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Sample records for gonorrhoeae genotyping analysis

  1. Antimicrobial susceptibility and genotyping analysis of Hungarian Neisseria gonorrhoeae strains in 2013.

    PubMed

    Nemes-Nikodém, Éva; Brunner, Alexandra; Pintér, Dóra; Mihalik, Noémi; Lengyel, György; Marschalkó, Márta; Kárpáti, Sarolta; Szabó, Dóra; Ostorházi, Eszter

    2014-12-01

    Emergence and spread of antimicrobial resistance in Neisseria gonorrhoeae is a major public health concern worldwide. The current study aims to determine the antimicrobial resistance in N. gonorrhoeae and associated molecular typing to enhance gonococcal antimicrobial surveillance in Hungary. In the National N. gonorrhoeae Reference Laboratory of Hungary 187 N. gonorrhoeae infections were detected in 2013, antibiograms were determined for all the isolated strains, and 52 (one index strain from every sexually contact related group) of them were also analysed by the N. gonorrhoeae multi-antigen sequence typing (NG-MAST) method. Twenty-two different NG-MAST sequence types (STs) were identified, of which 8 STs had not been previously described. In Hungary, the highly diversified gonococcal population displayed high resistance to penicillin, ciprofloxacin and tetracycline (the antimicrobials previously recommended for gonorrhoea treatment). Resistance to the currently recommended extended spectrum cephalosporines were rare: only two of the expected strains, an ST 1407 and an ST 210, had cefixime MIC above the resistance breakpoint. By the revision of our National Treatment Guideline, it must be considered, that the azithromycin resistance is about 60% among the four most frequently isolated STs in Hungary.

  2. 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

  3. Genotyping as a tool for antibiotic resistance surveillance of Neisseria gonorrhoeae in New Caledonia: evidence of a novel genotype associated with reduced penicillin susceptibility.

    PubMed

    Vernel-Pauillac, Frédérique; Nandi, Sobhan; Nicholas, Robert A; Goarant, Cyrille

    2008-09-01

    Antibiotic resistance in Neisseria gonorrhoeae continues to be a major concern in public health. Resistance of N. gonorrhoeae bacteria to penicillin G is widespread in most developed countries, which has necessitated a change to newer drugs for treatment of gonococcal infections. Recent reports indicate that resistance to these newer drugs is increasing, highlighting the need for accurate therapeutic recommendations. In some countries or communities, however, N. gonorrhoeae isolates are still susceptible to penicillin, so the use of this antibiotic for single-dose treatments of medically under-resourced patients is beneficial. In order to evaluate the adequacy and sustainability of this treatment approach, we explored the presence and prevalence of chromosomally mediated resistance determinants in N. gonorrhoeae isolates collected from 2005 to 2007 in New Caledonia. We developed two new real-time PCR assays targeting the penB and mtrR determinants, to be used together with a previously described duplex assay targeting the penA and ponA determinants. The results of this study provided evidence that neither the most-common mtrR determinants nor the most-resistance-associated penB alleles are currently circulating in New Caledonia, suggesting that penicillin should still be considered a valuable treatment strategy. Additionally, using our genotyping assay, we observed an unexpected penB genotype at a relatively high frequency that was associated with a decreased susceptibility to penicillin (average MIC, 0.15 mug/ml). Sequencing revealed that this genotype corresponded to an A102S mutation in the penB gene. The molecular tools developed in this study can be used successfully for prospective epidemiological monitoring and surveillance of penicillin susceptibility. PMID:18591264

  4. Phenotypic and genotypic properties of Neisseria gonorrhoeae isolates in Norway in 2009: antimicrobial resistance warrants an immediate change in national management guidelines.

    PubMed

    Hjelmevoll, S O; Golparian, D; Dedi, L; Skutlaberg, D H; Haarr, E; Christensen, A; Jørgensen, S; Nilsen, Ø J; Unemo, M; Skogen, V

    2012-06-01

    Despite rapidly diminishing treatment options for Neisseria gonorrhoeae and high levels of ciprofloxacin resistance worldwide, Norwegian guidelines still recommend ciprofloxacin as empirical treatment for gonorrhea. The present study aimed to characterize phenotypical and genotypical properties of N. gonorrhoeae isolates in Norway in 2009. All viable N. gonorrhoeae isolates (n = 114) from six university hospitals in Norway (2009) were collected, representing 42% of all notified gonorrhea cases. Epidemiological data were collected from the Norwegian Surveillance System for Communicable Diseases and linked to phenotypical and genotypical characteristics for each N. gonorrhoeae isolate. Resistance levels to the antimicrobials examined were: ciprofloxacin 78%, azithromycin 11%, cefixime 3.5%, ceftriaxone 1.8%, and spectinomycin 0%. The minimum inhibitory concentrations of gentamicin varied from 1.5 to 8 mg/L. Forty-one (36%) of the isolates were β-lactamase-producing, 17 displayed penA mosaic alleles, and 72 different N. gonorrhoeae multiantigen sequence types (ST; 37 novel) were identified. The most common ST was ST1407 (n = 11), containing penA mosaic allele. Four of these isolates displayed intermediate susceptibility/resistance to cefixime. The N. gonorrhoeae strains circulating in Norway were highly diverse. The level of ciprofloxacin resistance was high and the Norwegian management guidelines should promptly exclude ciprofloxacin as an empirical treatment option for gonorrhea. PMID:21960034

  5. Transcriptional and functional analysis of the Neisseria gonorrhoeae fur regulon

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To ensure survival in the host, bacteria have evolved strategies to acquire the essential element iron. In Neisseria gonorrhoeae, the ferric uptake regulator senses intracellular iron stores and acting as a repressor, directly regulates transcription of iron-responsive genes by binding to a conserve...

  6. Analysis of the sociodemography of gonorrhoea in Leeds, 1989-93.

    PubMed Central

    Lacey, C. J.; Merrick, D. W.; Bensley, D. C.; Fairley, I.

    1997-01-01

    OBJECTIVE: To investigate the epidemiology of gonorrhoea in an urban area in the United Kingdom. DESIGN: Analysis of all cases of gonorrhoea with regard to age, sex, ethnic group, and socioeconomic group with 1991 census data as a denominator. SETTING: Leeds, a comparatively large urban area (population around 700,000) in the United Kingdom. SUBJECTS: All residents of Leeds with culture proved cases of gonorrhoea during 1989-95. MAIN OUTCOME MEASURE: Relative risk of gonorrhoea. RESULTS: Sex, age, race, and socioeconomic group and area of residence were all independently predictive of risk of infection. Young black men aged 20-29 were at highest risk, with incidences of 3-4% per year. Black subjects were 10 times more likely than white subjects to acquire infection, and subjects from the most deprived socioeconomic areas were more than four times more likely than those from the most affluent areas to acquire infection. CONCLUSIONS: Different ethnic and socioeconomic groups vary in their risk of infection with gonorrhoea within an urban area. Targeted interventions and screening to reduce the incidence of sexually transmitted disease are now priorities. PMID:9185496

  7. Functional analysis of the Gonococcal Genetic Island of Neisseria gonorrhoeae.

    PubMed

    Pachulec, Emilia; Siewering, Katja; Bender, Tobias; Heller, Eva-Maria; Salgado-Pabon, Wilmara; Schmoller, Shelly K; Woodhams, Katelynn L; Dillard, Joseph P; van der Does, Chris

    2014-01-01

    Neisseria gonorrhoeae is an obligate human pathogen that is responsible for the sexually-transmitted disease gonorrhea. N. gonorrhoeae encodes a T4SS within the Gonococcal Genetic Island (GGI), which secretes ssDNA directly into the external milieu. Type IV secretion systems (T4SSs) play a role in horizontal gene transfer and delivery of effector molecules into target cells. We demonstrate that GGI-like T4SSs are present in other β-proteobacteria, as well as in α- and γ-proteobacteria. Sequence comparison of GGI-like T4SSs reveals that the GGI-like T4SSs form a highly conserved unit that can be found located both on chromosomes and on plasmids. To better understand the mechanism of DNA secretion by N. gonorrhoeae, we performed mutagenesis of all genes encoded within the GGI, and studied the effects of these mutations on DNA secretion. We show that genes required for DNA secretion are encoded within the yaa-atlA and parA-parB regions, while genes encoded in the yfeB-exp1 region could be deleted without any effect on DNA secretion. Genes essential for DNA secretion are encoded within at least four different operons. PMID:25340397

  8. Antigenic analysis of Neisseria gonorrhoeae by crossed immunoelectrophoresis.

    PubMed

    Smyth, C J; Friedman-Kien, A E; Salton, M R

    1976-04-01

    Crossed immunoelectrophoresis was used to study two complex antigenic preparations from Neisseria gonorrhoeae, one of cytoplasmic origin and the other derived by Triton X-100 extraction of isolated washed gonococcal envelopes, with the aim of developing suitable reference antigen-antibody systems that could be subsequently used to investigate the immune response to gonococcal infection and to monitor envelope preparations for cytoplasmic contamination. A number of parameters were investigated to optimized and standardize antigen preparation, e.g., harvesting and washing of gonococci, methods of bacterial disruption, and washing of envelopes. The effects of Triton X-100 concentration, initial total envelope protein concentration, and the composition, pH, and concentration of buffer on cell envelope extractability were studied to obviate the need to concentrate material before use in crossed immunoelectrophoresis. The electroendoosmotic properties of agarose were a major determining factor in resolving envelope antigens. From 25 to 30 immunoprecipitates were revealed in the envelope antigen-antibody system; 75 to 80 were revealed in the cytoplasmic sytem. Envelope immunoprecipitates with reduced nicotinamide adenine dinucleotide and lactate dehydrogenase activities were identified. Crossed immunoelectrophoresis with intermediate gels revealed the presence of antibodies in a preimmune rabbit antiserum pool to a distinctive fact-moving component in both the envelope and cytoplasmic antigen preparations. The intermediate gel technique also demonstrated that extensive washing of envelope preparations with buffer did not remove cytoplasmic ontamination completely. The method provides a much more sensitive means of monitoring the purity of envelope fractions than the use of single enzy,e markers as indexes of such contamination. The use of rabbit antisera raised to formolized gonococci in intermediate gels indicated that both reference antigen-antibody systems were of

  9. Antimicrobial susceptibility and molecular epidemiology of Neisseria gonorrhoeae in Germany.

    PubMed

    Horn, Nicole Nari; Kresken, Michael; Körber-Irrgang, Barbara; Göttig, Stephan; Wichelhaus, Cornelia; Wichelhaus, Thomas A

    2014-07-01

    Antimicrobial drug resistance in Neisseria gonorrhoeae has become an increasing public health problem. Hence, surveillance of resistance development is of crucial importance to implement adequate treatment guidelines. Data on the spread of antibiotic resistance among gonococcal isolates in Germany, however, is scarce. In a resistance surveillance study conducted by the Paul Ehrlich Society for Chemotherapy between October 2010 and December 2011, 23 laboratories all over Germany were requested to send N. gonorrhoeae isolates to the study laboratory in Frankfurt am Main. Species verification was performed biochemically using ApiNH and with Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS). Antimicrobial susceptibility testing was performed using the Etest method. For molecular epidemiological analysis, N. gonorrhoeae strains were genotyped by means of N. gonorrhoeae multi-antigen sequence typing. A total of 213 consecutive gonococcal isolates were analyzed in this nationwide study. Applying EUCAST breakpoints, high resistance rates were found for ciprofloxacin (74%) and tetracycline (41%). Penicillin non-susceptibility was detected in 80% of isolates. The rate of azithromycin resistance was 6%, while all strains were susceptible to spectinomycin, cefixime, and ceftriaxone. Molecular typing of gonococcal isolates revealed a great heterogeneity of 99 different sequence types (ST), but ST1407 predominated (n=39). This is the first comprehensive German multi-centre surveillance study on antibiotic susceptibility and molecular epidemiology of N. gonorrhoeae with implications for antibiotic choice for treatment of gonorrhoea. The World Health Organization supports the concept that an efficacious treatment of gonorrhoea results in at least 95% of infections being cured. Accordingly, as spectinomycin is not available on the German market, only the third generation cephalosporins cefixime and ceftriaxone are regarded as valuable drugs

  10. Analysis of penicillinase-producing Neisseria gonorrhoeae isolates in Madrid (Spain) from 1983-85.

    PubMed Central

    Fenoll, A.; Berrón, S.; Vázquez, J. A.

    1987-01-01

    Between April 1983 and December 1985, 576 strains of Neisseria gonorrhoeae were isolated in our laboratory from patients attending Sexually Transmitted Diseases (STD) clinics. Of these, 61 (10.6%) were penicillinase-producing. Studies on these strains by plasmid analysis, auxotyping and serogrouping showed that the predominant type strains harboured the Asian resistance plasmid, were prototrophic, and were of serogroup W II/W III. About half of the strains, both of the African and Asian type, harboured the transfer plasmid. Strains of serogroup W II/W III were less sensitive to tetracycline and cefoxitin than serogroup W I strains. Images Fig. 2 PMID:2960555

  11. Tetrapac (tpc), a novel genotype of Neisseria gonorrhoeae affecting epithelial cell invasion, natural transformation competence and cell separation.

    PubMed

    Fussenegger, M; Kahrs, A F; Facius, D; Meyer, T F

    1996-03-01

    We characterized a novel mutant phenotype (tetrapac, tpc) of Neisseria gonorrhoeae (Ngo) associated with a distinctive rough-colony morphology and bacterial growth in clusters of four. This phenotype, suggesting a defect in cell division, was isolated from a mutant library of Ngo MS11 generated with the phoA minitransposon TnMax4. The tpc mutant shows a 30% reduction in the overall murein hydrolase activity using Escherichia coli murein as substrate. Tetrapacs can be resolved by co-cultivation with wild-type Ngo, indicating that Tpc is a diffusible protein. Interestingly, Tpc is absolutely required for the natural transformation competence of piliated Ngo. Mutants in tpc grow normally, but show a approximately 10-fold reduction in their ability to invade human epithelial cells. The tpc sequence reveals an open reading frame of approximately 1 kb encoding a protein (Tpc) of 37 kDa. The primary gene product exhibits an N-terminal leader sequence typical of lipoproteins, but palmitoylation of Tpc could not be demonstrated. The ribosomal binding site of tpc is immediately downstream of the translational stop codon of the folC gene coding for an enzyme involved in folic acid biosynthesis and one-carbon metabolism. The tpc gene is probably co-transcribed from the folC promoter and a promoter located within the folC gene. The latter promoter sequence shares significant homology with E. coli gearbox consensus promoters. All three mutant phenotypes, i.e. the cell separation defect, the transformation deficiency and the defect in cell invasion can be restored by complementation of the mutant with an intact tpc gene. To some extent the tcp phenotype is reminiscent of iap in Listeria, lytA in Streptococcus pneumoniae and lyt in Bacillus subtilis, all of which are considered to represent murein hydrolase defects.

  12. SNP genotyping by heteroduplex analysis.

    PubMed

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

    2015-01-01

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

  13. Analysis of the sex ratio of reported gonorrhoea incidence in Shenzhen, China

    PubMed Central

    Xiong, Mingzhou; Lan, Lina; Feng, Tiejian; Zhao, Guanglu; Wang, Feng; Hong, Fuchang; Wu, Xiaobing; Zhang, Chunlai; Wen, Lizhang; Liu, Aizhong; Best, John McCulloch; Tang, Weiming

    2016-01-01

    Objective To assess the clinical process of gonorrhoea diagnosis and report in China, and to determine the difference of sex ratio between reported incidence based on reporting data and true diagnosis rate based on reference tests of gonorrhoea. Setting A total of 26 dermatology and sexually transmitted disease (STD) departments, 34 obstetrics-gynaecology clinics and 28 urology outpatient clinics selected from 34 hospitals of Shenzhen regarded as our study sites. Participants A total of 2754 participants were recruited in this study, and 2534 participants completed the questionnaire survey and provided genital tract secretion specimens. There were 1106 male and 1428 female participants. Eligible participants were patients who presented for outpatient STD care at the selected clinics for the first time in October 2012 were at least 18 years old, and were able to give informed consent. Outcome measures Untested rate, true-positive rate, false-negative rate and unreported rate of gonorrhoea, as well as reported gonorrhoea incidence sex ratio and true diagnosis sex ratio were calculated and used to describe the results. Results 2534 participants were enrolled in the study. The untested rate of gonorrhoea among females was significantly higher than that among males (female 88.1%, male 68.3%, p=0.001). The male-to-female sex ratios of untested rate, true-positive rate, false-negative rate and unreported rate were 1:1.3, 1.2:1, 1:1.6 and 1:1.4, respectively. The reported gonorrhoea incidence sex ratio of new diagnosed gonorrhoea was 19.8:1 (male vs female: 87/1106 vs 5/1420), while the true diagnosis sex ratio was 2.5:1 (male vs female: 161/1106 vs 84/1420). These data indicate that the sex ratio of reported gonorrhoea incidence has been overestimated by a factor of 7.9 (19.8/2.5). Conclusions We found the current reported gonorrhoea incidence and sex ratios to be inaccurate due to underestimations of gonorrhoea incidence, especially among women. PMID:26975933

  14. Multiplex Real-Time PCR Assay with High-Resolution Melting Analysis for Characterization of Antimicrobial Resistance in Neisseria gonorrhoeae.

    PubMed

    Donà, Valentina; Kasraian, Sara; Lupo, Agnese; Guilarte, Yuvia N; Hauser, Christoph; Furrer, Hansjakob; Unemo, Magnus; Low, Nicola; Endimiani, Andrea

    2016-08-01

    Resistance to antibiotics used against Neisseria gonorrhoeae infections is a major public health concern. Antimicrobial resistance (AMR) testing relies on time-consuming culture-based methods. Development of rapid molecular tests for detection of AMR determinants could provide valuable tools for surveillance and epidemiological studies and for informing individual case management. We developed a fast (<1.5-h) SYBR green-based real-time PCR method with high-resolution melting (HRM) analysis. One triplex and three duplex reactions included two sequences for N. gonorrhoeae identification and seven determinants of resistance to extended-spectrum cephalosporins (ESCs), azithromycin, ciprofloxacin, and spectinomycin. The method was validated by testing 39 previously fully characterized N. gonorrhoeae strains, 19 commensal Neisseria species strains, and an additional panel of 193 gonococcal isolates. Results were compared with results of culture-based AMR determination. The assay correctly identified N. gonorrhoeae and the presence or absence of the seven AMR determinants. There was some cross-reactivity with nongonococcal Neisseria species, and the detection limit was 10(3) to 10(4) genomic DNA (gDNA) copies/reaction. Overall, the platform accurately detected resistance to ciprofloxacin (sensitivity and specificity, 100%), ceftriaxone (sensitivity, 100%; specificity, 90%), cefixime (sensitivity, 92%; specificity, 94%), azithromycin (sensitivity and specificity, 100%), and spectinomycin (sensitivity and specificity, 100%). In conclusion, our methodology accurately detects mutations that generate resistance to antibiotics used to treat gonorrhea. Low assay sensitivity prevents direct diagnostic testing of clinical specimens, but this method can be used to screen collections of gonococcal isolates for AMR more quickly than current culture-based AMR testing. PMID:27225407

  15. Multiplex Real-Time PCR Assay with High-Resolution Melting Analysis for Characterization of Antimicrobial Resistance in Neisseria gonorrhoeae.

    PubMed

    Donà, Valentina; Kasraian, Sara; Lupo, Agnese; Guilarte, Yuvia N; Hauser, Christoph; Furrer, Hansjakob; Unemo, Magnus; Low, Nicola; Endimiani, Andrea

    2016-08-01

    Resistance to antibiotics used against Neisseria gonorrhoeae infections is a major public health concern. Antimicrobial resistance (AMR) testing relies on time-consuming culture-based methods. Development of rapid molecular tests for detection of AMR determinants could provide valuable tools for surveillance and epidemiological studies and for informing individual case management. We developed a fast (<1.5-h) SYBR green-based real-time PCR method with high-resolution melting (HRM) analysis. One triplex and three duplex reactions included two sequences for N. gonorrhoeae identification and seven determinants of resistance to extended-spectrum cephalosporins (ESCs), azithromycin, ciprofloxacin, and spectinomycin. The method was validated by testing 39 previously fully characterized N. gonorrhoeae strains, 19 commensal Neisseria species strains, and an additional panel of 193 gonococcal isolates. Results were compared with results of culture-based AMR determination. The assay correctly identified N. gonorrhoeae and the presence or absence of the seven AMR determinants. There was some cross-reactivity with nongonococcal Neisseria species, and the detection limit was 10(3) to 10(4) genomic DNA (gDNA) copies/reaction. Overall, the platform accurately detected resistance to ciprofloxacin (sensitivity and specificity, 100%), ceftriaxone (sensitivity, 100%; specificity, 90%), cefixime (sensitivity, 92%; specificity, 94%), azithromycin (sensitivity and specificity, 100%), and spectinomycin (sensitivity and specificity, 100%). In conclusion, our methodology accurately detects mutations that generate resistance to antibiotics used to treat gonorrhea. Low assay sensitivity prevents direct diagnostic testing of clinical specimens, but this method can be used to screen collections of gonococcal isolates for AMR more quickly than current culture-based AMR testing.

  16. Transcript analysis of nrrF, a Fur repressed sRNA of Neisseria gonorrhoeae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Like most microorganisms, Neisseria gonorrhoeae alters gene expression in response to iron availability. The ferric uptake regulator Fur has been shown to be involved in controlling this response, but the extent of this involvement remains unknown. It is known that in addition to working directly to...

  17. Crystallization and preliminary X-ray diffraction analysis of the multidrug efflux transporter NorM from Neisseria gonorrhoeae

    SciTech Connect

    Su, Chih-Chia; Long, Feng; McDermott, Gerry; Shafer, William M.; Yu, Edward W.

    2008-04-01

    The multidrug efflux transporter NorM from N. gonorrhoeae has been crystallized and X-ray diffraction data have been collected to a resolution of 6.5 Å. The crystallization and preliminary X-ray data analysis of the NorM multidrug efflux pump produced by Neisseria gonorrhoeae are reported. NorM is a cytoplasmic membrane protein that consists of 459 amino-acid residues. It is a member of the recently classified multidrug and toxic compound extrusion (MATE) family of transporters and recognizes a number of cationic toxic compounds such as ethidium bromide, acriflavin, 2-N-methylellipticinium and ciprofloxacin. Recombinant NorM protein was expressed in Escherichia coli and purified by metal-affinity and gel-filtration chromatography. The protein was crystallized using hanging-drop vapor diffusion. X-ray diffraction data were collected from cryocooled crystals at a synchrotron light source. The best crystal diffracted anisotropically to 3.8 Å and diffraction data were complete to 6.5 Å resolution. The space group was determined to be C2, with unit-cell parameters a = 81.5, b = 164.4, c = 111.5 Å.

  18. Genetic Resistance Determinants, In Vitro Time-Kill Curve Analysis and Pharmacodynamic Functions for the Novel Topoisomerase II Inhibitor ETX0914 (AZD0914) in Neisseria gonorrhoeae.

    PubMed

    Foerster, Sunniva; Golparian, Daniel; Jacobsson, Susanne; Hathaway, Lucy J; Low, Nicola; Shafer, William M; Althaus, Christian L; Unemo, Magnus

    2015-01-01

    Resistance in Neisseria gonorrhoeae to all available therapeutic antimicrobials has emerged and new efficacious drugs for treatment of gonorrhea are essential. The topoisomerase II inhibitor ETX0914 (also known as AZD0914) is a new spiropyrimidinetrione antimicrobial that has different mechanisms of action from all previous and current gonorrhea treatment options. In this study, the N. gonorrhoeae resistance determinants for ETX0914 were further described and the effects of ETX0914 on the growth of N. gonorrhoeae (ETX0914 wild type, single step selected resistant mutants, and efflux pump mutants) were examined in a novel in vitro time-kill curve analysis to estimate pharmacodynamic parameters of the new antimicrobial. For comparison, ciprofloxacin, azithromycin, ceftriaxone, and tetracycline were also examined (separately and in combination with ETX0914). ETX0914 was rapidly bactericidal for all wild type strains and had similar pharmacodynamic properties to ciprofloxacin. All selected resistant mutants contained mutations in amino acid codons D429 or K450 of GyrB and inactivation of the MtrCDE efflux pump fully restored the susceptibility to ETX0914. ETX0914 alone and in combination with azithromycin and ceftriaxone was highly effective against N. gonorrhoeae and synergistic interaction with ciprofloxacin, particularly for ETX0914-resistant mutants, was found. ETX0914, monotherapy or in combination with azithromycin (to cover additional sexually transmitted infections), should be considered for phase III clinical trials and future gonorrhea treatment.

  19. Genetic Resistance Determinants, In Vitro Time-Kill Curve Analysis and Pharmacodynamic Functions for the Novel Topoisomerase II Inhibitor ETX0914 (AZD0914) in Neisseria gonorrhoeae

    PubMed Central

    Foerster, Sunniva; Golparian, Daniel; Jacobsson, Susanne; Hathaway, Lucy J.; Low, Nicola; Shafer, William M.; Althaus, Christian L.; Unemo, Magnus

    2015-01-01

    Resistance in Neisseria gonorrhoeae to all available therapeutic antimicrobials has emerged and new efficacious drugs for treatment of gonorrhea are essential. The topoisomerase II inhibitor ETX0914 (also known as AZD0914) is a new spiropyrimidinetrione antimicrobial that has different mechanisms of action from all previous and current gonorrhea treatment options. In this study, the N. gonorrhoeae resistance determinants for ETX0914 were further described and the effects of ETX0914 on the growth of N. gonorrhoeae (ETX0914 wild type, single step selected resistant mutants, and efflux pump mutants) were examined in a novel in vitro time-kill curve analysis to estimate pharmacodynamic parameters of the new antimicrobial. For comparison, ciprofloxacin, azithromycin, ceftriaxone, and tetracycline were also examined (separately and in combination with ETX0914). ETX0914 was rapidly bactericidal for all wild type strains and had similar pharmacodynamic properties to ciprofloxacin. All selected resistant mutants contained mutations in amino acid codons D429 or K450 of GyrB and inactivation of the MtrCDE efflux pump fully restored the susceptibility to ETX0914. ETX0914 alone and in combination with azithromycin and ceftriaxone was highly effective against N. gonorrhoeae and synergistic interaction with ciprofloxacin, particularly for ETX0914-resistant mutants, was found. ETX0914, monotherapy or in combination with azithromycin (to cover additional sexually transmitted infections), should be considered for phase III clinical trials and future gonorrhea treatment. PMID:26696986

  20. Genetic Resistance Determinants, In Vitro Time-Kill Curve Analysis and Pharmacodynamic Functions for the Novel Topoisomerase II Inhibitor ETX0914 (AZD0914) in Neisseria gonorrhoeae.

    PubMed

    Foerster, Sunniva; Golparian, Daniel; Jacobsson, Susanne; Hathaway, Lucy J; Low, Nicola; Shafer, William M; Althaus, Christian L; Unemo, Magnus

    2015-01-01

    Resistance in Neisseria gonorrhoeae to all available therapeutic antimicrobials has emerged and new efficacious drugs for treatment of gonorrhea are essential. The topoisomerase II inhibitor ETX0914 (also known as AZD0914) is a new spiropyrimidinetrione antimicrobial that has different mechanisms of action from all previous and current gonorrhea treatment options. In this study, the N. gonorrhoeae resistance determinants for ETX0914 were further described and the effects of ETX0914 on the growth of N. gonorrhoeae (ETX0914 wild type, single step selected resistant mutants, and efflux pump mutants) were examined in a novel in vitro time-kill curve analysis to estimate pharmacodynamic parameters of the new antimicrobial. For comparison, ciprofloxacin, azithromycin, ceftriaxone, and tetracycline were also examined (separately and in combination with ETX0914). ETX0914 was rapidly bactericidal for all wild type strains and had similar pharmacodynamic properties to ciprofloxacin. All selected resistant mutants contained mutations in amino acid codons D429 or K450 of GyrB and inactivation of the MtrCDE efflux pump fully restored the susceptibility to ETX0914. ETX0914 alone and in combination with azithromycin and ceftriaxone was highly effective against N. gonorrhoeae and synergistic interaction with ciprofloxacin, particularly for ETX0914-resistant mutants, was found. ETX0914, monotherapy or in combination with azithromycin (to cover additional sexually transmitted infections), should be considered for phase III clinical trials and future gonorrhea treatment. PMID:26696986

  1. Multivariate Analysis of Genotype-Phenotype Association.

    PubMed

    Mitteroecker, Philipp; Cheverud, James M; Pavlicev, Mihaela

    2016-04-01

    With the advent of modern imaging and measurement technology, complex phenotypes are increasingly represented by large numbers of measurements, which may not bear biological meaning one by one. For such multivariate phenotypes, studying the pairwise associations between all measurements and all alleles is highly inefficient and prevents insight into the genetic pattern underlying the observed phenotypes. We present a new method for identifying patterns of allelic variation (genetic latent variables) that are maximally associated-in terms of effect size-with patterns of phenotypic variation (phenotypic latent variables). This multivariate genotype-phenotype mapping (MGP) separates phenotypic features under strong genetic control from less genetically determined features and thus permits an analysis of the multivariate structure of genotype-phenotype association, including its dimensionality and the clustering of genetic and phenotypic variables within this association. Different variants of MGP maximize different measures of genotype-phenotype association: genetic effect, genetic variance, or heritability. In an application to a mouse sample, scored for 353 SNPs and 11 phenotypic traits, the first dimension of genetic and phenotypic latent variables accounted for >70% of genetic variation present in all 11 measurements; 43% of variation in this phenotypic pattern was explained by the corresponding genetic latent variable. The first three dimensions together sufficed to account for almost 90% of genetic variation in the measurements and for all the interpretable genotype-phenotype association. Each dimension can be tested as a whole against the hypothesis of no association, thereby reducing the number of statistical tests from 7766 to 3-the maximal number of meaningful independent tests. Important alleles can be selected based on their effect size (additive or nonadditive effect on the phenotypic latent variable). This low dimensionality of the genotype-phenotype map

  2. Crystallization and preliminary X-ray diffraction analysis of the multidrug efflux transporter NorM from Neisseria gonorrhoeae

    SciTech Connect

    Su, C.C.; Long, F.; McDermott, G.; Shafer, W.M.; Yu, E.W.

    2008-06-03

    The crystallization and preliminary X-ray data analysis of the NorM multidrug efflux pump produced by Neisseria gonorrhoeae are reported. NorM is a cytoplasmic membrane protein that consists of 459 amino-acid residues. It is a member of the recently classified multidrug and toxic compound extrusion (MATE) family of transporters and recognizes a number of cationic toxic compounds such as ethidium bromide, acriflavin, 2-N-methylellipticinium and ciprofloxacin. Recombinant NorM protein was expressed in Escherichia coli and purified by metal-affinity and gel-filtration chromatography. The protein was crystallized using hanging-drop vapor diffusion. X-ray diffraction data were collected from cryocooled crystals at a synchrotron light source. The best crystal diffracted anisotropically to 3.8 {angstrom} and diffraction data were complete to 6.5 {angstrom} resolution. The space group was determined to be C2, with unit-cell parameters a = 81.5, b = 164.4, c = 111.5 {angstrom}.

  3. The laboratory diagnosis of Neisseria gonorrhoeae

    PubMed Central

    Ng, Lai-King; Martin, Irene E

    2005-01-01

    The present article describes the laboratory diagnosis of Neisseria gonorrhoeae by culturing of the organism from different types of clinical specimens followed by confirmatory tests. The success of culture methods requires good quality collection and transport of clinical specimens. The present guide describes the media requirements and cultural conditions for N gonorrhoeae growth and the characteristics for a presumptive identification of N gonorrhoeae. Confirmatory tests include biochemical tests, chromogenic enzyme substrate tests, immunoassays and nucleic acid methods. Nucleic acid detection methods include either amplification-based methods or nonamplification tests, and are increasingly used in clinical laboratories where a viable culture is not possible to obtain. Nucleic acid methods can also be used to detect the presence of low numbers in a specimen. Nucleic acid detection methods need confirmation with another amplification method or gene target. Controls must be included to ensure true positive and negative results, and to rule out nucleic acid contamination. Monitoring of antimicrobial susceptibilities of N gonorrhoeae is important to investigate treatment failure and to evaluate the efficacy of currently recommended therapies. Many methods for the characterization of N gonorrhoeae require cultures. The useful typing methods for determining strain relatedness include auxotyping, serotyping, plasmid profile analysis, DNA sequencing of the porB gene and pulsed-field gel electrophoresis. Quality assurance programs for diagnostic testing and antimicrobial susceptibility testing is reviewed. PMID:18159523

  4. Efficient genotype compression and analysis of large genetic variation datasets

    PubMed Central

    Layer, Ryan M.; Kindlon, Neil; Karczewski, Konrad J.; Quinlan, Aaron R.

    2015-01-01

    Genotype Query Tools (GQT) is a new indexing strategy that expedites analyses of genome variation datasets in VCF format based on sample genotypes, phenotypes and relationships. GQT’s compressed genotype index minimizes decompression for analysis, and performance relative to existing methods improves with cohort size. We show substantial (up to 443 fold) performance gains over existing methods and demonstrate GQT’s utility for exploring massive datasets involving thousands to millions of genomes. PMID:26550772

  5. Geomapping of chlamydia and gonorrhoea in Birmingham

    PubMed Central

    Shahmanesh, M.; Gayed, S.; Ashcroft, M.; Smith, R.; Roopnarainsingh, R.; Dunn, J.; Ross, J.

    2000-01-01

    Objective: To investigate if the core population hypothesis is applicable to patients with genital chlamydia infections. Design: Retrospective cross sectional study. Setting: Two genitourinary medicine (GUM) clinics in the city of Birmingham and eight adjacent clinics. Subjects: All patients with chlamydia (n = 665) or gonorrhoea (n = 584) attending between 1 October 1995 and 30 September 1996 with a postcode within the Birmingham health district. Controls were 727 patients seen in the same period with no infection. Methods: Postcodes were used to calculate population prevalence rates per 100 000 aged 15–65 in the 39 wards of the city and to estimate the socioeconomic status using the Super Profile (SP). Ethnic specific rates were also calculated. Data were obtained on gonorrhoea and chlamydia isolation from all the major laboratories of the city over the same time period. Results: GUM clinic attenders accounted for 67.6% and 82.5% of all chlamydia and gonorrhoea isolates reported by the laboratories and that were available for our epidemiological analysis. Both infections were more common in men and in black ethnic groups. However, patients with gonorrhoea only infection were more likely to be of black ethnicity than those with chlamydia only infection (p = 0.0001) and to have different SP distribution (p = 0.0001). On logistic regression age <20 years, male sex, black ethnicity, and living in neighbourhoods with SP J ("have nots") were predictive of both infections compared with controls. Overall chlamydia and gonorrhoea prevalence rates were 129 and 98.4 per 105 respectively. Corresponding rates for whites was 64.7 and 37.2 and for black ethnic groups 1105 and 1183 per 105 of each ethnic group. Eight adjacent wards accounted for 41% of the chlamydia and 66.5% of the gonorrhoea. Conclusion: In a large urban setting patients attending GUM clinics with chlamydia belong to core population groups with similar, but not identical, sociodemographic characteristics to

  6. Neisseria gonorrhoeae: a versatile pathogen.

    PubMed Central

    Easmon, C S; Ison, C A

    1987-01-01

    Neisseria gonorrhoeae is one of the most important causes of sexually transmitted disease. We do not fully understand the pathogenesis of infection with this organism, although recent improvements in immunological and molecular techniques have brought us closer to an answer. These techniques are now also being used to detect and identify N gonorrhoeae and to analyse the epidemiology of gonorrhoea. Plasmid and chromosomal mediated antibiotic resistance increases the difficulty of controlling gonorrhoea. Resistant strains occur all over the world and new patterns of resistance are still emerging. A better understanding of gonococcal pathogenicity is necessary for the development of an effective vaccine. Despite work on pili and outer membrane proteins no vaccine yet exists. The control of gonorrhoea still depends on diagnosis, treatment, and epidemiological control, facilities that are not widely available in many of those parts of the world where gonorrhoea is a major problem. PMID:3117850

  7. Spatial analysis to support geographic targeting of genotypes to environments

    PubMed Central

    Hyman, Glenn; Hodson, Dave; Jones, Peter

    2013-01-01

    Crop improvement efforts have benefited greatly from advances in available data, computing technology, and methods for targeting genotypes to environments. These advances support the analysis of genotype by environment interactions (GEI) to understand how well a genotype adapts to environmental conditions. This paper reviews the use of spatial analysis to support crop improvement research aimed at matching genotypes to their most appropriate environmental niches. Better data sets are now available on soils, weather and climate, elevation, vegetation, crop distribution, and local conditions where genotypes are tested in experimental trial sites. The improved data are now combined with spatial analysis methods to compare environmental conditions across sites, create agro-ecological region maps, and assess environment change. Climate, elevation, and vegetation data sets are now widely available, supporting analyses that were much more difficult even 5 or 10 years ago. While detailed soil data for many parts of the world remains difficult to acquire for crop improvement studies, new advances in digital soil mapping are likely to improve our capacity. Site analysis and matching and regional targeting methods have advanced in parallel to data and technology improvements. All these developments have increased our capacity to link genotype to phenotype and point to a vast potential to improve crop adaptation efforts. PMID:23515351

  8. Failure to control gonorrhoea

    PubMed Central

    Guthe, T.

    1961-01-01

    There is increasing evidence of emerging resistance of the gonococcus to antibiotics, particularly penicillin, but apparent resistance may sometimes be due to concomitant infection with micro-organisms other than the gonococcus, which are not susceptible to this antibiotic. There is also evidence that clinical complications, particularly in the female, are much more frequent than was previously believed to be the case. The incidence of gonorrhoea has increased significantly in many countries in spite of the availability of the ”ideal” drug, penicillin. The reasons for the failure to control gonorrhoea in the individual and in the mass of patients are discussed, taking into account a number of factors relating to emerging drug resistance of the gonococcus, insufficient criteria for diagnosis, the possibility of persistence of infection and increased frequency of reinfection. PMID:13709981

  9. argyle: An R Package for Analysis of Illumina Genotyping Arrays.

    PubMed

    Morgan, Andrew P

    2015-12-18

    Genotyping microarrays are an important and widely-used tool in genetics. I present argyle, an R package for analysis of genotyping array data tailored to Illumina arrays. The goal of the argyle package is to provide simple, expressive tools for nonexpert users to perform quality checks and exploratory analyses of genotyping data. To these ends, the package consists of a suite of quality-control functions, normalization procedures, and utilities for visually and statistically summarizing such data. Format-conversion tools allow interoperability with popular software packages for analysis of genetic data including PLINK, R/qtl and DOQTL. Detailed vignettes demonstrating common use cases are included as supporting information. argyle bridges the gap between the low-level tasks of quality control and high-level tasks of genetic analysis. It is freely available at https://github.com/andrewparkermorgan/argyle and has been submitted to Bioconductor.

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

    PubMed

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

    2015-06-01

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

  11. Mutational and biochemical analysis of cytochrome c', a nitric oxide-binding lipoprotein important for adaptation of Neisseria gonorrhoeae to oxygen-limited growth.

    PubMed

    Turner, Susan M; Moir, James W B; Griffiths, Lesley; Overton, Timothy W; Smith, Harry; Cole, Jeff A

    2005-06-01

    Neisseria gonorrhoeae is a prolific source of c-type cytochromes. Five of the constitutively expressed cytochromes are predicted, based on in silico analysis of the N. gonorrhoeae genome, to be components of the cytochrome bc1 complex, cytochrome c oxidase cbb3 or periplasmic cytochromes involved in electron transfer reactions typical of a bacterium with a microaerobic physiology. Cytochrome c peroxidase was previously shown to be a lipoprotein expressed only during oxygen-limited growth. The final c-type cytochrome, cytochrome c', similar to cytochrome c peroxidase, includes a lipobox required for targeting to the outer membrane. Maturation of cytochrome c' was partially inhibited by globomycin, an antibiotic that specifically inhibits signal peptidase II, resulting in the accumulation of the prolipoprotein in the cytoplasmic membrane. Disruption of the gonococcal cycP gene resulted in an extended lag phase during microaerobic growth in the presence but not in the absence of nitrite, suggesting that cytochrome c' protects the bacteria from NO generated by nitrite reduction during adaptation to oxygen-limited growth. The cytochrome c' gene was overexpressed in Escherichia coli and recombinant cytochrome c' was shown to be targeted to the outer membrane. Spectroscopic evidence is presented showing that gonococcal cytochrome c' is similar to previously characterized cytochrome c' proteins and that it binds NO in vitro. The demonstration that two of the seven gonococcal c-type cytochromes fulfil specialized functions and are outer membrane lipoproteins suggests that the localization of these lipoproteins close to the bacterial surface provides effective protection against external assaults from reactive oxygen and reactive nitrogen species.

  12. Proteomic analysis of Neisseria gonorrhoeae biofilms shows shift to anaerobic respiration and changes in nutrient transport and outermembrane proteins.

    PubMed

    Phillips, Nancy J; Steichen, Christopher T; Schilling, Birgit; Post, Deborah M B; Niles, Richard K; Bair, Thomas B; Falsetta, Megan L; Apicella, Michael A; Gibson, Bradford W

    2012-01-01

    Neisseria gonorrhoeae, the causative agent of gonorrhea, can form biofilms in vitro and in vivo. In biofilms, the organism is more resistant to antibiotic treatment and can serve as a reservoir for chronic infection. We have used stable isotope labeling by amino acids in cell culture (SILAC) to compare protein expression in biofilm and planktonic organisms. Two parallel populations of N. gonorrhoeae strain 1291, which is an arginine auxotroph, were grown for 48 h in continuous-flow chambers over glass, one supplemented with (13)C(6)-arginine for planktonic organisms and the other with unlabeled arginine for biofilm growth. The biofilm and planktonic cells were harvested and lysed separately, and fractionated into three sequential protein extracts. Corresponding heavy (H) planktonic and light (L) biofilm protein extracts were mixed and separated by 1D SDS-PAGE gels, and samples were extensively analyzed by liquid chromatography-mass spectrometry. Overall, 757 proteins were identified, and 152 unique proteins met a 1.5-fold cutoff threshold for differential expression with p-values <0.05. Comparing biofilm to planktonic organisms, this set included 73 upregulated and 54 downregulated proteins. Nearly a third of the upregulated proteins were involved in energy metabolism, with cell envelope proteins making up the next largest group. Of the downregulated proteins, the largest groups were involved in protein synthesis and energy metabolism. These proteomics results were compared with our previously reported results from transcriptional profiling of gonococcal biofilms using microarrays. Nitrite reductase and cytochrome c peroxidase, key enzymes required for anaerobic growth, were detected as highly upregulated in both the proteomic and transcriptomic datasets. These and other protein expression changes observed in the present study were consistent with a shift to anaerobic respiration in gonococcal biofilms, although changes in membrane proteins not explicitly related

  13. Genetic Manipulation of Neisseria gonorrhoeae.

    PubMed

    Dillard, Joseph P

    2011-11-01

    The sexually transmitted pathogen, Neisseria gonorrhoeae, undergoes natural transformation at high frequency. This property has led to the rapid dissemination of antibiotic resistance markers and to the panmictic structure of the gonococcal population. However, high-frequency transformation also makes N. gonorrhoeae one of the easiest bacterial species to manipulate genetically in the laboratory. Techniques have been developed that result in transformation frequencies >50%, allowing the identification of mutants by screening and without selection. Constructs have been created to take advantage of this high-frequency transformation, facilitating genetic mutation, complementation, and heterologous gene expression. Techniques are described for genetic manipulation of N. gonorrhoeae, as well as for growth of this fastidious organism.

  14. Genetic manipulation of Neisseria gonorrhoeae.

    PubMed

    Dillard, Joseph P

    2006-01-01

    The sexually-transmitted pathogen, Neisseria gonorrhoeae, undergoes natural transformation at high frequency. This property has led to the rapid dissemination of antibiotic resistance markers and to the panmictic structure of the gonococcal population. However, high frequency transformation also makes N. gonorrhoeae one of the easiest bacterial species to manipulate genetically in the laboratory. Techniques have been developed that result in transformation frequencies >50%, allowing the identification of mutants by screening and without selection. Constructs have been created to take advantage of this high frequency transformation, facilitating genetic mutation, complementation, and heterologous gene expression. Techniques are described for genetic manipulation of N. gonorrhoeae, as well as for growth of this fastidious organism.

