed by polymerase chain reaction amplification and allele-specific oligonucleotide hybridization the most polymorphic region of the HLA-DQ A1 gene. No differen