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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 Several of the unpublished DBCRs defined candidate breakpoints for nail-patella, Prader-Willi, and Schmidt syndromes, ataxia, and ulna aplasia.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. 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

  10. 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...

  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. 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

  15. 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

  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.

    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.

  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.

    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.

  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. 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

  10. 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

  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. 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

  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. 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

  14. 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

  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 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

  5. 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

  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. 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

  6. 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

  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. 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...

  14. 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

  15. 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...

  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 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  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. 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

  13. 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

  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. 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

  19. 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

  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

    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

  11. 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

  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. 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

  17. 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

  18. 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.

  19. 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

  20. 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

  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. 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

  11. 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

  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 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

  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

    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

  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. 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.

  18. 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

  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. 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

  7. 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

  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. 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

  17. 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

  18. 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

  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. 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

  6. 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

  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

    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

  11. 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

  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. 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

  7. 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

  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. 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

  14. 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 ...

  15. 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: ...

  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. 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

  18. 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

  19. 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

  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