  15. Gonorrhoea diagnostics: An update.

    PubMed

    Verma, R; Sood, S

    2016-01-01

    Diagnosis of gonorrhoea is an ongoing challenge. The organism is fastidious requiring meticulous collection and transport for successful cultivation. Asymptomatic infections are common which go undetected by conventional methods thereby leading to continued transmission and the risk of complications. The nucleic acid amplification tests, now increasingly used in developed countries, offer improved sensitivity compared to bacterial culture. However, these continue to suffer sequence related problems leading to false positive and false negative results. Further, these cannot be used for generation of data on antibiotic susceptibility because genetic markers of antibiotic resistance to recommended therapies have not been fully characterised. They are unaffordable in a setting like ours where reliance is placed on syndromic approach for sexually transmitted infection (STI) management. The use of syndromic approach has resulted in a considerable decline in the number of Neisseria gonorrhoeae isolates that have been cultured for diagnostic purposes. Many laboratories formerly doing so are no longer performing culture for gonococci, and the basic skills have been lost. There is a need to not only revive this skill but also adopt newer technologies that can aid in accurate diagnosis in a cost-effective manner. There is room for innovation that can facilitate the development of a point-of-care test for this bacterial STI. PMID:27080763

  16. Genetic diversity of popcorn genotypes using molecular analysis.

    PubMed

    Resh, F S; Scapim, C A; Mangolin, C A; Machado, M F P S; do Amaral, A T; Ramos, H C C; Vivas, M

    2015-08-19

    In this study, we analyzed dominant molecular markers to estimate the genetic divergence of 26 popcorn genotypes and evaluate whether using various dissimilarity coefficients with these dominant markers influences the results of cluster analysis. Fifteen random amplification of polymorphic DNA primers produced 157 amplified fragments, of which 65 were monomorphic and 92 were polymorphic. To calculate the genetic distances among the 26 genotypes, the complements of the Jaccard, Dice, and Rogers and Tanimoto similarity coefficients were used. A matrix of Dij values (dissimilarity matrix) was constructed, from which the genetic distances among genotypes were represented in a more simplified manner as a dendrogram generated using the unweighted pair-group method with arithmetic average. Clusters determined by molecular analysis generally did not group material from the same parental origin together. The largest genetic distance was between varieties 17 (UNB-2) and 18 (PA-091). In the identification of genotypes with the smallest genetic distance, the 3 coefficients showed no agreement. The 3 dissimilarity coefficients showed no major differences among their grouping patterns because agreement in determining the genotypes with large, medium, and small genetic distances was high. The largest genetic distances were observed for the Rogers and Tanimoto dissimilarity coefficient (0.74), followed by the Jaccard coefficient (0.65) and the Dice coefficient (0.48). The 3 coefficients showed similar estimations for the cophenetic correlation coefficient. Correlations among the matrices generated using the 3 coefficients were positive and had high magnitudes, reflecting strong agreement among the results obtained using the 3 evaluated dissimilarity coefficients.

  17. Genetic diversity of popcorn genotypes using molecular analysis.

    PubMed

    Resh, F S; Scapim, C A; Mangolin, C A; Machado, M F P S; do Amaral, A T; Ramos, H C C; Vivas, M

    2015-01-01

    In this study, we analyzed dominant molecular markers to estimate the genetic divergence of 26 popcorn genotypes and evaluate whether using various dissimilarity coefficients with these dominant markers influences the results of cluster analysis. Fifteen random amplification of polymorphic DNA primers produced 157 amplified fragments, of which 65 were monomorphic and 92 were polymorphic. To calculate the genetic distances among the 26 genotypes, the complements of the Jaccard, Dice, and Rogers and Tanimoto similarity coefficients were used. A matrix of Dij values (dissimilarity matrix) was constructed, from which the genetic distances among genotypes were represented in a more simplified manner as a dendrogram generated using the unweighted pair-group method with arithmetic average. Clusters determined by molecular analysis generally did not group material from the same parental origin together. The largest genetic distance was between varieties 17 (UNB-2) and 18 (PA-091). In the identification of genotypes with the smallest genetic distance, the 3 coefficients showed no agreement. The 3 dissimilarity coefficients showed no major differences among their grouping patterns because agreement in determining the genotypes with large, medium, and small genetic distances was high. The largest genetic distances were observed for the Rogers and Tanimoto dissimilarity coefficient (0.74), followed by the Jaccard coefficient (0.65) and the Dice coefficient (0.48). The 3 coefficients showed similar estimations for the cophenetic correlation coefficient. Correlations among the matrices generated using the 3 coefficients were positive and had high magnitudes, reflecting strong agreement among the results obtained using the 3 evaluated dissimilarity coefficients. PMID:26345916

  18. Potential impact of vaccination against Neisseria meningitidis on Neisseria gonorrhoeae in the United States: results from a decision-analysis model.

    PubMed

    Régnier, Stéphane A; Huels, Jasper

    2014-01-01

    Components in 4CMenB vaccine against Neisseria meningitidis serogroup B have shown to potentially cross-react with Neisseria gonorrhoeae. We modeled the theoretical impact of a US 4CMenB vaccination program on gonorrhea outcomes. A decision-analysis model was populated using published healthcare utilization and cost data. A two-dose adolescent vaccination campaign was assumed, with protective immunity starting at age 15 years and a base-case efficacy against gonorrhea of 20%. The 20%-efficacy level is an assumption since no clinical data have yet quantified the efficacy of 4CMenB against Neisseria gonorrhoea. Key outcome measures were reductions in gonorrhea and HIV infections, reduction in quality-adjusted life-years (QALYs) lost, and the economically justifiable price assuming a willingness-to-pay threshold of $75,000 per QALY gained. Adolescent vaccination with 4CMenB would prevent 83,167 (95% credible interval [CrI], 44,600-134,600) gonorrhea infections and decrease the number of HIV infections by 55 (95% CrI, 2-129) per vaccinated birth cohort in the USA. Excluding vaccination costs, direct medical costs for gonorrhea would reduce by $28.7 million (95% CrI, $6.8-$70.0 million), and income and productivity losses would reduce by $40.0 million (95% CrI, $8.2-$91.7 million). Approximately 83% of the reduction in lost productivity is generated by avoiding HIV infections. At a cost of $75,000 per QALY gained, and incremental to the vaccine's effect on meningococcal disease, a price of $26.10 (95% CrI, $9.10-$57.20) per dose, incremental to the price of the meningococcal vaccine, would be justified from the societal perspective. At this price, the net cost per infection averted would be $1,677 (95% CrI, $404-$2,564). Even if the cross-immunity of 4CMenB vaccine and gonorrhea is only 20%, the reduction in gonorrhea infections and associated costs would be substantial.

  19. Current and future antimicrobial treatment of gonorrhoea - the rapidly evolving Neisseria gonorrhoeae continues to challenge.

    PubMed

    Unemo, Magnus

    2015-08-21

    Neisseria gonorrhoeae has developed antimicrobial resistance (AMR) to all drugs previously and currently recommended for empirical monotherapy of gonorrhoea. In vitro resistance, including high-level, to the last option ceftriaxone and sporadic failures to treat pharyngeal gonorrhoea with ceftriaxone have emerged. In response, empirical dual antimicrobial therapy (ceftriaxone 250-1000 mg plus azithromycin 1-2 g) has been introduced in several particularly high-income regions or countries. These treatment regimens appear currently effective and should be considered in all settings where local quality assured AMR data do not support other therapeutic options. However, the dual antimicrobial regimens, implemented in limited geographic regions, will not entirely prevent resistance emergence and, unfortunately, most likely it is only a matter of when, and not if, treatment failures with also these dual antimicrobial regimens will emerge. Accordingly, novel affordable antimicrobials for monotherapy or at least inclusion in new dual treatment regimens, which might need to be considered for all newly developed antimicrobials, are essential. Several of the recently developed antimicrobials deserve increased attention for potential future treatment of gonorrhoea. In vitro activity studies examining collections of geographically, temporally and genetically diverse gonococcal isolates, including multidrug-resistant strains particularly with resistance to ceftriaxone and azithromycin, are important. Furthermore, understanding of effects and biological fitness of current and emerging (in vitro induced/selected and in vivo emerged) genetic resistance mechanisms for these antimicrobials, prediction of resistance emergence, time-kill curve analysis to evaluate antibacterial activity, appropriate mice experiments, and correlates between genetic and phenotypic laboratory parameters, and clinical treatment outcomes, would also be valuable. Subsequently, appropriately designed

  20. Current and future antimicrobial treatment of gonorrhoea - the rapidly evolving Neisseria gonorrhoeae continues to challenge.

    PubMed

    Unemo, Magnus

    2015-01-01

    Neisseria gonorrhoeae has developed antimicrobial resistance (AMR) to all drugs previously and currently recommended for empirical monotherapy of gonorrhoea. In vitro resistance, including high-level, to the last option ceftriaxone and sporadic failures to treat pharyngeal gonorrhoea with ceftriaxone have emerged. In response, empirical dual antimicrobial therapy (ceftriaxone 250-1000 mg plus azithromycin 1-2 g) has been introduced in several particularly high-income regions or countries. These treatment regimens appear currently effective and should be considered in all settings where local quality assured AMR data do not support other therapeutic options. However, the dual antimicrobial regimens, implemented in limited geographic regions, will not entirely prevent resistance emergence and, unfortunately, most likely it is only a matter of when, and not if, treatment failures with also these dual antimicrobial regimens will emerge. Accordingly, novel affordable antimicrobials for monotherapy or at least inclusion in new dual treatment regimens, which might need to be considered for all newly developed antimicrobials, are essential. Several of the recently developed antimicrobials deserve increased attention for potential future treatment of gonorrhoea. In vitro activity studies examining collections of geographically, temporally and genetically diverse gonococcal isolates, including multidrug-resistant strains particularly with resistance to ceftriaxone and azithromycin, are important. Furthermore, understanding of effects and biological fitness of current and emerging (in vitro induced/selected and in vivo emerged) genetic resistance mechanisms for these antimicrobials, prediction of resistance emergence, time-kill curve analysis to evaluate antibacterial activity, appropriate mice experiments, and correlates between genetic and phenotypic laboratory parameters, and clinical treatment outcomes, would also be valuable. Subsequently, appropriately designed

  1. Genotype Analysis of Bacillus anthracis Strains Circulating in Bangladesh.

    PubMed

    Rume, Farzana Islam; Affuso, Alessia; Serrecchia, Luigina; Rondinone, Valeria; Manzulli, Viviana; Campese, Emanuele; Di Taranto, Pietro; Biswas, Paritosh Kumar; Ahsan, Chowdhury Rafiqul; Yasmin, Mahmuda; Fasanella, Antonio; Hugh-Jones, Martin

    2016-01-01

    In Bangladesh, anthrax, caused by the bacterium Bacillus anthracis, is considered an endemic disease affecting ruminants with sporadic zoonotic occurrences in humans. Due to the lack of knowledge about risks from an incorrect removal of infected carcasses, the disease is not properly monitored, and because of the socio-economic conditions, the situation is under-reported and under-diagnosed. For sensitive species, anthrax represents a fatal outcome with sudden death and sometimes bleeding from natural orifices. The most common source of infection for ruminants is ingestion of spores during grazing in contaminated pastures or through grass and water contaminated with anthrax spores. Domestic cattle, sheep and goats can also become infected through contaminated bone meal (used as feed) originating from anthrax-infected carcasses. The present investigation was conducted to isolate B. anthracis organisms from 169 samples (73 soil, 1 tissue, 4 bone and 91 bone meal samples) collected from 12 different districts of Bangladesh. The sampling was carried out from 2012 to 2015. Twelve samples resulted positive for B. anthracis. Biomolecular analyses were conducted starting from the Canonical Single Nucleotide Polymorphism (CanSNP) to analyze the phylogenetic origin of strains. The analysis of genotype, obtained through the Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) with the analysis of 15 Variable Number Tandem Repeats (VNTR), demonstrated four different genotypes: two of them were previously identified in the district of Sirajganj. The sub-genotyping, conducted with Single Nucleotide Repeats analysis, revealed the presence of eight subgenotypes. The data of the present study concluded that there was no observed correlation between imported cattle feed and anthrax occurrence in Bangladesh and that the remarkable genetic variations of B. anthracis were found in the soil of numerous outbreaks in this country. PMID:27082248

  2. Genotype Analysis of Bacillus anthracis Strains Circulating in Bangladesh

    PubMed Central

    Rume, Farzana Islam; Affuso, Alessia; Serrecchia, Luigina; Rondinone, Valeria; Manzulli, Viviana; Campese, Emanuele; Di Taranto, Pietro; Biswas, Paritosh Kumar; Ahsan, Chowdhury Rafiqul; Yasmin, Mahmuda; Fasanella, Antonio; Hugh-Jones, Martin

    2016-01-01

    In Bangladesh, anthrax, caused by the bacterium Bacillus anthracis, is considered an endemic disease affecting ruminants with sporadic zoonotic occurrences in humans. Due to the lack of knowledge about risks from an incorrect removal of infected carcasses, the disease is not properly monitored, and because of the socio-economic conditions, the situation is under-reported and under-diagnosed. For sensitive species, anthrax represents a fatal outcome with sudden death and sometimes bleeding from natural orifices. The most common source of infection for ruminants is ingestion of spores during grazing in contaminated pastures or through grass and water contaminated with anthrax spores. Domestic cattle, sheep and goats can also become infected through contaminated bone meal (used as feed) originating from anthrax-infected carcasses. The present investigation was conducted to isolate B. anthracis organisms from 169 samples (73 soil, 1 tissue, 4 bone and 91 bone meal samples) collected from 12 different districts of Bangladesh. The sampling was carried out from 2012 to 2015. Twelve samples resulted positive for B. anthracis. Biomolecular analyses were conducted starting from the Canonical Single Nucleotide Polymorphism (CanSNP) to analyze the phylogenetic origin of strains. The analysis of genotype, obtained through the Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) with the analysis of 15 Variable Number Tandem Repeats (VNTR), demonstrated four different genotypes: two of them were previously identified in the district of Sirajganj. The sub-genotyping, conducted with Single Nucleotide Repeats analysis, revealed the presence of eight subgenotypes. The data of the present study concluded that there was no observed correlation between imported cattle feed and anthrax occurrence in Bangladesh and that the remarkable genetic variations of B. anthracis were found in the soil of numerous outbreaks in this country. PMID:27082248

  3. Antimicrobial susceptibility/resistance and genetic characteristics of Neisseria gonorrhoeae isolates from Poland, 2010-2012

    PubMed Central

    2014-01-01

    Background In Poland, gonorrhoea has been a mandatorily reported infection since 1948, however, the reported incidences are likely underestimated. No antimicrobial resistance (AMR) data for Neisseria gonorrhoeae has been internationally reported in nearly four decades, and data concerning genetic characteristics of N. gonorrhoeae are totally lacking. The aims of this study were to investigate the AMR to previously and currently recommended gonorrhoea treatment options, the main genetic resistance determinant (penA) for extended-spectrum cephalosporins (ESCs), and genotypic distribution of N. gonorrhoeae isolates in Poland in 2010-2012. Methods N. gonorrhoeae isolates cultured in 2010 (n = 28), 2011 (n = 92) and 2012 (n = 108) in Warsaw and Bialystok, Poland, were examined using antimicrobial susceptibility testing (Etest), pyrosequencing of penA and N. gonorrhoeae multi-antigen sequence typing (NG-MAST). Results The proportions of N. gonorrhoeae isolates showing resistance were as follows: ciprofloxacin 61%, tetracycline 43%, penicillin G 22%, and azithromycin 8.8%. No isolates resistant to ceftriaxone, cefixime or spectinomycin were found. However, the proportion of isolates with an ESC MIC = 0.125 mg/L, i.e. at the resistance breakpoint, increased significantly from none in 2010 to 9.3% and 19% in 2012 for ceftriaxone and cefixime, respectively. Furthermore, 3.1% of the isolates showed multidrug resistance, i.e., resistance to ciprofloxacin, penicillin G, azithromycin, and decreased susceptibility to cefixime (MIC = 0.125 mg/L). Seventy-six isolates (33%) possessed a penA mosaic allele and 14 isolates (6.1%) contained an A501V/T alteration in penicillin-binding protein 2. NG-MAST ST1407 (n = 58, 25% of isolates) was the most prevalent ST, which significantly increased from 2010 (n = 0) to 2012 (n = 46; 43%). Conclusions In Poland, the diversified gonococcal population displayed a high resistance to most antimicrobials

  4. Polymerase chain reaction-based screening for the ceftriaxone-resistant Neisseria gonorrhoeae F89 strain.

    PubMed

    Goire, N; Lahra, M M; Ohnishi, M; Hogan, T; Liminios, A E; Nissen, M D; Sloots, T P; Whiley, D M

    2013-04-04

    Emergence and spread of Neisseria gonorrhoeae resistant to extended spectrum cephalosporins is a major problem threatening treatment of gonorrhoea and is further highlighted by the recent report of a second ceftriaxone-resistant N. gonorrhoeae strain (F89) in Europe, initially observed in France and subsequently identified in Spain. N. gonorrhoeae antimicrobial resistance (AMR) surveillance has acquired new importance and molecular tools have the potential to enhance bacterial culture-based methods. In this study, we established a polymerase chain reaction (PCR) protocol for direct detection of the F89 strain. A key component of this screening protocol was the development of a hybridisation probe-based melting curve analysis assay (mosaic501-hybPCR) to detect the presence of an A501P substitution on the N. gonorrhoeae mosaic penicillin binding protein 2 (PBP2) sequence, an important characteristic of the F89 strain. The mosaic501-hybPCR was evaluated using plasmid-derived positive controls (n=3) and characterised gonococcal (n=33) and non-gonococcal (n=58) isolates. The protocol was then applied to 159 clinical specimens from Sydney, Australia, collected during the first half of the year 2012 that were N. gonorrhoeae PCR-positive. Overall, the results indicate that the PCR-based protocol is suitable for direct detection of the N. gonorrhoeae F89 strain in non-cultured clinical samples. It therefore provides an additional tool to aid investigations into the potential spread of F89 strain throughout Europe and elsewhere.

  5. Analysis of Variance Components for Genetic Markers with Unphased Genotypes.

    PubMed

    Wang, Tao

    2016-01-01

    An ANOVA type general multi-allele (GMA) model was proposed in Wang (2014) on analysis of variance components for quantitative trait loci or genetic markers with phased or unphased genotypes. In this study, by applying the GMA model, we further examine estimation of the genetic variance components for genetic markers with unphased genotypes based on a random sample from a study population. In one locus and two loci cases, we first derive the least square estimates (LSE) of model parameters in fitting the GMA model. Then we construct estimators of the genetic variance components for one marker locus in a Hardy-Weinberg disequilibrium population and two marker loci in an equilibrium population. Meanwhile, we explore the difference between the classical general linear model (GLM) and GMA based approaches in association analysis of genetic markers with quantitative traits. We show that the GMA model can retain the same partition on the genetic variance components as the traditional Fisher's ANOVA model, while the GLM cannot. We clarify that the standard F-statistics based on the partial reductions in sums of squares from GLM for testing the fixed allelic effects could be inadequate for testing the existence of the variance component when allelic interactions are present. We point out that the GMA model can reduce the confounding between the allelic effects and allelic interactions at least for independent alleles. As a result, the GMA model could be more beneficial than GLM for detecting allelic interactions.

  6. Analysis of Genotype-Phenotype Correlations in Human Holoprosencephaly

    PubMed Central

    Solomon, Benjamin D.; Mercier, Sandra; Vélez, Jorge I.; Pineda-Alvarez, Daniel E.; Wyllie, Adrian; Zhou, Nan; Dubourg, Christèle; David, Veronique; Odent, Sylvie; Roessler, Erich; Muenke, Maximilian

    2009-01-01

    Since the discovery of the first gene causing holoprosencephaly (HPE), over 500 patients with mutations in genes associated with non-chromosomal, non-syndromic HPE have been described, with detailed descriptions available in over 300. Comprehensive clinical analysis of these individuals allows examination for the presence of genotype-phenotype correlations. These correlations allow a degree of differentiation between patients with mutations in different HPE-associated genes and for the application of functional studies to determine intragenic correlations. These early correlations are an important advance in the understanding of the clinical aspects of this disease, and in general argue for continued analysis of the genetic and clinical findings of large cohorts of patients with rare diseases in order to better inform both basic biological insight and care and counseling for affected patients and families. PMID:20104608

  7. Phylogenetic analysis of human parvovirus B19, indicating two subgroups of genotype 1 in Vietnamese patients.

    PubMed

    Toan, Nguyen L; Duechting, Anja; Kremsner, Peter G; Song, Le H; Ebinger, Martin; Aberle, Susanne; Binh, Vu Q; Duy, Dinh Ng; Torresi, Joseph; Kandolf, Reinhard; Bock, C-Thomas

    2006-10-01

    Recently, three distinct genotypes (1, 2 and 3) of human parvovirus B19 (B19) have been identified. However, the characteristics and distribution of B19 genotypes in Vietnam have not been investigated. Phylogenetic analysis using 49 subgenomic NS1/VP1u regions and two coding NS1-VP1/VP2 regions has been applied to investigate the prevalence of B19 genotypes in Vietnamese patients co-infected with Hepatitis B virus. Genetic analysis of the subgenomic NS1/VP1u region of B19 revealed that two genotypes of B19 were identified in these populations, with predominance of genotype 1 (47/49, 96 %) followed by genotype 2 (2/49, 4 %), but not genotype 3. Further, phylogenetic analysis of subgenomic B19 genomes revealed two major subgroups within genotype 1 (B19-1A and B19-1B) with an estimated nucleotide difference of >5 % between each subgroup, forming different branches. The mean percentage of amino acid variation between subgroup B19-1A and B19-1B was >2 % of the NS1, VP1 and VP2 proteins. Our results indicated that two of the three known genotypes of B19 were present in Vietnamese patients, with genotype 1 predominating, and that this genotype can be classified into at least two subgroups, B19-1A and B19-1B.

  8. An Index of Information Content for Genotype Probabilities Derived from Segregation Analysis

    PubMed Central

    Kinghorn, B. P.

    1997-01-01

    A genotype probability index (GPI) is proposed to indicate the information content of genotype probabilities derived from a segregation analysis. Typically, some individuals are genotyped at a marker locus or a quantitative trait locus, and segregation analysis is used to make genotype inferences about ungenotyped relatives. Genotype probabilities for a two-allele autosomal locus are plotted on a triangular surface. The GPI has a value of zero at the point corresponding to Hardy-Weinberg frequencies, and a value of 100% at the vertices of the triangle. Trigonometric functions are used to help calculate intermediate index values. It is proposed that such an index can be useful to help identify which ungenotyped individuals or loci should be genotyped to maximize the benefit/cost of genotyping operations. PMID:9071600

  9. Genetic diversity analysis of highly incomplete SNP genotype data with imputations: an empirical assessment.

    PubMed

    Fu, Yong-Bi

    2014-05-01

    Genotyping by sequencing (GBS) recently has emerged as a promising genomic approach for assessing genetic diversity on a genome-wide scale. However, concerns are not lacking about the uniquely large unbalance in GBS genotype data. Although some genotype imputation has been proposed to infer missing observations, little is known about the reliability of a genetic diversity analysis of GBS data, with up to 90% of observations missing. Here we performed an empirical assessment of accuracy in genetic diversity analysis of highly incomplete single nucleotide polymorphism genotypes with imputations. Three large single-nucleotide polymorphism genotype data sets for corn, wheat, and rice were acquired, and missing data with up to 90% of missing observations were randomly generated and then imputed for missing genotypes with three map-independent imputation methods. Estimating heterozygosity and inbreeding coefficient from original, missing, and imputed data revealed variable patterns of bias from assessed levels of missingness and genotype imputation, but the estimation biases were smaller for missing data without genotype imputation. The estimates of genetic differentiation were rather robust up to 90% of missing observations but became substantially biased when missing genotypes were imputed. The estimates of topology accuracy for four representative samples of interested groups generally were reduced with increased levels of missing genotypes. Probabilistic principal component analysis based imputation performed better in terms of topology accuracy than those analyses of missing data without genotype imputation. These findings are not only significant for understanding the reliability of the genetic diversity analysis with respect to large missing data and genotype imputation but also are instructive for performing a proper genetic diversity analysis of highly incomplete GBS or other genotype data. PMID:24626289

  10. Temporal dynamics and subpopulation analysis of Theileria orientalis genotypes in cattle.

    PubMed

    Jenkins, C; Micallef, M; Alex, S M; Collins, D; Djordjevic, S P; Bogema, D R

    2015-06-01

    In Australia, outbreaks of clinical theileriosis caused by Theileria orientalis have been largely associated with the Ikeda genotype which can occur as a sole infection, or more commonly, as a mixture of genotypes. The most prevalent genotype, Chitose, frequently co-occurs with type Ikeda, however the role of this genotype in clinical disease has not been clearly established. Furthermore, the dynamics of individual genotypes in field infection of cattle have not been examined. In this study we developed quantitative PCR (qPCR) and genotyping methods to examine the role of the Chitose genotype in clinical disease and to investigate the temporal dynamics of T. orientalis Ikeda, Chitose and Buffeli genotypes in naïve animals introduced to a T. orientalis-endemic area. Analysis of the major piroplasm surface protein (MPSP) genes of Chitose isolates revealed the presence of two distinct phylogenetic clusters, Chitose A and Chitose B. A genotyping assay aimed at determining Chitose A/B allele frequency revealed that the Chitose A phylogenetic cluster is strongly associated with clinical disease but nearly always co-occurs with the Ikeda genotype. qPCR revealed that the Chitose genotype (particularly Chitose A), undergoes temporal switching in conjunction with the Ikeda genotype and contributes substantially to the overall parasite burden. The benign Buffeli genotype can also undergo temporal switching but levels of this genotype appear to remain low relative to the Ikeda and Chitose types. Interplay between vector and host immunological factors is presumed to be critical to the population dynamics observed in this study. Genotypic switching likely contributes to the persistence of T. orientalis in the host.

  11. [25S intron analysis followed by restriction enzyme digestion performed for genotyping Candida albicans isolates].

    PubMed

    Karahan, Zeynep Ceren; Saran, Begüm; Yenice, Sevinç; Ağırbaşlı, Handan; Arıkan Akan, Ozay; Tekeli, Alper

    2012-04-01

    Candida albicans is the most frequently encountered fungal pathogen especially in the immunocompromised hosts. Genotyping clinical microbial isolates is important for obtaining epidemiological data and for establishing appropriate infection control strategies in the hospital setting. 25S intron analysis is an easy and reliable method used for genotyping C.albicans strains. As it has a low discriminatory power, its use is limited in epidemiological studies. In this study, our aim was to genotype clinical C.albicans isolates by using 25S intron analysis followed by restriction enzyme digestion in order to develop a more discriminative genotyping system for C.albicans. A total of 260 clinical C.albicans strains isolated from various infection sites (121 blood, 69 sputum, 36 vaginal discharge, 26 wound, 8 urine samples) were genotyped by 25S intron analysis, and all the products obtained by polymerase chain reaction (PCR) were digested with HaeIII restriction enzyme. Discriminatory power of each method was calculated. Among the isolates 184 (70.8%) were classified as genotype A, 42 (16.2%) as genotype B, and 34 (13%) as genotype C by 25S intron analysis. Discriminatory power of the method was calculated as 0.46. HaeIII restriction of genotype A, B and C isolates produced ten, one, and five restriction patterns (genotypes), respectively. By the addition of restriction enzyme analysis, the number of genotypes obtained was increased to 16, and the discriminatory power of the method to 0.79. Combining different genotyping methods increases the discriminatory power by increasing the number of genotypes obtained. However, there is also a risk to split certain strains in different genotypes by the different methods used and this makes the genotypic evaluation more difficult. On the other hand, combining 25S intron analysis with restriction enzyme analysis increases the discriminatory power without introducing a totally different method, and makes the method more suitable for

  12. Cephalosporin Resistance in Neisseria gonorrhoeae

    PubMed Central

    Bala, Manju; Sood, Seema

    2010-01-01

    Gonorrhea, a disease of public health importance, not only leads to high incidence of acute infections and complications but also plays a major role in facilitating human immunodeficiency virus (HIV) acquisition and transmission. One of the major public health needs for gonorrhea control is appropriate, effective treatment. However, treatment options for gonorrhea are diminishing as Neisseria gonorrhoeae have developed resistance to several antimicrobial drugs such as sulfonamides, penicillin, tetracyclines and quinolones. Antimicrobial resistance (AMR) surveillance of N. gonorrhoeae helps establish and maintain the efficacy of standard treatment regimens. AMR surveillance should be continuous to reveal the emergence of new resistant strains, monitor the changing patterns of resistance, and be able to update treatment recommendations so as to assist in disease control. Current treatment guidelines recommend the use of single dose injectable or oral cephalosporins. The emergence and spread of cephalosporin resistant and multi drug resistant N. gonorrhoeae strains, represents a worrying trend that requires monitoring and investigation. Routine clinical laboratories need to be vigilant for the detection of such strains such that strategies for control and prevention could be reviewed and revised from time to time. It will be important to elucidate the genetic mechanisms responsible for decreased susceptibility and future resistance. There is also an urgent need for research of safe, alternative anti-gonococcal compounds that can be administered orally and have effective potency, allowing high therapeutic efficacy (greater than 95.0% cure rate). PMID:20927291

  13. Genotyping and molecular analysis of Enterocytozoon bieneusi isolated from immunocompromised patients in Iran.

    PubMed

    Mirjalali, Hamed; Mirhendi, Hossein; Meamar, Ahmad Reza; Mohebali, Mehdi; Askari, Zeynab; Mirsamadi, Elnaz Sadat; Rezaeian, Mostafa

    2015-12-01

    Microsporidia are known as opportunistic unicellular pathogens, particularly so in individuals with congenital or acquired immunodeficiency. Enterocytozoon bieneusi is one of the most common species infecting both immunocompromised and immunocompetent individuals. The aim of this study was to assess the distribution of E. bieneusi genotypes among immunocompromised patients in Iran. From 329 stool samples referred for parasitological analysis during 2011-2014, 14 samples from immunocompromised patients proving positive for E. bieneusi by SSU rDNA analysis were selected. Genotyping was carried out using specific primers targeting the Internal Transcribed Spacer (ITS) region. Subsequently, all samples were sequenced and results queried against the GenBank database. Moreover, sequences were subject to phylogenetic analysis. The expected amplification product was generated for all samples. Genotype D was identified in patients with HIV+/AIDS, transplant recipients, and cancer patients, while Genotype E was identified only in cancer and HIV+/AIDS patients. Phylogenetic analysis revealed that there was no relationship between genotypes and types of immunosuppression, whereas most genotype D isolates grouped with those described previously from cattle, horses, birds, and humans. E. bieneusi genotype D appears to be the most frequent genotype in immunocompromised patients, while Genotype E was observed only in HIV+/AIDS patients and cancer patients, not transplant recipients.

  14. Stemming the tide of drug-resistant Neisseria gonorrhoeae: the need for an individualized approach to treatment.

    PubMed

    Buono, Sean A; Watson, Tyler D; Borenstein, Lee A; Klausner, Jeffrey D; Pandori, Mark W; Godwin, Hilary A

    2015-02-01

    Drug-resistant Neisseria gonorrhoeae poses a significant public health challenge. In recent years, gonococci resistant to first- and second-line antibiotics have spread worldwide and new strains have developed that are increasingly resistant to third-generation cephalosporins, which are currently our last line of available treatments. Given the timeline required to develop new drugs or an effective vaccine for N. gonorrhoeae, a top priority is to use the drugs that are available as effectively as possible. Currently, clinical management of gonorrhoea is based upon treatment guidelines informed by international gonococcal antimicrobial susceptibility surveillance programmes. This approach, although currently the most practical, is subject to a number of limitations since surveillance data inherently provide population-level information. As a result, basing treatment guidelines on these data can result in the prescription of more aggressive or broader treatment than is needed by individual patients and hence inadvertently contribute to the development and spread of resistance to important drugs. Clearly, methods are needed that provide patient-specific drug susceptibility information in a time frame that would allow clinicians to prescribe individualized treatment regimens for gonorrhoea. Fortunately, in recent years, there have been a number of advances in the development of rapid methods for characterizing both the genotype and the drug resistance phenotype of N. gonorrhoeae strains. Here, we review these advances and propose additional studies that would help facilitate a transition towards an individualized treatment approach for gonorrhoea.

  15. Do the factors associated with successful contact tracing of patients with gonorrhoea and Chlamydia differ?

    PubMed Central

    Ross, J. D.; Sukthankar, A.; Radcliffe, K. W.; Andre, J.

    1999-01-01

    OBJECTIVE: To assess and compare factors which may be associated with successful contact tracing in patients with gonorrhoea and chlamydia. STUDY DESIGN: Prospective observational study of patients attending a genitourinary medicine clinic with a diagnosis of gonorrhoea or chlamydia. Multivariate analysis model including demographic, socioeconomic, and behavioural variables. RESULTS: The attendance of at least one sexual contact was associated with naming more contacts for patients with gonorrhoea (OR 1.44, 95% CI 1.04-2.01). A history of gonorrhoea was associated with successful contact tracing for patients with chlamydia (OR 1.46, 95% CI 1.12-1.9). Successful contact tracing, as defined by at least one confirmed contact attendance after the index case, was not associated with age, sex, sexual orientation, history of chlamydia, use of condoms, marital status, ethnicity, or socioeconomic status for either gonorrhoea or chlamydia. CONCLUSIONS: Differences in the composition of the core groups infected with gonorrhoea and chlamydia are not explained by differences in contact tracing success. In the clinic setting studied, the outcome of contact tracing was not associated with a variety of demographic, socioeconomic, and behaviour factors. 


 PMID:10448364

  16. Genonets server—a web server for the construction, analysis and visualization of genotype networks

    PubMed Central

    Khalid, Fahad; Aguilar-Rodríguez, José; Wagner, Andreas; Payne, Joshua L.

    2016-01-01

    A genotype network is a graph in which vertices represent genotypes that have the same phenotype. Edges connect vertices if their corresponding genotypes differ in a single small mutation. Genotype networks are used to study the organization of genotype spaces. They have shed light on the relationship between robustness and evolvability in biological systems as different as RNA macromolecules and transcriptional regulatory circuits. Despite the importance of genotype networks, no tool exists for their automatic construction, analysis and visualization. Here we fill this gap by presenting the Genonets Server, a tool that provides the following features: (i) the construction of genotype networks for categorical and univariate phenotypes from DNA, RNA, amino acid or binary sequences; (ii) analyses of genotype network topology and how it relates to robustness and evolvability, as well as analyses of genotype network topography and how it relates to the navigability of a genotype network via mutation and natural selection; (iii) multiple interactive visualizations that facilitate exploratory research and education. The Genonets Server is freely available at http://ieu-genonets.uzh.ch. PMID:27106055

  17. Phenotypic and genotypic analysis of variability in Aspergillus fumigatus.

    PubMed Central

    Rinyu, E; Varga, J; Ferenczy, L

    1995-01-01

    Sixty-one isolates and collection strains of Aspergillus fumigatus were compared for their phenotypic (morphological features and isoenzyme profiles) and genotypic (restriction enzyme-generated mitochondrial DNA and ribosomal DNA profiles and random amplified polymorphic DNA patterns) features. The examined strains exhibited highly variable colony morphologies and growth rates at different temperatures, but their micromorphologies and conidial diameters were characteristic of the species. Of the isoenzymes studied, the beta-arylesterase and phosphatase patterns were the most divergent, and the 61 strains could be classified into seven groups. The glucose 6-phosphate dehydrogenase and catalase isoenzyme patterns displayed only a limited variability, while the profiles of superoxide dismutase, lactate dehydrogenase, and glutamate dehydrogenase were highly conserved. The HaeIII-generated mitochondrial DNA patterns and SmaI-digested repetitive DNA and ribosomal DNA hybridization patterns of almost all strains were also invariable. The level of variation was much higher when random amplified polymorphic DNA analysis was applied. Although the patterns of the strains were very similar with most of the primers, the application of some primers made it possible to cluster the A. fumigatus isolates into several groups. The results indicate that the random amplified polymorphic DNA technique could be used more efficiently than isoenzyme analysis for typing A. fumigatus isolates. A good correlation was found between the dendrograms obtained from the isoenzyme and random amplified polymorphic DNA data, but the isoenzyme and amplified DNA patterns did not correlate with the pathogenicity, pigment production, or geographical origin of the strains.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8567884

  18. Identification of Coxiella burnetii genotypes in Croatia using multi-locus VNTR analysis.

    PubMed

    Račić, Ivana; Spičić, Silvio; Galov, Ana; Duvnjak, Sanja; Zdelar-Tuk, Maja; Vujnović, Anja; Habrun, Boris; Cvetnić, Zeljko

    2014-10-10

    Although Q fever affects humans and animals in Croatia, we are unaware of genotyping studies of Croatian strains of the causative pathogen Coxiella burnetii, which would greatly assist monitoring and control efforts. Here 3261 human and animal samples were screened for C. burnetii DNA by conventional PCR, and 335 (10.3%) were positive. Of these positive samples, 82 were genotyped at 17 loci using the relatively new method of multi-locus variable number tandem repeat analysis (MLVA). We identified 13 C. burnetii genotypes not previously reported anywhere in the world. Two of these 13 genotypes are typical of the continental part of Croatia and share more similarity with genotypes outside Croatia than with genotypes within the country. The remaining 11 novel genotypes are typical of the coastal part of Croatia and show more similarity to one another than to genotypes outside the country. Our findings shed new light on the phylogeny of C. burnetii strains and may help establish MLVA as a standard technique for Coxiella genotyping.

  19. Phylogenetic Analysis of Hepatitis B Virus Genotypes Circulating in Different Risk Groups of Panama, Evidence of the Introduction of Genotype A2 in the Country

    PubMed Central

    Martínez, Alexander A.; Zaldívar, Yamitzel; Arteaga, Griselda; de Castillo, Zoila; Ortiz, Alma; Mendoza, Yaxelis; Castillero, Omar; Castillo, Juan A.; Cristina, Juan; Pascale, Juan M.

    2015-01-01

    The Hepatitis B Virus (HBV) can cause acute or chronic infection it is also associated with the development of liver cancer, thousands of new infections occur on a yearly basis, and many of these cases are located in certain areas of the Caribbean and Latin America. In these areas, the HBV prevalence is still high which makes this virus a serious public health concern to the entire region. Studies performed in Panama suggest a complex pattern in the distribution of HBV among the country’s different risk groups. We use phylogenetic analysis in order to determine which HBV genotypes were circulating in these specific groups; for this we used a fragment of the PreS2/2 region of the HBV genome. Subsequently whole HBV genome sequences were used for Bayesian analysis of phylodynamics and phylogeography. Two main genotypes were found: genotype A (54.5%) and genotype F (45.5%). There was a difference in the distribution of genotypes according to risk groups: 72.9% of high risk groups were associated to genotype A, and 55.0% of samples of genotype F were associated to the low risk group (p<0.002). The Bayesian analysis of phylogeny-traits association revealed a statistically significant geographical association (p<0.0001) with both genotypes and different regions of the country. The Bayesian time of most recent common ancestor analysis (tMRCA) revealed a recent tMRCA for genotype A2 circulating in Panama (1997, 95% HPD: 1986—2005), when it is compared with Panamanian genotype F1c sequences (1930, 95% HPD: 1810 – 2005). These results suggest a possible change in the distribution of HBV genotypes in Panama and Latin America as a whole. They also serve to encourage the implementation of vaccination programs in high-risk groups, in order to prevent an increase in the number of new HBV cases in Latin America and worldwide. PMID:26230260

  20. Analysis of Helicobacter pylori Genotypes in Afghani and Iranian Isolates

    PubMed Central

    Dabrii, Hossein; Bolfion, Mehdi; Mirsalehian, Akbar; Rezadehbashi, Maryam; Jafari, Fereshteh; Shokrzadeh, Leila; Sahebekhtiari, Navid; Zojaji, Homayon; Yamaoka, Yoshio; Mirsattari, Darioush; Zali, Mohammad Reza

    2011-01-01

    The geographical variation in Helicobacter pylori genotypes is an observed phenomenon. Cytotoxin associated genes A (cagA) and E (cagE), and vacuolating cytotoxin (vacA) genotypes of H. pylori are associated with peptic ulcer disease (PUD). This study compared the distribution of these genotypes in Iranian and Afghani isolates and their association with clinical outcomes. H. pylori infected patients, as proven by positive culture, were recruited prospectively. A total of 70 patients, 55 Iranian (26 men and 29 women, mean age 48±18 years) and 15 Afghani immigrants (13 men and 2 women, mean age 34.8±11 years) living in Tehran, Iran were enrolled in this study. DNA was extracted from isolated H. pylori and polymerase chain reaction was carried out to determine the cagA and cagE status and vacA alleles. The number of gastric cancer, peptic ulcer and gastritis cases was 11, 23 and 36, respectively. The cagA positive isolates were more common in Iranian (67%) than Afghani isolates (60%). cagE was positive in 53% of Afghani compared to 51% of Iranian isolates. The most common vacA s-region genotype was s1; 80% in Afghani and 67% in Iranian. The s1m1 was a frequently observed genotype in Afghani strains (53%) while s1m2 (47%) was more common in strains isolated from Iranian patients. There is a difference in the H. pylori strains between Iranian and Afghani groups, for instance Iranian isolates were similar to European isolates while Afghani isolates were similar to isolates from India. However, there was no significant association between cagA, cagE and vacA genotypes and clinical outcomes in Iranian and Afghani patients. PMID:20568532

  1. Will targeting oropharyngeal gonorrhoea delay the further emergence of drug-resistant Neisseria gonorrhoeae strains?

    PubMed

    Lewis, D A

    2015-06-01

    Gonorrhoea is an important sexually transmitted infection associated with serious complications and enhanced HIV transmission. Oropharyngeal infections are often asymptomatic and will only be detected by screening. Gonococcal culture has low sensitivity (<50%) for detecting oropharyngeal gonorrhoea, and, although not yet approved commercially, nucleic acid amplification tests (NAAT) are the assay of choice. Screening for oropharyngeal gonorrhoea should be performed in high-risk populations, such as men-who-have-sex-with-men(MSM). NAATs have a poor positive predictive value when used in low-prevalence populations. Gonococci have repeatedly thwarted gonorrhoea control efforts since the first antimicrobial agents were introduced. The oropharyngeal niche provides an enabling environment for horizontal transfer of genetic material from commensal Neisseria and other bacterial species to Neisseria gonorrhoeae. This has been the mechanism responsible for the generation of mosaic penA genes, which are responsible for most of the observed cases of resistance to extended-spectrum cephalosporins (ESC). As antimicrobial-resistant gonorrhoea is now an urgent public health threat, requiring improved antibiotic stewardship, laboratory-guided recycling of older antibiotics may help reduce ESC use. Future trials of antimicrobial agents for gonorrhoea should be powered to test their efficacy at the oropharynx as this is the anatomical site where treatment failure is most likely to occur. It remains to be determined whether a combination of frequent screening of high-risk individuals and/or laboratory-directed fluoroquinolone therapy of oropharyngeal gonorrhoea will delay the further emergence of drug-resistant N. gonorrhoeae strains.

  2. Efficiency control in large-scale genotyping using analysis of variance.

    PubMed

    Spijker, Geert T; Bruinenberg, Marcel; te Meerman, Gerard J

    2005-01-01

    The efficiency of the genotyping process is determined by many simultaneous factors. In actual genotyping, a production run is often preceded by small-scale experiments to find optimal conditions. We propose to use statistical analysis of production run data as well, to gain insight into factors important for the outcome of genotyping. As an example, we show that analysis of variance (ANOVA) applied to the first-pass results of a genetic study reveals important determinants of genotyping success. The largest factor limiting genotyping appeared to be interindividual variation among DNA samples, explaining 20% of the variance, and a smaller reaction volume, sizing failure, and differences among markers all explained approximately 10%. Other potentially important factors, such as sample position within the plate and reusing electrophoresis matrix, appeared to be of minor influence. About 55% of the total variance could be explained by systematic factors. These results show that ANOVA can provide valuable feedback to improve genotyping efficiency. We propose to adjust genotype production runs using principles of experimental design in order to maximize genotyping efficiency at little additional cost.

  3. Oral Immunization of Rabbits with S. enterica Typhimurium Expressing Neisseria gonorrhoeae Filamentous Phage Φ6 Induces Bactericidal Antibodies Against N. gonorrhoeae

    PubMed Central

    Piekarowicz, Andrzej; Kłyż, Aneta; Majchrzak, Michał; Stein, Daniel C.

    2016-01-01

    All Neisseria gonorrhoeae strains whose DNA sequences have been determined possess filamentous phage DNA sequences. To ascertain if phage encoded proteins could form the basis of a gonococcal vaccine, rabbits were orally infected with S. enterica Typhimurium strain χ3987 harboring phagemid NgoΦ6 fm. The elicited sera contained large quantities of anti-phage IgG and IgA antibodies that bound to the surface of N. gonorrhoeae cells, as shown by indirect fluorescent analysis and flow cytometry. The elicited sera was able to bind to several phage proteins. The sera also had bactericidal activity. These data demonstrate that N. gonorrhoeae filamentous phage can induce antibodies with anti-gonococcal activity and that phage proteins may be a candidate for vaccine development. PMID:26939573

  4. Oral Immunization of Rabbits with S. enterica Typhimurium Expressing Neisseria gonorrhoeae Filamentous Phage Φ6 Induces Bactericidal Antibodies Against N. gonorrhoeae.

    PubMed

    Piekarowicz, Andrzej; Kłyż, Aneta; Majchrzak, Michał; Stein, Daniel C

    2016-01-01

    All Neisseria gonorrhoeae strains whose DNA sequences have been determined possess filamentous phage DNA sequences. To ascertain if phage encoded proteins could form the basis of a gonococcal vaccine, rabbits were orally infected with S. enterica Typhimurium strain χ3987 harboring phagemid NgoΦ6 fm. The elicited sera contained large quantities of anti-phage IgG and IgA antibodies that bound to the surface of N. gonorrhoeae cells, as shown by indirect fluorescent analysis and flow cytometry. The elicited sera was able to bind to several phage proteins. The sera also had bactericidal activity. These data demonstrate that N. gonorrhoeae filamentous phage can induce antibodies with anti-gonococcal activity and that phage proteins may be a candidate for vaccine development. PMID:26939573

  5. Genotypic analysis of primary and metastatic cutaneous melanoma.

    PubMed

    Rao, U N M; Jones, M W; Finkelstein, S D

    2003-01-01

    Microdissection genotyping was performed on 16 cases of melanoma, including two cutaneous and one lymph node metastases. Three benign nevi were used as controls. Where possible, tumor was microdissected at several sites. Genotyping involved assessment of loss of heterozygosity [LOH]), which was accomplished using a panel of nine polymorphic tetranucleotide microsatellites. Polymerase chain reaction was performed on the normal tissue sample to establish microsatellite heterozygous status. Informative markers were then tested on microdissected lesional tissue and scored for the presence and extent of allelic imbalance (AI). Microsatellite informativeness varied from 33% to 66%. Benign nevi were without AI. All invasive melanomas manifested acquired allelic loss, which involved 75% or 100% of the markers shown to be informative for each subject. Eleven of 13 (84%) primary melanomas demonstrated intratumoral heterogeneity of AI consistent with development of tumor subclones with differing genotypic profiles within thin as well as thick melanomas. Although a consistent pattern did not emerge among the markers, LOH of 9p21 (D9S254) occurred in 60% (9/15) of the cases followed by 40% of cases displaying LOH of 1p34, p53, 10q (MXI1), and 10q23 (D10S520) and 25% with 5q21 (D5S 592) abnormalities. A third of the cases including the metastatic foci demonstrated two different patterns of AI affecting alternative alleles of the same genomic marker within different parts of the melanoma. Two melanomas in situ did not display LOH of any markers in the informative cases although the in situ component in the invasive tumors had allelic losses that were in part similar to the invasive areas. The results of this study support the expanded use of microdissection genotyping and explore other markers to define the unique mutational profile for malignant melanoma that may complement other histologic characteristics of melanoma. PMID:12550756

  6. Validation of a blood group genotyping method based on high-resolution melting curve analysis.

    PubMed

    Gong, Tianxiang; Hong, Ying; Wang, Naihong; Fu, Xuemei; Zhou, Changhua

    2014-01-01

    The detection of polymorphism is the basis of blood group genotyping and phenotype prediction. Genotyping may be useful to determine blood groups when serologic results are unclear. The development and application of different methods for blood group genotyping may be needed as a substitute for blood group typing. The purpose of this study is to establish an approach for blood group genotyping based on a melting curve analysis of real-time polymerase chain reaction (PCR). Using DNA extracted from whole blood, we developed and validated a DNA typing method for detecting DO*01/DO*02, DO*01/DI*02, LU*01/LU*02, and GYPB*03/GYBP*04 alleles using a melting curve analysis. All assays were confirmed with a commercial reagent containing sequence-specific primers (PCR-SSP), and a cohort of the samples was confirmed with sequencing. Results for all blood groups were within the range of specificity and assay variability. Genotypes of 300 blood donors were fully consistent with PCR-SSP data. The obtained genotype distribution is in complete concordance with existing data for the Chinese population. There are several advantages for this approach of blood group genotyping: lower contamination rates with PCR products in this laboratory, ease of performance, automation potential, and rapid cycling time.

  7. Entire genome sequence analysis of genotype IX Newcastle disease viruses reveals their early-genotype phylogenetic position and recent-genotype genome size

    PubMed Central

    2011-01-01

    Background Six nucleotide (nt) insertion in the 5'-noncoding region (NCR) of the nucleoprotein (NP) gene of Newcaslte disease virus (NDV) is considered to be a genetic marker for recent genotypes of NDV, which emerged after 1960. However, F48-like NDVs from China, identified a 6-nt insert in the NP gene, have been previously classified into genotype III or genotype IX. Results In order to clarify their phylogenetic position and explore the origin of NDVs with the 6-nt insert and its significance in NDV evolution, we determined the entire genome sequences of five F48-like viruses isolated in China between 1946 and 2002 by RT-PCR amplification of overlapping fragments of full-length genome and rapid amplification of cDNA ends. All the five NDV isolates shared the same genome size of 15,192-nt with the recent genotype V-VIII viruses whereas they had the highest homology with early genotype III and IV isolates. Conclusions The unique characteristic of the genome size and phylogenetic position of F48-like viruses warrants placing them in a separate geno-group, genotype IX. Results in this study also suggest that genotype IX viruses most likely originate from a genotype III virus by insertion of a 6-nt motif in the 5'-NCR of the NP gene which had occurred as early as in 1940 s, and might be the common origin of genotype V-VIII viruses. PMID:21396134

  8. AFLP analysis and zebra disease resistance identification of 40 sisal genotypes in China.

    PubMed

    Gao, Jianming; Luoping; Guo, Chaoming; Li, Jinzhi; Liu, Qiaolian; Chen, Helong; Zhang, Shiqing; Zheng, Jinlong; Jiang, Chenji; Dai, Zhenzhen; Yi, Kexian

    2012-05-01

    Sisal is the most important fiber crop in tropical and subtropical areas in China and the world. Zebra disease is a serious threat to the main cultivar Agave hybrid No.11648 (H.11648) worldwide. To select germplasm materials with zebra disease resistance for breeding, the fluorescent amplified fragment length polymorphism (AFLP) technique was used to make a cluster analysis of the genetic relationships of 40 sisal genotypes grown in China, and Phytophthora nicotianae was used to inoculate the 40 genotypes to identify their resistance to zebra disease. As a result, the similarity coefficient among 40 sisal genotypes was found to be 0.44-0.83 and the 40 genotypes show different levels of disease resistance. According to the AFLP analysis, the disease resistance and chromosomal ploidy, it can be reasoned that, A. attenuata var. marginata, Dong 109, Nan ya 1 and A. attenuata are suitable for hybridization with H.11648 to breed a new disease-resistant variety. PMID:22327644

  9. AFLP analysis and zebra disease resistance identification of 40 sisal genotypes in China.

    PubMed

    Gao, Jianming; Luoping; Guo, Chaoming; Li, Jinzhi; Liu, Qiaolian; Chen, Helong; Zhang, Shiqing; Zheng, Jinlong; Jiang, Chenji; Dai, Zhenzhen; Yi, Kexian

    2012-05-01

    Sisal is the most important fiber crop in tropical and subtropical areas in China and the world. Zebra disease is a serious threat to the main cultivar Agave hybrid No.11648 (H.11648) worldwide. To select germplasm materials with zebra disease resistance for breeding, the fluorescent amplified fragment length polymorphism (AFLP) technique was used to make a cluster analysis of the genetic relationships of 40 sisal genotypes grown in China, and Phytophthora nicotianae was used to inoculate the 40 genotypes to identify their resistance to zebra disease. As a result, the similarity coefficient among 40 sisal genotypes was found to be 0.44-0.83 and the 40 genotypes show different levels of disease resistance. According to the AFLP analysis, the disease resistance and chromosomal ploidy, it can be reasoned that, A. attenuata var. marginata, Dong 109, Nan ya 1 and A. attenuata are suitable for hybridization with H.11648 to breed a new disease-resistant variety.

  10. Preliminary analysis of Plasmodium vivax genotypes isolated in southeastern Turkey.

    PubMed

    Döşkaya, Aysu Değirmenci; Döşkaya, Mert; Caner, Ayşe; Gül, Kadri; Nergiz, Şebnem; Can, Hüseyin; Gürüz, Yüksel

    2015-06-01

    Plasmodium vivax is the most common cause of malaria worldwide as well as southeastern Turkey. After the implementation of a successful national elimination program that the local malaria cases were not reported in 2011, malaria returned to county of Savur located in southeastern Turkey in summer of 2012. The present study aimed to determine the prevalent P. vivax genotypes isolated from southeastern Turkey. Genetic polymorphism in P. vivax CSP gene was analyzed by PCR-RFLP to assess the ratio of VK210 and VK247 types. Blood samples were obtained from 15 patients who lived in southeastern between 2005-2006. According to the results, VK210 type was detected in 10 samples (66.6%), VK247 type was observed in three samples (20%). Remaining two samples showed mixed infection (13.3%). The results of the present study first time showed the ratio of P. vivax genotypes in southeastern Turkey before the elimination in 2011. The results of the present study will be enable researchers to compare the new isolates with the previously detected ones and design new treatment and/elimination strategies.

  11. [Determination of hepatitis B virus genotypes by DNA sequence analysis in patients from Ankara, Turkey].

    PubMed

    Külah, Canan; Cirak, Meltem Yalinay

    2010-04-01

    Hepatitis B virus (HBV) genotypes vary depending on the geographical region. The HBV genotype determined in Turkey has been genotype D which is found as the homogenously disseminated single genotype. The aim of this study was to determine HBV genotypes in a group of HBV infected patients who were admitted to a university hospital in Ankara, Turkey. Serum samples from HBsAg positive and anti-HBs negative 84 (52 male, 32 female) patients with HBV infection were included into the study. Anti-HBc was positive in 95.2%, HBeAg was positive in 47.6% and anti-HBe was positive in 11.9% of the patients. Mean HBV-DNA levels of the patients were 5.7 x 10(7) +/- 4.6 x 10(7) IU/ml; mean ALT levels were 131 +/- 171 IU/ml and mean AST levels were 98 +/- 170 IU/ml. HBV-DNA was extracted from serum by the phenol-chloroform method and PCR was performed to amplify the S gene region of HBV-DNA. Cycle sequencing of PCR products was performed by a commercial "Cy5/Cy5.5 Dye Primer Cycle Sequencing Kit" (Visible Genetics, Canada) based on dideoxy chain termination method. The sequences were read and analyzed in an automated fluorescence-based DNA-sequencing system (Long-Read Tower System, Visible Genetics, Canada). The nucleotide sequences of the patient samples were compared with the previously reported sequences in gene bank for each genotype. According to the comparative analysis of S-sequences of all patient samples with the published sequences of the genotypes in gene bank, all of the 84 hepatitis B strains (100%) were shown to be related to D genotypic group, subtype ayw. A phylogenetic analysis was performed and phylogenetic trees were constructed using programs in the PHYLIP phylogeny inference package. The patient samples clustered within the genotypic group D. According to these results, the main HBV genotype in our patients was genotype D in accordance with the previous molecular epidemiologic information on HBV in this geographic area. HBV genotype determination may help to

  12. Breakpoint analysis: Precise localization of genetic markers by means of nonstatistical computation using relatively few genotypes

    SciTech Connect

    Elsner, T.I.; Albertsen, H.; Gerken, S.C.; Cartwright, P.; White, R.

    1995-02-01

    Placing new markers on a previously existing genetic map by using conventional methods of multilocus linkage analysis requires that a large number of reference families be genotyped. This paper presents a methodology for placing new markers on existing genetic maps by genotyping only a few individuals in a selected subset of the reference panel. We show that by identifying meiotic breakpoint events within existing genetic maps and genotyping individuals who exhibit these events, along with one nonrecombinant sibling and their parents, we can determine precise locations for new markers even within subcentimorgan chromosomal regions. This method also improves detection of errors in genotyping and assists in the observation of chromosome behavior in specific regions. 31 refs., 9 figs.

  13. Antimicrobial susceptibility testing of Neisseria gonorrhoeae and implications for epidemiology and therapy.

    PubMed Central

    Fekete, T

    1993-01-01

    Antimicrobial susceptibility testing (AST) of Neisseria gonorrhoeae has been under development since the early days of antimicrobial agents. However, it is rarely applied to clinical isolates today. The history of the various in vitro tests to determine the susceptibility of N. gonorrhoeae to antibiotics is rich with evidence that these results predict response to therapy for almost all agents tested. Further, AST is a useful and important aspect of strain characterization and disease epidemiology in conjunction with the more specific but laborious techniques of auxotyping, serotyping, and plasmid analysis. Current technology has overcome many of the objections to AST for N. gonorrhoeae with standardization of test media and the development of an accurate disk diffusion AST method that is suited to most clinical laboratories regardless of volume or level of technical expertise. Ironically, the very low level of resistance to the current primary treatment strategy in the United States, ceftriaxone or another potent cephalosporin, makes the use of AST somewhat superfluous. PMID:8457978

  14. Comparative analysis of gene expression in response to cold stress in diverse rice genotypes.

    PubMed

    Moraes de Freitas, Gabriela Peres; Basu, Supratim; Ramegowda, Venkategowda; Braga, Eugenia Bolacel; Pereira, Andy

    2016-02-26

    Cold stress is a major factor affecting rice (Oryza sativa) growth and productivity, limiting its distribution worldwide. Rice production is affected primarily due to its vulnerability to cold stress at seedling stage, as well as reproductive stage leading to spikelet sterility. We report here the analysis of 21 diverse rice genotypes from the USDA mini-core collection for cold tolerance and categorized their tolerance levels on the basis of reduction in growth measured by root and shoot length. The screening identified 12 cold tolerant genotypes from which six tolerant genotypes were characterized at the vegetative stage for cold tolerance and gas-exchange parameters. Two tolerant and two sensitive genotypes were used further for gene expression analysis. Lipid Transfer Protein (LTP) genes showed a clear difference in expression between cold tolerant and sensitive genotypes suggesting that they are good candidates for engineering cold tolerance in rice. Nipponbare was identified as a cold tolerant genotype with stress tolerance mechanism potentially operating via both ABA dependent and independent pathways. PMID:26855133

  15. Analysis of genotype diversity and evolution of Dengue virus serotype 2 using complete genomes

    PubMed Central

    Waman, Vaishali P.; Kolekar, Pandurang; Ramtirthkar, Mukund R.; Kale, Mohan M.

    2016-01-01

    Background Dengue is one of the most common arboviral diseases prevalent worldwide and is caused by Dengue viruses (genus Flavivirus, family Flaviviridae). There are four serotypes of Dengue Virus (DENV-1 to DENV-4), each of which is further subdivided into distinct genotypes. DENV-2 is frequently associated with severe dengue infections and epidemics. DENV-2 consists of six genotypes such as Asian/American, Asian I, Asian II, Cosmopolitan, American and sylvatic. Comparative genomic study was carried out to infer population structure of DENV-2 and to analyze the role of evolutionary and spatiotemporal factors in emergence of diversifying lineages. Methods Complete genome sequences of 990 strains of DENV-2 were analyzed using Bayesian-based population genetics and phylogenetic approaches to infer genetically distinct lineages. The role of spatiotemporal factors, genetic recombination and selection pressure in the evolution of DENV-2 is examined using the sequence-based bioinformatics approaches. Results DENV-2 genetic structure is complex and consists of fifteen subpopulations/lineages. The Asian/American genotype is observed to be diversified into seven lineages. The Asian I, Cosmopolitan and sylvatic genotypes were found to be subdivided into two lineages, each. The populations of American and Asian II genotypes were observed to be homogeneous. Significant evidence of episodic positive selection was observed in all the genes, except NS4A. Positive selection operational on a few codons in envelope gene confers antigenic and lineage diversity in the American strains of Asian/American genotype. Selection on codons of non-structural genes was observed to impact diversification of lineages in Asian I, cosmopolitan and sylvatic genotypes. Evidence of intra/inter-genotype recombination was obtained and the uncertainty in classification of recombinant strains was resolved using the population genetics approach. Discussion Complete genome-based analysis revealed that the

  16. Analysis of genotype diversity and evolution of Dengue virus serotype 2 using complete genomes

    PubMed Central

    Waman, Vaishali P.; Kolekar, Pandurang; Ramtirthkar, Mukund R.; Kale, Mohan M.

    2016-01-01

    Background Dengue is one of the most common arboviral diseases prevalent worldwide and is caused by Dengue viruses (genus Flavivirus, family Flaviviridae). There are four serotypes of Dengue Virus (DENV-1 to DENV-4), each of which is further subdivided into distinct genotypes. DENV-2 is frequently associated with severe dengue infections and epidemics. DENV-2 consists of six genotypes such as Asian/American, Asian I, Asian II, Cosmopolitan, American and sylvatic. Comparative genomic study was carried out to infer population structure of DENV-2 and to analyze the role of evolutionary and spatiotemporal factors in emergence of diversifying lineages. Methods Complete genome sequences of 990 strains of DENV-2 were analyzed using Bayesian-based population genetics and phylogenetic approaches to infer genetically distinct lineages. The role of spatiotemporal factors, genetic recombination and selection pressure in the evolution of DENV-2 is examined using the sequence-based bioinformatics approaches. Results DENV-2 genetic structure is complex and consists of fifteen subpopulations/lineages. The Asian/American genotype is observed to be diversified into seven lineages. The Asian I, Cosmopolitan and sylvatic genotypes were found to be subdivided into two lineages, each. The populations of American and Asian II genotypes were observed to be homogeneous. Significant evidence of episodic positive selection was observed in all the genes, except NS4A. Positive selection operational on a few codons in envelope gene confers antigenic and lineage diversity in the American strains of Asian/American genotype. Selection on codons of non-structural genes was observed to impact diversification of lineages in Asian I, cosmopolitan and sylvatic genotypes. Evidence of intra/inter-genotype recombination was obtained and the uncertainty in classification of recombinant strains was resolved using the population genetics approach. Discussion Complete genome-based analysis revealed that the

  17. Current and future treatment options for gonorrhoea.

    PubMed

    Ison, Catherine A; Deal, Carolyn; Unemo, Magnus

    2013-12-01

    The delivery of effective antimicrobial therapy is essential for public health control of gonorrhoea, in the absence of a suitable vaccine. The antimicrobial agent chosen should have high efficacy and quality, lack toxicity and give >95% success when given empirically. Guidelines, which are informed by surveillance data, are used to aid clinicians in their choice of appropriate agent. Historically, gonorrhoea treatment has been delivered as a single, directly observed dose but this has resulted in failure of successive antimicrobial agents which have been replaced by a new antimicrobial to which resistance has been rare or non-existing. Following the drift towards decreased susceptibility and treatment failure to the extended spectrum cephalosporins, and the lack of 'new' alternative antimicrobials, the threat of difficult to treat or untreatable gonorrhoea has emerged. The challenge of maintaining gonorrhoea as a treatable infection has resulted in national, regional and global response or action plans. This review discusses different approaches to the future treatment of gonorrhoea including; use of ceftriaxone, the injectable cephalosporin at increased dosage; dual antimicrobial therapy; use of drugs developed for other infections and use of older agents, directed by rapid point of care tests, to susceptible infections. Finally, it is considered whether the time is right to readdress the possibility of developing an effective gonococcal vaccine, given the major advances in our understanding of natural infection, molecular pathogenesis and the revolution in molecular biology techniques.

  18. Will targeting oropharyngeal gonorrhoea delay the further emergence of drug-resistant Neisseria gonorrhoeae strains?

    PubMed

    Lewis, D A

    2015-06-01

    Gonorrhoea is an important sexually transmitted infection associated with serious complications and enhanced HIV transmission. Oropharyngeal infections are often asymptomatic and will only be detected by screening. Gonococcal culture has low sensitivity (<50%) for detecting oropharyngeal gonorrhoea, and, although not yet approved commercially, nucleic acid amplification tests (NAAT) are the assay of choice. Screening for oropharyngeal gonorrhoea should be performed in high-risk populations, such as men-who-have-sex-with-men(MSM). NAATs have a poor positive predictive value when used in low-prevalence populations. Gonococci have repeatedly thwarted gonorrhoea control efforts since the first antimicrobial agents were introduced. The oropharyngeal niche provides an enabling environment for horizontal transfer of genetic material from commensal Neisseria and other bacterial species to Neisseria gonorrhoeae. This has been the mechanism responsible for the generation of mosaic penA genes, which are responsible for most of the observed cases of resistance to extended-spectrum cephalosporins (ESC). As antimicrobial-resistant gonorrhoea is now an urgent public health threat, requiring improved antibiotic stewardship, laboratory-guided recycling of older antibiotics may help reduce ESC use. Future trials of antimicrobial agents for gonorrhoea should be powered to test their efficacy at the oropharynx as this is the anatomical site where treatment failure is most likely to occur. It remains to be determined whether a combination of frequent screening of high-risk individuals and/or laboratory-directed fluoroquinolone therapy of oropharyngeal gonorrhoea will delay the further emergence of drug-resistant N. gonorrhoeae strains. PMID:25911525

  19. Analysis of amino acid sequences of penicillin-binding protein 2 in clinical isolates of Neisseria gonorrhoeae with reduced susceptibility to cefixime and ceftriaxone.

    PubMed

    Osaka, Kazuyoshi; Takakura, Tadakazu; Narukawa, Kayo; Takahata, Masahiro; Endo, Katsuhisa; Kiyota, Hiroshi; Onodera, Shoichi

    2008-06-01

    Neisseria gonorrhoeae strains with reduced susceptibility to cefixime and ceftriaxone, with minimum inhibitory concentrations (MICs) of cefixime of 0.125-0.25 microg/ml and ceftriaxone of 0.031-0.125 microg/ml, were isolated from male urethritis patients in Tokyo, Japan, in 2006. The amino acid sequences of PenA, penicillin-binding protein 2, in these strains were of two types: PenA mosaic and nonmosaic strains. In the PenA mosaic strain, some regions in the transpeptidase-encoding domain in PenA were similar to those of Neisseria perflava/sicca, Neisseria cinerea, Neisseria flavescens, Neisseria polysaccharea, and Neisseria meningitidis. In the PenA nonmosaic strain, there was a mutation of Ala-501 to Val in PenA. In addition, we performed homology modeling of PenA wild-type and mosaic strains and compared them. The results of the modeling studies suggested that reduced susceptibility to cephems such as cefixime and ceftriaxone is due to a conformational alteration of the beta-lactam-binding pocket. These results also indicated that the mosaic structures and the above point mutation in PenA make a major contribution to the reduced susceptibility to cephem antibiotics.

  20. Time-Motion Analysis of Four Automated Systems for the Detection of Chlamydia trachomatis and Neisseria gonorrhoeae by Nucleic Acid Amplification Testing.

    PubMed

    Williams, James A; Eddleman, Laura; Pantone, Amy; Martinez, Regina; Young, Stephen; Van Der Pol, Barbara

    2014-08-01

    Next-generation diagnostics for Chlamydia trachomatis and Neisseria gonorrhoeae are available on semi- or fully-automated platforms. These systems require less hands-on time than older platforms and are user friendly. Four automated systems, the ABBOTT m2000 system, Becton Dickinson Viper System with XTR Technology, Gen-Probe Tigris DTS system, and Roche cobas 4800 system, were evaluated for total run time, hands-on time, and walk-away time. All of the systems evaluated in this time-motion study were able to complete a diagnostic test run within an 8-h work shift, instrument setup and operation were straightforward and uncomplicated, and walk-away time ranged from approximately 90 to 270 min in a head-to-head comparison of each system. All of the automated systems provide technical staff with increased time to perform other tasks during the run, offer easy expansion of the diagnostic test menu, and have the ability to increase specimen throughput. PMID:24196751

  1. Update on Quinolone Resistance in Neisseria gonorrhoeae.

    PubMed

    Zenilman, Jonathan M.

    2002-04-01

    Quinolones are widely used for treating gonococcal infections, typically in single-dose, oral regimens. However, in the 1990s, quinolone-resistant Neisseria gonorrhoeae emerged, potentially compromising the utility of this drug class. In the past year, these strains have widely disseminated, accounting for over half of isolates in parts of Southeast Asia. The molecular mechanism of resistance has been localized to multiple mutations in genes coding for the bacterial DNA gyrase and topoisomerase enzymes. These mutations accumulate until the minimum inhibitory concentration is 4.0 g/mL or more, which in clinical studies appears to be the threshold for clinical treatment failure. Quinolone-resistant N. gonorrhoeae is independent from other plasmid- and chromosomally-mediated resistance determinants; nearly all isolates to date have been sensitive to cephalosporins and spectinomycin. Nevertheless, designing public health strategies to contain quinolone-resistant N. gonorrhoeae will be difficult. PMID:11927047

  2. A FORTRAN program for analysis of genotypic frequencies and description of the breeding structure of populations.

    PubMed

    Black, W C; Krafsur, E S

    1985-08-01

    A FORTRAN program, "Genestats" was designed to analyse genotypic and allelic frequencies in subpopulations. The genotypes of individuals gathered from electrophoretic analysis at one or more loci are submitted. The program subsequently calculates allele frequencies, determines if significant heterogeneity exists among subpopulations, tests for departures from random mating in subpopulations and calculates F-statistics. A description of the statistical methods is provided. Printout from analysis of allozyme data collected from field subpopulations of the house fly (Musca domestica L.) is provided to illustrate and evaluate the computational methods.

  3. Bacterial hemagglutination by Neisseria gonorrhoeae.

    PubMed Central

    Koransky, J R; Scales, R W; Kraus, S J

    1975-01-01

    Direct bacterial hemagglutination was investigated with 20 clinical isolates of Neisseria gonorrhoeae. The hemagglutination tests were performed by both a macrotechnique with glass slides and a microtechnique with autotrays. Only organisms from form type 1 or 2 colonies caused hemagglutination. There was no statistical difference at a 10% or higher level in hemagglutination powers of type 1 and type 2 organisms, of male urethral and female cervical isolates, and of the eight major human blood types (ABO-Rh). Of seven erythrocyte species tested, only human cells were agglutinated. D-Mannose did not prevent the agglutination. Rabbit antigonococcal serum and high-titer antigonococcal human sera inhibited the hemagglutination. The results suggest the pili are the mediators of hemagglutination and that their specific agglutination of human erythrocytes may be a correlate of their adherence to human mucosal cells in natural infection. Also, although the procedure is presently insensitive, it is possible to detect human antigonococcal antibody by inhibition of direct bacterial hemagglutination. Images PMID:809353

  4. Molecular Taxonomic Evidence for Two Distinct Genotypes of Mycobacterium yongonense via Genome-Based Phylogenetic Analysis

    PubMed Central

    Kim, Byoung-Jun; Kim, Bo-Ram; Lee, So-Young; Kim, Ga-Na; Kook, Yoon-Hoh; Kim, Bum-Joon

    2016-01-01

    Recently, we introduced a distinct Mycobacterium intracellulare INT-5 genotype, distantly related to other genotypes of M. intracellulare (INT-1 to -4). The aim of this study is to determine the exact taxonomic status of the M. intracellulare INT-5 genotype via genome-based phylogenetic analysis. To this end, genome sequences of the two INT-5 strains, MOTT-H4Y and MOTT-36Y were compared with M. intracellulare ATCC 13950T and Mycobacterium yongonense DSM 45126T. Our phylogenetic analysis based on complete genome sequences, multi-locus sequence typing (MLST) of 35 target genes, and single nucleotide polymorphism (SNP) analysis indicated that the two INT-5 strains were more closely related to M. yongonense DSM 45126T than the M. intracellulare strains. These results suggest their taxonomic transfer from M. intracellulare into M. yongonense. Finally, we selected 5 target genes (argH, dnaA, deaD, hsp65, and recF) and used SNPs for the identification of M. yongonese strains from other M. avium complex (MAC) strains. The application of the SNP analysis to 14 MAC clinical isolates enabled the selective identification of 4 M. yongonense clinical isolates from the other MACs. In conclusion, our genome-based phylogenetic analysis showed that the taxonomic status of two INT-5 strains, MOTT-H4Y and MOTT-36Y should be revised into M. yongonense. Our results also suggest that M. yongonense could be divided into 2 distinct genotypes (the Type I genotype with the M. parascrofulaceum rpoB gene and the Type II genotype with the M. intracellulare rpoB gene) depending on the presence of the lateral gene transfer of rpoB from M. parascrofulaceum. PMID:27031100

  5. Online tools for polyphasic analysis of Mycobacterium tuberculosis complex genotyping data: now and next.

    PubMed

    Weniger, Thomas; Krawczyk, Justina; Supply, Philip; Harmsen, Dag; Niemann, Stefan

    2012-06-01

    Molecular diagnostics and genotyping of pathogens have become indispensable tools in clinical microbiology and disease surveillance. For isolates of the Mycobacterium tuberculosis complex (MTBC, causative agents of tuberculosis), multilocus variable number tandem repeat analysis (MLVA) targeting mycobacterial interspersed repetitive units (MIRU) has been internationally adopted as the new standard, portable, reproducible, and discriminatory typing method. Here, we review new sets of specialized web based bioinformatics tools that have become available for analyzing MLVA data especially in combination with other, complementary genotyping markers (polyphasic analysis). Currently, there are only two databases available that are not restricted to store one kind of genotyping data only, namely SITVIT/SpolDB4 and MIRU-VNTRplus. SITVIT/SpolDB4 (http://www.pasteur-guadeloupe.fr:8081/SITVITDemo) contains spoligotyping data from a large number of strains of diverse origin. However, besides options to query the data, the actual version of SITVIT/SpolDB4 offers no functionality for more complex analysis e.g. tree-based analysis. In comparison, the MIRU-VNTRplus web application (http://www.miru-vntrplus.org), represents a freely accessible service that enables users to analyze genotyping data of their strains alone or in comparison with a currently limited but well characterized reference database of strains representing the major MTBC lineages. Data (MLVA-, spoligotype-, large sequence polymorphism, and single nucleotide polymorphism) can be visualized and analyzed using just one genotyping method or a weighted combination of several markers. A variety of analysis tools are available such as creation of phylogenetic and minimum spanning trees, semi-automated phylogenetic lineage identification based on comparison with the reference database and mapping of geographic information. To facilitate scientific communication, a universal, expanding genotype nomenclature (MLVA MtbC15

  6. Microsatellite based analysis of genetic diversity of popular black pepper genotypes in South India.

    PubMed

    Joy, Nisha; Prasanth, V P; Soniya, E V

    2011-08-01

    The genotypes of black pepper are morphologically and genotypically highly diverse and carry all the cumulative variations inherited and maintained through generations. The present study describes the Simple Sequence Repeat (SSR) or microsatellite based assessment of genetic diversity among forty popular genotypes and four different species of black pepper in Southern region of India. For isolation of SSR primers, our earlier attempts with enrichment strategies like 'Triplex affinity capture' did not extract a single SSR primer due to close proximity of restriction sites to the SSR motif. Hence we developed a 'Sequential Reverse Genome Walking (SRGW)' strategy with better enrichment efficiency of 72% that generated seven new SSR primers. Genotyping precisely discriminated majority of genotypes which indicated that the SSR primers are very informative. A total of 62 alleles with an average of 15.5 alleles over 4 loci were identified. All the SSR primers showed an average Polymorphism Information Content (PIC) value of 0.85. The estimated average Shared Allele Frequency ranged between 1.57 and 20.12%. The PCA plot revealed four closely related individual groups and identified Karimunda, Wild pepper and a local landrace 'local b' as the most divergent genotypes. Cluster analysis exposed the genetic relatedness between hybrids and selections with other known cultivars. The introduction of black pepper from South India to Malaysia was emphasized from the observation of genetic similarity of Malaysian cultivar 'Kuching' with other indigenous popular cultivars. The study was first to portray the precise genetic relatedness among the major indigenous genotypes of black pepper. PMID:21874534

  7. Prevalence of Hepatitis C Virus Genotypes in Iranian Patients: A Systematic Review and Meta-Analysis

    PubMed Central

    Khodabandehloo, Mazaher; Roshani, Daem

    2014-01-01

    Context: Hepatitis C virus (HCV) is a global public health problem and a major etiology of chronic liver disease, which may develop into cirrhosis and hepatocellular carcinoma. Genotypes of HCV indicate the route of acquisition, the clinical outcome, response to treatment, prognosis and control strategies. Objectives: The aim of this study was to estimate the overall prevalence and trend of HCV genotypes or subtypes in Iran Data Sources: A literature review was done for papers reporting HCV genotypes in Iranian patients in PubMed, Magiran, IranMedex, Scientific Information Databank, and Google scholar databases. Study Selection: Data were selected according to inclusion and exclusion criteria. Data Extraction: Data were abstracted by two independent authors. Data were analyzed based on random-effects model using the Meta R. Pooled statistical software. Prevalence of HCV genotypes in cities and provinces of Iran with 95% confidence interval (CI) were calculated. Results: Fifty-three articles published between 1999 and 31 June 2014 including 22952 HCV infected individuals were included in the meta-analysis. Subtype 1a was predominant with a rate of 39% (95% CI: 34-44%); followed by subtype 3a, 32% (95% CI: 26-39%); subtype 1b, 13% (95% CI: 10-15%); genotype 4, 5.18% (95% CI: 3.27-7.5%); and genotype 2, 3.6% (95% CI: 1.6-8.3%). Untypeable HCV had a rate of 0.11% (95% CI: 0.07-0.16%). Conclusions: The most frequent subtypes of HCV in Iran were 1a, 3a and 1b, respectively. This frequency differed in various provinces of Iran and fluctuated with time. It is important to determine the distribution of HCV genotypes in different geographical areas and its trend with time for epidemiological and patients’ management purposes. PMID:25685164

  8. A new cytoplasmic male sterile genotype in the sugar beet Beta vulgaris L.: a molecular analysis.

    PubMed

    Mann, V; McIntosh, L; Theurer, C; Hirschberg, J

    1989-08-01

    Mitochondrial DNA (mtDNA) from fertile (N) and possibly new cytoplasmic male sterile (CMS) genotypes was studied in the sugar beet Beta vulgaris L. It was found by restriction endonuclease analysis that BMC-CMS, a cytoplasm that was derived from the wild beet Beta maritima, contained a unique type of mtDNA which is distinguishable from both the N and S-CMS, the only other CMS genotype that is currently availabe in B. vulgaris L. The organization of three genes: coxI, coxII and cob, was analyzed by hybridization with heterologous probes from maize. These genes have a similar structure in N and BMC-CMS that is different from S-CMS. It is concluded that BMC-CMS is a novel CMS genotype in the sugar beet.

  9. Performance and Verification of a Real-Time PCR Assay Targeting the gyrA Gene for Prediction of Ciprofloxacin Resistance in Neisseria gonorrhoeae

    PubMed Central

    Hemarajata, P.; Yang, S.; Soge, O. O.; Klausner, J. D.

    2016-01-01

    In the United States, 19.2% of Neisseria gonorrhoeae isolates are resistant to ciprofloxacin. We evaluated a real-time PCR assay to predict ciprofloxacin susceptibility using residual DNA from the Roche Cobas 4800 CT/NG assay. The results of the assay were 100% concordant with agar dilution susceptibility test results for 100 clinical isolates. Among 76 clinical urine and swab specimens positive for N. gonorrhoeae by the Cobas assay, 71% could be genotyped. The test took 1.5 h to perform, allowing the physician to receive results in time to make informed clinical decisions. PMID:26739156

  10. Performance and Verification of a Real-Time PCR Assay Targeting the gyrA Gene for Prediction of Ciprofloxacin Resistance in Neisseria gonorrhoeae.

    PubMed

    Hemarajata, P; Yang, S; Soge, O O; Humphries, R M; Klausner, J D

    2016-03-01

    In the United States, 19.2% of Neisseria gonorrhoeae isolates are resistant to ciprofloxacin. We evaluated a real-time PCR assay to predict ciprofloxacin susceptibility using residual DNA from the Roche Cobas 4800 CT/NG assay. The results of the assay were 100% concordant with agar dilution susceptibility test results for 100 clinical isolates. Among 76 clinical urine and swab specimens positive for N. gonorrhoeae by the Cobas assay, 71% could be genotyped. The test took 1.5 h to perform, allowing the physician to receive results in time to make informed clinical decisions. PMID:26739156

  11. Multilocus phylogeographical analysis of Trypanosoma (Megatrypanum) genotypes from sympatric cattle and water buffalo populations supports evolutionary host constraint and close phylogenetic relationships with genotypes found in other ruminants.

    PubMed

    Garcia, Herakles A; Rodrigues, Adriana C; Martinkovic, Franjo; Minervino, Antonio H H; Campaner, Marta; Nunes, Vânia L B; Paiva, Fernando; Hamilton, Patrick B; Teixeira, Marta M G

    2011-11-01

    Species of the subgenus Trypanosoma (Megatrypanum) have been reported in cattle and other domestic and wild ruminants worldwide. A previous study in Brazil found at least four genotypes infecting cattle (Bos taurus), but only one in water buffalo (Bubalus bubalis). However, the small number of isolates examined from buffalo, all inhabiting nearby areas, has precluded evaluation of their diversity, host associations and geographical structure. To address these questions, we evaluated the genetic diversity and phylogeographical patterns of 25 isolates from water buffalo and 28 from cattle from four separate locations in Brazil and Venezuela. Multigene phylogenetic analyses of ssrRNA, internal transcribed spacer of rDNA (ITSrDNA), 5SrRNA, glycosomal glyceraldehyde 3-phosphate dehydrogenase (gGAPDH), mitochondrial cytochrome b (Cyt b), spliced leader (SL) and cathepsin L-like (CATL) sequences positioned all isolates from sympatric and allopatric buffalo populations into the highly homogeneous genotype TthIA, while the cattle isolates were assigned to three different genotypes, all distinct from TthIA. Polymorphisms in all of these sequences separated the trypanosomes infecting water buffalo, cattle, sheep, antelope and deer, and suggested that they correspond to separate species. Congruent phylogenies inferred with all genes indicated a predominant clonal structure of the genotypes. The multilocus analysis revealed one monophyletic assemblage formed exclusively by trypanosomes of ruminants, which corresponds to the subgenus T. (Megatrypanum). The high degree of host specificity, evidenced by genotypes exclusive to each ruminant species and lack of genotype shared by different host species, suggested that the evolutionary history of trypanosomes of this subgenus was strongly constrained by their ruminant hosts. However, incongruence between ruminant and trypanosome phylogenies did not support host-parasite co-evolution, indicating that host switches have occurred across

  12. Full-length sequence analysis of hepatitis E virus isolates: showing potential determinants of virus genotype and identity.

    PubMed

    Yang, Dong; Jiang, Mei; Jin, Min; Qiu, Zhigang; Cui, Weihong; Shen, Zhiqiang; Li, Bo; Gong, Lianfeng; Chen, Zhaoli; Wang, Xinwei; Li, Jun-Wen

    2013-12-01

    The complete genome sequence of a genotype 4 strain of hepatitis E virus (CH-YT-HEV02) from a patient (in Yantai, China) has been determined. Phylogenetic analysis showed that CH-YT-HEV02 belongs to genotype 4, subtype 4a. However, the phylogenetic analysis indicated that it was most closely related to JKO-CHiSai98C (AB197673) strain, sharing only 91.6% sequence identity with it. Judging from the phylogenetic tree based on the full-length nucleotide sequences of all 70 genotype 4 HEV isolates retrieved from GenBank up to May, 2013, the CH-YT-HEV02 isolates could serve as a Yantai-indigenous strain. A broader comparison with other genotype isolates revealed that there are a few conserved amino acids in the HVR region of different HEV genotypes, and two amino acid motifs in ORF2 and ORF3 might serve as signatures of genotype diversity of HEV.

  13. Fluorescent directed heteroduplex analysis enhances PCR-based DQA1 and DQB1 genotyping

    SciTech Connect

    Zimmerman, P.A.; Mansfield, E.S.; Miyasaki, T.

    1994-09-01

    We previously showed how directed heteroduplex analysis (DHDA) simplifies DQA1 and DQB1 genotyping and have used the technique to identify a new DQA1 allele (DQA{sup *}0502, which has a single nucleotide difference from DQA1{sup *}0501). In DHDA, labeled probes are mixed with unlabeled PCR products amplified from patient genomic DNA. After controlled re-annealing, allelic heteroduplexes are resolved on polyacrylamide gels (5%, 2.7 M urea). To utilize fluorescence imaging for detecting the heteroduplexes in HLA-typing, probes are labeled by PCR amplification using locus-specific generic primers and gels scanned using the Fluorimager{trademark} 575 (Molecular Dynamics, Inc.). We generate 2-color DHDA probes using locus-specific PCR primers 5{prime}-end labeled with the fluorochromes FAM (positive-strand primer) and JOE (negative-strand primer) (Perkin-Elmer). Genotypic analysis within families obtained from the CEPH repository have been performed by fluorescence-based DHDA. Results to date show 100% concordance between DHDA and sequence-specific oligonucleotide probe (SSOP) genotyping. Fluorescence-based DHDA is performed with fewer probes than SSOP (1 set of locus-specific probes for DHDA and 10 SSOP probes for DQA1 typing or 13 SSOP probes for DQB1 typing). In addition, fluorescent DHDA allows rapid assessment of genotype, aproximately four hours from receipt of sample to typing result. These results suggest that fluorescent DHDA may facilitate DNA-based HLA-typing within the time constraints required for solid organ transplantation.

  14. UV-visible scanning spectrophotometry and chemometric analysis as tools for carotenoids analysis in cassava genotypes (Manihot esculenta Crantz).

    PubMed

    Moresco, Rodolfo; Uarrota, Virgílio Gavicho; Pereira, Aline; Tomazzoli, Maíra Maciel; Nunes, Eduardo da C; Peruch, Luiz Augusto Martins; Gazzola, Jussara; Costa, Christopher; Rocha, Miguel; Maraschin, Marcelo

    2015-10-21

    In this study, the metabolomics characterization focusing on the carotenoid composition of ten cassava (Manihot esculenta) genotypes cultivated in southern Brazil by UV-visible scanning spectrophotometry and reverse phase-high performance liquid chromatography was performed. Cassava roots rich in β-carotene are an important staple food for populations with risk of vitamin A deficiency. Cassava genotypes with high pro-vitamin A activity have been identified as a strategy to reduce the prevalence of deficiency of this vitamin. The data set was used for the construction of a descriptive model by chemometric analysis. The genotypes of yellow-fleshed roots were clustered by the higher concentrations of cis-β-carotene and lutein. Inversely, cream-fleshed roots genotypes were grouped precisely due to their lower concentrations of these pigments, as samples rich in lycopene (red-fleshed) differed among the studied genotypes. The analytical approach (UV-Vis, HPLC, and chemometrics) used showed to be efficient for understanding the chemodiversity of cassava genotypes, allowing to classify them according to important features for human health and nutrition.

  15. Molecular epidemiology of Neisseria gonorrhoeae in Amsterdam, The Netherlands, shows distinct heterosexual and homosexual networks.

    PubMed

    Kolader, Marion-Eliëtte; Dukers, Nicole H T M; van der Bij, Akke K; Dierdorp, Mirjam; Fennema, Johan S A; Coutinho, Roel A; Bruisten, Sylvia M

    2006-08-01

    Molecular typing, added to epidemiological data, can better identify transmission patterns of gonorrhea in Western countries, where the incidence has recently been rising. From September 2002 to September 2003, patients with a laboratory-confirmed diagnosis of gonorrhea at the Clinic for Sexually Transmitted Infections in Amsterdam, The Netherlands, were subjected to a questionnaire pertaining to sexual risk behavior and sexual partners in the 6 months prior to the diagnosis. The Neisseria gonorrhoeae isolates were all genotyped using PCR-restriction fragment length polymorphism of the porin and opacity genes. All patients with a completed questionnaire and genotyped isolates were included in the study. We obtained 885 N. gonorrhoeae isolates from 696 patients that revealed 88 clusters and 46 unique genotypes. Patients infected at multiple anatomical sites with one or more strains and patients infected several times during the study period were shown to pursue high-risk sexual behavior and were considered core groups. There were 11 clusters of > or =20 patients; in seven clusters, 81% to 100% of patients were men who have sex with men (MSM), three clusters contained 87 to 100% heterosexual men and women, and one cluster was formed by equal proportions of MSM and heterosexual male and female patients. However, the various clusters differed in characteristics such as types of coinfections, numbers of sexual partners, Internet use to seek sexual partners, and locations of sexual encounters. Molecular epidemiology of gonococcal isolates in Amsterdam revealed core groups and clusters of MSM and heterosexual patients that probably indicate distinct transmission networks.

  16. Neisseria gonorrhoeae and fosfomycin: Past, present and future.

    PubMed

    Tesh, Lauren D; Shaeer, Kristy M; Cho, Jonathan C; Estrada, Sandy J; Huang, Vanthida; Bland, Christopher M; DiMondi, V Paul; Potter, Alicia N; Hussein, Gamal; Bookstaver, P Brandon

    2015-09-01

    Drug-resistant Neisseria gonorrhoeae has become a global health concern that requires immediate attention. Due to increasing resistance to cephalosporins, pursuing novel alternatives for treating N. gonorrhoeae infections is paramount. Whilst new drug development is often cumbersome, reviving antiquated antibiotic agents for treatment of modern infections has become prevalent in clinical practice. Fosfomycin exhibits bactericidal activity through a unique mechanism of action, and a variety of organisms including N. gonorrhoeae are susceptible. In vitro studies have demonstrated that fosfomycin can retain activity against ceftriaxone-resistant N. gonorrhoeae; however, it remains unclear whether there is synergy between fosfomycin and other antibiotics. Clinical investigations evaluating fosfomycin for the treatment of N. gonorrhoeae infections are confounded by methodological limitations, none the less they do provide some perspective on its potential role in therapy. Future studies are needed to establish a safe, convenient and effective fosfomycin regimen for treating N. gonorrhoeae infections.

  17. Neisseria gonorrhoeae and fosfomycin: Past, present and future.

    PubMed

    Tesh, Lauren D; Shaeer, Kristy M; Cho, Jonathan C; Estrada, Sandy J; Huang, Vanthida; Bland, Christopher M; DiMondi, V Paul; Potter, Alicia N; Hussein, Gamal; Bookstaver, P Brandon

    2015-09-01

    Drug-resistant Neisseria gonorrhoeae has become a global health concern that requires immediate attention. Due to increasing resistance to cephalosporins, pursuing novel alternatives for treating N. gonorrhoeae infections is paramount. Whilst new drug development is often cumbersome, reviving antiquated antibiotic agents for treatment of modern infections has become prevalent in clinical practice. Fosfomycin exhibits bactericidal activity through a unique mechanism of action, and a variety of organisms including N. gonorrhoeae are susceptible. In vitro studies have demonstrated that fosfomycin can retain activity against ceftriaxone-resistant N. gonorrhoeae; however, it remains unclear whether there is synergy between fosfomycin and other antibiotics. Clinical investigations evaluating fosfomycin for the treatment of N. gonorrhoeae infections are confounded by methodological limitations, none the less they do provide some perspective on its potential role in therapy. Future studies are needed to establish a safe, convenient and effective fosfomycin regimen for treating N. gonorrhoeae infections. PMID:26145201

  18. Validation of Genotyping-By-Sequencing Analysis in Populations of Tetraploid Alfalfa by 454 Sequencing

    PubMed Central

    Rocher, Solen; Jean, Martine; Castonguay, Yves; Belzile, François

    2015-01-01

    Genotyping-by-sequencing (GBS) is a relatively low-cost high throughput genotyping technology based on next generation sequencing and is applicable to orphan species with no reference genome. A combination of genome complexity reduction and multiplexing with DNA barcoding provides a simple and affordable way to resolve allelic variation between plant samples or populations. GBS was performed on ApeKI libraries using DNA from 48 genotypes each of two heterogeneous populations of tetraploid alfalfa (Medicago sativa spp. sativa): the synthetic cultivar Apica (ATF0) and a derived population (ATF5) obtained after five cycles of recurrent selection for superior tolerance to freezing (TF). Nearly 400 million reads were obtained from two lanes of an Illumina HiSeq 2000 sequencer and analyzed with the Universal Network-Enabled Analysis Kit (UNEAK) pipeline designed for species with no reference genome. Following the application of whole dataset-level filters, 11,694 single nucleotide polymorphism (SNP) loci were obtained. About 60% had a significant match on the Medicago truncatula syntenic genome. The accuracy of allelic ratios and genotype calls based on GBS data was directly assessed using 454 sequencing on a subset of SNP loci scored in eight plant samples. Sequencing depth in this study was not sufficient for accurate tetraploid allelic dosage, but reliable genotype calls based on diploid allelic dosage were obtained when using additional quality filtering. Principal Component Analysis of SNP loci in plant samples revealed that a small proportion (<5%) of the genetic variability assessed by GBS is able to differentiate ATF0 and ATF5. Our results confirm that analysis of GBS data using UNEAK is a reliable approach for genome-wide discovery of SNP loci in outcrossed polyploids. PMID:26115486

  19. Validation of Genotyping-By-Sequencing Analysis in Populations of Tetraploid Alfalfa by 454 Sequencing.

    PubMed

    Rocher, Solen; Jean, Martine; Castonguay, Yves; Belzile, François

    2015-01-01

    Genotyping-by-sequencing (GBS) is a relatively low-cost high throughput genotyping technology based on next generation sequencing and is applicable to orphan species with no reference genome. A combination of genome complexity reduction and multiplexing with DNA barcoding provides a simple and affordable way to resolve allelic variation between plant samples or populations. GBS was performed on ApeKI libraries using DNA from 48 genotypes each of two heterogeneous populations of tetraploid alfalfa (Medicago sativa spp. sativa): the synthetic cultivar Apica (ATF0) and a derived population (ATF5) obtained after five cycles of recurrent selection for superior tolerance to freezing (TF). Nearly 400 million reads were obtained from two lanes of an Illumina HiSeq 2000 sequencer and analyzed with the Universal Network-Enabled Analysis Kit (UNEAK) pipeline designed for species with no reference genome. Following the application of whole dataset-level filters, 11,694 single nucleotide polymorphism (SNP) loci were obtained. About 60% had a significant match on the Medicago truncatula syntenic genome. The accuracy of allelic ratios and genotype calls based on GBS data was directly assessed using 454 sequencing on a subset of SNP loci scored in eight plant samples. Sequencing depth in this study was not sufficient for accurate tetraploid allelic dosage, but reliable genotype calls based on diploid allelic dosage were obtained when using additional quality filtering. Principal Component Analysis of SNP loci in plant samples revealed that a small proportion (<5%) of the genetic variability assessed by GBS is able to differentiate ATF0 and ATF5. Our results confirm that analysis of GBS data using UNEAK is a reliable approach for genome-wide discovery of SNP loci in outcrossed polyploids.

  20. Changing Antimicrobial Resistance Profiles among Neisseria gonorrhoeae Isolates in Italy, 2003 to 2012

    PubMed Central

    Carannante, Anna; Renna, Giovanna; Dal Conte, Ivano; Ghisetti, Valeria; Matteelli, Alberto; Prignano, Grazia; Impara, Giampaolo; Cusini, Marco; D'Antuono, Antonietta; Vocale, Caterina; Antonetti, Raffaele; Gaino, Marina; Busetti, Marina; Latino, Maria Agnese; Mencacci, Antonella; Bonanno, Carmen; Cava, Maria Carmela; Giraldi, Cristina

    2014-01-01

    The emergence of Neisseria gonorrhoeae isolates displaying resistance to antimicrobial agents is a major public health concern and a serious issue related to the occurrence of further untreatable gonorrhea infections. A retrospective analysis on 1,430 N. gonorrhoeae isolates, collected from 2003 through 2012, for antimicrobial susceptibility by Etest and molecular characterization by Neisseria gonorrhoeae multiantigen sequence typing (NG-MAST) was carried out in Italy. Azithromycin-resistant gonococci decreased from 14% in 2007 to 2.2% in 2012. Similarly, isolates with high MICs to cefixime (>0.125 mg/liter) decreased from 11% in 2008 to 3.3% in 2012. The ciprofloxacin resistance rate remains quite stable, following an increasing trend up to 64% in 2012. The percentage of penicillinase-producing N. gonorrhoeae (PPNG) significantly declined from 77% in 2003 to 7% in 2012. A total of 81 multidrug-resistant (MDR) gonococci were identified, showing 11 different antimicrobial resistance patterns. These were isolated from men who have sex with men (MSM) and from heterosexual patients. Two sequence types (STs), ST661 and ST1407, were the most common. Genogroup 1407, which included cefixime-, ciprofloxacin-, and azithromycin-resistant isolates, was found. In conclusion, a change in the antimicrobial resistance profiles among gonococci was identified in Italy together with a percentage of MDR isolates. PMID:25070110

  1. Genotyping of classical swine fever virus using high-resolution melt analysis.

    PubMed

    Titov, Ilya; Tsybanov, Sodnom; Malogolovkin, Alexander

    2015-11-01

    Discrimination between different field and vaccine strains of classical swine fever virus (CSFV) is crucial for meaningful disease diagnosis and epidemiological investigation. In this study, a rapid method for differentiating vaccine strains and outbreak CSFV isolates by combined RT-PCR and high-resolution melt (HRM) analysis has been developed. The assay is based on PCR amplification of short fragments from the most variable region of CSFVgene E2, followed by HRM analysis of amplicons. Real-Time PCR/HRM for CSFV detection and differentiation analysis has sensitivity comparable to RT-qPCR and genotyping resolution comparable to E2 nucleotide sequencing. This assay in one step enables rapid and sensitive identification and genotype discrimination of CSFV in field samples, and thus will be valuable for CSF outbreak response and disease control. PMID:26300371

  2. Analysis of linear and non-linear genotype × environment interaction

    PubMed Central

    Yang, Rong-Cai

    2014-01-01

    The usual analysis of genotype × environment interaction (G × E) is based on the linear regression of genotypic performance on environmental changes (e.g., classic stability analysis). This linear model may often lead to lumping together of the non-linear responses to the whole range of environmental changes from suboptimal and super optimal conditions, thereby lowering the power of detecting G × E variation. On the other hand, the G × E is present when the magnitude of the genetic effect differs across the range of environmental conditions regardless of whether the response to environmental changes is linear or non-linear. The objectives of this study are: (i) explore the use of four commonly used non-linear functions (logistic, parabola, normal and Cauchy functions) for modeling non-linear genotypic responses to environmental changes and (ii) to investigate the difference in the magnitude of estimated genetic effects under different environmental conditions. The use of non-linear functions was illustrated through the analysis of one data set taken from barley cultivar trials in Alberta, Canada (Data A) and the examination of change in effect sizes is through the analysis another data set taken from the North America Barley Genome Mapping Project (Data B). The analysis of Data A showed that the Cauchy function captured an average of >40% of total G × E variation whereas the logistic function captured less G × E variation than the linear function. The analysis of Data B showed that genotypic responses were largely linear and that strong QTL × environment interaction existed as the positions, sizes and directions of QTL detected differed in poor vs. good environments. We conclude that (i) the non-linear functions should be considered when analyzing multi-environmental trials with a wide range of environmental variation and (ii) QTL × environment interaction can arise from the difference in effect sizes across environments. PMID:25101112

  3. High Diversity of Hepatitis B Virus Genotypes in Panamanian Blood Donors: A Molecular Analysis of New Variants

    PubMed Central

    Martínez, Alexander A.; Zaldivar, Yamitzel Y.; De Castillo, Zoila; Ortiz, Alma Y.; Mendoza, Yaxelis; Cristina, Juan; Pascale, Juan M.

    2014-01-01

    Hepatitis B Virus (HBV) is an infectious agent that causes more than half of the cases of liver disease and cancer in the world. Globally there are around 250 million people chronically infected with this virus. Despite 16% of the cases of liver disease in Central America are caused by HBV, the information regarding its genetic diversity, genotypes and circulation is scarce. The purpose of this study was to evaluate the genetic variability of the HBV genotypes from HBV-DNA positive samples obtained from screening blood donors at the Social Security System of Panama and to estimate its possible origin. From 59,696 blood donors tested for HBV infection during 2010–2012, there were 74 HBV-DNA positive subjects. Analysis of the partial PreS2-S region of 27 sequences shows that 21% of the infections were caused by genotype A, 3% by genotype D and 76% by genotype F. In addition, we were able to confirm circulation of six sub-genotypes A1, A2, A3, D4, F3, F1 and a proposed new sub-genotype denominated F5pan. We found a confinement of sub-genotypes F1 and F5pan to the western area of Panama. The tMRCA analysis suggests a simultaneous circulation of previously described sub-genotypes rather than recent introductions of the Panamanian sub-genotypes in the country. Moreover, these results highlight the need of more intensive research of the HBV strains circulating in the region at the molecular level. In conclusion, Panama has a high HBV genotype diversity that includes a new proposed sub-genotype, an elevated number of PreCore-Core mutations, and confinement of these variants in a specific geographical location. PMID:25093674

  4. Phylogenetic Analysis and Epidemic History of Hepatitis C Virus Genotype 2 in Tunisia, North Africa

    PubMed Central

    Rajhi, Mouna; Ghedira, Kais; Chouikha, Anissa; Djebbi, Ahlem; Cheikh, Imed; Ben Yahia, Ahlem; Sadraoui, Amel; Hammami, Walid; Azouz, Msaddek; Ben Mami, Nabil; Triki, Henda

    2016-01-01

    HCV genotype 2 (HCV-2) has a worldwide distribution with prevalence rates that vary from country to country. High genetic diversity and long-term endemicity were suggested in West African countries. A global dispersal of HCV-2 would have occurred during the 20th century, especially in European countries. In Tunisia, genotype 2 was the second prevalent genotype after genotype 1 and most isolates belong to subtypes 2c and 2k. In this study, phylogenetic analyses based on the NS5B genomic sequences of 113 Tunisian HCV isolates from subtypes 2c and 2k were carried out. A Bayesian coalescent-based framework was used to estimate the origin and the spread of these subtypes circulating in Tunisia. Phylogenetic analyses of HCV-2c sequences suggest the absence of country-specific or time-specific variants. In contrast, the phylogenetic grouping of HCV-2k sequences shows the existence of two major genetic clusters that may represent two distinct circulating variants. Coalescent analysis indicated a most recent common ancestor (tMRCA) of Tunisian HCV-2c around 1886 (1869–1902) before the introduction of HCV-2k in 1901 (1867–1931). Our findings suggest that the introduction of HCV-2c in Tunisia is possibly a result of population movements between Tunisia and European population following the French colonization. PMID:27100294

  5. Phylogenetic Analysis and Epidemic History of Hepatitis C Virus Genotype 2 in Tunisia, North Africa.

    PubMed

    Rajhi, Mouna; Ghedira, Kais; Chouikha, Anissa; Djebbi, Ahlem; Cheikh, Imed; Ben Yahia, Ahlem; Sadraoui, Amel; Hammami, Walid; Azouz, Msaddek; Ben Mami, Nabil; Triki, Henda

    2016-01-01

    HCV genotype 2 (HCV-2) has a worldwide distribution with prevalence rates that vary from country to country. High genetic diversity and long-term endemicity were suggested in West African countries. A global dispersal of HCV-2 would have occurred during the 20th century, especially in European countries. In Tunisia, genotype 2 was the second prevalent genotype after genotype 1 and most isolates belong to subtypes 2c and 2k. In this study, phylogenetic analyses based on the NS5B genomic sequences of 113 Tunisian HCV isolates from subtypes 2c and 2k were carried out. A Bayesian coalescent-based framework was used to estimate the origin and the spread of these subtypes circulating in Tunisia. Phylogenetic analyses of HCV-2c sequences suggest the absence of country-specific or time-specific variants. In contrast, the phylogenetic grouping of HCV-2k sequences shows the existence of two major genetic clusters that may represent two distinct circulating variants. Coalescent analysis indicated a most recent common ancestor (tMRCA) of Tunisian HCV-2c around 1886 (1869-1902) before the introduction of HCV-2k in 1901 (1867-1931). Our findings suggest that the introduction of HCV-2c in Tunisia is possibly a result of population movements between Tunisia and European population following the French colonization.

  6. Phylogenetic Analysis and Epidemic History of Hepatitis C Virus Genotype 2 in Tunisia, North Africa.

    PubMed

    Rajhi, Mouna; Ghedira, Kais; Chouikha, Anissa; Djebbi, Ahlem; Cheikh, Imed; Ben Yahia, Ahlem; Sadraoui, Amel; Hammami, Walid; Azouz, Msaddek; Ben Mami, Nabil; Triki, Henda

    2016-01-01

    HCV genotype 2 (HCV-2) has a worldwide distribution with prevalence rates that vary from country to country. High genetic diversity and long-term endemicity were suggested in West African countries. A global dispersal of HCV-2 would have occurred during the 20th century, especially in European countries. In Tunisia, genotype 2 was the second prevalent genotype after genotype 1 and most isolates belong to subtypes 2c and 2k. In this study, phylogenetic analyses based on the NS5B genomic sequences of 113 Tunisian HCV isolates from subtypes 2c and 2k were carried out. A Bayesian coalescent-based framework was used to estimate the origin and the spread of these subtypes circulating in Tunisia. Phylogenetic analyses of HCV-2c sequences suggest the absence of country-specific or time-specific variants. In contrast, the phylogenetic grouping of HCV-2k sequences shows the existence of two major genetic clusters that may represent two distinct circulating variants. Coalescent analysis indicated a most recent common ancestor (tMRCA) of Tunisian HCV-2c around 1886 (1869-1902) before the introduction of HCV-2k in 1901 (1867-1931). Our findings suggest that the introduction of HCV-2c in Tunisia is possibly a result of population movements between Tunisia and European population following the French colonization. PMID:27100294

  7. The “3 in 1” Study: Pooling Self-Taken Pharyngeal, Urethral, and Rectal Samples into a Single Sample for Analysis for Detection of Neisseria gonorrhoeae and Chlamydia trachomatis in Men Who Have Sex with Men

    PubMed Central

    White, J. A.; Fish, R.; Carrick, G.; Brima, N.; Copas, A.; Robinson, A.; Gilson, R.; Mercey, D.; Benn, P.

    2015-01-01

    Triple-site testing (using pharyngeal, rectal, and urethral/first-void urine samples) for Neisseria gonorrhoeae and Chlamydia trachomatis using nucleic acid amplification tests detects greater numbers of infections among men who have sex with men (MSM). However, triple-site testing represents a cost pressure for services. MSM over 18 years of age were eligible if they requested testing for sexually transmitted infections (STIs), reported recent sexual contact with either C. trachomatis or N. gonorrhoeae, or had symptoms of an STI. Each patient underwent standard-of-care (SOC) triple-site testing, and swabs were taken to form a pooled sample (PS) (pharyngeal, rectal, and urine specimens). The PS was created using two methods during different periods at one clinic, but we analyzed the data in combination because the sensitivity of the two methods did not differ significantly for C. trachomatis (P = 0.774) or N. gonorrhoeae (P = 0.163). The sensitivity of PS testing (92%) was slightly lower than that of SOC testing (96%) for detecting C. trachomatis (P = 0.167). For N. gonorrhoeae, the sensitivity of PS testing (90%) was significantly lower than that of SOC testing (99%) (P < 0.001). When pharynx-only infections were excluded, the sensitivity of PS testing to detect N. gonorrhoeae infections increased to 94%. Our findings show that pooling of self-taken samples could be an effective and cost-saving method, with high negative predictive values. (Interim results of this study were presented at the BASHH 2013 summer meeting.) PMID:26719439

  8. The "3 in 1" Study: Pooling Self-Taken Pharyngeal, Urethral, and Rectal Samples into a Single Sample for Analysis for Detection of Neisseria gonorrhoeae and Chlamydia trachomatis in Men Who Have Sex with Men.

    PubMed

    Sultan, B; White, J A; Fish, R; Carrick, G; Brima, N; Copas, A; Robinson, A; Gilson, R; Mercey, D; Benn, P

    2016-03-01

    Triple-site testing (using pharyngeal, rectal, and urethral/first-void urine samples) for Neisseria gonorrhoeae and Chlamydia trachomatis using nucleic acid amplification tests detects greater numbers of infections among men who have sex with men (MSM). However, triple-site testing represents a cost pressure for services. MSM over 18 years of age were eligible if they requested testing for sexually transmitted infections (STIs), reported recent sexual contact with either C. trachomatis or N. gonorrhoeae, or had symptoms of an STI. Each patient underwent standard-of-care (SOC) triple-site testing, and swabs were taken to form a pooled sample (PS) (pharyngeal, rectal, and urine specimens). The PS was created using two methods during different periods at one clinic, but we analyzed the data in combination because the sensitivity of the two methods did not differ significantly for C. trachomatis (P = 0.774) or N. gonorrhoeae (P = 0.163). The sensitivity of PS testing (92%) was slightly lower than that of SOC testing (96%) for detecting C. trachomatis (P = 0.167). For N. gonorrhoeae, the sensitivity of PS testing (90%) was significantly lower than that of SOC testing (99%) (P < 0.001). When pharynx-only infections were excluded, the sensitivity of PS testing to detect N. gonorrhoeae infections increased to 94%. Our findings show that pooling of self-taken samples could be an effective and cost-saving method, with high negative predictive values. (Interim results of this study were presented at the BASHH 2013 summer meeting.). PMID:26719439

  9. Phylogenetic analysis of hepatitis D viruses indicating a new genotype I subgroup among African isolates.

    PubMed Central

    Zhang, Y Y; Tsega, E; Hansson, B G

    1996-01-01

    Genetic analysis was performed on 13 hepatitis D virus (HDV) isolates from Ethiopia, Somalia, Jordan, Kuwait, Bulgaria, Moldavia, and Sweden. The complete nucleotide sequence and genomic organization are described for the first time for two African HDV isolates. Phylogenetic analysis showed all the African isolates to be intrarelated and to form a novel group within HDV genotype I; the suggested designation for this group is IC. The genetic distance to previously described type I isolates was about 0.15. The HDV genotype I isolates (total of 22 examined) phylogenetically formed three clusters, each of them corresponding to certain geographic regions; the "western" group consisted of six HDV isolates from western Europe and the United States plus one from Kuwait; the "eastern" group consisted of two isolates from Moldavia and one each from Bulgaria, Nauru, mainland China, and Taiwan; and the "African-Middle East" group consisted of six HDV isolates from Ethiopia and one from Somalia, Jordan, and Lebanon. PMID:8940442

  10. Proteomics of Neisseria gonorrhoeae: the treasure hunt for countermeasures against an old disease.

    PubMed

    Baarda, Benjamin I; Sikora, Aleksandra E

    2015-01-01

    Neisseria gonorrhoeae is an exquisitely adapted, strictly human pathogen and the causative agent of the sexually transmitted infection gonorrhea. This ancient human disease remains a serious problem, occurring at high incidence globally and having a major impact on reproductive and neonatal health. N. gonorrhoeae is rapidly evolving into a superbug and no effective vaccine exists to prevent gonococcal infections. Untreated or inadequately treated gonorrhea can lead to severe sequelae, including pelvic inflammatory disease and infertility in women, epididymitis in men, and sight-threatening conjunctivitis in infants born to infected mothers. Therefore, there is an immediate need for accelerated research toward the identification of molecular targets for development of drugs with new mechanisms of action and preventive vaccine(s). Global proteomic approaches are ideally suited to guide these studies. Recent quantitative proteomics (SILAC, iTRAQ, and ICAT) have illuminated the pathways utilized by N. gonorrhoeae to adapt to different lifestyles and micro-ecological niches within the host, while comparative 2D SDS-PAGE analysis has been used to elucidate spectinomycin resistance mechanisms. Further, high-throughput examinations of cell envelopes and naturally released membrane vesicles have unveiled the ubiquitous and differentially expressed proteins between temporally and geographically diverse N. gonorrhoeae isolates. This review will focus on these different approaches, emphasizing the role of proteomics in the search for vaccine candidates. Although our knowledge of N. gonorrhoeae has been expanded, still far less is known about this bacterium than the closely related N. meningitidis, where genomics- and proteomics-driven studies have led to the successful development of vaccines. PMID:26579097

  11. Proteomics of Neisseria gonorrhoeae: the treasure hunt for countermeasures against an old disease

    PubMed Central

    Baarda, Benjamin I.; Sikora, Aleksandra E.

    2015-01-01

    Neisseria gonorrhoeae is an exquisitely adapted, strictly human pathogen and the causative agent of the sexually transmitted infection gonorrhea. This ancient human disease remains a serious problem, occurring at high incidence globally and having a major impact on reproductive and neonatal health. N. gonorrhoeae is rapidly evolving into a superbug and no effective vaccine exists to prevent gonococcal infections. Untreated or inadequately treated gonorrhea can lead to severe sequelae, including pelvic inflammatory disease and infertility in women, epididymitis in men, and sight-threatening conjunctivitis in infants born to infected mothers. Therefore, there is an immediate need for accelerated research toward the identification of molecular targets for development of drugs with new mechanisms of action and preventive vaccine(s). Global proteomic approaches are ideally suited to guide these studies. Recent quantitative proteomics (SILAC, iTRAQ, and ICAT) have illuminated the pathways utilized by N. gonorrhoeae to adapt to different lifestyles and micro-ecological niches within the host, while comparative 2D SDS-PAGE analysis has been used to elucidate spectinomycin resistance mechanisms. Further, high-throughput examinations of cell envelopes and naturally released membrane vesicles have unveiled the ubiquitous and differentially expressed proteins between temporally and geographically diverse N. gonorrhoeae isolates. This review will focus on these different approaches, emphasizing the role of proteomics in the search for vaccine candidates. Although our knowledge of N. gonorrhoeae has been expanded, still far less is known about this bacterium than the closely related N. meningitidis, where genomics- and proteomics-driven studies have led to the successful development of vaccines. PMID:26579097

  12. Genotyping of Enteric Adenoviruses by Using Single-Stranded Conformation Polymorphism Analysis and Heteroduplex Mobility Assay

    PubMed Central

    Soares, Caroline C.; Volotão, Eduardo M.; Albuquerque, Maria Carolina M.; Nozawa, Carlos M.; Linhares, Rosa Elisa C.; Volokhov, Dmitriy; Chizhikov, Vladimir; Lu, Xiaoyan; Erdman, Dean; Santos, Norma

    2004-01-01

    Single-stranded conformation polymorphism (SSCP) analysis and heteroduplex mobility assays (HMAs) were used to identify and genotype enteric adenoviruses (EAd). The results were compared to those of restriction endonuclease assays, species-specific PCRs, and direct nucleotide sequence analyses. Of the 31 stool samples tested, 15 isolates were identified as EAd and 7 were identified as nonenteric Ad by all methods. An agreement of 100% was found between the SSCP and HMA results. PMID:15071032

  13. Limitation of high-resolution melting curve analysis for genotyping simple sequence repeats in sheep.

    PubMed

    Yang, M; Yue, Y J; Guo, T T; Han, J L; Liu, J B; Guo, J; Sun, X P; Feng, R L; Wu, Y Y; Wang, C F; Wang, L P; Yang, B H

    2014-04-08

    Variation in microsatellite or simple sequence repeat (SSR) loci has, until recently, relied heavily on the use of gel-based methods that can be both time consuming and difficult to genotype. Non gel-based systems are therefore important to increase simplicity and improve turn-around time without compromising assay sensitivity and accuracy. In this report, we assessed the latest of the non-gel-based methods, high-resolution melting (HRM) curve analysis. HRM is a technique that monitors exactly the decreasing fluorescence of intercalating dye in the process of dissociation of double-stranded DNA. The measurement immediately follows polymerase chain reaction in a one-step, closed-tube method. Four SSR loci of different complexity in sheep, namely MAF209, MCM140, CB226, and SRCRSP5, were assessed using the LightScanners System with LC Greens PLUS DNA binding dye. In order to improve the accuracy of genotyping, we applied internal oligo nucleotide calibrators while performing HRM. DNA polymorphisms were previously identified using capillary electrophoresis analysis (CE). The result showed that CE detected more genotypes than HRM in the same loci regardless of the level of polymorphism at the SSR loci. We demonstrate current limitations of the HRM method for the analysis of SSR loci.

  14. First Neisseria gonorrhoeae strain with resistance to cefixime causing gonorrhoea treatment failure in Austria, 2011.

    PubMed

    Unemo, M; Golparian, D; Stary, A; Eigentler, A

    2011-10-27

    We describe the first cefixime-resistant Neisseria gonorrhoeae strain in Austria that caused treatment failure.It follows the first five cases in Europe of cefixime treatment failure, reported in Norway in 2010 and the United Kingdom in 2011. Effective treatment of gonorrhoea is crucial for public health control and, at present, requires substantially enhanced awareness, more frequent test-of-cure, interaction with experts after therapeutic failure, tracing and therapy of contacts, and surveillance of gonococcal antimicrobial resistance and treatment failures worldwide.

  15. Antimicrobial Resistance and Neisseria gonorrhoeae Multiantigen Sequence Typing Profile of Neisseria gonorrhoeae in New Delhi, India.

    PubMed

    Mahajan, Neeraj; Sood, Seema; Singh, Rajendra; Kapil, Arti; Das, Bimal Kumar; Sreenivas, Vishnubhatla; Kar, Hemanta Kumar; Sharma, Vinod Kumar

    2016-08-01

    Molecular epidemiology of 100 consecutive gonococcal isolates collected between April 2010 and October 2013 from New Delhi was investigated using Neisseria gonorrhoeae multiantigen sequence typing (NG-MAST) along with its association with antimicrobial resistance profiles. Neisseria gonorrhoeae isolates were assigned into 60 different sequence types and 43 (71.6%) were novel. Sole representation was seen in 76.6% sequence types. There was significant association between ST6058 and resistance to penicillin (P = 0.00) and tetracycline (P = 0.002). PMID:27414684

  16. Cefixime and ceftriaxone susceptibility of Neisseria gonorrhoeae in Italy from 2006 to 2010.

    PubMed

    Carannante, A; Prignano, G; Cusini, M; Matteelli, A; Dal Conte, I; Ghisetti, V; D'Antuono, A; Cavrini, F; Antonetti, R; Stefanelli, P

    2012-06-01

    Neisseria gonorrhoeae resistance to cephalosporins, the currently recommended treatment, and treatment failures with cefixime have been reported worldwide. The purposes of the present study were (i) to examine the susceptibility of N. gonorrhoeae isolates isolated in Italy from 2006 through 2010 to cefixime (n = 293) taking into account both European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Clinical And Laboratory Standards Institute (CLSI) criteria for categorization; (ii) to determine the contribution to decreased/resistant susceptibility of mutations in the penA, mtrR, ponA and porB1b genes in a subsample of isolates; and (iii) to genotype the isolates showing decreased susceptibility or resistance to cefixime, by N. gonorrhoeae multi-antigen sequence typing (NG-MAST) and by pulsed-field gel electrophoresis (PFGE) to identify the predominant genotypes. Minimum inhibitory concentrations (MICs) were determined by the E-test and agar dilution method on 293 isolates and results were interpreted according to both EUCAST 2010 (MIC R >0.12 mg/L) and CLSI 2008 (MIC R >0.25 mg/L) criteria. All isolates showed full susceptibility to ceftriaxone, whereas those with a MIC for cefixime ≥0.125 mg/L were on the increase from 2008 through 2010. The same penA gene alterations were found among isolates with MICs close to the EUCAST breakpoint as the resistant ones, and they belong to ST1407. Seven isolates, belonging to various sequence types, showed a different por allele, though similar to the por 908 allele present in ST1407. PFGE divided strains ST1407 into two main groups confirming their genetic relationship.

  17. Hierarchical Modeling and Differential Expression Analysis for RNA-seq Experiments with Inbred and Hybrid Genotypes

    PubMed Central

    Lithio, Andrew; Nettleton, Dan

    2016-01-01

    The performance of inbred and hybrid genotypes is of interest in plant breeding and genetics. High-throughput sequencing of RNA (RNA-seq) has proven to be a useful tool in the study of the molecular genetic responses of inbreds and hybrids to environmental stresses. Commonly used experimental designs and sequencing methods lead to complex data structures that require careful attention in data analysis. We demonstrate an analysis of RNA-seq data from a split-plot design involving drought stress applied to two inbred genotypes and two hybrids formed by crosses between the inbreds. Our generalized linear modeling strategy incorporates random effects for whole-plot experimental units and uses negative binomial distributions to allow for overdispersion in count responses for split-plot experimental units. Variations in gene length and base content, as well as differences in sequencing intensity across experimental units, are also accounted for. Hierarchical modeling with thoughtful parameterization and prior specification allows for borrowing of information across genes to improve estimation of dispersion parameters, genotype effects, treatment effects, and interaction effects of primary interest. PMID:27110090

  18. Meta-analysis of the association between GSTT1 null genotype and risk of nasopharyngeal carcinoma in Chinese.

    PubMed

    Jin, Bin; Dong, Pin; Li, Keyong; Shen, Bin; Xie, Jin

    2014-01-01

    Glutathione S-transferase T1 (GSTT1) null genotype has been proven to be associated with risks of many cancers. There were also many studies assessing on the association between GSTT1 null genotype and nasopharyngeal carcinoma risk in Chinese, but the findings from those studies were inconsistent. We performed a meta-analysis to provide a more precise assessment on the effect of GSTT1 null genotype on nasopharyngeal carcinoma risk. The PubMed and Wanfang databases were searched to identify eligible case-control studies on the association between GSTT1 null genotype and risk of nasopharyngeal carcinoma in Chinese. The pooled odds ratios (OR) with corresponding 95% confidence intervals (95% CI) were used to assess the association. Eight case-control studies with a total of 3,702 individuals were finally included in the meta-analysis. Meta-analysis of a total of eight studies showed that GSTT1 null genotype was significantly associated with increased risk of nasopharyngeal carcinoma in Chinese (OR = 2.27; 95% CI 1.41-3.67; P = 0.001). The finding from cumulative meta-analysis showed that there was a trend of more obvious association between GSTT1 null genotype and risk of nasopharyngeal carcinoma in Chinese as data accumulated by publication year. Therefore, the GSTT1 null genotype is significantly associated with increased risk of nasopharyngeal carcinoma in Chinese.

  19. Analysis of the Molecular Evolution of Hepatitis B Virus Genotypes in Symptomatic Acute Infections in Argentina

    PubMed Central

    Rodrigo, María Belén; Mojsiejczuk, Laura Noelia; Torres, Carolina; Sevic, Ina; González López Ledesma, María Mora; Perez, Paula Soledad; Bouzas, María Belén; Galdame, Omar; Marciano, Sebastián; Fainboim, Hugo; Flichman, Diego Martín; Campos, Rodolfo Héctor

    2016-01-01

    Hepatitis B virus (HBV) is a globally distributed human pathogen that leads to both self-limited and chronic infections. At least eight genotypes (A-H) with distinct geographical allocations and phylodynamic behaviors have been described. They differ substantially in many virological and probably some clinical parameters. The aim of this study was to analyze full-length HBV genome sequences from individuals with symptomatic acute HBV infections using phylogenetic and coalescent methods. The phylogenetic analysis resulted in the following subgenotype distribution: F1b (52.7%), A2 (18.2%), F4 (18.2%) and A1, B2, D3 and F2a 1.8% each. These results contrast with those previously reported from chronic infections, where subgenotypes F1b, F4, A2 and genotype D were evenly distributed. This differential distribution might be related to recent internal migrations and/or intrinsic biological features of each viral genotype that could impact on the probability of transmission. The coalescence analysis showed that after a diversification process started in the 80s, the current sequences of subgenotype F1b were grouped in at least four highly supported lineages, whereas subgenotype F4 revealed a more limited diversification pattern with most lineages without offspring in the present. In addition, the genetic characterization of the studied sequences showed that only two of them presented mutations of clinical relevance at S codifyng region and none at the polymerase catalytic domains. Finally, since the acute infections could be an expression of the genotypes currently being transmitted to new hosts, the predominance of subgenotype F1b might have epidemiological, as well as, clinical relevance due to its potential adverse disease outcome among the chronic cases. PMID:27433800

  20. Review and International Recommendation of Methods for Typing Neisseria gonorrhoeae Isolates and Their Implications for Improved Knowledge of Gonococcal Epidemiology, Treatment, and Biology

    PubMed Central

    Unemo, Magnus; Dillon, Jo-Anne R.

    2011-01-01

    Summary: Gonorrhea, which may become untreatable due to multiple resistance to available antibiotics, remains a public health problem worldwide. Precise methods for typing Neisseria gonorrhoeae, together with epidemiological information, are crucial for an enhanced understanding regarding issues involving epidemiology, test of cure and contact tracing, identifying core groups and risk behaviors, and recommending effective antimicrobial treatment, control, and preventive measures. This review evaluates methods for typing N. gonorrhoeae isolates and recommends various methods for different situations. Phenotypic typing methods, as well as some now-outdated DNA-based methods, have limited usefulness in differentiating between strains of N. gonorrhoeae. Genotypic methods based on DNA sequencing are preferred, and the selection of the appropriate genotypic method should be guided by its performance characteristics and whether short-term epidemiology (microepidemiology) or long-term and/or global epidemiology (macroepidemiology) matters are being investigated. Currently, for microepidemiological questions, the best methods for fast, objective, portable, highly discriminatory, reproducible, typeable, and high-throughput characterization are N. gonorrhoeae multiantigen sequence typing (NG-MAST) or full- or extended-length porB gene sequencing. However, pulsed-field gel electrophoresis (PFGE) and Opa typing can be valuable in specific situations, i.e., extreme microepidemiology, despite their limitations. For macroepidemiological studies and phylogenetic studies, DNA sequencing of chromosomal housekeeping genes, such as multilocus sequence typing (MLST), provides a more nuanced understanding. PMID:21734242

  1. Purification and biochemical characterization of DnaK and its transcriptional activator RpoH from Neisseria gonorrhoeae.

    PubMed

    Narayanan, Shalini; Beckham, Simone A; Davies, John K; Roujeinikova, Anna

    2014-12-01

    DnaK plays a central role in stress response in the important human pathogen Neisseria gonorrhoeae. The genes encoding the DnaK chaperone machine (DnaK/DnaJ/GrpE) in N. gonorrhoeae are transcribed from RpoH (σ(32))-dependent promoters. In this study, we cloned, purified and biochemically characterised N. gonorrhoeae DnaK (NgDnaK) and RpoH. The NgDnaK and RpoH sequences are 73 and 50 % identical to the sequences of their respective E. coli counterparts. Similar to EcDnaK, nucleotide-free NgDnaK exists as a mix of monomers, dimers and higher oligomeric species in solution, and dissociates into monomers on addition of ATP. Like E. coli σ(32), RpoH of N. gonorrhoeae is monomeric in solution. Kinetic analysis of the basal ATPase activity of purified NgDnaK revealed a V max of 193 pmol phosphate released per minute per microgram DnaK (which is significantly higher than reported basal ATPase activity of EcDnaK), and the turnover number against ATP was 0.4 min(-1) under our assay conditions. Nucleotide-free NgDnaK bound a short model substrate, NR-peptide, with micromolar affinity close to that reported for EcDnaK. Our analysis showed that interaction between N. gonorrhoeae RpoH and DnaK appears to be ATP-dependent and non-specific, in stark contrast to the E. coli DnaK system where σ(32) and DnaK interact as monomers even in the absence of ATP. Sequence comparison showed that the DnaK-binding site of σ(32) is not conserved in RpoH. Our findings suggest that the mechanism of DnaK/RpoH recognition in N. gonorrhoeae is different from that in E. coli. PMID:25156536

  2. Exhaustive Analysis of a Genotype Space Comprising 10(15 )Central Carbon Metabolisms Reveals an Organization Conducive to Metabolic Innovation.

    PubMed

    Hosseini, Sayed-Rzgar; Barve, Aditya; Wagner, Andreas

    2015-08-01

    All biological evolution takes place in a space of possible genotypes and their phenotypes. The structure of this space defines the evolutionary potential and limitations of an evolving system. Metabolism is one of the most ancient and fundamental evolving systems, sustaining life by extracting energy from extracellular nutrients. Here we study metabolism's potential for innovation by analyzing an exhaustive genotype-phenotype map for a space of 10(15) metabolisms that encodes all possible subsets of 51 reactions in central carbon metabolism. Using flux balance analysis, we predict the viability of these metabolisms on 10 different carbon sources which give rise to 1024 potential metabolic phenotypes. Although viable metabolisms with any one phenotype comprise a tiny fraction of genotype space, their absolute numbers exceed 10(9) for some phenotypes. Metabolisms with any one phenotype typically form a single network of genotypes that extends far or all the way through metabolic genotype space, where any two genotypes can be reached from each other through a series of single reaction changes. The minimal distance of genotype networks associated with different phenotypes is small, such that one can reach metabolisms with novel phenotypes--viable on new carbon sources--through one or few genotypic changes. Exceptions to these principles exist for those metabolisms whose complexity (number of reactions) is close to the minimum needed for viability. Increasing metabolic complexity enhances the potential for both evolutionary conservation and evolutionary innovation.

  3. Exhaustive Analysis of a Genotype Space Comprising 1015 Central Carbon Metabolisms Reveals an Organization Conducive to Metabolic Innovation

    PubMed Central

    Hosseini, Sayed-Rzgar; Barve, Aditya; Wagner, Andreas

    2015-01-01

    All biological evolution takes place in a space of possible genotypes and their phenotypes. The structure of this space defines the evolutionary potential and limitations of an evolving system. Metabolism is one of the most ancient and fundamental evolving systems, sustaining life by extracting energy from extracellular nutrients. Here we study metabolism’s potential for innovation by analyzing an exhaustive genotype-phenotype map for a space of 1015 metabolisms that encodes all possible subsets of 51 reactions in central carbon metabolism. Using flux balance analysis, we predict the viability of these metabolisms on 10 different carbon sources which give rise to 1024 potential metabolic phenotypes. Although viable metabolisms with any one phenotype comprise a tiny fraction of genotype space, their absolute numbers exceed 109 for some phenotypes. Metabolisms with any one phenotype typically form a single network of genotypes that extends far or all the way through metabolic genotype space, where any two genotypes can be reached from each other through a series of single reaction changes. The minimal distance of genotype networks associated with different phenotypes is small, such that one can reach metabolisms with novel phenotypes – viable on new carbon sources – through one or few genotypic changes. Exceptions to these principles exist for those metabolisms whose complexity (number of reactions) is close to the minimum needed for viability. Increasing metabolic complexity enhances the potential for both evolutionary conservation and evolutionary innovation. PMID:26252881

  4. Detection and identification of genotypes of Prototheca zopfii in clinical samples by quantitative PCR analysis.

    PubMed

    Onozaki, Masanobu; Makimura, Koichi; Satoh, Kazuo; Hasegawa, Atsuhiko

    2013-01-01

    In this study, a specific quantitative PCR system for the detection and identification of Prototheca zopfii genotypes was developed using a TaqMan(®) MGB probe and ResoLight dye. The P. zopfii-specific primers 18PZF1 and 18PZR1 were generated on the basis of the alignment of the small subunit ribosomal DNA domain base sequences of the genera Chlorella and Prototheca obtained from DDBJ/EMBL/GenBank, and the TaqMan(®) MGB probe PZP1 was designed corresponding to this amplification region. Analysis of the melting curves of the amplicons using ResoLight dye was able to differentiate between P. zopfii genotypes 1 and 2. The specificity of this detection system was examined using strains from a culture collection (28 strains) and clinical isolates (140 strains). The TaqMan(®) MGB probe amplicon was detected only in reference strains of P. zopfii (n = 12) and clinical isolates (n = 135). Ninety-two clinical specimens from cows with mastitis (36 samples) and healthy controls (56 samples) were also tested. All isolates from milk samples (n = 92) and clinical isolates (n = 135) were identified as P. zopfii genotype 2. PMID:24047735

  5. A model system for QTL analysis: Effects of alcohol dehydrogenase genotype on alcohol pharmacokinetics

    SciTech Connect

    Martin, N.G.; Nightingale, B.; Whitfield, J.B.

    1994-09-01

    There is much interest in the detection of quantitative trait loci (QTL) - major genes which affect quantitative phenotypes. The relationship of polymorphism at known alcohol metabolizing enzyme loci to alcohol pharmacokinetics is a good model system. The three class I alcohol dehydrogenase genes are clustered on chromosome 4 and protein electrophoresis has revealed polymorphisms at the ADH2 and ADH3 loci. While different activities of the isozymes have been demonstrated in vitro, little work has been done in trying to relate ADH polymorphism to variation in ethanol metabolism in vivo. We previously measured ethanol metabolism and psychomotor reactivity in 206 twin pairs and demonstrated that most of the repeatable variation was genetic. We have now recontacted the twins to obtain DNA samples and used PCR with allele specific primers to type the ADH2 and ADH3 polymorphisms in 337 individual twins. FISHER has been used to estimate fixed effects of typed polymorphisms simultaneously with remaining linked and unlinked genetic variance. The ADH2*1-2 genotypes metabolize ethanol faster and attain a lower peak blood alcohol concentration than the more common ADH2*1-1 genotypes, although less than 3% of the variance is accounted for. There is no effect of ADH3 genotype. However, sib-pair linkage analysis suggests that there is a linked polymorphism which has a much greater effect on alcohol metabolism that those typed here.

  6. Array-based genotyping and expression analysis of barley cv. Maythorpe and Golden Promise

    PubMed Central

    Walia, Harkamal; Wilson, Clyde; Condamine, Pascal; Ismail, Abdelbagi M; Xu, Jin; Cui, Xinping; Close, Timothy J

    2007-01-01

    Background Golden Promise is a salt-tolerant spring barley closely related to Maythorpe. Salt tolerance in Golden Promise has been attributed to a single mutation at the Ari-e locus (on 5H) resulting from irradiation of Maythorpe. Golden Promise accumulates lower shoot Na+ compared to Maythorpe when growing under saline conditions. This study focused on elucidating the genetic basis and mechanisms involved in this difference. Results The level of polymorphism between the two genotypes was explored using the Barley1 GeneChip for single feature polymorphisms (SFPs) and an oligonucleotide pool assay for single nucleotide polymorphisms (SNPs). Polymorphism analyses revealed three haplotype blocks spanning 6.4 cM on chromosome 1H, 23.7 cM on chromosome 4H and 3.0 cM on 5H. The Barley1 GeneChip was used to examine transcript abundance in different tissues and stages during development. Several genes within the polymorphic haplotype blocks were differentially regulated. Additionally, a more global difference in the jasmonic acid pathway regulation was detected between the two genotypes. Conclusion The results confirm that Golden Promise and Maythorpe are genetically very closely related but establish that they are not isogenic, as previously reported, due to three polymorphic haplotype blocks. Transcriptome analysis indicates that the response of the two genotypes to salinity stress is quite different. Additionally, the response to salinity stress in the roots and shoot tissue is strikingly different. PMID:17394671

  7. Fully automated analysis of multi-resolution four-channel micro-array genotyping data

    NASA Astrophysics Data System (ADS)

    Abbaspour, Mohsen; Abugharbieh, Rafeef; Podder, Mohua; Tebbutt, Scott J.

    2006-03-01

    We present a fully-automated and robust microarray image analysis system for handling multi-resolution images (down to 3-micron with sizes up to 80 MBs per channel). The system is developed to provide rapid and accurate data extraction for our recently developed microarray analysis and quality control tool (SNP Chart). Currently available commercial microarray image analysis applications are inefficient, due to the considerable user interaction typically required. Four-channel DNA microarray technology is a robust and accurate tool for determining genotypes of multiple genetic markers in individuals. It plays an important role in the state of the art trend where traditional medical treatments are to be replaced by personalized genetic medicine, i.e. individualized therapy based on the patient's genetic heritage. However, fast, robust, and precise image processing tools are required for the prospective practical use of microarray-based genetic testing for predicting disease susceptibilities and drug effects in clinical practice, which require a turn-around timeline compatible with clinical decision-making. In this paper we have developed a fully-automated image analysis platform for the rapid investigation of hundreds of genetic variations across multiple genes. Validation tests indicate very high accuracy levels for genotyping results. Our method achieves a significant reduction in analysis time, from several hours to just a few minutes, and is completely automated requiring no manual interaction or guidance.

  8. Gonorrhoea in 21st century--international and Polish situation.

    PubMed

    Serwin, Agnieszka Beata; Koper, Marta; Unemo, Magnus

    2014-01-01

    Gonorrhoea, according to the latest World Health Organization (WHO) estimates in 2008, is the most frequent bacterial sexually transmitted infection globally, accounting for 106.1 million new cases among adults. Of those cases, 3.4 (3.2%) million were in the WHO European Region. In the European Union and European Economic Area, the incidence of reported cases was 12.6 per 100,000 inhabitants in 2011. The highest incidences were noted in the United Kingdom (37.1), Latvia (24.4) and Ireland (18.6). However, in Poland from 2000 to 2011 the reported incidence declined and was only 0.8-0.9 per 100,000 inhabitants in 2011, that might indicate a suboptimal diagnostics and incomplete case reporting and epidemiological surveillance. A study surveying the diagnostics for gonorrhoea and the case reporting system, including the local and national epidemiological surveillance, in Poland is recommended. The high resistance in Neisseria gonorrhoeae to nearly all antimicrobials introduced for treatment of gonorrhoea is an exceedingly serious problem globally. A few years ago the first extensively-drug resistant N. gonorrhoeae strains with high-level resistance to ceftriaxone, the last remaining option for first-line empirical monotherapy, were reported. Due to this emergent situation, in 2012 the WHO and the European Centre for Disease Prevention and Control (ECDC) launched a global action plan and regional response plan, respectively, to combat the spread of multidrug resistant N. gonorrhoeae. Additionally, an updated European guideline on the diagnosis and treatment of gonorrhoea, recommending treatment with ceftriaxone together with azithromycin, was published in 2012. Worryingly, no antimicrobial susceptibility data for N. gonorrhoeae strains circulating in Poland have been internationally reported in several decades. It is imperative to implement some regular and quality assured antimicrobial susceptibility surveillance for N. gonorrhoeae in Poland and the official Polish

  9. TASSEL-GBS: a high capacity genotyping by sequencing analysis pipeline.

    PubMed

    Glaubitz, Jeffrey C; Casstevens, Terry M; Lu, Fei; Harriman, James; Elshire, Robert J; Sun, Qi; Buckler, Edward S

    2014-01-01

    Genotyping by sequencing (GBS) is a next generation sequencing based method that takes advantage of reduced representation to enable high throughput genotyping of large numbers of individuals at a large number of SNP markers. The relatively straightforward, robust, and cost-effective GBS protocol is currently being applied in numerous species by a large number of researchers. Herein we describe a bioinformatics pipeline, TASSEL-GBS, designed for the efficient processing of raw GBS sequence data into SNP genotypes. The TASSEL-GBS pipeline successfully fulfills the following key design criteria: (1) Ability to run on the modest computing resources that are typically available to small breeding or ecological research programs, including desktop or laptop machines with only 8-16 GB of RAM, (2) Scalability from small to extremely large studies, where hundreds of thousands or even millions of SNPs can be scored in up to 100,000 individuals (e.g., for large breeding programs or genetic surveys), and (3) Applicability in an accelerated breeding context, requiring rapid turnover from tissue collection to genotypes. Although a reference genome is required, the pipeline can also be run with an unfinished "pseudo-reference" consisting of numerous contigs. We describe the TASSEL-GBS pipeline in detail and benchmark it based upon a large scale, species wide analysis in maize (Zea mays), where the average error rate was reduced to 0.0042 through application of population genetic-based SNP filters. Overall, the GBS assay and the TASSEL-GBS pipeline provide robust tools for studying genomic diversity.

  10. TASSEL-GBS: a high capacity genotyping by sequencing analysis pipeline.

    PubMed

    Glaubitz, Jeffrey C; Casstevens, Terry M; Lu, Fei; Harriman, James; Elshire, Robert J; Sun, Qi; Buckler, Edward S

    2014-01-01

    Genotyping by sequencing (GBS) is a next generation sequencing based method that takes advantage of reduced representation to enable high throughput genotyping of large numbers of individuals at a large number of SNP markers. The relatively straightforward, robust, and cost-effective GBS protocol is currently being applied in numerous species by a large number of researchers. Herein we describe a bioinformatics pipeline, TASSEL-GBS, designed for the efficient processing of raw GBS sequence data into SNP genotypes. The TASSEL-GBS pipeline successfully fulfills the following key design criteria: (1) Ability to run on the modest computing resources that are typically available to small breeding or ecological research programs, including desktop or laptop machines with only 8-16 GB of RAM, (2) Scalability from small to extremely large studies, where hundreds of thousands or even millions of SNPs can be scored in up to 100,000 individuals (e.g., for large breeding programs or genetic surveys), and (3) Applicability in an accelerated breeding context, requiring rapid turnover from tissue collection to genotypes. Although a reference genome is required, the pipeline can also be run with an unfinished "pseudo-reference" consisting of numerous contigs. We describe the TASSEL-GBS pipeline in detail and benchmark it based upon a large scale, species wide analysis in maize (Zea mays), where the average error rate was reduced to 0.0042 through application of population genetic-based SNP filters. Overall, the GBS assay and the TASSEL-GBS pipeline provide robust tools for studying genomic diversity. PMID:24587335

  11. TASSEL-GBS: A High Capacity Genotyping by Sequencing Analysis Pipeline

    PubMed Central

    Glaubitz, Jeffrey C.; Casstevens, Terry M.; Lu, Fei; Harriman, James; Elshire, Robert J.; Sun, Qi; Buckler, Edward S.

    2014-01-01

    Genotyping by sequencing (GBS) is a next generation sequencing based method that takes advantage of reduced representation to enable high throughput genotyping of large numbers of individuals at a large number of SNP markers. The relatively straightforward, robust, and cost-effective GBS protocol is currently being applied in numerous species by a large number of researchers. Herein we describe a bioinformatics pipeline, tassel-gbs, designed for the efficient processing of raw GBS sequence data into SNP genotypes. The tassel-gbs pipeline successfully fulfills the following key design criteria: (1) Ability to run on the modest computing resources that are typically available to small breeding or ecological research programs, including desktop or laptop machines with only 8–16 GB of RAM, (2) Scalability from small to extremely large studies, where hundreds of thousands or even millions of SNPs can be scored in up to 100,000 individuals (e.g., for large breeding programs or genetic surveys), and (3) Applicability in an accelerated breeding context, requiring rapid turnover from tissue collection to genotypes. Although a reference genome is required, the pipeline can also be run with an unfinished “pseudo-reference” consisting of numerous contigs. We describe the tassel-gbs pipeline in detail and benchmark it based upon a large scale, species wide analysis in maize (Zea mays), where the average error rate was reduced to 0.0042 through application of population genetic-based SNP filters. Overall, the GBS assay and the tassel-gbs pipeline provide robust tools for studying genomic diversity. PMID:24587335

  12. Genotypic analysis of Staphylococcus aureus from milk of dairy cows with mastitis in Argentina.

    PubMed Central

    Buzzola, F. R.; Quelle, L.; Gomez, M. I.; Catalano, M.; Steele-Moore, L.; Berg, D.; Gentilini, E.; Denamiel, G.; Sordelli, D. O.

    2001-01-01

    Staphylococcus aureus is the most prevalent pathogen causing mastitis of dairy ruminants. This study was developed to ascertain the genotypes and genealogical relationship among strains isolated from milk of bovines with mastitis in Argentina. Molecular epidemiological analysis of S. aureus was performed on 112 isolates from 21 districts. Clonality was assessed by SmaI pulsed-field gel electrophoresis (PFGE) typing, automated EcoRI ribotyping and restriction enzyme analysis of plasmid (REAP) DNA profiles. A total of 22 band patterns distributed in four clusters were found by SmaI PFGE analysis. The similarity of clusters 2, 3 and 4 with cluster 1 was 0.73, 0.69 and 0.33, respectively, and 101 of 112 isolates belonged in cluster 1. PFGE band patterns from 42 isolates within cluster I were indistinguishable from each other (type A). The second largest group of isolates with indistinguishable PFGE band patterns was subtype A11, which was composed of 19 isolates. Automated ribotyping assigned the 112 isolates into 13 ribotypes. Among these, the most prevalent ribotypes I and VI were composed of 49 and 35 isolates respectively. Although there was certain correspondence between PFGE genotypes and ribotypes, further discrimination was achieved by combining both methods. REAP DNA profile analysis was useful to provide even further discrimination between isolates with identical PFGE genotype and ribotype. The most prevalent S. aureus strains A/I and A11/VI were widely distributed in the country and were not restricted to individual nearby locations. Prevalence of these two strains varied consecutively within a period of 8 years. Whether the shift in type prevalence was due to selection of a phenotypic trait remains undisclosed. PMID:11467802

  13. Mutation of the Conserved Calcium-Binding Motif in Neisseria gonorrhoeae PilC1 Impacts Adhesion but Not Piliation

    PubMed Central

    Cheng, Yuan; Johnson, Michael D. L.; Burillo-Kirch, Christine; Mocny, Jeffrey C.; Anderson, James E.; Garrett, Christopher K.; Redinbo, Matthew R.

    2013-01-01

    Neisseria gonorrhoeae PilC1 is a member of the PilC family of type IV pilus-associated adhesins found in Neisseria species and other type IV pilus-producing genera. Previously, a calcium-binding domain was described in the C-terminal domains of PilY1 of Pseudomonas aeruginosa and in PilC1 and PilC2 of Kingella kingae. Genetic analysis of N. gonorrhoeae revealed a similar calcium-binding motif in PilC1. To evaluate the potential significance of this calcium-binding region in N. gonorrhoeae, we produced recombinant full-length PilC1 and a PilC1 C-terminal domain fragment. We show that, while alterations of the calcium-binding motif disrupted the ability of PilC1 to bind calcium, they did not grossly affect the secondary structure of the protein. Furthermore, we demonstrate that both full-length wild-type PilC1 and full-length calcium-binding-deficient PilC1 inhibited gonococcal adherence to cultured human cervical epithelial cells, unlike the truncated PilC1 C-terminal domain. Similar to PilC1 in K. kingae, but in contrast to the calcium-binding mutant of P. aeruginosa PilY1, an equivalent mutation in N. gonorrhoeae PilC1 produced normal amounts of pili. However, the N. gonorrhoeae PilC1 calcium-binding mutant still had partial defects in gonococcal adhesion to ME180 cells and genetic transformation, which are both essential virulence factors in this human pathogen. Thus, we conclude that calcium binding to PilC1 plays a critical role in pilus function in N. gonorrhoeae. PMID:24002068

  14. Sequence-independent amplification coupled with DNA microarray analysis for detection and genotyping of noroviruses.

    PubMed

    Hu, Yuan; Yan, Huijun; Mammel, Mark; Chen, Haifeng

    2015-12-01

    Noroviruses (NoVs) have high levels of genetic sequence diversities, which lead to difficulties in designing robust universal primers to efficiently amplify specific viral genomes for molecular analysis. We here described the practicality of sequence-independent amplification combined with DNA microarray analysis for simultaneous detection and genotyping of human NoVs in fecal specimens. We showed that single primer isothermal linear amplification (Ribo-SPIA) of genogroup I (GI) and genogroup II (GII) NoVs could be run through the same amplification protocol without the need to design and use any virus-specific primers. Related virus could be subtyped by the unique pattern of hybridization with the amplified product to the microarray. By testing 22 clinical fecal specimens obtained from acute gastroenteritis cases as blinded samples, 2 were GI positive and 18 were GII positive as well as 2 negative for NoVs. A NoV GII positive specimen was also identified as having co-occurrence of hepatitis A virus. The study showed that there was 100 % concordance for positive NoV detection at genogroup level between the results of Ribo-SPIA/microarray and the phylogenetic analysis of viral sequences of the capsid gene. In addition, 85 % genotype agreement was observed for the new assay compared to the results of phylogenetic analysis. PMID:26556029

  15. Sequence-independent amplification coupled with DNA microarray analysis for detection and genotyping of noroviruses.

    PubMed

    Hu, Yuan; Yan, Huijun; Mammel, Mark; Chen, Haifeng

    2015-12-01

    Noroviruses (NoVs) have high levels of genetic sequence diversities, which lead to difficulties in designing robust universal primers to efficiently amplify specific viral genomes for molecular analysis. We here described the practicality of sequence-independent amplification combined with DNA microarray analysis for simultaneous detection and genotyping of human NoVs in fecal specimens. We showed that single primer isothermal linear amplification (Ribo-SPIA) of genogroup I (GI) and genogroup II (GII) NoVs could be run through the same amplification protocol without the need to design and use any virus-specific primers. Related virus could be subtyped by the unique pattern of hybridization with the amplified product to the microarray. By testing 22 clinical fecal specimens obtained from acute gastroenteritis cases as blinded samples, 2 were GI positive and 18 were GII positive as well as 2 negative for NoVs. A NoV GII positive specimen was also identified as having co-occurrence of hepatitis A virus. The study showed that there was 100 % concordance for positive NoV detection at genogroup level between the results of Ribo-SPIA/microarray and the phylogenetic analysis of viral sequences of the capsid gene. In addition, 85 % genotype agreement was observed for the new assay compared to the results of phylogenetic analysis.

  16. Identification of sRNAs expressed by the human pathogen Neisseria gonorrhoeae under disparate growth conditions.

    PubMed

    McClure, Ryan; Tjaden, Brian; Genco, Caroline

    2014-01-01

    In the last several years, bacterial gene regulation via small RNAs (sRNAs) has been recognized as an important mechanism controlling expression of essential proteins that are critical to bacterial growth and metabolism. Technologies such as RNA-seq are rapidly expanding the field of sRNAs and are enabling a global view of the "sRNAome" of several bacterial species. While numerous sRNAs have been identified in a variety of both Gram-negative and Gram-positive bacteria, only a very small number have been fully characterized in the human pathogen Neisseria gonorrhoeae, the etiological agent of the STD gonorrhea. Here we present the first analysis of N. gonorrhoeae specifically focused on the identification of sRNAs through RNA-seq analysis of the organism cultured under different in vitro growth conditions. Using a new computational program, Rockhopper, to analyze prokaryotic RNA-seq data obtained from N. gonorrhoeae we identified several putative sRNAs and confirmed their expression and size through Northern blot analysis. In addition, RNA was collected from four different growth conditions (iron replete and deplete, as well as with and without co-culture with human endocervical cells). Many of the putative sRNAs identified shoed varying expression levels relative to the different growth conditions examine or were detected only under certain conditions but not others. Comparisons of identified sRNAs with the regulatory pattern of putative mRNA targets revealed possible functional roles for these sRNAs. These studies are the first to carry out a global analysis of N. gonorrhoeae specifically focused on sRNAs and show that RNA-mediated regulation may be an important mechanism of gene control in this human pathogen. PMID:25221548

  17. Restriction Site Tiling Analysis: accurate discovery and quantitative genotyping of genome-wide polymorphisms using nucleotide arrays

    PubMed Central

    2010-01-01

    High-throughput genotype data can be used to identify genes important for local adaptation in wild populations, phenotypes in lab stocks, or disease-related traits in human medicine. Here we advance microarray-based genotyping for population genomics with Restriction Site Tiling Analysis. The approach simultaneously discovers polymorphisms and provides quantitative genotype data at 10,000s of loci. It is highly accurate and free from ascertainment bias. We apply the approach to uncover genomic differentiation in the purple sea urchin. PMID:20403197

  18. Association between glutathione S-transferase M1 null genotype and risk of gallbladder cancer: a meta-analysis.

    PubMed

    Sun, Hong-Li; Han, Bing; Zhai, Hong-Peng; Cheng, Xin-Hua; Ma, Kai

    2014-01-01

    Glutathione S-transferases (GSTs) are a family of enzymes which are involved in the detoxification of potential carcinogens. Glutathione S-transferase M1 (GSTM1) null genotype can impair the enzyme activity of GSTs and is suspected to increase the susceptibility to gallbladder cancer. Previous studies investigating the association between GSTM1 null genotype and risk of gallbladder cancer reported inconsistent findings. To quantify the association between GSTM1 null genotype and risk of gallbladder cancer, we performed a meta-analysis of published studies. We searched PubMed, Embase, and Wanfang databases for all possible studies. We estimated the pooled odds ratio (OR) with its 95% confidence interval (95% CI) to assess the association. Meta-analysis of total included studies showed that GSTM1 null genotype was not associated with gallbladder cancer risk (OR = 1.13, 95% CI 0.88-1.46, P = 0.332). Subgroup analysis by ethnicity showed that there was no association between GSTM1 null genotype and risk of gallbladder cancer in both Caucasians and Asians. However, meta-analysis of studies with adjusted estimations showed that GSTM1 null genotype was associated with increased risk of gallbladder cancer (OR = 1.46, 95% CI 1.02-2.09, P = 0.038). Thus, this meta-analysis shows that GSTM1 null genotype is likely to be associated with risk of gallbladder cancer. More studies with well design and large sample size are needed to further validate the association between GSTM1 null genotype and gallbladder cancer.

  19. Genotypic change of porcine circovirus type 2 on Japanese pig farms as revealed by restriction fragment length polymorphism analysis.

    PubMed

    Takahagi, Yoichi; Nishiyama, Yasutaka; Toki, Shinji; Yonekita, Taro; Morimatsu, Fumiki; Murakami, Hiroshi

    2008-06-01

    Porcine circovirus type 2 (PCV2) has been recognized as the causal agent of postweaning multisystemic wasting syndrome and can be divided into two major genotypic groups. We developed a method of restriction fragment length polymorphism (RFLP) analysis of PCV2 open reading frame 2 for easy discrimination between the two major groups. Genotyping of PCV2 isolates from 10 Japanese commercial pig farms was performed, and the analysis revealed that both PCV2 groups and at least five RFLP types of PCV2 are prevalent in Japan. On two farms, the genotypes of the PCV2 isolates in the spring of 2007 were different from those in the autumn of 2006. One genotype may have become dominant within only six months on these farms.

  20. Genotyping of black grouse MHC class II B using reference Strand-Mediated Conformational Analysis (RSCA)

    PubMed Central

    2011-01-01

    Background The Major Histocompatibility Complex (MHC) is a cluster of genes involved in the vertebrate immune system and includes loci with an extraordinary number of alleles. Due to the complex evolution of MHC genes, alleles from different loci within the same MHC class can be very similar and therefore difficult to assign to separate loci. Consequently, single locus amplification of MHC genes is hard to carry out in species with recently duplicated genes in the same MHC class, and multiple MHC loci have to be genotyped simultaneously. Since amplified alleles have the same length, accurate genotyping is difficult. Reference Strand-Mediated Conformational Analysis (RSCA), which is increasingly used in studies of natural populations with multiple MHC genes, is a genotyping method capable to provide high resolution and accuracy in such cases. Findings We adapted the RSCA method to genotype multiple MHC class II B (BLB) genes in black grouse (Tetrao tetrix), a non-model galliform bird species, using a 96-Capillary Array Electrophoresis, the MegaBACE™ 1000 DNA Analysing System (GE Healthcare). In this study we used fluorescently labelled reference strands from both black grouse and hazel grouse and observed good agreement between RSCA and cloning/sequencing since 71 alleles were observed by cloning/sequencing and 76 alleles by RSCA among the 24 individuals included in the comparison. At the individual level however, there was a trend towards more alleles scored with RSCA (1-6 per individual) than cloning/sequencing (1-4 per individual). In 63% of the pair-wise comparison, the identical allele was scored in RSCA as in cloning/sequencing. Nine out of 24 individuals had the same number of alleles in RSCA as in cloning/sequencing. Our RSCA protocol allows a faster RSCA genotyping than presented in many other RSCA studies. Conclusions In this study, we have developed the RSCA typing method further to work on a 96-Capillary Array Electrophoresis (MegaBACE™ 1000). Our

  1. Correlates of gonorrhoea among African Americans in North Carolina.

    PubMed

    Doherty, Irene A; Adimora, Adaora A; Schoenbach, Victor J; Aral, Sevgi O

    2007-02-01

    Rates of gonorrhoea (GC) infections are highest among African Americans in the USA, but risk factors for GC in this population have not been well defined. The purpose of this study was to study these risk factors. We used secondary analysis of cross-sectional data from a population-based case-control study of African Americans, aged 18-59 years, in rural North Carolina, USA. A history of past infection was 2.4 times (95% confidence interval [CI]: 1.3, 4.4) as likely among men (35%) than women (18%). Among men, a history of gonorrhoea was associated with earlier age at first intercourse (adjusted Odds Ratio [aOR] = 2.0 95% CI [1.2, 3.4]), substance use (aOR = 4.4 95% CI [1.1, 17.9]), and having a partner who had been incarcerated (aOR = 18.1 [2.8, 118.0]). Among women, factors associated with GC included diagnosis of another STD (aOR = 5.9 [1.9, 16.9]), lifetime number of sex partners (aOR = 1.9 [1.1, 3.4]), and a sex partner who used drugs (aOR = 4.1 (1.3, 12.9)). In conclusion, African Americans with a history of GC are likely to have traditional STD risk factors, including partners at high risk for sexually transmitted disease (STD). STD screening is warranted upon entry to treatment facilities for substance abuse or correctional facilities.

  2. Polyomavirus JCV excretion and genotype analysis in HIV-infected patients receiving highly active antiretroviral therapy

    NASA Technical Reports Server (NTRS)

    Lednicky, John A.; Vilchez, Regis A.; Keitel, Wendy A.; Visnegarwala, Fehmida; White, Zoe S.; Kozinetz, Claudia A.; Lewis, Dorothy E.; Butel, Janet S.

    2003-01-01

    OBJECTIVE: To assess the frequency of shedding of polyomavirus JC virus (JCV) genotypes in urine of HIV-infected patients receiving highly active antiretroviral therapy (HAART). METHODS: Single samples of urine and blood were collected prospectively from 70 adult HIV-infected patients and 68 uninfected volunteers. Inclusion criteria for HIV-infected patients included an HIV RNA viral load < 1000 copies, CD4 cell count of 200-700 x 106 cells/l, and stable HAART regimen. PCR assays and sequence analysis were carried out using JCV-specific primers against different regions of the virus genome. RESULTS: JCV excretion in urine was more common in HIV-positive patients but not significantly different from that of the HIV-negative group [22/70 (31%) versus 13/68 (19%); P = 0.09]. HIV-positive patients lost the age-related pattern of JCV shedding (P = 0.13) displayed by uninfected subjects (P = 0.01). Among HIV-infected patients significant differences in JCV shedding were related to CD4 cell counts (P = 0.03). Sequence analysis of the JCV regulatory region from both HIV-infected patients and uninfected volunteers revealed all to be JCV archetypal strains. JCV genotypes 1 (36%) and 4 (36%) were the most common among HIV-infected patients, whereas type 2 (77%) was the most frequently detected among HIV-uninfected volunteers. CONCLUSION: These results suggest that JCV shedding is enhanced by modest depressions in immune function during HIV infection. JCV shedding occurred in younger HIV-positive persons than in the healthy controls. As the common types of JCV excreted varied among ethnic groups, JCV genotypes associated with progressive multifocal leukoencephalopathy may reflect demographics of those infected patient populations.

  3. Genetic Variation, Heritability, and Diversity Analysis of Upland Rice (Oryza sativa L.) Genotypes Based on Quantitative Traits.

    PubMed

    Tuhina-Khatun, Mst; Hanafi, Mohamed M; Rafii Yusop, Mohd; Wong, M Y; Salleh, Faezah M; Ferdous, Jannatul

    2015-01-01

    Upland rice is important for sustainable crop production to meet future food demands. The expansion in area of irrigated rice faces limitations due to water scarcity resulting from climate change. Therefore, this research aimed to identify potential genotypes and suitable traits of upland rice germplasm for breeding programmes. Forty-three genotypes were evaluated in a randomised complete block design with three replications. All genotypes exhibited a wide and significant variation for 22 traits. The highest phenotypic and genotypic coefficient of variation was recorded for the number of filled grains/panicle and yields/plant (g). The highest heritability was found for photosynthetic rate, transpiration rate, stomatal conductance, intercellular CO₂, and number of filled grains/panicle and yields/plant (g). Cluster analysis based on 22 traits grouped the 43 rice genotypes into five clusters. Cluster II was the largest and consisted of 20 genotypes mostly originating from the Philippines. The first four principle components of 22 traits accounted for about 72% of the total variation and indicated a wide variation among the genotypes. The selected best trait of the number of filled grains/panicle and yields/plant (g), which showed high heritability and high genetic advance, could be used as a selection criterion for hybridisation programmes in the future. PMID:26258135

  4. Genetic Variation, Heritability, and Diversity Analysis of Upland Rice (Oryza sativa L.) Genotypes Based on Quantitative Traits.

    PubMed

    Tuhina-Khatun, Mst; Hanafi, Mohamed M; Rafii Yusop, Mohd; Wong, M Y; Salleh, Faezah M; Ferdous, Jannatul

    2015-01-01

    Upland rice is important for sustainable crop production to meet future food demands. The expansion in area of irrigated rice faces limitations due to water scarcity resulting from climate change. Therefore, this research aimed to identify potential genotypes and suitable traits of upland rice germplasm for breeding programmes. Forty-three genotypes were evaluated in a randomised complete block design with three replications. All genotypes exhibited a wide and significant variation for 22 traits. The highest phenotypic and genotypic coefficient of variation was recorded for the number of filled grains/panicle and yields/plant (g). The highest heritability was found for photosynthetic rate, transpiration rate, stomatal conductance, intercellular CO₂, and number of filled grains/panicle and yields/plant (g). Cluster analysis based on 22 traits grouped the 43 rice genotypes into five clusters. Cluster II was the largest and consisted of 20 genotypes mostly originating from the Philippines. The first four principle components of 22 traits accounted for about 72% of the total variation and indicated a wide variation among the genotypes. The selected best trait of the number of filled grains/panicle and yields/plant (g), which showed high heritability and high genetic advance, could be used as a selection criterion for hybridisation programmes in the future.

  5. History and epidemiology of antibiotic susceptibilities of Neisseria gonorrhoeae.

    PubMed

    Shigemura, Katsumi; Fujisawa, Masato

    2015-01-01

    Neisseria gonorrhoeae is a common causative microorganism of male urethritis. The most important problem with this infectious disease is antibiotic resistance. For instance, in the 1980's-1990's, most studies showed almost 100% susceptibility of N. gonorrhoeae to the representative cephalosporins, cefixime and cefpodoxime. By the late 1990s, the reported susceptibility decreased to 93.3-100% and further decreased to 82.9-100% in the early 2000's. However, reported susceptibility was revived to 95.8-100% in the late 2000's to 2010's. The susceptibility of N. gonorrhoeae to penicillins varied in different countries and regions. A 2002 Japanese study showed a resistance ratio of about 30% and while Laos, China and Korea showed 80-100% resistance. Fluoroquinolones have shown a dramatic change in their effect on N. gonorrhoeae. In the early 1990's, 0.3-1.3% of N. gonorrhoeae showed low susceptibility or resistance to ciprofloxacin in the US but this figure jumped to 9.5% by 1999. In Asia, N. gonorrhoeae ciprofloxacin resistance or lower susceptibility was about 80-90% in the early 2000's and this trend continues to the present day. Azithromycin is currently the possible last weapon for N. gonorrhoeae treatment per oral administration. The susceptibility of N. gonorrhoeae to azithromycin was 100% in Indonesia in 2004 and the latest study from Germany showed 6% resistance in strains from 2010-2011. This review summarizes the history and epidemiology of N. gonorrhoeae antibiotic susceptibilities, for which the most frequently used antibiotics vary between countries or regions.

  6. Diallel cross analysis of plesiomorphic traits in Triticum aestivum L. genotypes.

    PubMed

    Shehzad, M; Hussain, S B; Qureshi, M K; Akbar, M; Javed, M; Imran, H M; Manzoor, S A

    2015-10-28

    We conducted a 5 x 5 complete diallel cross experiment in bread wheat (Triticum aestivum) with the genotypes 6309, Chkwal-50, Dhrabi, Bhkhar-02, and FS-08. Our objective was to evaluate the type of gene action and the general and specific combining abilities required for various morphological traits in wheat. The results of analysis of variance revealed highly significant differences among genotypes for all the investigated traits. The results of joint regression analysis showed that the data for all the investigated traits fitted a simple additive dominance model. Graphical representation of variance and covariance suggested that most of the investigated traits were controlled by overdominance gene action. However, the peduncle length and plant height were controlled by additive gene action. Variety 6309 carried the highest number of dominant genes for the number of spikelets per spike, number of tillers per plant, plant height, number of fertile tillers per plant, and grain yield per plant. Chakwal-50 carried the highest number of recessive genes for grain yield per plant, number of tillers per plant, number of grains per spike, number of fertile tillers per plant, and plant height. Chakwal-50 and 6309 were the best general combiners for number of spikelets per spike, number of grains per spike, grain yield per plant, 1000-grain weight, number of fertile tillers per plant, and number of tillers per plant. On other hand, 6309 performed well in specific crosses with Chakwal-50, FS-08, and Bhakhar-02 for spike length and number of tillers per plant.

  7. Uses of Staphylococcus aureus GeneChips in Genotyping and Genetic Composition Analysis

    PubMed Central

    Dunman, P. M.; Mounts, W.; McAleese, F.; Immermann, F.; Macapagal, D.; Marsilio, E.; McDougal, L.; Tenover, F. C.; Bradford, P. A.; Petersen, P. J.; Projan, S. J.; Murphy, E.

    2004-01-01

    Understanding the relatedness of strains within a bacterial species is essential for monitoring reservoirs of antimicrobial resistance and for epidemiological studies. Pulsed-field gel electrophoresis (PFGE), ribotyping, and multilocus sequence typing are commonly used for this purpose. However, these techniques are either nonquantitative or provide only a limited estimation of strain relatedness. Moreover, they cannot extensively define the genes that constitute an organism. In the present study, 21 oxacillin-resistant Staphylococcus aureus (ORSA) isolates, representing eight major ORSA lineages, and each of the seven strains for which the complete genomic sequence is publicly available were genotyped using a novel GeneChip-based approach. Strains were also subjected to PFGE and ribotyping analysis. GeneChip results provided a higher level of discrimination among isolates than either ribotyping or PFGE, although strain clustering was similar among the three techniques. In addition, GeneChip signal intensity cutoff values were empirically determined to provide extensive data on the genetic composition of each isolate analyzed. Using this technology it was shown that strains could be examined for each element represented on the GeneChip, including virulence factors, antimicrobial resistance determinants, and agr type. These results were validated by PCR, growth on selective media, and detailed in silico analysis of each of the sequenced genomes. Collectively, this work demonstrates that GeneChips provide extensive genotyping information for S. aureus strains and may play a major role in epidemiological studies in the future where correlating genes with particular disease phenotypes is critical. PMID:15365023

  8. High-throughput SNP-genotyping analysis of the relationships among Ponto-Caspian sturgeon species

    PubMed Central

    Rastorguev, Sergey M; Nedoluzhko, Artem V; Mazur, Alexander M; Gruzdeva, Natalia M; Volkov, Alexander A; Barmintseva, Anna E; Mugue, Nikolai S; Prokhortchouk, Egor B

    2013-01-01

    Abstract Legally certified sturgeon fisheries require population protection and conservation methods, including DNA tests to identify the source of valuable sturgeon roe. However, the available genetic data are insufficient to distinguish between different sturgeon populations, and are even unable to distinguish between some species. We performed high-throughput single-nucleotide polymorphism (SNP)-genotyping analysis on different populations of Russian (Acipenser gueldenstaedtii), Persian (A. persicus), and Siberian (A. baerii) sturgeon species from the Caspian Sea region (Volga and Ural Rivers), the Azov Sea, and two Siberian rivers. We found that Russian sturgeons from the Volga and Ural Rivers were essentially indistinguishable, but they differed from Russian sturgeons in the Azov Sea, and from Persian and Siberian sturgeons. We identified eight SNPs that were sufficient to distinguish these sturgeon populations with 80% confidence, and allowed the development of markers to distinguish sturgeon species. Finally, on the basis of our SNP data, we propose that the A. baerii-like mitochondrial DNA found in some Russian sturgeons from the Caspian Sea arose via an introgression event during the Pleistocene glaciation. In the present study, the high-throughput genotyping analysis of several sturgeon populations was performed. SNP markers for species identification were defined. The possible explanation of the baerii-like mitotype presence in some Russian sturgeons in the Caspian Sea was suggested. PMID:24567827

  9. HIV infection among U.S. Army and Air Force military personnel: sociodemographic and genotyping analysis.

    PubMed

    Singer, Darrell E; Bautista, Christian T; O'Connell, Robert J; Sanders-Buell, Eric; Agan, Brian K; Kijak, Gustavo H; Hakre, Shilpa; Sanchez, Jose L; Sateren, Warren B; McCutchan, Francine E; Michael, Nelson L; Scott, Paul T

    2010-08-01

    Since 1985, the U.S. Department of Defense has periodically screened all military personnel for HIV allowing for the monitoring of the infection in this dynamic cohort population. A nested case-control study was performed to study sociodemographics, overseas assignment, and molecular analysis of HIV. Cases were newly identified HIV infections among U.S. Army and Air Force military personnel from 2000 to 2004. Controls were frequency matched to cases by gender and date of case first positive HIV screening test. Genotyping analysis was performed using high-throughput screening assays and partial genome sequencing. HIV was significantly associated with black race [odds ratio (OR) = 6.65], single marital status (OR = 4.45), and age (OR per year = 1.07). Ninety-seven percent were subtype B and 3% were non-B subtypes (A3, CRF01_AE, A/C recombinant, G, CRF02_AG). Among cases, overseas assignment in the period at risk prior to their first HIV-positive test was associated with non-B HIV subtype infection (OR = 8.44). Black and single military personnel remain disproportionately affected by HIV infection. Most non-B HIV subtypes were associated with overseas assignment. Given the increased frequency and length of assignments, and the expanding HIV genetic diversity observed in this population, there is a need for active HIV genotyping surveillance and a need to reinforce primary HIV prevention efforts.

  10. A thermonuclease of Neisseria gonorrhoeae enhances bacterial escape from killing by neutrophil extracellular traps.

    PubMed

    Juneau, Richard A; Stevens, Jacqueline S; Apicella, Michael A; Criss, Alison K

    2015-07-15

    Acute gonorrhea is characterized by neutrophilic inflammation that is insufficient to clear Neisseria gonorrhoeae. Activated neutrophils release extracellular traps (NETs), which are composed of chromatin and decorated with antimicrobial proteins. The N. gonorrhoeae NG0969 open reading frame contains a gene (nuc) that encodes a putatively secreted thermonuclease (Nuc) that contributes to biofilm remodeling. Here, we report that Nuc degrades NETs to help N. gonorrhoeae resist killing by neutrophils. Primary human neutrophils released NETs after exposure to N. gonorrhoeae, but NET integrity declined over time with Nuc-containing bacteria. Recombinant Nuc and conditioned medium from Nuc-containing N. gonorrhoeae degraded human neutrophil DNA and NETs. NETs were found to have antimicrobial activity against N. gonorrhoeae, and Nuc expression enhanced N. gonorrhoeae survival in the presence of neutrophils that released NETs. We propose that Nuc enables N. gonorrhoeae to escape trapping and killing by NETs during symptomatic infection, highlighting Nuc as a multifunctional virulence factor for N. gonorrhoeae.

  11. 2012 European guideline on the diagnosis and treatment of gonorrhoea in adults.

    PubMed

    Bignell, C; Unemo, M

    2013-02-01

    Gonorrhoea is a major public health concern globally. Of particularly grave concern is that resistance to the extended-spectrum cephalosporins has emerged during the most recent years. This guideline provides recommendations regarding the diagnosis and treatment of gonorrhoea in Europe. Compared to the outdated 2009 European gonorrhoea guideline, this 2012 European gonorrhoea guideline provides up-to-date guidance on, broader indications for testing and treatment of gonorrhoea;the introduction of dual antimicrobial therapy (ceftriaxone 500 mg and azithromycin 2 g) for uncomplicated gonorrhoea when the antimicrobial sensitivity is unknown; recommendation of test of cure in all gonorrhoea cases to ensure eradication of infection and identify emerging resistance; and recommendations to identify, verify and report failures with recommended treatment regimens. Optimisations of the testing, diagnostics, antimicrobial treatment and follow-up of gonorrhoea patients are crucial in controlling the emergent spread of cephalosporin-resistant and multidrug-resistant gonorrhoea.

  12. Resistance to peroxynitrite in Neisseria gonorrhoeae.

    PubMed

    Barth, Kenneth R; Isabella, Vincent M; Wright, Lori F; Clark, Virginia L

    2009-08-01

    Neisseria gonorrhoeae encodes a number of important genes that aid in survival during times of oxidative stress. The same immune cells capable of oxygen-dependent killing mechanisms also have the capacity to generate reactive nitrogen species (RNS) that may function antimicrobially. F62 and eight additional gonococcal strains displayed a high level of resistance to peroxynitrite, while Neisseria meningitidis and Escherichia coli showed a four- to seven-log and a four-log decrease in viability, respectively. Mutation of gonococcal orthologues that are known or suspected to be involved in RNS defence in other bacteria (ahpC, dnrN and msrA) resulted in no loss of viability, suggesting that N. gonorrhoeae has a novel mechanism of resistance to peroxynitrite. Whole-cell extracts of F62 prevented the oxidation of dihydrorhodamine, and decomposition of peroxynitrite was not dependent on ahpC, dnrN or msrA. F62 grown in co-culture with E. coli strain DH10B was shown to protect E. coli viability 10-fold. Also, peroxynitrite treatment of F62 did not result in accumulation of nitrated proteins, suggesting that an active peroxynitrite reductase is responsible for peroxynitrite decomposition rather than a protein sink for amino acid modification. PMID:19406894

  13. Cladistic analysis of genotype data-application to GAW15 Problem 3

    PubMed Central

    Jung, Hsuan; Zhao, Keyan; Marjoram, Paul

    2007-01-01

    Given the increasing size of modern genetic data sets and, in particular, the move towards genome-wide studies, there is merit in considering analyses that gain computational efficiency by being more heuristic in nature. With this in mind, we present results of cladistic analyses methods on the Genetic Analysis Workshop 15 Problem 3 simulated data (answers known). Our analysis attempts to capture similarities between individuals using a series of trees, and then looks for regions in which mutations on those trees can successfully explain a phenotype of interest. Existing varieties of such algorithms assume haplotypes are known, or have been inferred, an assumption that is often unrealistic for genome-wide data. We therefore present an extension of these methods that can successfully analyze genotype, rather than haplotype, data. PMID:18466467

  14. Cladistic analysis of genotype data-application to GAW15 Problem 3.

    PubMed

    Jung, Hsuan; Zhao, Keyan; Marjoram, Paul

    2007-01-01

    Given the increasing size of modern genetic data sets and, in particular, the move towards genome-wide studies, there is merit in considering analyses that gain computational efficiency by being more heuristic in nature. With this in mind, we present results of cladistic analyses methods on the Genetic Analysis Workshop 15 Problem 3 simulated data (answers known). Our analysis attempts to capture similarities between individuals using a series of trees, and then looks for regions in which mutations on those trees can successfully explain a phenotype of interest. Existing varieties of such algorithms assume haplotypes are known, or have been inferred, an assumption that is often unrealistic for genome-wide data. We therefore present an extension of these methods that can successfully analyze genotype, rather than haplotype, data.

  15. Molecular identification of mumps virus genotypes from clinical samples: standardized method of analysis.

    PubMed

    Palacios, G; Jabado, O; Cisterna, D; de Ory, F; Renwick, N; Echevarria, J E; Castellanos, A; Mosquera, M; Freire, M C; Campos, R H; Lipkin, W I

    2005-04-01

    A sensitive nested reverse transcription-PCR assay, targeting a short fragment of the gene encoding the small hydrophobic protein (SH gene), was developed to allow rapid characterization of mumps virus in clinical samples. The sensitivity and specificity of the assay were established using representative genotypes A, B, C, D, E, and F. Mumps virus RNA was characterized directly from cerebrospinal fluid (CSF) samples and in extracts of mumps virus isolates from patients with various clinical syndromes. Direct sequencing of products and subsequent phylogenetic analysis enabled genetic classification. A simple web-based system of sequence analysis was established. The study also allowed characterization of mumps virus strains from Argentina as part of a new subgenotype. This PCR assay for characterization of mumps infections coupled to a web-based analytical program provides a rapid method for identification of known and novel strains.

  16. Adaptability and Stability Study of Selected Sweet Sorghum Genotypes for Ethanol Production under Different Environments Using AMMI Analysis and GGE Biplots

    PubMed Central

    Cheruiyot, Erick Kimutai; Othira, Jacktone Odongo; Njuguna, Virginia Wanjiku; Macharia, Joseph Kinyoro; Owuoche, James; Oyier, Moses; Kange, Alex Machio

    2016-01-01

    The genotype and environment interaction influences the selection criteria of sorghum (Sorghum bicolor) genotypes. Eight sweet sorghum genotypes were evaluated at five different locations in two growing seasons of 2014. The aim was to determine the interaction between genotype and environment on cane, juice, and ethanol yield and to identify best genotypes for bioethanol production in Kenya. The experiments were conducted in a randomized complete block design replicated three times. Sorghum canes were harvested at hard dough stage of grain development and passed through rollers to obtain juice that was then fermented to obtain ethanol. Cane, juice, and ethanol yield was analyzed using the additive main effect and multiplication interaction model (AMMI) and genotype plus genotype by environment (GGE) biplot. The combined analysis of variance of cane and juice yield of sorghum genotypes showed that sweet sorghum genotypes were significantly (P < 0.05) affected by environments (E), genotypes (G) and genotype by environment interaction (GEI). GGE biplot showed high yielding genotypes EUSS10, ACFC003/12, SS14, and EUSS11 for cane yield; EUSS10, EUSS11, and SS14 for juice yield; and EUSS10, SS04, SS14, and ACFC003/12 for ethanol yield. Genotype SS14 showed high general adaptability for cane, juice, and ethanol yield. PMID:27777968

  17. Effect of genotype x alcoholism interaction on linkage analysis of an alcoholism-related quantitative phenotype.

    PubMed

    Arya, Rector; Dyer, Thomas D; Warren, Diane M; Jenkinson, Christopher P; Duggirala, Ravindranath; Almasy, Laura

    2005-01-01

    Studies have shown that genetic and environmental factors and their interactions affect several alcoholism phenotypes. Genotype x alcoholism (GxA) interaction refers to the environmental (alcoholic and non-alcoholic) influences on the autosomal genes contributing to variation in an alcoholism-related quantitative phenotype. The purpose of this study was to examine the effects of GxA interaction on the detection of linkage for alcoholism-related phenotypes. We used phenotypic and genotypic data from the Collaborative Study on the Genetics of Alcoholism relating to 1,388 subjects as part of Genetic Analysis Workshop 14 problem 1. We analyzed the MXDRNK phenotype to detect GxA interaction using SOLAR. Upon detecting significant interaction, we conducted variance-component linkage analyses using microsatellite marker data. For maximum number of drinks per a 24 hour period, the highest LODs were observed on chromosomes 1, 4, and 13 without GxA interaction. Interaction analysis yielded four regions on chromosomes 1, 4, 13, and 15. On chromosome 4, a maximum LOD of 1.5 at the same location as the initial analysis was obtained after incorporating GxA interaction effects. However, after correcting for extra parameters, the LOD score was reduced to a corrected LOD of 1.1, which is similar to the LOD observed in the non-interaction analysis. Thus, we see little differences in LOD scores, while some linkage regions showed large differences in the magnitudes of estimated quantitative trait loci heritabilities between the alcoholic and non-alcoholic groups. These potential hints of differences in genetic effect may influence future analyses of variants under these linkage peaks.

  18. Large-scale phenome analysis defines a behavioral signature for Huntington's disease genotype in mice.

    PubMed

    Alexandrov, Vadim; Brunner, Dani; Menalled, Liliana B; Kudwa, Andrea; Watson-Johnson, Judy; Mazzella, Matthew; Russell, Ian; Ruiz, Melinda C; Torello, Justin; Sabath, Emily; Sanchez, Ana; Gomez, Miguel; Filipov, Igor; Cox, Kimberly; Kwan, Mei; Ghavami, Afshin; Ramboz, Sylvie; Lager, Brenda; Wheeler, Vanessa C; Aaronson, Jeff; Rosinski, Jim; Gusella, James F; MacDonald, Marcy E; Howland, David; Kwak, Seung

    2016-08-01

    Rapid technological advances for the frequent monitoring of health parameters have raised the intriguing possibility that an individual's genotype could be predicted from phenotypic data alone. Here we used a machine learning approach to analyze the phenotypic effects of polymorphic mutations in a mouse model of Huntington's disease that determine disease presentation and age of onset. The resulting model correlated variation across 3,086 behavioral traits with seven different CAG-repeat lengths in the huntingtin gene (Htt). We selected behavioral signatures for age and CAG-repeat length that most robustly distinguished between mouse lines and validated the model by correctly predicting the repeat length of a blinded mouse line. Sufficient discriminatory power to accurately predict genotype required combined analysis of >200 phenotypic features. Our results suggest that autosomal dominant disease-causing mutations could be predicted through the use of subtle behavioral signatures that emerge in large-scale, combinatorial analyses. Our work provides an open data platform that we now share with the research community to aid efforts focused on understanding the pathways that link behavioral consequences to genetic variation in Huntington's disease. PMID:27376585

  19. Comparative analysis of Papaver somniferum genotypes having contrasting latex and alkaloid profiles.

    PubMed

    Chaturvedi, Nidarshana; Singh, Mridula; Shukla, Ashutosh K; Shasany, Ajit K; Shanker, Karuna; Lal, Raj K; Khanuja, Suman P S

    2014-07-01

    Papaver somniferum produces therapeutically useful benzylisoquinoline alkaloids (BIAs) like papaverine, thebaine, codeine, and morphine that accumulate in its capsular latex. Morphine is a potent analgesic but is also abused as a narcotic, which has increased the demand for non-narcotic thebaine that can be converted into various analgesics. To curtail the narcotic menace, many distinct genotypes of the plant have been developed that are deficient in morphine and/or latex. Sujata is one such latex-less low alkaloid-producing variety developed from the alkaloid-rich gum harvest variety Sampada. Its utility for gene prospecting and studying differential gene regulation responsible for its low alkaloid, nutritive seed oil, and latex-less phenotype has been exploited in this study. BIA profiling of Sujata and Sampada capsules at the early and late stages indicated that except for thebaine, Sujata had a depressed alkaloid phenotype as compared to Sampada. Comparative transcript-based analysis of the two genotypes was carried out in the early stage capsule (higher thebaine) using subtractive hybridization and microarray. Interrogation of a P. somniferum array yielded many differentially expressing transcripts. Their homology-based annotation classified them into categories--latex related, oil/lipid related, alkaloid related, cell wall related, and others. These leads will be useful to characterize the highly sought after Sujata phenotype.

  20. Genotype analysis of faecal and blood isolates of Salmonella dublin from humans in England and Wales.

    PubMed Central

    Chowdry, N.; Threlfall, E. J.; Rowe, B.; Stanley, J.

    1993-01-01

    An analysis of genotype was made for representative strains of Salmonella dublin. The collection consisted primarily of strains isolated from humans in England and Wales, and were of both intestinal and extra-intestinal origin. Three genetic elements were characterized by DNA hybridization. They were the spvBC genes, extrachromosomal virulence determinants, the salmonella-specific insertion sequence IS200, and the 16S ribosomal RNA genes, a phylogenetic marker. Two clones of S. dublin (SdRI and SdRII) which shared an identical IS200 profile, were identified on the basis of restriction fragment length polymorphism at the 16S rRNA locus. With one exception, all strains harboured a 52 MDa plasmid which contained a conserved 3.7 kbp Hind III fragment homologous to the spvBC mouse-virulence genes of S. typhimurium. However, a single plasmid-free strain of SdRI, isolated from a patient with septicaemia exhibited no spc homology. In SdRI there was no observable genotype distinction between strains causing gastroenteritis or bacteraemia. In contrast, none of the strains of SdRII were from cases of bacteraemia, and all human isolates of this clone were from cases of gastroenteritis. Images Fig. 1 Fig. 2 PMID:8097167

  1. Hepatic manifestations of tuberous sclerosis complex: a genotypic and phenotypic analysis.

    PubMed

    Black, M E; Hedgire, S S; Camposano, S; Paul, E; Harisinghani, M; Thiele, E A

    2012-12-01

    Hepatic manifestations of tuberous sclerosis complex: a genotypic and phenotypic analysis. A retrospective review of the clinical records and radiological images of 205 patients with tuberous sclerosis complex (TSC) was performed to evaluate the prevalence and progression of hepatic lesions; examine the association of hepatic phenotype with genotype, age, and gender; and investigate the relationships between hepatic, renal, and pulmonary involvement. Hepatic angiomyolipomas (AML), cysts, and other benign lesions were identified in 30% of the cohort, and some lesions grew significantly over time. However, no patient had clinical symptoms or complications from hepatic lesions. TSC2 patients exhibited a higher frequency of AML compared to TSC1 patients (p = 0.037), and patients with no mutation identified exhibited a higher frequency of cysts compared to TSC2 patients (p = 0.023). Age was positively correlated with frequency of hepatic involvement (p < 0.001), whereas hepatic phenotype was independent of gender. Presence of hepatic AML was associated with presence of renal AML (p = 0.001). These findings confirm a high rate of asymptomatic hepatic lesions in TSC and further characterize the TSC phenotype.

  2. Neisseria gonorrhoeae DNA recombination and repair enzymes protect against oxidative damage caused by hydrogen peroxide.

    PubMed

    Stohl, Elizabeth A; Seifert, H Steven

    2006-11-01

    The strict human pathogen Neisseria gonorrhoeae is exposed to oxidative damage during infection. N. gonorrhoeae has many defenses that have been demonstrated to counteract oxidative damage. However, recN is the only DNA repair and recombination gene upregulated in response to hydrogen peroxide (H(2)O(2)) by microarray analysis and subsequently shown to be important for oxidative damage protection. We therefore tested the importance of RecA and DNA recombination and repair enzymes in conferring resistance to H(2)O(2) damage. recA mutants, as well as RecBCD (recB, recC, and recD) and RecF-like pathway mutants (recJ, recO, and recQ), all showed decreased resistance to H(2)O(2). Holliday junction processing mutants (ruvA, ruvC, and recG) showed decreased resistance to H(2)O(2) resistance as well. Finally, we show that RecA protein levels did not increase as a result of H(2)O(2) treatment. We propose that RecA, recombinational DNA repair, and branch migration are all important for H(2)O(2) resistance in N. gonorrhoeae but that constitutive levels of these enzymes are sufficient for providing protection against oxidative damage by H(2)O(2). PMID:16936020

  3. Antimicrobial susceptibility pattern of Neisseria gonorrhoeae in western Austria.

    PubMed

    Allerberger, F; Kofler, H; Brezinka, C; Guggenbichler, J P; Dierich, M P

    1993-01-01

    From January to October 1992 24 Neisseria gonorrhoeae isolates from clinical specimens were collected at the Federal Public Health Laboratory in Innsbruck (Austria) and screened for resistance to penicillin G, erythromycin, tetracycline, spectinomycin, ceftriaxone, cefuroxime, ciprofloxacine, and silver nitrate. Patients originated from the Austrian provinces Salzburg, Tirol, and Vorarlberg, and presented with manifest gonorrhoea. Two of 24 isolates were penicillinase-producing N. gonorrhoeae. Both strains were isolated from men who had just returned from Thailand or Kenya. The isolate from Africa was also resistant to tetracycline. Five of 24 infections were acquired abroad, sex tourism being involved in four cases. The antimicrobial resistance pattern found in gonococci in western Austria revealed that topical silver nitrate and erythromycin are equally acceptable for use in prophylaxis of neonatal ophthalmia. Penicillin is still the drug of choice in the treatment of endemic infections. If gonorrhoea has been acquired abroad, especially in Asia or Africa, ceftriaxone, spectinomycin or ciprofloxazine are recommended for therapy. PMID:8333204

  4. Genotyping of French Bacillus anthracis Strains Based on 31-Loci Multi Locus VNTR Analysis: Epidemiology, Marker Evaluation, and Update of the Internet Genotype Database

    PubMed Central

    Thierry, Simon; Tourterel, Christophe; Le Flèche, Philippe; Derzelle, Sylviane; Dekhil, Neira; Mendy, Christiane; Colaneri, Cécile; Vergnaud, Gilles; Madani, Nora

    2014-01-01

    Background Bacillus anthracis is known to have low genetic variability. In spite of this lack of diversity, multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) and single nucleotide polymorphisms (SNPs) including the canonical SNPs assay (canSNP) have proved to be highly effective to differentiate strains. Five different MLVA schemes based on a collection of 31 VNTR loci (MLVA8, MLVA15, MLVA20, MLVA25 and MLVA31) with increased resolving power have been described. Results MLVA31 was applied to characterize the French National Reference Laboratory collection. The total collection of 130 strains is resolved in 35 genotypes. The 119 veterinary and environmental strains collection in France were resolved into 26 genotypes belonging to three canSNP lineages and four MLVA clonal complexes (CCs) with particular geographical clustering. A subset of seven loci (MLVA7) is proposed to constitute a first line assay. The loci are compatible with moderate resolution equipment such as agarose gel electrophoresis and show a good congruence value with MLVA31. The associated MLVA and SNP data was imported together with published genotyping data by taking advantage of major enhancements to the MLVAbank software and web site. Conclusions The present report provides a wide coverage of the genetic diversity of naturally occurring B. anthracis strains in France as can be revealed by MLVA. The data obtained suggests that once such coverage is achieved, it becomes possible to devise optimized first-line MLVA assays comprising a sufficiently low number of loci to be typed either in one multiplex PCR or on agarose gels. Such a selection of seven loci is proposed here, and future similar investigations in additional countries will indicate to which extend the same selection can be used worldwide as a common minimum set. It is hoped that this approach will contribute to an efficient and low-cost routine surveillance of important pathogens for biosecurity such as B. anthracis

  5. Multiple-Locus Variable-Number Tandem-Repeat Analysis Genotyping of Human Brucella Isolates from Turkey▿†

    PubMed Central

    Kılıç, Selçuk; Ivanov, Ivan N.; Durmaz, Rıza; Bayraktar, Mehmet Refik; Ayaşlıoğlu, Ergin; Uyanık, M. Hamidullah; Alışkan, Hikmet; Yaşar, Ekrem; Bayramoğlu, Gülçin; Arslantürk, Ahmet; Vergnaud, Gilles; Kantardjiev, Todor V.

    2011-01-01

    A multiple-locus variable-number tandem-repeat analysis (MLVA) was applied to investigate the epidemiological relationship and genetic diversity among 162 human Brucella isolates collected from all geographic regions of Turkey in an 8-year period (2001 to 2008). The isolates were genotyped by using an MLVA assay developed in Orsay, France (MLVA-16Orsay) including eight minisatellite (panel 1) and eight microsatellite (panel 2, subdivided into 2A and 2B) markers. Panels 1 and 2A distinguish 14 genotypes; two of these represented 85% of the strains. Panel 2B displayed a very high discriminatory power. Three loci from panel 2B had diversity index values higher than 0.74. MLVA-16Orsay yielded 105 genotypes; 73 were represented by a unique isolate, and 32 included two to eight isolates. The isolates from different patients within the same outbreak or from the same patient before first-line therapy and after relapse showed identical genotypes. A number of MLVA genotypes appeared to be partially restricted to some geographic areas and displayed no annual variation, possibly reflecting persistence of genotypes in certain areas for a time span of at least a decade. This study, representing the first molecular typing results of human Brucella isolates from Turkey, indicated that Turkish human Brucella melitensis isolates were most closely related to the neighboring countries' isolates included in the East Mediterranean group. PMID:21795514

  6. Multiple-locus variable-number tandem-repeat analysis genotyping of human Brucella isolates from Turkey.

    PubMed

    Kiliç, Selçuk; Ivanov, Ivan N; Durmaz, Riza; Bayraktar, Mehmet Refik; Ayaslioglu, Ergin; Uyanik, M Hamidullah; Aliskan, Hikmet; Yasar, Ekrem; Bayramoglu, Gülçin; Arslantürk, Ahmet; Vergnaud, Gilles; Kantardjiev, Todor V

    2011-09-01

    A multiple-locus variable-number tandem-repeat analysis (MLVA) was applied to investigate the epidemiological relationship and genetic diversity among 162 human Brucella isolates collected from all geographic regions of Turkey in an 8-year period (2001 to 2008). The isolates were genotyped by using an MLVA assay developed in Orsay, France (MLVA-16(Orsay)) including eight minisatellite (panel 1) and eight microsatellite (panel 2, subdivided into 2A and 2B) markers. Panels 1 and 2A distinguish 14 genotypes; two of these represented 85% of the strains. Panel 2B displayed a very high discriminatory power. Three loci from panel 2B had diversity index values higher than 0.74. MLVA-16(Orsay) yielded 105 genotypes; 73 were represented by a unique isolate, and 32 included two to eight isolates. The isolates from different patients within the same outbreak or from the same patient before first-line therapy and after relapse showed identical genotypes. A number of MLVA genotypes appeared to be partially restricted to some geographic areas and displayed no annual variation, possibly reflecting persistence of genotypes in certain areas for a time span of at least a decade. This study, representing the first molecular typing results of human Brucella isolates from Turkey, indicated that Turkish human Brucella melitensis isolates were most closely related to the neighboring countries' isolates included in the East Mediterranean group.

  7. Null genotypes of GSTM1 and GSTT1 contribute to increased risk of diabetes mellitus: a meta-analysis.

    PubMed

    Zhang, Jingwen; Liu, Hu; Yan, Hongyi; Huang, Guoliang; Wang, Bin

    2013-04-15

    Diabetes mellitus (DM) is a common disease which results from various causes including genetic and environmental factors. Glutathione S-Transferase M1 (GSTM1) and Glutathione S-Transferase T1 (GSTT1) genes are polymorphic in human and the null genotypes lead to the absence of enzyme function. Many studies assessed the associations between GSTM1/GSTT1 null genotypes and DM risk but reported conflicting results. In order to get a more precise estimate of the associations of GSTM1/GSTT1 null genotypes with DM risk, we performed this meta-analysis. Published literature from PubMed, Embase and China Biology Medicine (CBM) databases was searched for eligible studies. Pooled odds ratios (OR) and corresponding 95% confidence intervals (95%CI) were calculated using a fixed- or random-effects model. 11 publications (a total of 2577 cases and 4572 controls) were finally included into this meta-analysis. Meta-analyses indicated that null genotypes of GSTM1/GSTT1 and dual null genotype of GSTM1-GSTT1 were all associated with increased risk of DM (GSTM1: OR random-effects=1.60, 95%CI 1.10-2.34, POR=0.014; GSTT1: OR random-effects=1.47, 95%CI 1.12-1.92, POR=0.005; GSTM1-GSTT1: OR fixed-effects=1.83, 95%CI 1.30-2.59, POR=0.001). Subgroup by ethnicity suggested significant associations between null genotypes of GSTM1 and GSTT1 and DM risk among Asians (GSTM1: OR random-effects=1.77, 95%CI 1.24-2.53, POR=0.002; GSTT1: OR random-effects=1.58, 95%CI 1.09-2.27, POR=0.015). This meta-analysis suggests null genotypes of GSTM1/GSTT1 and dual null genotype of GSTM1-GSTT1 are all associated with increased risk of DM, and null genotypes of GSTM1/GSTT1 and dual null genotype of GSTM1-GSTT1 are potential biomarkers of DM.

  8. Nucleotide sequence composition and method for detection of neisseria gonorrhoeae

    SciTech Connect

    Lo, A.; Yang, H.L.

    1990-02-13

    This patent describes a composition of matter that is specific for {ital Neisseria gonorrhoeae}. It comprises: at least one nucleotide sequence for which the ratio of the amount of the sequence which hybridizes to chromosomal DNA of {ital Neisseria gonorrhoeae} to the amount of the sequence which hybridizes to chromosomal DNA of {ital Neisseria meningitidis} is greater than about five. The ratio being obtained by a method described.

  9. The Genotype-Tissue Expression (GTEx) pilot analysis: Multitissue gene regulation in humans

    PubMed Central

    2015-01-01

    Understanding the functional consequences of genetic variation, and how it affects complex human disease and quantitative traits, remains a critical challenge for biomedicine. We present an analysis of RNA sequencing data from 1641 samples across 43 tissues from 175 individuals, generated as part of the pilot phase of the Genotype-Tissue Expression (GTEx) project. We describe the landscape of gene expression across tissues, catalog thousands of tissue-specific and shared regulatory expression quantitative trait loci (eQTL) variants, describe complex network relationships, and identify signals from genome-wide association studies explained by eQTLs. These findings provide a systematic understanding of the cellular and biological consequences of human genetic variation and of the heterogeneity of such effects among a diverse set of human tissues. PMID:25954001

  10. Genetic Diversity and Geographic Population Structure of Bovine Neospora caninum Determined by Microsatellite Genotyping Analysis

    PubMed Central

    Regidor-Cerrillo, Javier; Díez-Fuertes, Francisco; García-Culebras, Alicia; Moore, Dadín P.; González-Warleta, Marta; Cuevas, Carmen; Schares, Gereon; Katzer, Frank; Pedraza-Díaz, Susana; Mezo, Mercedes; Ortega-Mora, Luis M.

    2013-01-01

    The cyst-forming protozoan parasite Neosporacaninum is one of the main causes of bovine abortion worldwide and is of great economic importance in the cattle industry. Recent studies have revealed extensive genetic variation among N. caninum isolates based on microsatellite sequences (MSs). MSs may be suitable molecular markers for inferring the diversity of parasite populations, molecular epidemiology and the basis for phenotypic variations in N. caninum, which have been poorly defined. In this study, we evaluated nine MS markers using a panel of 11 N. caninum-derived reference isolates from around the world and 96 N. caninum bovine clinical samples and one ovine clinical sample collected from four countries on two continents, including Spain, Argentina, Germany and Scotland, over a 10-year period. These markers were used as molecular tools to investigate the genetic diversity, geographic distribution and population structure of N. caninum. Multilocus microsatellite genotyping based on 7 loci demonstrated high levels of genetic diversity in the samples from all of the different countries, with 96 microsatellite multilocus genotypes (MLGs) identified from 108 N. caninum samples. Geographic sub-structuring was present in the country populations according to pairwise FST. Principal component analysis (PCA) and Neighbor Joining tree topologies also suggested MLG segregation partially associated with geographical origin. An analysis of the MLG relationships, using eBURST, confirmed that the close genetic relationship observed between the Spanish and Argentinean populations may be the result of parasite migration (i.e., the introduction of novel MLGs from Spain to South America) due to cattle movement. The eBURST relationships also revealed genetically different clusters associated with the abortion. The presence of linkage disequilibrium, the co-existence of specific MLGs to individual farms and eBURST MLG relationships suggest a predominant clonal propagation for

  11. Analysis of population structure and genetic diversity of Egyptian and exotic rice (Oryza sativa L.) genotypes.

    PubMed

    Salem, Khaled F M; Sallam, Ahmed

    2016-01-01

    Understanding the population structure and genetic diversity is a very important goal to improve the economic value of crops. In rice, a loss of genetic diversity in the last few centuries is observed. To address this challenge, a set of 22 lines from three different regions - India (two), and Philippines (six), and Egypt (14) - were used to assess the genetic diversity and the features of population structure. These genotypes were analyzed using 106 SSR alleles that showed a clear polymorphism among the lines. Genetic diversity was estimated based on the number of different alleles, polymorphism information content (PIC), and gene diversity. A total of 106 SSR alleles was identified from the 23 SSR loci and used to study the population structure and carry out a cluster analysis. All SSR loci showed a wide range of the number of different alleles extended from two (one loci) to seven alleles (three loci). Five and eight loci showed high PIC and gene diversity (≥0.70), respectively. The results of population structure are in agreement with cluster analysis results. Both analyses revealed two different subpopulations (G1 and G2) with different genetic properties in number of private alleles, number of different alleles (Na), number of effective alleles (Ne), expected heterozygosity (He), and Shannon's Information Index (SII). Our findings indicate that five SSR loci (RM 111, RM 307, RM 22, RM 19, and RM 271) could be used in breeding programs to enhance the marker-assisted selection through QTL mapping and association studies. A high genetic diversity found between genotypes which can be exploited to improve and produce rice cultivars for important traits (e.g. high agronomic features and tolerance to biotic or/and abiotic stresses).

  12. Characterization of the Novel DNA Gyrase Inhibitor AZD0914: Low Resistance Potential and Lack of Cross-Resistance in Neisseria gonorrhoeae

    PubMed Central

    Kutschke, Amy; Otterson, Linda G.; McLaughlin, Robert E.; Whiteaker, James D.; Lewis, Lisa A.; Su, Xiaohong; Huband, Michael D.; Gardner, Humphrey; Mueller, John P.

    2014-01-01

    The unmet medical need for novel intervention strategies to treat Neisseria gonorrhoeae infections is significant and increasing, as rapidly emerging resistance in this pathogen is threatening to eliminate the currently available treatment options. AZD0914 is a novel bacterial gyrase inhibitor that possesses potent in vitro activities against isolates with high-level resistance to ciprofloxacin and extended-spectrum cephalosporins, and it is currently in clinical development for the treatment of N. gonorrhoeae infections. The propensity to develop resistance against AZD0914 was examined in N. gonorrhoeae and found to be extremely low, a finding supported by similar studies with Staphylococcus aureus. The genetic characterization of both first-step and second-step mutants that exhibited decreased susceptibilities to AZD0914 identified substitutions in the conserved GyrB TOPRIM domain, confirming DNA gyrase as the primary target of AZD0914 and providing differentiation from fluoroquinolones. The analysis of available bacterial gyrase and topoisomerase IV structures, including those bound to fluoroquinolone and nonfluoroquinolone inhibitors, has allowed the rationalization of the lack of cross-resistance that AZD0914 shares with fluoroquinolones. Microbiological susceptibility data also indicate that the topoisomerase inhibition mechanisms are subtly different between N. gonorrhoeae and other bacterial species. Taken together, these data support the progression of AZD0914 as a novel treatment option for the oral treatment of N. gonorrhoeae infections. PMID:25534723

  13. Characterization of the novel DNA gyrase inhibitor AZD0914: low resistance potential and lack of cross-resistance in Neisseria gonorrhoeae.

    PubMed

    Alm, Richard A; Lahiri, Sushmita D; Kutschke, Amy; Otterson, Linda G; McLaughlin, Robert E; Whiteaker, James D; Lewis, Lisa A; Su, Xiaohong; Huband, Michael D; Gardner, Humphrey; Mueller, John P

    2015-03-01

    The unmet medical need for novel intervention strategies to treat Neisseria gonorrhoeae infections is significant and increasing, as rapidly emerging resistance in this pathogen is threatening to eliminate the currently available treatment options. AZD0914 is a novel bacterial gyrase inhibitor that possesses potent in vitro activities against isolates with high-level resistance to ciprofloxacin and extended-spectrum cephalosporins, and it is currently in clinical development for the treatment of N. gonorrhoeae infections. The propensity to develop resistance against AZD0914 was examined in N. gonorrhoeae and found to be extremely low, a finding supported by similar studies with Staphylococcus aureus. The genetic characterization of both first-step and second-step mutants that exhibited decreased susceptibilities to AZD0914 identified substitutions in the conserved GyrB TOPRIM domain, confirming DNA gyrase as the primary target of AZD0914 and providing differentiation from fluoroquinolones. The analysis of available bacterial gyrase and topoisomerase IV structures, including those bound to fluoroquinolone and nonfluoroquinolone inhibitors, has allowed the rationalization of the lack of cross-resistance that AZD0914 shares with fluoroquinolones. Microbiological susceptibility data also indicate that the topoisomerase inhibition mechanisms are subtly different between N. gonorrhoeae and other bacterial species. Taken together, these data support the progression of AZD0914 as a novel treatment option for the oral treatment of N. gonorrhoeae infections.

  14. Characterization of the novel DNA gyrase inhibitor AZD0914: low resistance potential and lack of cross-resistance in Neisseria gonorrhoeae.

    PubMed

    Alm, Richard A; Lahiri, Sushmita D; Kutschke, Amy; Otterson, Linda G; McLaughlin, Robert E; Whiteaker, James D; Lewis, Lisa A; Su, Xiaohong; Huband, Michael D; Gardner, Humphrey; Mueller, John P

    2015-03-01

    The unmet medical need for novel intervention strategies to treat Neisseria gonorrhoeae infections is significant and increasing, as rapidly emerging resistance in this pathogen is threatening to eliminate the currently available treatment options. AZD0914 is a novel bacterial gyrase inhibitor that possesses potent in vitro activities against isolates with high-level resistance to ciprofloxacin and extended-spectrum cephalosporins, and it is currently in clinical development for the treatment of N. gonorrhoeae infections. The propensity to develop resistance against AZD0914 was examined in N. gonorrhoeae and found to be extremely low, a finding supported by similar studies with Staphylococcus aureus. The genetic characterization of both first-step and second-step mutants that exhibited decreased susceptibilities to AZD0914 identified substitutions in the conserved GyrB TOPRIM domain, confirming DNA gyrase as the primary target of AZD0914 and providing differentiation from fluoroquinolones. The analysis of available bacterial gyrase and topoisomerase IV structures, including those bound to fluoroquinolone and nonfluoroquinolone inhibitors, has allowed the rationalization of the lack of cross-resistance that AZD0914 shares with fluoroquinolones. Microbiological susceptibility data also indicate that the topoisomerase inhibition mechanisms are subtly different between N. gonorrhoeae and other bacterial species. Taken together, these data support the progression of AZD0914 as a novel treatment option for the oral treatment of N. gonorrhoeae infections. PMID:25534723

  15. Extending Rare-Variant Testing Strategies: Analysis of Noncoding Sequence and Imputed Genotypes

    PubMed Central

    Zawistowski, Matthew; Gopalakrishnan, Shyam; Ding, Jun; Li, Yun; Grimm, Sara; Zöllner, Sebastian

    2010-01-01

    Next Generation Sequencing Technology has revolutionized our ability to study the contribution of rare genetic variation to heritable traits. However, existing single-marker association tests are underpowered for detecting rare risk variants. A more powerful approach involves pooling methods that combine multiple rare variants from the same gene into a single test statistic. Proposed pooling methods can be limited because they generally assume high-quality genotypes derived from deep-coverage sequencing, which may not be available. In this paper, we consider an intuitive and computationally efficient pooling statistic, the cumulative minor-allele test (CMAT). We assess the performance of the CMAT and other pooling methods on datasets simulated with population genetic models to contain realistic levels of neutral variation. We consider study designs ranging from exon-only to whole-gene analyses that contain noncoding variants. For all study designs, the CMAT achieves power comparable to that of previously proposed methods. We then extend the CMAT to probabilistic genotypes and describe application to low-coverage sequencing and imputation data. We show that augmenting sequence data with imputed samples is a practical method for increasing the power of rare-variant studies. We also provide a method of controlling for confounding variables such as population stratification. Finally, we demonstrate that our method makes it possible to use external imputation templates to analyze rare variants imputed into existing GWAS datasets. As proof of principle, we performed a CMAT analysis of more than 8 million SNPs that we imputed into the GAIN psoriasis dataset by using haplotypes from the 1000 Genomes Project. PMID:21070896

  16. Shuttle mutagenesis of Neisseria gonorrhoeae: pilin null mutations lower DNA transformation competence.

    PubMed Central

    Seifert, H S; Ajioka, R S; Paruchuri, D; Heffron, F; So, M

    1990-01-01

    The method of shuttle mutagenesis has been extended to Neisseria gonorrhoeae. We have constructed a defective mini-Tn3 derivative that encodes chloramphenicol resistance in both N. gonorrhoeae and Escherichia coli and selected for mutations in the chloramphenicol resistance gene that express higher levels of antibiotic resistance in N. gonorrhoeae. Isogenic N. gonorrhoeae strains that differ only in pilin expression were constructed and used to test the effect of pilin null mutations on DNA transformation competence. PMID:2152910

  17. Towards measles elimination: Phylogenetic analysis of measles viruses in Turkey (2012-2013) and identification of genotype D8.

    PubMed

    Kalaycioglu, Atila T; Yolbakan, Sultan; Guldemir, Dilek; Korukluoglu, Gulay; Coskun, Aslihan; Cosgun, Yasemin; Durmaz, Riza

    2016-11-01

    Molecular characterization of different measles virus (MV) strains is essential to combat the disease. Sixty measles MV strains were obtained from throat swabs or urine of patients in Turkey between 2012 and 2013 and characterized. MV RNA sequences (n = 60) were analysed for 456 nucleotides representing hypervariable domain of the nucleoprotein (N) gene. Of the 60 strains analysed 53 were the D8 genotype, 6 were B3, 1 was D4, and 1 was A. This report describes MV genotype D8 that was involved in a measles outbreak in Turkey. Sequences of most genotype D8 strains (n = 51) were identical to the sequence of variant D8-Frankfurt-Main, which has been associated with outbreaks throughout Europe. Despite the lack of epidemiologic information, a phylogenetic analysis suggested that the genotype D8 MV may have been brought to Turkey from elsewhere. Phylogenetic and epidemiological findings suggested that strains identified in tourists and associated with importation included one strain of genotype D8, one strain of genotype B3, and one strain of genotype D4. These findings from the 2012 to 2013 outbreak in Turkey confirm that pockets of unimmunised individuals are making the country susceptible to measles outbreaks. To prevent further outbreaks, deliberate and sustained effort must be made to reach, and immunise susceptible age groups. Towards measles elimination process, continued molecular surveillance of measles strains in Turkey will help identify transmission patterns of virus and evaluate vaccination efforts. J. Med. Virol. 88:1867-1873, 2016. © 2016 Wiley Periodicals, Inc. PMID:27089242

  18. Primate genotyping via high resolution melt analysis: rapid and reliable identification of color vision status in wild lemurs.

    PubMed

    Jacobs, Rachel L; Spriggs, Amanda N; MacFie, Tammie S; Baden, Andrea L; Irwin, Mitchell T; Wright, Patricia C; Louis, Edward E; Lawler, Richard R; Mundy, Nicholas I; Bradley, Brenda J

    2016-10-01

    Analyses of genetic polymorphisms can aid our understanding of intra- and interspecific variation in primate sociality, ecology, and behavior. Studies of primate opsin genes are prime examples of this, as single nucleotide variants (SNVs) in the X-linked opsin gene underlie variation in color vision. For primate species with polymorphic trichromacy, genotyping opsin SNVs can generally indicate whether individual primates are red-green color-blind (denoted homozygous M or homozygous L) or have full trichromatic color vision (heterozygous ML). Given the potential influence of color vision on behavior and fitness, characterizing the color vision status of study subjects is becoming commonplace for many primate field projects. Such studies traditionally involve a multi-step sequencing-based method that can be costly and time-consuming. Here we present a new reliable, rapid, and relatively inexpensive method for characterizing color vision in primate populations using high resolution melt analysis (HRMA). Using lemurs as a case study, we characterized variation at exons 3 and/or 5 of the X-linked opsin gene for 87 individuals representing nine species. We scored opsin genotypes and color vision status using both traditional sequencing-based methods as well as our novel melting-curve based HRMA protocol. For each species, the melting curves of varying genotypes (homozygous M, homozygous L, heterozygous ML) differed in melting temperature and/or shape. Melting curves for each sample were consistent across replicates, and genotype-specific melting curves were consistent across DNA sources (blood vs. feces). We show that opsin genotypes can be quickly and reliably scored using HRMA once lab-specific reference curves have been developed based on known genotypes. Although the protocol presented here focuses on genotyping lemur opsin loci, we also consider the larger potential for applying this approach to various types of genetic studies of primate populations. PMID:27271303

  19. Primate genotyping via high resolution melt analysis: rapid and reliable identification of color vision status in wild lemurs.

    PubMed

    Jacobs, Rachel L; Spriggs, Amanda N; MacFie, Tammie S; Baden, Andrea L; Irwin, Mitchell T; Wright, Patricia C; Louis, Edward E; Lawler, Richard R; Mundy, Nicholas I; Bradley, Brenda J

    2016-10-01

    Analyses of genetic polymorphisms can aid our understanding of intra- and interspecific variation in primate sociality, ecology, and behavior. Studies of primate opsin genes are prime examples of this, as single nucleotide variants (SNVs) in the X-linked opsin gene underlie variation in color vision. For primate species with polymorphic trichromacy, genotyping opsin SNVs can generally indicate whether individual primates are red-green color-blind (denoted homozygous M or homozygous L) or have full trichromatic color vision (heterozygous ML). Given the potential influence of color vision on behavior and fitness, characterizing the color vision status of study subjects is becoming commonplace for many primate field projects. Such studies traditionally involve a multi-step sequencing-based method that can be costly and time-consuming. Here we present a new reliable, rapid, and relatively inexpensive method for characterizing color vision in primate populations using high resolution melt analysis (HRMA). Using lemurs as a case study, we characterized variation at exons 3 and/or 5 of the X-linked opsin gene for 87 individuals representing nine species. We scored opsin genotypes and color vision status using both traditional sequencing-based methods as well as our novel melting-curve based HRMA protocol. For each species, the melting curves of varying genotypes (homozygous M, homozygous L, heterozygous ML) differed in melting temperature and/or shape. Melting curves for each sample were consistent across replicates, and genotype-specific melting curves were consistent across DNA sources (blood vs. feces). We show that opsin genotypes can be quickly and reliably scored using HRMA once lab-specific reference curves have been developed based on known genotypes. Although the protocol presented here focuses on genotyping lemur opsin loci, we also consider the larger potential for applying this approach to various types of genetic studies of primate populations.

  20. Genotyping and Phylogenetic Analysis of Giardia duodenalis Isolates from Turkish Children

    PubMed Central

    Tamer, Gulden Sonmez; Kasap, Murat; Er, Doganhan Kadir

    2015-01-01

    Background Giardiasis is caused by the intestinal protozoan parasite Giardia duodenalis (synonyms: G. lamblia, G. intestinalis), which is one of the most frequent parasites that infect Turkish children. However, molecular characterization of G. duodenalis in Turkey is relatively scarce. The present work aimed at genotyping G. duodenalis isolates from Turkey using molecular techniques. Material/Methods In the present study, 145 fecal samples from children were collected to search for the presence of Giardia by microscopy and PCR screening. PCR generated a 384 bp fragment for β-giardin. The PCR products were sequenced and the sequences were subjected to phylogenetic analysis by using PHYLIP. Results Based on the phylogenetic analysis of the sequences, assemblage A, B, and mixed subtypes were determined. Of 22 isolates, 11 were identified as assemblage A (50%), 7 were assemblage B (31.8%), and 4 were assemblage AB (18.2%). Association between G. duodenalis assemblages and the epidemiological data was analyzed. No correlation was found between symptoms and infection with specific assemblages (P>0.05), but we found statistically significant association between age and the assemblage AB (P=0.001). Conclusions The association between G. duodenalis and the epidemiologic data were analyzed. Since assemblage A is the more prevalent subgroup compared with assemblage B, this subgroup might be responsible for common Giardia infections in Turkey. This is the first study that included a detailed phylogenetic analysis of Giardia strains from Turkey. PMID:25689970

  1. Direct genotyping of Toxoplasma gondii from amniotic fluids based on B1 gene polymorphism using minisequencing analysis

    PubMed Central

    2013-01-01

    Background Because some Toxoplasma gondii genotypes may be more virulent in pregnant women, discriminating between them appears valuable. Currently, the main genotyping method is based on single copy microsatellite markers, which limit direct genotyping from amniotic fluids (AFs) to samples with a high parasitic load. We investigated whether the multicopy gene B1 could type the parasite with a higher sensitivity. To estimate the amplifiable DNA present in AFs, we first compared three different PCR assays used for Toxoplasma infection diagnosis: the P30-PCR, targeting the single copy gene P30; the B1-PCR, targeting the repeated B1 gene; and RE-PCR, targeting the repeated element. Results Of the 1792 AFs analyzed between 2008 and 2011, 73 were RE-PCR positive. Of those, 49 (67.1%) were P30-PCR and B1-PCR positive, and 14 (19.2%) additional AFs were B1-PCR positive only. All 63 BI-positive AFs (France n = 49; overseas n = 14) could be genotyped based on an analysis of eight nucleotide polymorphisms (SNPs) located within the B1 gene. Following high-resolution melting (HRM) analysis, minisequencing was carried out for each of the eight SNPs. DNA from six reference strains was included in the study, and AFs were assigned to one of the three major lineages (Types I, II, and III). In total, 26 genotypes were observed, and the hierarchical clustering distinguished two clades in lineages II (IIa, n = 30 and IIb, n = 4) and III (IIIa n = 23 and IIIb n = 6). There was an overrepresentation of overseas isolates in Clade IIb (4/4, 100%) and Clade IIIa (8/22; 36.4%) (p <0.0001), whereas medical interruption and fetal death were overrepresented in Clade IIb (2/4, 50%) and Clade IIIa (4/23, 17.4%) (p = 0.049). Conclusions Although the current genotyping system cannot pretend to replace multilocus typing, we clearly show that targeting the multicopy B1 gene yields a genotyping capacity of AFs around 20% better than when single copy targets are used. The

  2. The application of XRF and PIXE in the analysis of rice shoot and compositional screening of genotypes

    NASA Astrophysics Data System (ADS)

    Bado, S.; Padilla-Alvarez, R.; Migliori, A.; Forster, B. P.; Jaksic, M.; Diawara, Y.; Kaiser, R.; Laimer, M.

    2016-03-01

    The analytical performance of Particle Induced X-ray Emission (PIXE) and X-ray Fluorescence (XRF) techniques was assessed in the determination of fourteen elements (Na, Mg, P, S, Cl, K, Ca, Mn, Fe, Cu, Zn, Br, Rb and Sr) in plant samples. The quality of the results - in terms of accuracy, associated uncertainty and correlation between the two methods - was evaluated with regard to their usability for compositional classification of different rice genotypes with known tolerance levels to salinity stress. Plant uptake of essential elements was explored by Principal Component Analysis, which illuminated patterns between treatments (salt and control treatments) and across the rice genotypes tested.

  3. TASSEL-GBS: a high capacity genotyping by sequencing analysis pipeline

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genotyping by sequencing (GBS) is a next generation sequencing based method that takes advantage of reduced representation to enable high throughput genotyping of large numbers of individuals at a large number of SNP markers. The relatively straightforward, robust, and cost-effective GBS protocol is...

  4. [GENOTYPING OF THE BURKHOLDERIA MALLEI STRAINS BASED ON DIFFERENT REGION ANALYSIS].

    PubMed

    Bondareva, O S; Savchenko, S S; Tkachenko, G A; Ledeneva, M L; Lemasova, L V; Antonov, V A

    2016-01-01

    Development of the genotyping methods of glanders agent is urgent due to its high pathogenicity, lack of effective preventive measures and threat of the use of Burkholderia mallei as a biological weapon. In this work we proposed a scheme for the typing of the B. mallei strains based on different region analysis (DFR). The choice of variable loci differentially presented in various strains of glanders agents was performed by analyzing annotated whole-genome sequences of the B. mallei strains. Primers and fluorescence probes were designed for 9 selected loci. The amplification conditions for different regions were optimized in two variants: with electrophoretic detection and hybridization-fluorescence detection in the strip format. The possibility of applying the DFR analysis to genetic characterization of strains was assessed in 14 B. mallei strains. The genetic profiles of the studied B. mallei strains revealed that the developed DFR-typing scheme was characterized by high discrimination power (Hunter-Gaston index value was 0.92), reproducibility, rapidity, easy interpretation, and applicability for epidemiological surveillance of glanders.

  5. [GENOTYPING OF THE BURKHOLDERIA MALLEI STRAINS BASED ON DIFFERENT REGION ANALYSIS].

    PubMed

    Bondareva, O S; Savchenko, S S; Tkachenko, G A; Ledeneva, M L; Lemasova, L V; Antonov, V A

    2016-01-01

    Development of the genotyping methods of glanders agent is urgent due to its high pathogenicity, lack of effective preventive measures and threat of the use of Burkholderia mallei as a biological weapon. In this work we proposed a scheme for the typing of the B. mallei strains based on different region analysis (DFR). The choice of variable loci differentially presented in various strains of glanders agents was performed by analyzing annotated whole-genome sequences of the B. mallei strains. Primers and fluorescence probes were designed for 9 selected loci. The amplification conditions for different regions were optimized in two variants: with electrophoretic detection and hybridization-fluorescence detection in the strip format. The possibility of applying the DFR analysis to genetic characterization of strains was assessed in 14 B. mallei strains. The genetic profiles of the studied B. mallei strains revealed that the developed DFR-typing scheme was characterized by high discrimination power (Hunter-Gaston index value was 0.92), reproducibility, rapidity, easy interpretation, and applicability for epidemiological surveillance of glanders. PMID:27183720

  6. Genotyping of Klebsiella Pneumonia Strains Isolated from Eldly Inpatients by Multiple-locus Variable-number Tandem-repeat Analysis.

    PubMed

    Zhang, Yuan-Yuan; Xu, Ya-Ping; DU, Peng-Cheng; Qiang, Yu-Jun; Zhang, Wen; Chen, Chen; Yu, Ji-Hong; Guo, Jun

    2016-08-01

    Objective To investigate the genotype of klebsiella pneumonia strains isolated from eldly inpatients by multiple-locus variable-number tandem-repeat analysis. Methods Totally 184 klebsiella pneumonia strains,isolated from eldly inpatients,were collected,and their genome DNA were extracted. The polymorphism of 7 variable-number tandem-repeat locus in the DNA samples was analyzed by multiple primers polymerase chain reaction and capillary electrophoresis. The clustering analysis of genotyping was carried out with the BioNumerics 5.1 software. Results A total of 139 genotypes were identified in 184 klebsiella pneumonia clinical strains,showing obvious genetic polymorphisms. With clustering analysis of genotypes,all the strains were categorized into three gene clusters (genogroups 1,2,and 3). The genogroup 1 was the biggest cluster,containing 93.06% of the isolated strains. Conclusion There was a predominant cluster in the klebsiella pneumonia strains isolated from eldly inpatients in our center,and the major source of klebsiella pneumonia infection remained the nosocomial infection. PMID:27594157

  7. Analysis of Mycobacterium tuberculosis Genotypic Lineage Distribution in Chile and Neighboring Countries.

    PubMed

    Lagos, Jaime; Couvin, David; Arata, Loredana; Tognarelli, Javier; Aguayo, Carolina; Leiva, Tamara; Arias, Fabiola; Hormazabal, Juan Carlos; Rastogi, Nalin; Fernández, Jorge

    2016-01-01

    Tuberculosis (TB), caused by the pathogen Mycobacterium tuberculosis (MTB), remains a disease of high importance to global public health. Studies into the population structure of MTB have become vital to monitoring possible outbreaks and also to develop strategies regarding disease control. Although Chile has a low incidence of MTB, the current rates of migration have the potential to change this scenario. We collected and analyzed a total of 458 M. tuberculosis isolates (1 isolate per patient) originating from all 15 regions of Chile. The isolates were genotyped using the spoligotyping method and the data obtained were analyzed and compared with the SITVIT2 database. A total of 169 different patterns were identified, of which, 119 patterns (408 strains) corresponded to Spoligotype International Types (SITs) and 50 patterns corresponded to orphan strains. The most abundantly represented SITs/lineages were: SIT53/T1 (11.57%), SIT33/LAM3 (9.6%), SIT42/LAM9 (9.39%), SIT50/H3 (5.9%), SIT37/T3 (5%); analysis of the spoligotyping minimum spanning tree as well as spoligoforest were suggestive of a recent expansion of SIT42, SIT50 and SIT37; all of which potentially evolved from SIT53. The most abundantly represented lineages were LAM (40.6%), T (34.1%) and Haarlem (13.5%). LAM was more prevalent in the Santiago (43.6%) and Concepción (44.1%) isolates, rather than the Iquique (29.4%) strains. The proportion of X lineage was appreciably higher in Iquique and Concepción (11.7% in both) as compared to Santiago (1.6%). Global analysis of MTB lineage distribution in Chile versus neighboring countries showed that evolutionary recent lineages (LAM, T and Haarlem) accounted together for 88.2% of isolates in Chile, a pattern which mirrored MTB lineage distribution in neighboring countries (n = 7378 isolates recorded in SITVIT2 database for Peru, Brazil, Paraguay, and Argentina; and published studies), highlighting epidemiological advantage of Euro-American lineages in this region

  8. Analysis of Mycobacterium tuberculosis Genotypic Lineage Distribution in Chile and Neighboring Countries

    PubMed Central

    Lagos, Jaime; Couvin, David; Arata, Loredana; Tognarelli, Javier; Aguayo, Carolina; Leiva, Tamara; Arias, Fabiola; Hormazabal, Juan Carlos; Rastogi, Nalin; Fernández, Jorge

    2016-01-01

    Tuberculosis (TB), caused by the pathogen Mycobacterium tuberculosis (MTB), remains a disease of high importance to global public health. Studies into the population structure of MTB have become vital to monitoring possible outbreaks and also to develop strategies regarding disease control. Although Chile has a low incidence of MTB, the current rates of migration have the potential to change this scenario. We collected and analyzed a total of 458 M. tuberculosis isolates (1 isolate per patient) originating from all 15 regions of Chile. The isolates were genotyped using the spoligotyping method and the data obtained were analyzed and compared with the SITVIT2 database. A total of 169 different patterns were identified, of which, 119 patterns (408 strains) corresponded to Spoligotype International Types (SITs) and 50 patterns corresponded to orphan strains. The most abundantly represented SITs/lineages were: SIT53/T1 (11.57%), SIT33/LAM3 (9.6%), SIT42/LAM9 (9.39%), SIT50/H3 (5.9%), SIT37/T3 (5%); analysis of the spoligotyping minimum spanning tree as well as spoligoforest were suggestive of a recent expansion of SIT42, SIT50 and SIT37; all of which potentially evolved from SIT53. The most abundantly represented lineages were LAM (40.6%), T (34.1%) and Haarlem (13.5%). LAM was more prevalent in the Santiago (43.6%) and Concepción (44.1%) isolates, rather than the Iquique (29.4%) strains. The proportion of X lineage was appreciably higher in Iquique and Concepción (11.7% in both) as compared to Santiago (1.6%). Global analysis of MTB lineage distribution in Chile versus neighboring countries showed that evolutionary recent lineages (LAM, T and Haarlem) accounted together for 88.2% of isolates in Chile, a pattern which mirrored MTB lineage distribution in neighboring countries (n = 7378 isolates recorded in SITVIT2 database for Peru, Brazil, Paraguay, and Argentina; and published studies), highlighting epidemiological advantage of Euro-American lineages in this region

  9. GSTM1 Null Genotype and GSTP1 Ile105Val Polymorphism Are Associated with Alzheimer's Disease: a Meta-Analysis.

    PubMed

    Wang, Mo; Li, Yu; Lin, Lulu; Song, Guijun; Deng, Teng

    2016-03-01

    Published studies on the associations between glutathione S-transferase (GST) polymorphisms and Alzheimer's disease reported controversial findings. A meta-analysis of published studies was performed to assess the associations between polymorphisms of GSTM1, GSTT1 and GSTP1, and Alzheimer's disease. PubMed, Embase, and other databases were searched for case-control on the associations between polymorphisms of GSTM1, GSTT1 and GSTP1, and Alzheimer's disease. The odds ratio (OR) and 95% confidence interval (95% CI) were used to assess the associations. Eleven articles were finally included into the meta-analysis, including eight studies on GSTM1 null genotype, six studies on GSTT1 null genotype, and six studies on GSTP1 Ile105Val polymorphism. Overall, GSTM1 null genotype was associated with increased risk of Alzheimer's disease (fixed effect OR = 1.34, 95% CI 1.10-1.64, P = 0.004). GSTT1 null genotype was not associated with risk of Alzheimer's disease (random effect OR = 1.15, 95% CI 0.68-1.92, P = 0.60). Besides, GSTP1 Ile105Val polymorphism was significantly associated with increased risk of Alzheimer's disease (Val vs Ile: OR = 1.45, 95% CI 1.05-1.99, P = 0.023; ValVal vs IleIle: OR = 1.87, 95% CI 1.30-2.69, P = 0.001; ValVal vs IleIle + IleVal: OR = 1.76, 95% CI 1.24-2.51, P = 0.002). No obvious risk of publication bias was observed in the meta-analysis. GSTM1 null genotype and GSTP1 Ile105Val polymorphism are associated with increased risk of Alzheimer's disease. More studies with large sample size are needed to validate the findings in the meta-analysis.

  10. Screening of pregnant women attending the antenatal care clinic of a tertiary hospital in eastern Saudi Arabia for Chlamydia trachomatis and Neisseria gonorrhoeae infections

    PubMed Central

    Alzahrani, Alhusain J.; Obeid, Obeid E.; Hassan, Manal I.; Almulhim, Abdalaziz A.

    2010-01-01

    Inroduction: Of the “top ten” sexually transmitted infections, Chlamydia trachomatis and Neisseria gonorrhoeae are ranked second and fifth, respectively, worldwide. Aim: The aim of this study was to screen the pregnant women for C. trachomatis and N. gonorrhoeae infections and to detect antimicrobial resistance pattern of N. gonorrhoeae. Materials and Methods: This study was a prospective, hospital-based analysis of a random sample of pregnant women visiting the antenatal clinic of a tertiary hospital in eastern Saudi Arabia. Endocervical and high vaginal swabs were collected both from pregnant women and female patients attending gynecology clinic with lower genital tract infection (control group). C. trachomatis antigen was detected using enzyme-linked immunosorbent assay (ELISA). N. gonorrhoeae was detected by culture and identification of isolates, and antimicrobial susceptibility testing was performed. Statistical Package for Social Sciences (SPSS) version 13.0 and Chi-square test were used for statistical analysis. Results: C. trachomatis antigen was detected in 10.5% (10/95) and 34.4% (35/102) of pregnant women and control group, respectively (P < 0.001). The isolation rate of N. gonorrhoeae among pregnant women was 0.0% compared to 7.8% (8/102) among the control group (P < 0.01). N. gonorrhoeae were resistant to penicillin (62.5%), tetracycline (50%), ampicillin (25%), amoxycillin–clavulinic acid (25%) and ciprofloxacin (37.5%), while they were susceptible to cefepime, ceftriaxone, ceftazidime, spectinomycin, and cefuroxime. Conclusion: Screening of pregnant women for C. trachomatis infection should be included in the antenatal care in this area. The detection rate of both organisms among the control group highlights the importance of preventive strategies. Certain antibiotics previously used in treating gonorrhea are no longer effective. PMID:21716786

  11. Emerging cephalosporin and multidrug-resistant gonorrhoea in Europe.

    PubMed

    Cole, M J; Spiteri, G; Chisholm, S A; Hoffmann, S; Ison, C A; Unemo, M; Van de Laar, M

    2014-11-13

    Neisseria gonorrhoeae has consistently developed resistance to antimicrobials used therapeutically for gonorrhoea and few antimicrobials remain for effective empiric first-line therapy. Since 2009 the European gonococcal antimicrobial surveillance programme (Euro-GASP) has been running as a sentinel surveillance system across Member States of the European Union (EU) and European Economic Area (EEA) to monitor antimicrobial susceptibility in N. gonorrhoeae. During 2011, N. gonorrhoeae isolates were collected from 21 participating countries, and 7.6% and 0.5% of the examined gonococcal isolates had in vitro resistance to cefixime and ceftriaxone, respectively. The rate of ciprofloxacin and azithromycin resistance was 48.7% and 5.3%, respectively. Two (0.1%) isolates displayed high-level resistance to azithromycin, i.e. a minimum inhibitory concentration (MIC) ≥256 mg/L. The current report further highlights the public health need to implement the European response plan, including further strengthening of Euro-GASP, to control and manage the threat of multidrug resistant N. gonorrhoeae.

  12. Emerging cephalosporin and multidrug-resistant gonorrhoea in Europe.

    PubMed

    Cole, M J; Spiteri, G; Chisholm, S A; Hoffmann, S; Ison, C A; Unemo, M; Van de Laar, M

    2014-01-01

    Neisseria gonorrhoeae has consistently developed resistance to antimicrobials used therapeutically for gonorrhoea and few antimicrobials remain for effective empiric first-line therapy. Since 2009 the European gonococcal antimicrobial surveillance programme (Euro-GASP) has been running as a sentinel surveillance system across Member States of the European Union (EU) and European Economic Area (EEA) to monitor antimicrobial susceptibility in N. gonorrhoeae. During 2011, N. gonorrhoeae isolates were collected from 21 participating countries, and 7.6% and 0.5% of the examined gonococcal isolates had in vitro resistance to cefixime and ceftriaxone, respectively. The rate of ciprofloxacin and azithromycin resistance was 48.7% and 5.3%, respectively. Two (0.1%) isolates displayed high-level resistance to azithromycin, i.e. a minimum inhibitory concentration (MIC) ≥256 mg/L. The current report further highlights the public health need to implement the European response plan, including further strengthening of Euro-GASP, to control and manage the threat of multidrug resistant N. gonorrhoeae. PMID:25411689

  13. Genotyping and phylogenetic analysis of bovine viral diarrhea virus isolates from BVDV infected alpacas in North America.

    PubMed

    Kim, Sung G; Anderson, Renee R; Yu, Jin Z; Zylich, Nancy C; Kinde, Hailu; Carman, Suzanne; Bedenice, Daniela; Dubovi, Edward J

    2009-05-12

    Over a three-year period, 2004-2007, greater than 12,000 alpacas in the United States were screened by real-time RT-PCR to identify alpacas persistently infected (PI) with bovine viral diarrhea virus (BVDV). A total of 46 BVD viruses were isolated from PI alpacas or diagnostic samples from alpacas. Forty-three US alpaca BVDV isolates and 3 Canadian isolates were analyzed by comparison of nucleotide sequences of two viral genomic regions, the 5'-UTR and the N(pro) gene to determine their genetic relatedness. All 46 alpaca BVDV isolates from 8 different states of the US and Canada were genotype 1b with > or =99% nt identity in the 290-base 5'-UTR region with the exception of one Canadian isolate. In contrast, 21 bovine BVDV isolates collected during the same period were grouped into the typical 3 genotypes, 1a, 1b, and 2, respectively. Forty five alpaca BVDV isolates formed a distinctive cluster separated from closely related bovine genotype 1b isolates by phylogenetic analysis of the 5'-UTR region. Comparison of the 504-base N(pro) gene sequences of 32 alpaca isolates also assigned them all to type 1b in a similar fashion as observed with the 5'-UTR region. The results suggest that unique genotypes of bovine BVDV 1b may be maintained in the alpaca population even though camelids are susceptible to infection by other genotypes. Further studies are needed to address why alpacas were predominantly infected with genotype 1b BVDV isolates and how bovine BVD viruses evolved to infect alpacas.

  14. Cold tolerance in rice germinating seeds revealed by deep RNAseq analysis of contrasting indica genotypes.

    PubMed

    Dametto, Andressa; Sperotto, Raul A; Adamski, Janete M; Blasi, Édina A R; Cargnelutti, Denise; de Oliveira, Luiz F V; Ricachenevsky, Felipe K; Fregonezi, Jeferson N; Mariath, Jorge E A; da Cruz, Renata P; Margis, Rogério; Fett, Janette P

    2015-09-01

    Rice productivity is largely affected by low temperature, which can be harmful throughout plant development, from germination to grain filling. Germination of indica rice cultivars under cold is slow and not uniform, resulting in irregular emergence and small plant population. To identify and characterize novel genes involved in cold tolerance during the germination stage, two indica rice genotypes (sister lines previously identified as cold-tolerant and cold-sensitive) were used in parallel transcriptomic analysis (RNAseq) under cold treatment (seeds germinating at 13 °C for 7 days). We detected 1,361 differentially expressed transcripts. Differences in gene expression found by RNAseq were confirmed for 11 selected genes using RT-qPCR. Biological processes enhanced in the cold-tolerant seedlings include: cell division and expansion (confirmed by anatomical sections of germinating seeds), cell wall integrity and extensibility, water uptake and membrane transport capacity, sucrose synthesis, generation of simple sugars, unsaturation of membrane fatty acids, wax biosynthesis, antioxidant capacity (confirmed by histochemical staining of H2O2), and hormone and Ca(2+)-signaling. The cold-sensitive seedlings respond to low temperature stress increasing synthesis of HSPs and dehydrins, along with enhanced ubiquitin/proteasome protein degradation pathway and polyamine biosynthesis. Our findings can be useful in future biotechnological approaches aiming to cold tolerance in indica rice.

  15. Genotype Analysis of Escherichia coli Strains Isolated from Children and Chickens Living in Close Contact

    PubMed Central

    Kariuki, S.; Gilks, C.; Kimari, J.; Obanda, A.; Muyodi, J.; Waiyaki, P.; Hart, C. A.

    1999-01-01

    Escherichia coli isolates from rectal swabs from 62 chickens and stools from 42 children living in close contact with chickens on the same farms in Kiambu district, Kenya, were compared for their genetic relatedness. Antibiotic susceptibility profiles broadly categorized isolates from the children and from the chickens into two separate clusters: the majority (144; 85.5%) of the E. coli isolates from children were multidrug resistant, while the majority (216; 87.1%) of the E. coli isolates from chickens were either fully susceptible or resistant only to tetracycline. Sixty- and 100- to 110-MDA plasmids were found to encode the transferable resistance to co-trimoxazole and tetracycline. HindIII restriction endonuclease digestion of the 60- and 100- to 110-MDA plasmids produced four distinct patterns for isolates from children and three distinct patterns for isolates from chickens. XbaI digestion of genomic DNA followed by pulsed-field gel electrophoresis (PFGE) analysis produced 14 distinct clusters. There were six distinct PFGE clusters among the isolates from children, while among the isolates from chickens there were seven distinct clusters. Only one PFGE cluster contained isolates from both children and chickens, with the isolates displaying an approximately 60% coefficient of similarity. This study showed that although several different genotypes of E. coli were isolated from children and chickens from the same farms, the E. coli strains from these two sources were distinct. PMID:9925570

  16. PGMRA: a web server for (phenotype x genotype) many-to-many relation analysis in GWAS.

    PubMed

    Arnedo, Javier; del Val, Coral; de Erausquin, Gabriel Alejandro; Romero-Zaliz, Rocío; Svrakic, Dragan; Cloninger, Claude Robert; Zwir, Igor

    2013-07-01

    It has been proposed that single nucleotide polymorphisms (SNPs) discovered by genome-wide association studies (GWAS) account for only a small fraction of the genetic variation of complex traits in human population. The remaining unexplained variance or missing heritability is thought to be due to marginal effects of many loci with small effects and has eluded attempts to identify its sources. Combination of different studies appears to resolve in part this problem. However, neither individual GWAS nor meta-analytic combinations thereof are helpful for disclosing which genetic variants contribute to explain a particular phenotype. Here, we propose that most of the missing heritability is latent in the GWAS data, which conceals intermediate phenotypes. To uncover such latent information, we propose the PGMRA server that introduces phenomics--the full set of phenotype features of an individual--to identify SNP-set structures in a broader sense, i.e. causally cohesive genotype-phenotype relations. These relations are agnostically identified (without considering disease status of the subjects) and organized in an interpretable fashion. Then, by incorporating a posteriori the subject status within each relation, we can establish the risk surface of a disease in an unbiased mode. This approach complements-instead of replaces-current analysis methods. The server is publically available at http://phop.ugr.es/fenogeno.

  17. Cold tolerance in rice germinating seeds revealed by deep RNAseq analysis of contrasting indica genotypes.

    PubMed

    Dametto, Andressa; Sperotto, Raul A; Adamski, Janete M; Blasi, Édina A R; Cargnelutti, Denise; de Oliveira, Luiz F V; Ricachenevsky, Felipe K; Fregonezi, Jeferson N; Mariath, Jorge E A; da Cruz, Renata P; Margis, Rogério; Fett, Janette P

    2015-09-01

    Rice productivity is largely affected by low temperature, which can be harmful throughout plant development, from germination to grain filling. Germination of indica rice cultivars under cold is slow and not uniform, resulting in irregular emergence and small plant population. To identify and characterize novel genes involved in cold tolerance during the germination stage, two indica rice genotypes (sister lines previously identified as cold-tolerant and cold-sensitive) were used in parallel transcriptomic analysis (RNAseq) under cold treatment (seeds germinating at 13 °C for 7 days). We detected 1,361 differentially expressed transcripts. Differences in gene expression found by RNAseq were confirmed for 11 selected genes using RT-qPCR. Biological processes enhanced in the cold-tolerant seedlings include: cell division and expansion (confirmed by anatomical sections of germinating seeds), cell wall integrity and extensibility, water uptake and membrane transport capacity, sucrose synthesis, generation of simple sugars, unsaturation of membrane fatty acids, wax biosynthesis, antioxidant capacity (confirmed by histochemical staining of H2O2), and hormone and Ca(2+)-signaling. The cold-sensitive seedlings respond to low temperature stress increasing synthesis of HSPs and dehydrins, along with enhanced ubiquitin/proteasome protein degradation pathway and polyamine biosynthesis. Our findings can be useful in future biotechnological approaches aiming to cold tolerance in indica rice. PMID:26259169

  18. Congenital stationary night blindness: an analysis and update of genotype-phenotype correlations and pathogenic mechanisms.

    PubMed

    Zeitz, Christina; Robson, Anthony G; Audo, Isabelle

    2015-03-01

    Congenital stationary night blindness (CSNB) refers to a group of genetically and clinically heterogeneous retinal disorders. Seventeen different genes with more than 360 different mutations and more than 670 affected alleles have been associated with CSNB, including genes coding for proteins of the phototransduction cascade, those important for signal transmission from the photoreceptors to the bipolar cells or genes involved in retinoid recycling in the retinal pigment epithelium. This article describes the phenotypic characteristics of different forms of CSNB that are necessary for accurate diagnosis and to direct and improve genetic testing. An overview of classical and recent methods used to identify specific CSNB genotypes is provided and a meta-analysis of all previously published and novel data is performed to determine the prevalence of disease-causing mutations. Studies of the underlying molecular pathogenic mechanisms based on cell culture techniques and animal studies are outlined. The article highlights how the study of CSNB has increased understanding of the mechanisms of visual signalling in the retina, likely to prove important in developing future treatments for CSNB and other retinal disorders. PMID:25307992

  19. Pili-taxis: Clustering of Neisseria gonorrhoeae bacteria

    NASA Astrophysics Data System (ADS)

    Taktikos, Johannes; Zaburdaev, Vasily; Biais, Nicolas; Stark, Holger; Weitz, David A.

    2012-02-01

    The first step of colonization of Neisseria gonorrhoeae bacteria, the etiological agent of gonorrhea, is the attachment to human epithelial cells. The attachment of N. gonorrhoeae bacteria to surfaces or other cells is primarily mediated by filamentous appendages, called type IV pili (Tfp). Cycles of elongation and retraction of Tfp are responsible for a common bacterial motility called twitching motility which allows the bacteria to crawl over surfaces. Experimentally, N. gonorrhoeae cells initially dispersed over a surface agglomerate into round microcolonies within hours. It is so far not known whether this clustering is driven entirely by the Tfp dynamics or if chemotactic interactions are needed. Thus, we investigate whether the agglomeration may stem solely from the pili-mediated attraction between cells. By developing a statistical model for pili-taxis, we try to explain the experimental measurements of the time evolution of the mean cluster size, number of clusters, and area fraction covered by the cells.

  20. Incidence, age distribution and complications of gonorrhoea in Sweden*

    PubMed Central

    Gisslén, H.; Hellgren, L.; Starck, V.

    1961-01-01

    The incidence of gonorrhoea in Sweden has shown a tendency to increase in recent years, particularly in the age-groups 15-19 and 20-24 years. However, an extrapolation of the annual incidence curve to 1970 for the 15-19-year age-group suggests that some deceleration in that increase may be expected. Study of gonococcal complications seen at Göteborg hospitals during the period 1950-59 indicates that the frequency of gonococcal salpingitis is higher than had previously been reported; this is attributed to the inclusion in the material studied of patients from departments other than those exclusively concerned with the venereal diseases. The authors conclude that, in view of the high frequency of gonorrhoea in the young and of gonococcal complications, gonorrhoea in Sweden cannot be considered an unimportant public health problem. PMID:13705767

  1. Testing commercial sex workers for chlamydia and gonorrhoea on outreach.

    PubMed

    Macauley, S; Creighton, S

    2009-06-01

    To assess the feasibility of testing indoor commercial sex workers (CSW) for Chlamydia trachomatis and Neisseria gonorrhoeae in an outreach setting. All CSW seen on outreach over a 6-week period were offered self-taken vulval swabs for chlamydia and gonorrhoea testing. Feasibility was assessed by all the outreach workers on a standardised proforma. Of the 93 women offered the service, 40 accepted, of whom five (12%) had not previously accessed sexual health services. The majority of women declining the service had recently attended a sexual health clinic. Three cases of chlamydia and one of gonorrhoea were diagnosed. The cost per sexually transmitted infection (STI) was pound 392.50. Most of this group of women were knowledgeable about sexual health and were already having regular check-ups, but a significant minority did not know how to access STI care. Offering STI testing on outreach was feasible and cost effective. PMID:19155241

  2. Coverage recommendation for genotyping analysis of highly heterologous species using next-generation sequencing technology

    PubMed Central

    Song, Kai; Li, Li; Zhang, Guofan

    2016-01-01

    Next-generation sequencing (NGS) technology is being applied to an increasing number of non-model species and has been used as the primary approach for accurate genotyping in genetic and evolutionary studies. However, inferring genotypes from sequencing data is challenging, particularly for organisms with a high degree of heterozygosity. This is because genotype calls from sequencing data are often inaccurate due to low sequencing coverage, and if this is not accounted for, genotype uncertainty can lead to serious bias in downstream analyses, such as quantitative trait locus mapping and genome-wide association studies. Here, we used high-coverage reference data sets from Crassostrea gigas to simulate sequencing data with different coverage, and we evaluate the influence of genotype calling rate and accuracy as a function of coverage. Having initially identified the appropriate parameter settings for filtering to ensure genotype accuracy, we used two different single-nucleotide polymorphism (SNP) calling pipelines, single-sample and multi-sample. We found that a coverage of 15× was suitable for obtaining sufficient numbers of SNPs with high accuracy. Our work provides guidelines for the selection of sequence coverage when using NGS to investigate species with a high degree of heterozygosity and rapid decay of linkage disequilibrium. PMID:27760996

  3. Integrated genotype calling and association analysis of SNPs, common copy number polymorphisms and rare CNVs

    PubMed Central

    Korn, Joshua M; Kuruvilla, Finny G; McCarroll, Steven A; Wysoker, Alec; Nemesh, James; Cawley, Simon; Hubbell, Earl; Veitch, Jim; Collins, Patrick J; Darvishi, Katayoon; Lee, Charles; Nizzari, Marcia M; Gabriel, Stacey B; Purcell, Shaun; Daly, Mark J; Altshuler, David

    2009-01-01

    Accurate and complete measurement of single nucleotide (SNP) and copy number (CNV) variants, both common and rare, will be required to understand the role of genetic variation in disease. We present Birdsuite, a four-stage analytical framework instantiated in software for deriving integrated and mutually consistent copy number and SNP genotypes. The method sequentially assigns copy number across regions of common copy number polymorphisms (CNPs), calls genotypes of SNPs, identifies rare CNVs via a hidden Markov model (HMM), and generates an integrated sequence and copy number genotype at every locus (for example, including genotypes such as A-null, AAB and BBB in addition to AA, AB and BB calls). Such genotypes more accurately depict the underlying sequence of each individual, reducing the rate of apparent mendelian inconsistencies. The Birdsuite software is applied here to data from the Affymetrix SNP 6.0 array. Additionally, we describe a method, implemented in PLINK, to utilize these combined SNP and CNV genotypes for association testing with a phenotype. PMID:18776909

  4. Phenotype-Genotype Association Analysis of ACTH-Secreting Pituitary Adenoma and Its Molecular Link to Patient Osteoporosis

    PubMed Central

    Wang, Renzhi; Yang, Yakun; Sheng, Miaomiao; Bu, Dechao; Huang, Fengming; Liu, Xiaohai; Zhou, Cuiqi; Dai, Congxin; Sun, Bowen; Zhu, Jindong; Qiao, Yi; Yao, Yong; Zhu, Huijuan; Lu, Lin; Pan, Hui; Feng, Ming; Deng, Kan; Xing, Bing; Lian, Wei; Zhao, Yi; Jiang, Chengyu

    2016-01-01

    Adrenocorticotrophin (ACTH)-secreting pituitary adenoma, also known as Cushing disease (CD), is rare and causes metabolic syndrome, cardiovascular disease and osteoporosis due to hypercortisolism. However, the molecular pathogenesis of CD is still unclear because of a lack of human cell lines and animal models. Here, we study 106 clinical characteristics and gene expression changes from 118 patients, the largest cohort of CD in a single-center. RNA deep sequencing is used to examine genotypic changes in nine paired female ACTH-secreting pituitary adenomas and adjacent nontumorous pituitary tissues (ANPT). We develop a novel analysis linking disease clinical characteristics and whole transcriptomic changes, using Pearson Correlation Coefficient to discover a molecular network mechanism. We report that osteoporosis is distinguished from the phenotype and genotype analysis. A cluster of genes involved in osteoporosis is identified using Pearson correlation coefficient analysis. Most of the genes are reported in the bone related literature, confirming the feasibility of phenotype-genotype association analysis, which could be used in the analysis of almost all diseases. Secreted phosphoprotein 1 (SPP1), collagen type I α 1 chain (COL1A1), 5′-nucleotidase ecto (NT5E), HtrA serine peptidase 1 (HTRA1) and angiopoietin 1 (ANGPT1) and their signalling pathways are shown to be involved in osteoporosis in CD patients. Our discoveries provide a molecular link for osteoporosis in CD patients, and may open new potential avenues for osteoporosis intervention and treatment. PMID:27690016

  5. Alterations in peptidoglycan of Neisseria gonorrhoeae induced by sub-MICs of beta-lactam antibiotics.

    PubMed Central

    Garcia-Bustos, J F; Dougherty, T J

    1987-01-01

    Exposure of Neisseria gonorrhoeae to sub-MICs of selected beta-lactam antibiotics caused distortion of normal cell morphology. Analysis of the peptidoglycan indicated that the cells were accumulating increased quantities of disaccharide pentapeptide in their cell walls. The O-acetylated form of the disaccharide pentapeptide was not detected among the major peaks. The correlation of antibiotic binding to gonococcal penicillin-binding protein 2 and accumulation of non-O-acetylated disaccharide pentapeptide suggested an explanation for the previously observed relationship of penicillin-binding protein 2 and O-acetylation of peptidoglycan. PMID:3105447

  6. Diagnosis of Neisseria gonorrhoeae using molecular beacon.

    PubMed

    Sachdev, Divya; Patel, Achchhe Lal; Sonkar, Subash Chandra; Kumari, Indu; Saluja, Daman

    2015-01-01

    Neisseria gonorrhoeae is an important sexually transmitted diseases (STD) causing pathogen worldwide. Due to absence of an affordable diagnostic assay, routine screening of gonococcal infection becomes impossible in developing countries where infection rates are maximum. Treatment is given on the basis of symptoms alone which leads to spread of infection. Thus, development of a rapid, sensitive, specific, and PCR based visual diagnostic assay suitable for developing countries, required for better disease management, is aimed at in present study. Endocervical swabs were collected from patients visiting gynecology department of various hospitals in Delhi. In-house PCR based assay was developed and modified to visual assay using molecular beacon for end-point detection. It was evaluated against Roche AMPLICOR NG kit and rmp gene. Specificity of beacon was confirmed by competition experiments. Diagnostic test was 98.21% specific and 99.59% sensitive whereas negative and positive predicted value were 99.40% and 98.78%, respectively. We also observed that twice the concentration (2X) of premix was stable at 4°C for 4 months and dry swab samples gave concordant results with that of wet swabs. These features make the test best suitable for routine diagnosis of genital infections in developing countries.

  7. Seminal plasma initiates a Neisseria gonorrhoeae transmission state.

    PubMed

    Anderson, Mark T; Dewenter, Lena; Maier, Berenike; Seifert, H Steven

    2014-03-04

    Niche-restricted pathogens are evolutionarily linked with the specific biological fluids that are encountered during infection. Neisseria gonorrhoeae causes the genital infection gonorrhea and is exposed to seminal fluid during sexual transmission. Treatment of N. gonorrhoeae with seminal plasma or purified semen proteins lactoferrin, serum albumin, and prostate-specific antigen each facilitated type IV pilus-mediated twitching motility of the bacterium. Motility in the presence of seminal plasma was characterized by high velocity and low directional persistence. In addition, infection of epithelial cells with N. gonorrhoeae in the presence of seminal plasma resulted in enhanced microcolony formation. Close association of multiple pili in the form of bundles was also disrupted after seminal plasma treatment leading to an increase in the number of single pilus filaments on the bacterial surface. Thus, exposure of N. gonorrhoeae to seminal plasma is proposed to alter bacterial motility and aggregation characteristics to influence the processes of transmission and colonization. IMPORTANCE There are greater than 100 million estimated new cases of gonorrhea annually worldwide. Research characterizing the mechanisms of pathogenesis and transmission of Neisseria gonorrhoeae is important for developing new prevention strategies, since antibiotic resistance of the organism is becoming increasingly prevalent. Our work identifies seminal plasma as a mediator of N. gonorrhoeae twitching motility and microcolony formation through functional modification of the type IV pilus. These findings provide insight into motility dynamics and epithelial cell colonization under conditions that are relevant to sexual transmission. Type IV pili are common virulence factors with diverse functions among bacterial pathogens, and this work identifies interactions between type IV pili and the host environment. Finally, this work illustrates the importance of the host environment and niche

  8. Genotype relative risks: Methods for design and analysis of candidate-gene association studies

    SciTech Connect

    Shaid, D.J.; Sommer, S.S. )

    1993-11-01

    Design and analysis methods are presented for studying the association of a candidate gene with a disease by using parental data in place of nonrelated controls. This alternating design eliminates spurious differences in allele frequencies between cases and nonrelated controls resulting from different ethnic origins and population stratification for these two groups. The authors present analysis methods which are based on two genetic relative risks: (1) the relative risk of disease for homozygotes with two copies of the candidate gene versus homozygotes without the candidate gene and (2) the relative risk for heterozygotes with one copy of the candidate gene versus homozygotes without the candidate gene. In addition to estimating the magnitude of these relative risks, likelihood methods allow specific hypotheses to be tested, namely, a test for overall association of the candidate gene with disease, as well as specific genetic hypotheses, such as dominant or recessive inheritance. Two likelihood methods are presented: (1) a likelihood method appropriate when Hardy-Weinberg equilibrium holds and (2) a likelihood method in which the authors condition on parental genotype data when Hardy-Weinberg equilibrium does not hold. The results for the relative efficiency of these two methods suggest that the conditional approach may at times be preferable, even when equilibrium holds. Sample-size and power calculations are presented for a multitiered design. Tier 1 detects the presence of an abnormal sequence for a postulated candidate gene among a small group of cases. Tier 2 tests for association of the abnormal variant with disease, such as by the likelihood methods presented. Tier 3 confirms positive results from tier 2. Results indicate that required sample sizes are smaller when expression of disease is recessive, rather than dominant, and that, for recessive disease and large relative risks, necessary sample sizes may be feasible. 19 refs., 2 figs., 2 tabs.

  9. Gonorrhoea in inner London: results of a cross sectional study.

    PubMed Central

    Low, N.; Daker-White, G.; Barlow, D.; Pozniak, A. L.

    1997-01-01

    OBJECTIVES: To estimate population based incidence rates of gonorrhoea in an inner London area and examine relations with age, ethnic group, and socioeconomic deprivation. DESIGN: Cross sectional study. SETTING: 11 departments of genitourinary medicine in south and central London. SUBJECTS: 1978 first episodes of gonorrhoea diagnosed in 1994 and 1995 in residents of 73 electoral wards in the boroughs of Lambeth, Southwark, and Lewisham who attended any of the departments of genitourinary medicine. MAIN OUTCOME MEASURES: Yearly age, sex, and ethnic group specific rates of gonorrhoea per 100,000 population aged 15-59 years; rate ratios for the effects of age and ethnic group on gonorrhoea rates in women and men before and after adjustment for confounding factors. RESULTS: Overall incidence rates of gonorrhoea in residents of Lambeth, Southwark, and Lewisham were 138.3 cases yearly per 100,000 women and 291.9 cases yearly per 100,000 men aged 15-59 years. At all ages gonorrhoea rates were higher in non-white minority ethnic groups. Rate ratios for the effect of age adjusted for ethnic group and underprivilege were 15.2 (95% confidence interval 11.6 to 19.7) for women and 2.0 (1.7 to 2.5) for men aged 15-19 years compared with those over 30. After deprivation score and age were taken into account, women from black minority groups were 10.5 (8.6 to 12.8) times as likely and men 11.0 (9.7 to 12.6) times as likely as white people to experience gonorrhoea. CONCLUSIONS: Gonorrhoea rates in Lambeth, Southwark, and Lewisham in 1994-5 were six to seven times higher than for England and Wales one year earlier. The presentation of national trends thus hides the disproportionate contribution of ongoing endemic transmission in the study area. Teenage women and young adult men, particularly those from black minority ethnic groups, are the most heavily affected, even when socioeconomic underprivilege is taken into account. There is urgent need for resources for culturally

  10. Simple and rapid human papillomavirus genotyping method by restriction fragment length polymorphism analysis with two restriction enzymes.

    PubMed

    Chen, Linghan; Watanabe, Ken; Haruyama, Takahiro; Kobayashi, Nobuyuki

    2013-07-01

    Cervical cancer, the third most common cancer that affects women worldwide, is caused by the human papillomavirus (HPV) and is treatable when detected at an early stage. To date, more than 100 different HPV types have been described, and the development of simple, low-cost, and accurate methods to distinguish HPV genotypes is highly warranted. In this study, an HPV genotyping assay based on polymerase chain reaction (PCR) was evaluated. This method involved the use of MY09/11 primers followed by restriction fragment length polymorphism (RFLP) analysis with the restriction enzymes HpyCH4V and NlaIII. Cervical specimens preserved using CytoRich Blue fluid were collected from 1,134 female volunteers for HPV detection, and 1,111 valid samples were amplified using PCR. The PCR method was sensitive enough to detect 25 copies of HPV18, and three copies of HPV16. Out of 202 PCR-positive samples, HPV genotypes were determined in 189 samples (93.6%) by this RFLP method. Results were then evaluated further by capillary sequencing method. Concordant results between the two tests were as high as 96.0%. Thirteen samples, which tested negative with RFLP, were verified as non-specific amplifications with PCR. In conclusion, this PCR-RFLP method using restriction enzymes HpyCH4V and NlaIII is simple, non-labor intensive, and is applicable for the inexpensive determination of HPV genotypes in clinical samples.

  11. Improved Genotyping Vaccine and Wild-Type Poliovirus Strains by Restriction Fragment Length Polymorphism Analysis: Clinical Diagnostic Implications

    PubMed Central

    Georgopoulou, Amalia; Markoulatos, Panayotis; Spyrou, Niki; Vamvakopoulos, Nicholas C.

    2000-01-01

    The combination of preventive vaccination and diagnostic typing of viral isolates from patients with clinical poliomyelitis constitutes our main protective shield against polioviruses. The restriction fragment length polymorphism (RFLP) adaptation of the reverse transcriptase (RT)-PCR methodology has advanced diagnostic genotyping of polioviruses, although further improvements are definitely needed. We report here on an improved RFLP procedure for the genotyping of polioviruses. A highly conserved segment within the 5′ noncoding region of polioviruses was selected for RT-PCR amplification by the UC53-UG52 primer pair with the hope that it would be most resistant to the inescapable genetic alteration-drift experienced by the other segments of the viral genome. Complete inter- and intratypic genotyping of polioviruses by the present RFLP method was accomplished with a minimum set of four restriction endonucleases (HaeIII, DdeI, NcoI, and AvaI). To compensate for potential genetic drift within the recognition sites of HaeIII, DdeI, or NcoI in atypical clinical samples, the RFLP patterns generated with HpaII and StyI as replacements were analyzed. The specificity of the method was also successfully assessed by RFLP analysis of 55 reference nonpoliovirus enterovirus controls. The concerted implementation of these conditional protocols for diagnostic inter- and intratypic genotyping of polioviruses was evaluated with 21 clinical samples with absolute success. PMID:11101561

  12. Genotypic and Phenotypic Analysis of Dairy Lactococcus lactis Biodiversity in Milk: Volatile Organic Compounds as Discriminating Markers

    PubMed Central

    Dhaisne, Amandine; Guellerin, Maeva; Laroute, Valérie; Laguerre, Sandrine; Le Bourgeois, Pascal; Loubiere, Pascal

    2013-01-01

    The diversity of nine dairy strains of Lactococcus lactis subsp. lactis in fermented milk was investigated by both genotypic and phenotypic analyses. Pulsed-field gel electrophoresis and multilocus sequence typing were used to establish an integrated genotypic classification. This classification was coherent with discrimination of the L. lactis subsp. lactis bv. diacetylactis lineage and reflected clonal complex phylogeny and the uniqueness of the genomes of these strains. To assess phenotypic diversity, 82 variables were selected as important dairy features; they included physiological descriptors and the production of metabolites and volatile organic compounds (VOCs). Principal-component analysis (PCA) demonstrated the phenotypic uniqueness of each of these genetically closely related strains, allowing strain discrimination. A method of variable selection was developed to reduce the time-consuming experimentation. We therefore identified 20 variables, all associated with VOCs, as phenotypic markers allowing discrimination between strain groups. These markers are representative of the three metabolic pathways involved in flavor: lipolysis, proteolysis, and glycolysis. Despite great phenotypic diversity, the strains could be divided into four robust phenotypic clusters based on their metabolic orientations. Inclusion of genotypic diversity in addition to phenotypic characters in the classification led to five clusters rather than four being defined. However, genotypic characters make a smaller contribution than phenotypic variables (no genetic distances selected among the most contributory variables). This work proposes an original method for the phenotypic differentiation of closely related strains in milk and may be the first step toward a predictive classification for the manufacture of starters. PMID:23709512

  13. Linkage disequilibrium mapping via cladistic analysis of phase-unknown genotypes and inferred haplotypes in the Genetic Analysis Workshop 14 simulated data.

    PubMed

    Durrant, Caroline; Morris, Andrew P

    2005-01-01

    We recently described a method for linkage disequilibrium (LD) mapping, using cladistic analysis of phased single-nucleotide polymorphism (SNP) haplotypes in a logistic regression framework. However, haplotypes are often not available and cannot be deduced with certainty from the unphased genotypes. One possible two-stage approach is to infer the phase of multilocus genotype data and analyze the resulting haplotypes as if known. Here, haplotypes are inferred using the expectation-maximization (EM) algorithm and the best-guess phase assignment for each individual analyzed. However, inferring haplotypes from phase-unknown data is prone to error and this should be taken into account in the subsequent analysis. An alternative approach is to analyze the phase-unknown multilocus genotypes themselves. Here we present a generalization of the method for phase-known haplotype data to the case of unphased SNP genotypes. Our approach is designed for high-density SNP data, so we opted to analyze the simulated dataset. The marker spacing in the initial screen was too large for our method to be effective, so we used the answers provided to request further data in regions around the disease loci and in null regions. Power to detect the disease loci, accuracy in localizing the true site of the locus, and false-positive error rates are reported for the inferred-haplotype and unphased genotype methods. For this data, analyzing inferred haplotypes outperforms analysis of genotypes. As expected, our results suggest that when there is little or no LD between a disease locus and the flanking region, there will be no chance of detecting it unless the disease variant itself is genotyped.

  14. Linkage disequilibrium mapping via cladistic analysis of phase-unknown genotypes and inferred haplotypes in the Genetic Analysis Workshop 14 simulated data

    PubMed Central

    Durrant, Caroline; Morris, Andrew P

    2005-01-01

    We recently described a method for linkage disequilibrium (LD) mapping, using cladistic analysis of phased single-nucleotide polymorphism (SNP) haplotypes in a logistic regression framework. However, haplotypes are often not available and cannot be deduced with certainty from the unphased genotypes. One possible two-stage approach is to infer the phase of multilocus genotype data and analyze the resulting haplotypes as if known. Here, haplotypes are inferred using the expectation-maximization (EM) algorithm and the best-guess phase assignment for each individual analyzed. However, inferring haplotypes from phase-unknown data is prone to error and this should be taken into account in the subsequent analysis. An alternative approach is to analyze the phase-unknown multilocus genotypes themselves. Here we present a generalization of the method for phase-known haplotype data to the case of unphased SNP genotypes. Our approach is designed for high-density SNP data, so we opted to analyze the simulated dataset. The marker spacing in the initial screen was too large for our method to be effective, so we used the answers provided to request further data in regions around the disease loci and in null regions. Power to detect the disease loci, accuracy in localizing the true site of the locus, and false-positive error rates are reported for the inferred-haplotype and unphased genotype methods. For this data, analyzing inferred haplotypes outperforms analysis of genotypes. As expected, our results suggest that when there is little or no LD between a disease locus and the flanking region, there will be no chance of detecting it unless the disease variant itself is genotyped. PMID:16451556

  15. Complete genome sequence analysis identifies a new genotype of brassica yellows virus that infects cabbage and radish in China.

    PubMed

    Zhang, Xiao-Yan; Xiang, Hai-Ying; Zhou, Cui-Ji; Li, Da-Wei; Yu, Jia-Lin; Han, Cheng-Gui

    2014-08-01

    For brassica yellows virus (BrYV), proposed to be a member of a new polerovirus species, two clearly distinct genotypes (BrYV-A and BrYV-B) have been described. In this study, the complete nucleotide sequences of two BrYV isolates from radish and Chinese cabbage were determined. Sequence analysis suggested that these isolates represent a new genotype, referred to here as BrYV-C. The full-length sequences of the two BrYV-C isolates shared 93.4-94.8 % identity with BrYV-A and BrYV-B. Further phylogenetic analysis showed that the BrYV-C isolates formed a subgroup that was distinct from the BrYV-A and BrYV-B isolates based on all of the proteins except P5.

  16. Complete genome sequence analysis identifies a new genotype of brassica yellows virus that infects cabbage and radish in China.

    PubMed

    Zhang, Xiao-Yan; Xiang, Hai-Ying; Zhou, Cui-Ji; Li, Da-Wei; Yu, Jia-Lin; Han, Cheng-Gui

    2014-08-01

    For brassica yellows virus (BrYV), proposed to be a member of a new polerovirus species, two clearly distinct genotypes (BrYV-A and BrYV-B) have been described. In this study, the complete nucleotide sequences of two BrYV isolates from radish and Chinese cabbage were determined. Sequence analysis suggested that these isolates represent a new genotype, referred to here as BrYV-C. The full-length sequences of the two BrYV-C isolates shared 93.4-94.8 % identity with BrYV-A and BrYV-B. Further phylogenetic analysis showed that the BrYV-C isolates formed a subgroup that was distinct from the BrYV-A and BrYV-B isolates based on all of the proteins except P5. PMID:24599564

  17. An integrative variant analysis pipeline for accurate genotype/haplotype inference in population NGS data

    PubMed Central

    Wang, Yi; Lu, James; Yu, Jin; Gibbs, Richard A.; Yu, Fuli

    2013-01-01

    Next-generation sequencing is a powerful approach for discovering genetic variation. Sensitive variant calling and haplotype inference from population sequencing data remain challenging. We describe methods for high-quality discovery, genotyping, and phasing of SNPs for low-coverage (approximately 5×) sequencing of populations, implemented in a pipeline called SNPTools. Our pipeline contains several innovations that specifically address challenges caused by low-coverage population sequencing: (1) effective base depth (EBD), a nonparametric statistic that enables more accurate statistical modeling of sequencing data; (2) variance ratio scoring, a variance-based statistic that discovers polymorphic loci with high sensitivity and specificity; and (3) BAM-specific binomial mixture modeling (BBMM), a clustering algorithm that generates robust genotype likelihoods from heterogeneous sequencing data. Last, we develop an imputation engine that refines raw genotype likelihoods to produce high-quality phased genotypes/haplotypes. Designed for large population studies, SNPTools' input/output (I/O) and storage aware design leads to improved computing performance on large sequencing data sets. We apply SNPTools to the International 1000 Genomes Project (1000G) Phase 1 low-coverage data set and obtain genotyping accuracy comparable to that of SNP microarray. PMID:23296920

  18. Gender, genotype, and phenotype differences in Smith-Magenis syndrome: a meta-analysis of 105 cases.

    PubMed

    Edelman, E A; Girirajan, S; Finucane, B; Patel, P I; Lupski, J R; Smith, A C M; Elsea, S H

    2007-06-01

    Smith-Magenis syndrome (SMS) is a multisystem disorder characterized by developmental delay and mental retardation, a distinctive behavioral phenotype, and sleep disturbance. We undertook a comprehensive meta-analysis to identify genotype-phenotype relationships to further understand the clinical variability and genetic factors involved in SMS. Clinical and molecular information on 105 patients with SMS was obtained through research protocols and a review of the literature and analyzed using Fisher's exact test with two-tailed p values. Several differences in these groups of patients were identified based on genotype and gender. Patients with RAI1 mutation were more likely to exhibit overeating, obesity, polyembolokoilamania, self-hugging, muscle cramping, and dry skin and less likely to have short stature, hearing loss, frequent ear infections, and heart defects when compared with patients with deletion, while a subset of small deletion cases with deletions spanning from TNFRSF13B to MFAP4 was less likely to exhibit brachycephaly, dental anomalies, iris abnormalities, head-banging, and hyperactivity. Significant differences between genders were also identified, with females more likely to have myopia, eating/appetite problems, cold hands and feet, and frustration with communication when compared with males. These results confirm previous findings and identify new genotype-phenotype associations including differences in the frequency of short stature, hearing loss, ear infections, obesity, overeating, heart defects, self-injury, self-hugging, dry skin, seizures, and hyperactivity among others based on genotype. Additional studies are required to further explore the relationships between genotype and phenotype and any potential discrepancies in health care and parental attitudes toward males and females with SMS. PMID:17539903

  19. Gender, genotype, and phenotype differences in Smith-Magenis syndrome: a meta-analysis of 105 cases.

    PubMed

    Edelman, E A; Girirajan, S; Finucane, B; Patel, P I; Lupski, J R; Smith, A C M; Elsea, S H

    2007-06-01

    Smith-Magenis syndrome (SMS) is a multisystem disorder characterized by developmental delay and mental retardation, a distinctive behavioral phenotype, and sleep disturbance. We undertook a comprehensive meta-analysis to identify genotype-phenotype relationships to further understand the clinical variability and genetic factors involved in SMS. Clinical and molecular information on 105 patients with SMS was obtained through research protocols and a review of the literature and analyzed using Fisher's exact test with two-tailed p values. Several differences in these groups of patients were identified based on genotype and gender. Patients with RAI1 mutation were more likely to exhibit overeating, obesity, polyembolokoilamania, self-hugging, muscle cramping, and dry skin and less likely to have short stature, hearing loss, frequent ear infections, and heart defects when compared with patients with deletion, while a subset of small deletion cases with deletions spanning from TNFRSF13B to MFAP4 was less likely to exhibit brachycephaly, dental anomalies, iris abnormalities, head-banging, and hyperactivity. Significant differences between genders were also identified, with females more likely to have myopia, eating/appetite problems, cold hands and feet, and frustration with communication when compared with males. These results confirm previous findings and identify new genotype-phenotype associations including differences in the frequency of short stature, hearing loss, ear infections, obesity, overeating, heart defects, self-injury, self-hugging, dry skin, seizures, and hyperactivity among others based on genotype. Additional studies are required to further explore the relationships between genotype and phenotype and any potential discrepancies in health care and parental attitudes toward males and females with SMS.

  20. Spectrum of mutations and genotype-phenotype analysis in Noonan syndrome patients with RIT1 mutations.

    PubMed

    Yaoita, Masako; Niihori, Tetsuya; Mizuno, Seiji; Okamoto, Nobuhiko; Hayashi, Shion; Watanabe, Atsushi; Yokozawa, Masato; Suzumura, Hiroshi; Nakahara, Akihiko; Nakano, Yusuke; Hokosaki, Tatsunori; Ohmori, Ayumi; Sawada, Hirofumi; Migita, Ohsuke; Mima, Aya; Lapunzina, Pablo; Santos-Simarro, Fernando; García-Miñaúr, Sixto; Ogata, Tsutomu; Kawame, Hiroshi; Kurosawa, Kenji; Ohashi, Hirofumi; Inoue, Shin-Ichi; Matsubara, Yoichi; Kure, Shigeo; Aoki, Yoko

    2016-02-01

    RASopathies are autosomal dominant disorders caused by mutations in more than 10 known genes that regulate the RAS/MAPK pathway. Noonan syndrome (NS) is a RASopathy characterized by a distinctive facial appearance, musculoskeletal abnormalities, and congenital heart defects. We have recently identified mutations in RIT1 in patients with NS. To delineate the clinical manifestations in RIT1 mutation-positive patients, we further performed a RIT1 analysis in RASopathy patients and identified 7 RIT1 mutations, including two novel mutations, p.A77S and p.A77T, in 14 of 186 patients. Perinatal abnormalities, including nuchal translucency, fetal hydrops, pleural effusion, or chylothorax and congenital heart defects, are observed in all RIT1 mutation-positive patients. Luciferase assays in NIH 3T3 cells demonstrated that the newly identified RIT1 mutants, including p.A77S and p.A77T, and the previously identified p.F82V, p.T83P, p.Y89H, and p.M90I, enhanced Elk1 transactivation. Genotype-phenotype correlation analyses of previously reported NS patients harboring RIT1, PTPN11, SOS1, RAF1, and KRAS revealed that hypertrophic cardiomyopathy (56 %) was more frequent in patients harboring a RIT1 mutation than in patients harboring PTPN11 (9 %) and SOS1 mutations (10 %). The rates of hypertrophic cardiomyopathy were similar between patients harboring RIT1 mutations and patients harboring RAF1 mutations (75 %). Short stature (52 %) was less prevalent in patients harboring RIT1 mutations than in patients harboring PTPN11 (71 %) and RAF1 (83 %) mutations. These results delineate the clinical manifestations of RIT1 mutation-positive NS patients: high frequencies of hypertrophic cardiomyopathy, atrial septal defects, and pulmonary stenosis; and lower frequencies of ptosis and short stature. PMID:26714497

  1. Molecular characterization of quinolone-resistant Neisseria gonorrhoeae isolates from Brazil.

    PubMed

    Uehara, Aline A; Amorin, Efigênia L T; Ferreira, Maria de Fátima; Andrade, Claudia F; Clementino, Maysa B M; de Filippis, Ivano; Neves, Felipe P G; Pinto, Tatiana de C A; Teixeira, Lúcia M; Giambiagi-Demarval, Marcia; Fracalanzza, Sérgio E L

    2011-12-01

    Despite the rapid spread of antibiotic resistance among gonococci worldwide, limited reports are available from Brazilian locations. In the present study, 25 quinolone-resistant Neisseria gonorrhoeae (QRNG) strains isolated in Rio de Janeiro, Brazil, were characterized by phenotypic and molecular methods, including analysis of mutations in the gyrA and parC genes. They represented 16.5% of the N. gonorrhoeae isolates obtained during a survey performed from 2006 to 2010. A trend for increasing resistance to ciprofloxacin was observed in the period investigated. The most prevalent pattern of mutation observed among QRNG isolates, Ser-91 to Phe and Asp-95 to Gly in gyrA and Ser-87 to Arg in parC, was detected in 40% of the isolates exhibiting MICs ranging from 4 to >32 μg/ml. Rare types of mutations were found in the gyrA gene (Gln-102 to His [12%] and Asp-95 to Tyr [4%]) and in the parC gene (Ser-88 to Thr [4%]). The genetic relationship of the QRNG isolates, evaluated by pulsed-field gel electrophoresis, suggested that the increase in the frequencies of the QRNG isolates in Rio de Janeiro, Brazil, may have arisen as a result of simultaneous spread of two clonal groups. The results also indicate that fluoroquinolones may no longer be used as first line antibiotics for the treatment of gonorrhea in Rio de Janeiro, and that programs for antimicrobial susceptibility surveillance of N. gonorrhoeae should also be implemented in other regions of Brazil. PMID:21976763

  2. Expression Quantitative Trait Loci Analysis Identifies Associations Between Genotype and Gene Expression in Human Intestine

    PubMed Central

    KABAKCHIEV, BOYKO; SILVERBERG, MARK S.

    2013-01-01

    . CONCLUSIONS eQTL analysis of intestinal tissue supports findings that some eQTL remain stable across cell types, whereas others are specific to the sampled location. Our findings confirm and expand the number of known genotypes associated with expression and could help elucidate mechanisms of intestinal disease. PMID:23474282

  3. Analysis of exome sequence in 604 trios for recessive genotypes in schizophrenia.

    PubMed

    Rees, E; Kirov, G; Walters, J T; Richards, A L; Howrigan, D; Kavanagh, D H; Pocklington, A J; Fromer, M; Ruderfer, D M; Georgieva, L; Carrera, N; Gormley, P; Palta, P; Williams, H; Dwyer, S; Johnson, J S; Roussos, P; Barker, D D; Banks, E; Milanova, V; Rose, S A; Chambert, K; Mahajan, M; Scolnick, E M; Moran, J L; Tsuang, M T; Glatt, S J; Chen, W J; Hwu, H-G; Neale, B M; Palotie, A; Sklar, P; Purcell, S M; McCarroll, S A; Holmans, P; Owen, M J; O'Donovan, M C

    2015-01-01

    Genetic associations involving both rare and common alleles have been reported for schizophrenia but there have been no systematic scans for rare recessive genotypes using fully phased trio data. Here, we use exome sequencing in 604 schizophrenia proband-parent trios to investigate the role of recessive (homozygous or compound heterozygous) nonsynonymous genotypes in the disorder. The burden of recessive genotypes was not significantly increased in probands at either a genome-wide level or in any individual gene after adjustment for multiple testing. At a system level, probands had an excess of nonsynonymous compound heterozygous genotypes (minor allele frequency, MAF ⩽ 1%) in voltage-gated sodium channels (VGSCs; eight in probands and none in parents, P = 1.5 × 10(-)(4)). Previous findings of multiple de novo loss-of-function mutations in this gene family, particularly SCN2A, in autism and intellectual disability provide biological and genetic plausibility for this finding. Pointing further to the involvement of VGSCs in schizophrenia, we found that these genes were enriched for nonsynonymous mutations (MAF ⩽ 0.1%) in cases genotyped using an exome array, (5585 schizophrenia cases and 8103 controls), and that in the trios data, synaptic proteins interacting with VGSCs were also enriched for both compound heterozygosity (P = 0.018) and de novo mutations (P = 0.04). However, we were unable to replicate the specific association with compound heterozygosity at VGSCs in an independent sample of Taiwanese schizophrenia trios (N = 614). We conclude that recessive genotypes do not appear to make a substantial contribution to schizophrenia at a genome-wide level. Although multiple lines of evidence, including several from this study, suggest that rare mutations in VGSCs contribute to the disorder, in the absence of replication of the original findings regarding compound heterozygosity, this conclusion requires evaluation in a larger sample of trios. PMID:26196440

  4. Analysis of exome sequence in 604 trios for recessive genotypes in schizophrenia

    PubMed Central

    Rees, E; Kirov, G; Walters, J T; Richards, A L; Howrigan, D; Kavanagh, D H; Pocklington, A J; Fromer, M; Ruderfer, D M; Georgieva, L; Carrera, N; Gormley, P; Palta, P; Williams, H; Dwyer, S; Johnson, J S; Roussos, P; Barker, D D; Banks, E; Milanova, V; Rose, S A; Chambert, K; Mahajan, M; Scolnick, E M; Moran, J L; Tsuang, M T; Glatt, S J; Chen, W J; Hwu, H-G; Faraone, Stephen V; Roe, Cheri A; Chandler, Sharon D; Liu, Chih-Min; Liu, Chen-Chung; Yeh, Ling-Ling; Ouyang, Wen-Chen; Chan, Hung-Yu; Chen, Chun-Ying; Neale, B M; Palotie, A; Sklar, P; Purcell, S M; McCarroll, S A; Holmans, P; Owen, M J; O'Donovan, M C

    2015-01-01

    Genetic associations involving both rare and common alleles have been reported for schizophrenia but there have been no systematic scans for rare recessive genotypes using fully phased trio data. Here, we use exome sequencing in 604 schizophrenia proband–parent trios to investigate the role of recessive (homozygous or compound heterozygous) nonsynonymous genotypes in the disorder. The burden of recessive genotypes was not significantly increased in probands at either a genome-wide level or in any individual gene after adjustment for multiple testing. At a system level, probands had an excess of nonsynonymous compound heterozygous genotypes (minor allele frequency, MAF ⩽1%) in voltage-gated sodium channels (VGSCs; eight in probands and none in parents, P=1.5 × 10−4). Previous findings of multiple de novo loss-of-function mutations in this gene family, particularly SCN2A, in autism and intellectual disability provide biological and genetic plausibility for this finding. Pointing further to the involvement of VGSCs in schizophrenia, we found that these genes were enriched for nonsynonymous mutations (MAF ⩽0.1%) in cases genotyped using an exome array, (5585 schizophrenia cases and 8103 controls), and that in the trios data, synaptic proteins interacting with VGSCs were also enriched for both compound heterozygosity (P=0.018) and de novo mutations (P=0.04). However, we were unable to replicate the specific association with compound heterozygosity at VGSCs in an independent sample of Taiwanese schizophrenia trios (N=614). We conclude that recessive genotypes do not appear to make a substantial contribution to schizophrenia at a genome-wide level. Although multiple lines of evidence, including several from this study, suggest that rare mutations in VGSCs contribute to the disorder, in the absence of replication of the original findings regarding compound heterozygosity, this conclusion requires evaluation in a larger sample of trios. PMID:26196440

  5. Epidemiological characterization of Neisseria gonorrhoeae by lectins.

    PubMed Central

    Schalla, W O; Whittington, W L; Rice, R J; Larsen, S A

    1985-01-01

    A total of 101 isolates of penicillinase-producing and non-penicillinase-producing Neisseria gonorrhoeae with known nutritional requirements, plasmid content, and serovars, were examined for lectin agglutination patterns. These isolates were from outbreaks in Georgia, California, Hawaii, and Pennsylvania. Cell suspensions made from 16- to 18-h cultures were mixed with 14 different lectins, and the resultant agglutination patterns were classified as agglutination groups. Among the 101 isolates tested, 24 different agglutination groups were demonstrated. Of the organisms tested, 55% were located in 3 of the 24 groups, and 86% of the isolates reacted with the lectins Trichosanthes kinlowii, Griffonia simplicifolia I, peanut agglutinin, soybean agglutinin, potato agglutinin, and wheat germ agglutinin. One isolate did not react with peanut or potato agglutinin, five isolates lacked reactivity with potato agglutinin, and six isolates did not react with wheat germ agglutinin. Of the wheat germ-negative isolates, four were from Pennsylvania and were identical with regard to auxotype, plasmid content, serovar, and lectin group. The other two wheat germ-negative isolates were from California and were unrelated by the same criteria to the four Pennsylvania isolates and to each other. Among the isolates tested, there were no differences in lectin groups with regard to the sex of the patient. In the Georgia collection, agglutination with one lectin group was confined to isolates of serogroup IA. This association was not observed for the other geographic areas. Some isolates showing identical auxotype, plasmid content, and serovars could be differentiated based on lectin agglutination patterns, whereas other isolates were identical by all testing criteria. PMID:3930560

  6. Long-Range HIV Genotyping Using Viral RNA and Proviral DNA for Analysis of HIV Drug Resistance and HIV Clustering

    PubMed Central

    Novitsky, Vlad; Zahralban-Steele, Melissa; McLane, Mary Fran; Moyo, Sikhulile; van Widenfelt, Erik; Gaseitsiwe, Simani; Makhema, Joseph

    2015-01-01

    The goal of the study was to improve the methodology of HIV genotyping for analysis of HIV drug resistance and HIV clustering. Using the protocol of Gall et al. (A. Gall, B. Ferns, C. Morris, S. Watson, M. Cotten, M. Robinson, N. Berry, D. Pillay, and P. Kellam, J Clin Microbiol 50:3838–3844, 2012, doi:10.1128/JCM.01516-12), we developed a robust methodology for amplification of two large fragments of viral genome covering about 80% of the unique HIV-1 genome sequence. Importantly, this method can be applied to both viral RNA and proviral DNA amplification templates, allowing genotyping in HIV-infected subjects with suppressed viral loads (e.g., subjects on antiretroviral therapy [ART]). The two amplicons cover critical regions across the HIV-1 genome (including pol and env), allowing analysis of mutations associated with resistance to protease inhibitors, reverse transcriptase inhibitors (nucleoside reverse transcriptase inhibitors [NRTIs] and nonnucleoside reverse transcriptase inhibitors [NNRTIs]), integrase strand transfer inhibitors, and virus entry inhibitors. The two amplicons generated span 7,124 bp, providing substantial sequence length and numbers of informative sites for comprehensive phylogenic analysis and greater refinement of viral linkage analyses in HIV prevention studies. The long-range HIV genotyping from proviral DNA was successful in about 90% of 212 targeted blood specimens collected in a cohort where the majority of patients had suppressed viral loads, including 65% of patients with undetectable levels of HIV-1 RNA loads. The generated amplicons could be sequenced by different methods, such as population Sanger sequencing, single-genome sequencing, or next-generation ultradeep sequencing. The developed method is cost-effective—the cost of the long-range HIV genotyping is under $140 per subject (by Sanger sequencing)—and has the potential to enable the scale up of public health HIV prevention interventions. PMID:26041893

  7. Long-Range HIV Genotyping Using Viral RNA and Proviral DNA for Analysis of HIV Drug Resistance and HIV Clustering.

    PubMed

    Novitsky, Vlad; Zahralban-Steele, Melissa; McLane, Mary Fran; Moyo, Sikhulile; van Widenfelt, Erik; Gaseitsiwe, Simani; Makhema, Joseph; Essex, M

    2015-08-01

    The goal of the study was to improve the methodology of HIV genotyping for analysis of HIV drug resistance and HIV clustering. Using the protocol of Gall et al. (A. Gall, B. Ferns, C. Morris, S. Watson, M. Cotten, M. Robinson, N. Berry, D. Pillay, and P. Kellam, J Clin Microbiol 50:3838-3844, 2012, doi:10.1128/JCM.01516-12), we developed a robust methodology for amplification of two large fragments of viral genome covering about 80% of the unique HIV-1 genome sequence. Importantly, this method can be applied to both viral RNA and proviral DNA amplification templates, allowing genotyping in HIV-infected subjects with suppressed viral loads (e.g., subjects on antiretroviral therapy [ART]). The two amplicons cover critical regions across the HIV-1 genome (including pol and env), allowing analysis of mutations associated with resistance to protease inhibitors, reverse transcriptase inhibitors (nucleoside reverse transcriptase inhibitors [NRTIs] and nonnucleoside reverse transcriptase inhibitors [NNRTIs]), integrase strand transfer inhibitors, and virus entry inhibitors. The two amplicons generated span 7,124 bp, providing substantial sequence length and numbers of informative sites for comprehensive phylogenic analysis and greater refinement of viral linkage analyses in HIV prevention studies. The long-range HIV genotyping from proviral DNA was successful in about 90% of 212 targeted blood specimens collected in a cohort where the majority of patients had suppressed viral loads, including 65% of patients with undetectable levels of HIV-1 RNA loads. The generated amplicons could be sequenced by different methods, such as population Sanger sequencing, single-genome sequencing, or next-generation ultradeep sequencing. The developed method is cost-effective-the cost of the long-range HIV genotyping is under $140 per subject (by Sanger sequencing)-and has the potential to enable the scale up of public health HIV prevention interventions. PMID:26041893

  8. Neisseria gonorrhoeae suppresses dendritic cell-induced, antigen-dependent CD4 T cell proliferation.

    PubMed

    Zhu, Weiyan; Ventevogel, Melissa S; Knilans, Kayla J; Anderson, James E; Oldach, Laurel M; McKinnon, Karen P; Hobbs, Marcia M; Sempowski, Gregory D; Duncan, Joseph A

    2012-01-01

    Neisseria gonorrhoeae is the second most common sexually transmitted bacterial pathogen worldwide. Diseases associated with N. gonorrhoeae cause localized inflammation of the urethra and cervix. Despite this inflammatory response, infected individuals do not develop protective adaptive immune responses to N. gonorrhoeae. N. gonorrhoeae is a highly adapted pathogen that has acquired multiple mechanisms to evade its host's immune system, including the ability to manipulate multiple immune signaling pathways. N. gonorrhoeae has previously been shown to engage immunosuppressive signaling pathways in B and T lymphocytes. We have now found that N. gonorrhoeae also suppresses adaptive immune responses through effects on antigen presenting cells. Using primary, murine bone marrow-derived dendritic cells and lymphocytes, we show that N. gonorrhoeae-exposed dendritic cells fail to elicit antigen-induced CD4+ T lymphocyte proliferation. N. gonorrhoeae exposure leads to upregulation of a number of secreted and dendritic cell surface proteins with immunosuppressive properties, particularly Interleukin 10 (IL-10) and Programmed Death Ligand 1 (PD-L1). We also show that N. gonorrhoeae is able to inhibit dendritic cell- induced proliferation of human T-cells and that human dendritic cells upregulate similar immunosuppressive molecules. Our data suggest that, in addition to being able to directly influence host lymphocytes, N. gonorrhoeae also suppresses development of adaptive immune responses through interactions with host antigen presenting cells. These findings suggest that gonococcal factors involved in host immune suppression may be useful targets in developing vaccines that induce protective adaptive immune responses to this pathogen.

  9. Recovery of Neisseria gonorrhoeae from 4 commercially available transport systems.

    PubMed

    Papp, John R; Henning, Tara; Khubbar, Manjeet; Kalve, Valdis; Bhattacharyya, Sanjib; Travanty, Emily; Xavier, Karen; Jones, Kelly; Rudrik, James T; Gaynor, Anne; Hagan, Celia

    2016-10-01

    Four commercial transport systems for the recovery of Neisseria gonorrhoeae were evaluated in support of the need to obtain culture isolates for the detection of antimicrobial resistance. Bacterial recovery from the InTray GC system was superior with minimal loss of viability in contrast to non-nutritive transport systems. PMID:27489119

  10. [Cultivation of Neisseria gonorrhoeae in an L-929 cell culture].

    PubMed

    Nurusheva, S M; Lebedeva, M V; Shcherbakova, N I

    1985-12-01

    N. gonorrhoeae strain b has been found to be capable of retaining its viability in medium 199 with 10% of inactivated cattle serum added and in monolayer cell culture L-929 in the above medium. The characteristics obtained in the present investigation permit simulating the mixed association of gonococci and chlamydiae in the culture system used in this work.

  11. Serological classification of Neisseria gonorrhoeae with monoclonal antibodies.

    PubMed Central

    Tam, M R; Buchanan, T M; Sandström, E G; Holmes, K K; Knapp, J S; Siadak, A W; Nowinski, R C

    1982-01-01

    Hybrid cells producing monoclonal antibodies against antigens of Neisseria gonorrhoeae were obtained by the polyethylene glycol-mediated fusion of mouse myeloma cells and lymphocytes from mice immunized with gonococcal protein I or outer membrane proteins. From four fusions, 16 phenotypically stable, independently cloned hybrid cell lines were selected for continued study. Each of the cell lines produced a characteristically different monoclonal antibody which reacted in immunoprecipitation assays with a unique antigenic determinant on protein I of the outer membrane complex of the bacteria. In antibody binding, immunofluorescence, and coagglutination assays these antibodies each reacted with a restricted group of N. gonorrhoeae strains. None of the monoclonal antibodies reacted with 17 other different species of Neisseria or with Branhamella catarrhalis. When tested on 34 N. gonorrhoeae reference serotyping strains, the monoclonal antibodies demonstrated serological relationships between the strains which paralleled those observed with conventional polyvalent antisera. These antibodies now provide standardized reagents for the rapid and precise serological characterization of many strains of N. gonorrhoeae. Images FIG. 1 FIG. 2 FIG. 3 FIG. 4 FIG. 5 PMID:6807844

  12. Isolated pharyngeal Neisseria gonorrhoeae in heterosexual male contacts.

    PubMed

    Thomson-Glover, Rebecca; Brown, Ruth; Edirisinghe, Damitha N

    2013-12-01

    Pharyngeal infection with Neisseria gonorrhoeae (NG) in heterosexual men is thought to be of low prevalence and the value of routinely testing this group of patients is uncertain. We present two cases of NG, isolated only in the pharynx, in asymptomatic heterosexual male contacts. The presence of pharyngeal NG was found irrespective of direct oropharyngeal sexual exposure.

  13. DNA stability of Chlamydia trachomatis and Neisseria gonorrhoeae in urine.

    PubMed

    Le Guern, Rémi; Miaux, Brigitte; Pischedda, Patricia; Herwegh, Stéphanie; Courcol, René

    2016-07-01

    We evaluated the DNA stability of Chlamydia trachomatis and Neisseria gonorrhoeae in 55 urine samples. Crossing threshold (Ct) values were highly similar after 3 to 14 days at room temperature (+0.002, P = 0.99). Consequently, it does not seem necessary to transfer urine specimens into a transport medium in less than 24 hours as recommended by manufacturers. PMID:27130478

  14. Pharyngeal Gonorrhoea in Women: An Important Reservoir for Increasing Neisseria gonorrhoea Prevalence in Urban Australian Heterosexuals?

    PubMed Central

    Lusk, M. Josephine; Uddin, Ruby N. N.; Lahra, Monica M.; Garden, Frances L.; Kundu, Ratan L.; Konecny, Pam

    2013-01-01

    We aim to characterize sexual behavioral aspects of heterosexual Neisseria gonorrhoea (NG) acquisition in two Sexually Transmitted Diseases clinics in Sydney, Australia, in 2008–2012. Of 167 NG cases, 102 were heterosexually acquired with a trend of increasing NG prevalence in heterosexuals from 1.1% (95% CI 0.6–2.1) in 2008 to 3.0% (95% CI 2.0–4.0) in 2012 (P = 0.027). Of heterosexual male cases, unprotected fellatio was the likely sexual activity for NG acquisition in 21/69 (30.4%) and commercial sex work (CSW) contact the likely source in 28/69 (40.6%). NG prevalence overall in CSW (2.2%) was not significantly higher than in non-CSW (1.2%) (P = 0.15), but in 2012 there was a significant increase in NG prevalence in CSW (8.6%) compared to non-CSW (1.6%) (P < 0.001). Pharyngeal NG was found in 9/33 (27.3%) female cases. Decreased susceptibility to ceftriaxone (MIC ≥ 0.03 mg/L) occurred in 2.5% NG isolates, none heterosexually acquired. All were azithromycin susceptible. A significant trend of increasing prevalence of heterosexual gonorrhoea in an urban Australian STD clinic setting is reported. We advocate maintenance of NG screening in women, including pharyngeal screening in all women with partner change who report fellatio, as pharyngeal NG may be an important reservoir for heterosexual transmission. Outreach to CSW should be enhanced. PMID:26316970

  15. Evolutionary analysis of rubella viruses in mainland China during 2010-2012: endemic circulation of genotype 1E and introductions of genotype 2B.

    PubMed

    Zhu, Zhen; Rivailler, Pierre; Abernathy, Emily; Cui, Aili; Zhang, Yan; Mao, Naiyin; Xu, Songtao; Zhou, Shujie; Lei, Yue; Wang, Yan; Zheng, Huanying; He, Jilan; Chen, Ying; Li, Chongshan; Bo, Fang; Zhao, Chunfang; Chen, Meng; Lu, Peishan; Li, Fangcai; Gu, Suyi; Gao, Hui; Guo, Yu; Chen, Hui; Feng, Daxing; Wang, Shuang; Tang, Xiaomin; Lei, Yake; Feng, Yan; Deng, Lili; Gong, Tian; Fan, Lixia; Xu, Wenbo; Icenogle, Joseph

    2015-01-23

    Rubella remains a significant burden in mainland China. In this report, 667 viruses collected in 24 of 31 provinces of mainland China during 2010-2012 were sequenced and analyzed, significantly extending previous reports on limited numbers of viruses collected before 2010. Only viruses of genotypes 1E and 2B were found. Genotype 1E viruses were found in all 24 provinces. Genotype 1E viruses were likely introduced into mainland China around 1997 and endemic transmission of primarily one lineage became established. Viruses reported here from 2010-2012 are largely in a single cluster within this lineage. Genotype 2B viruses were rarely detected in China prior to 2010. This report documents a previously undetected 2B lineage, which likely became endemic in eastern provinces of China between 2010 and 2012. Bayesian analyses were performed to estimate the evolutionary rates and dates of appearance of the genotype 1E and 2B viral linages in China. A skyline plot of viral population diversity did not provide evidence of reduction of diversity as a result of vaccination, but should be useful as a baseline for such reductions as vaccination programs for rubella become widespread in mainland China.

  16. Evolutionary analysis of rubella viruses in mainland China during 2010–2012: endemic circulation of genotype 1E and introductions of genotype 2B

    PubMed Central

    Zhu, Zhen; Rivailler, Pierre; Abernathy, Emily; Cui, Aili; Zhang, Yan; Mao, Naiyin; Xu, Songtao; Zhou, Shujie; Lei, Yue; Wang, Yan; Zheng, Huanying; He, Jilan; Chen, Ying; Li, Chongshan; Bo, Fang; Zhao, Chunfang; Chen, Meng; Lu, Peishan; Li, Fangcai; Gu, Suyi; Gao, Hui; Guo, Yu; Chen, Hui; Feng, Daxing; Wang, Shuang; Tang, Xiaomin; Lei, Yake; Feng, Yan; Deng, Lili; Gong, Tian; Fan, Lixia; Xu, Wenbo; Icenogle, Joseph; Chen, Xia; Tian, Hong; Ma, Yan; Liu, Leng; Liu, Li; Liu, Jianfeng; Fu, Hong; Yang, Yuying; Ma, Yujie; Zhao, Hua; Huang, Fang; Hu, Ying; Zhang, Hong; Tian, Xiaoling; Du, Hui; Ma, Xuemin; Zhang, Zhenying; Xu, Jin; Zhou, Jianhui; Ye, Xufang; Li, Jing; Lu, Yiyu; Liu, Wei; Zhang, Yanni; Zhao, Shengcang; Ba, Zhuoma

    2015-01-01

    Rubella remains a significant burden in mainland China. In this report, 667 viruses collected in 24 of 31 provinces of mainland China during 2010–2012 were sequenced and analyzed, significantly extending previous reports on limited numbers of viruses collected before 2010. Only viruses of genotypes 1E and 2B were found. Genotype 1E viruses were found in all 24 provinces. Genotype 1E viruses were likely introduced into mainland China around 1997 and endemic transmission of primarily one lineage became established. Viruses reported here from 2010–2012 are largely in a single cluster within this lineage. Genotype 2B viruses were rarely detected in China prior to 2010. This report documents a previously undetected 2B lineage, which likely became endemic in eastern provinces of China between 2010 and 2012. Bayesian analyses were performed to estimate the evolutionary rates and dates of appearance of the genotype 1E and 2B viral linages in China. A skyline plot of viral population diversity did not provide evidence of reduction of diversity as a result of vaccination, but should be useful as a baseline for such reductions as vaccination programs for rubella become widespread in mainland China. PMID:25613734

  17. Identification and complete genome sequence analysis of a genotype XIV Newcastle disease virus from Nigeria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The first complete genome sequence of a strain of Newcastle disease virus from genotype XIV is reported here. Strain duck/Nigeria/NG-695/KG.LOM.11-16/2009 was isolated from an apparently healthy domestic duck from a live bird market in Kogi State, Nigeria, in 2009. This strain is classified as a m...

  18. Combining quantitative trait loci analysis with physiological models to predict genotype-specific transpiration rates.

    PubMed

    Reuning, Gretchen A; Bauerle, William L; Mullen, Jack L; McKay, John K

    2015-04-01

    Transpiration is controlled by evaporative demand and stomatal conductance (gs ), and there can be substantial genetic variation in gs . A key parameter in empirical models of transpiration is minimum stomatal conductance (g0 ), a trait that can be measured and has a large effect on gs and transpiration. In Arabidopsis thaliana, g0 exhibits both environmental and genetic variation, and quantitative trait loci (QTL) have been mapped. We used this information to create a genetically parameterized empirical model to predict transpiration of genotypes. For the parental lines, this worked well. However, in a recombinant inbred population, the predictions proved less accurate. When based only upon their genotype at a single g0 QTL, genotypes were less distinct than our model predicted. Follow-up experiments indicated that both genotype by environment interaction and a polygenic inheritance complicate the application of genetic effects into physiological models. The use of ecophysiological or 'crop' models for predicting transpiration of novel genetic lines will benefit from incorporating further knowledge of the genetic control and degree of independence of core traits/parameters underlying gs variation.

  19. Affordable Hands-On DNA Sequencing and Genotyping: An Exercise for Teaching DNA Analysis to Undergraduates

    ERIC Educational Resources Information Center

    Shah, Kushani; Thomas, Shelby; Stein, Arnold

    2013-01-01

    In this report, we describe a 5-week laboratory exercise for undergraduate biology and biochemistry students in which students learn to sequence DNA and to genotype their DNA for selected single nucleotide polymorphisms (SNPs). Students use miniaturized DNA sequencing gels that require approximately 8 min to run. The students perform G, A, T, C…

  20. Multilocus variable-number tandem-repeat analysis for genotyping of Shigella sonnei strains isolated from pediatric patients

    PubMed Central

    Ranjbar, Reza; Memariani, Mojtaba

    2015-01-01

    Aim: The aims of this study were to characterize Iranian Shigella sonnei strains isolated from pediatric cases and evaluate the utility of multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) for genotyping of local S. sonnei strains. Background: S.  sonnei has become the dominant species in certain parts of Iran. Although PFGE is still a gold standard for genotyping and source tracking of food-borne pathogens, it is laborious, expensive, time-consuming, and often difficult to interpret. However, MLVA is a PCR-based method, which is rapid, relatively inexpensive and easy to perform. Patients and methods: A total of 47 S. sonnei isolates were obtained from sporadic cases of pediatric shigellosis in Tehran, Iran, during the years 2002-2003 (n=10) and 2008-2010 (n=37). The patients suffered from acute diarrhea and had evidence of more than three episodes of watery, loose, or bloody stools per day. A MLVA scheme based on 7 VNTR loci was established to assess the diversity of 47 S. sonnei isolates. Results: Based on the results, it was clear that the S. sonnei isolates were heterogeneous. Overall, 47 S. sonnei isolates were discriminated into 21 different genotypes. Analysis of the MLVA profiles using a minimum spanning tree (MST) algorithm showed the usefulness of the MLVA assay in discriminating S. sonnei isolates collected over different time periods. However, no correlation was found between the MLVA genotypes and age, gender or clinical symptoms of the patients. Conclusion: It is assumed that our S. sonnei isolates are derived from a limited number of clones that undergo minor genetic changes in the course of time. The present study has provided some valuable insights into the genetic relatedness of S. sonnei in Tehran, Iran. PMID:26328045

  1. Prevalence and Genotype Distribution of HPV Infection in China: Analysis of 51,345 HPV Genotyping Results from China's Largest CAP Certified Laboratory

    PubMed Central

    Zeng, Zhengyu; Yang, Huaitao; Li, Zaibo; He, Xuekui; Griffith, Christopher C.; Chen, Xiamen; Guo, Xiaolei; Zheng, Baowen; Wu, Shangwei; Zhao, Chengquan

    2016-01-01

    Introduction: The prevalence of cervical Human Papillomavirus (HPV) infection varies greatly worldwide and data regarding HPV prevalence and genotypes in China are limited. Methods: HPV testing results were retrospectively examined at KingMed Diagnostics, the largest independent pathology laboratory in China, from January 2011 to June 2014. All testing was performed using the 26 HPV Genotyping Panel of TellgenplexTM xMAP™ HPV DNA Test assay (TELLGEN, Shanghai, China). Overall prevalence, age-specific prevalence and genotype distributions were analyzed. Results: A total of 51,345 samples were tested and the overall HPV prevalence was 26%, with 21.12% positive for high risk (HR) HPV and 8.37% positive for low risk HPV. 80% of HPV positive cases were positive for a single HPV type. The three most common HR HPV types detected were HPV-52, -16, and -58, in descending order. HPV-18 was only the 6th most common type. When women were divided into three age groups: <30, 30-49, ≥50 years, HR HPV had the highest prevalence rate in women <30 years, and the lowest rate in women 30-49 years of age. The distribution of HR HPV genotypes also varied among these three age groups. Conclusions: To the best of our knowledge, this is largest routine clinical practice report of HPV prevalence and genotypes in a population of women having limited cervical cancer screening. HPV-52 was the most prevalent HR HPV type in this population of women followed by HPV-16 and HPV-58. The overall and age-specific prevalence and genotype distribution of HR HPV are different in this Chinese population compared to that reported from Western countries. PMID:27326245

  2. Surveillance of antibiotic resistance in Neisseria gonorrhoeae in the WHO Western Pacific and South East Asian Regions, 2010.

    PubMed

    Lahra, Monica M

    2012-03-01

    The World Health Organization (WHO) Gonococcal Antimicrobial Surveillance Programme (GASP) has conducted continuous surveillance of antimicrobial resistance in Neisseria gonorrhoeae in the WHO Western Pacific Region (WPR) to optimise antibiotic treatment and control of gonococcal disease since 1992. From 2007, this has been enhanced by the inclusion of data from the WHO South East Asian Region (SEAR). Over time, there has been recruitment of additional centres in both regions. This report provides an analysis of antimicrobial resistance in N. gonorrhoeae in the WHO WPR and SEAR derived from results of the 2010 GASP surveillance. In 2010 there were 9,744 N. gonorrhoeae isolates examined for their susceptibility to one or more of the antibiotics used for the treatment of gonorrhoea, incorporating External Quality Assurance controlled methods, from reporting centres in 19 countries and/or jurisdictions. A high proportion of penicillin and quinolone resistance was again detected amongst isolates tested in the 'Asian' countries of WHO WPR and SEAR. In contrast, lower levels of penicillin and quinolone resistance were reported from the Pacific Islands of Fiji and New Caledonia. The proportion of gonococci reported as having 'decreased susceptibility' to the third-generation cephalosporin antibiotic ceftriaxone varied widely, ranging from 1.3% to 55.8%. There is a continued need for revision and clarification of some of the in vitro criteria that are currently used to categorise the clinical importance of gonococci with different ceftriaxone and oral cephalosporin MIC levels, and to relate these to treatment outcome. Azithromycin resistance was very low in most countries reporting, except in Mongolia where it was 34%. The number of instances of spectinomycin resistance remained low. A high proportion of strains tested continued to exhibit high-level plasmid mediated resistance to tetracyclines. The continuing emergence and spread of antibiotic resistant gonococci in and

  3. Identification of self-incompatibility genotypes of apricot (Prunus armeniaca L.) by S-allele-specific PCR analysis.

    PubMed

    Jie, Qi; Shupeng, Gai; Jixiang, Zhang; Manru, Gu; Huairui, Shu

    2005-08-01

    A cDNA of 417 bp encoding an S-RNase gene, named PA S3, was isolated from apricot, Prunus aremeniaca. Nine S-alleles, S1-S9, were recognized by S-allele-specific PCR and confirmed by Southern blot analysis using PA S3 as probe. The S-genotypes of the six cultivars were determined and the results of self- and cross-pollination tests among the six cultivars were consistent with the predicted S-haplotypes by PCR analysis.

  4. Novel Genes Related to Ceftriaxone Resistance Found among Ceftriaxone-Resistant Neisseria gonorrhoeae Strains Selected In Vitro

    PubMed Central

    Gong, Zijian; Liu, Min; Hua, Zhengshuang; Sun, Yayin; Xu, Qingfang; Xia, Yue; Zhao, Yue; Xie, Xiaoyuan

    2016-01-01

    The emergence of ceftriaxone-resistant Neisseria gonorrhoeae is currently a global public health concern. However, the mechanism of ceftriaxone resistance is not yet fully understood. To investigate the potential genes related to ceftriaxone resistance in Neisseria gonorrhoeae, we subcultured six gonococcal strains with increasing concentrations of ceftriaxone and isolated the strains that became resistant. After analyzing several frequently reported genes involved in ceftriaxone resistance, we found only a single mutation in penA (A501V). However, differential analysis of the genomes and transcriptomes between pre- and postselection strains revealed many other mutated genes as well as up- and downregulated genes. Transformation of the mutated penA gene into nonresistant strains increased the MIC between 2.0- and 5.3-fold, and transformation of mutated ftsX increased the MIC between 3.3- and 13.3-fold. Genes encoding the ABC transporters FarB, Tfq, Hfq, and ExbB were overexpressed, while pilM, pilN, and pilQ were downregulated. Furthermore, the resistant strain developed cross-resistance to penicillin and cefuroxime, had an increased biochemical metabolic rate, and presented fitness defects such as prolonged growth time and downregulated PilMNQ. In conclusion, antimicrobial pressure could result in the emergence of ceftriaxone resistance, and the evolution of resistance of Neisseria gonorrhoeae to ceftriaxone is a complicated process at both the pretranscriptional and posttranscriptional levels, involving several resistance mechanisms of increased efflux and decreased entry. PMID:26787702

  5. Genotypes of Canine Distemper Virus Determined by Analysis of the Hemagglutinin Genes of Recent Isolates from Dogs in Japan

    PubMed Central

    Mochizuki, Masami; Hashimoto, Michiru; Hagiwara, Shigeyuki; Yoshida, Yoshio; Ishiguro, Shinryo

    1999-01-01

    Canine distemper of domestic dogs is caused by canine distemper virus (CDV), a member of the morbilliviruses. It has been a highly contagious disease of great veterinary importance for centuries, but for the last several decades it has been controlled satisfactorily by modified live vaccines. In the 1990s, however, it was described that CDV strains genetically different from vaccine strains may have caused the disease in vaccinated dogs. The highest antigenic variation is found in the H protein. Therefore, in the present study, hemagglutinin (H) genes obtained from current vaccines and field isolates and amplified directly from clinical specimens were genetically analyzed by restriction fragment length polymorphism assay and sequencing. Phylogenetic analysis of the H-gene amino acid sequences revealed that at least two CDV genotypes are circulating among dogs in Japan; one is a genotype to which almost all Japanese CDV isolates belong and the other has not been previously described. Both are separate and independent from the other lineages or genotypes of vaccine strains, as well as European and U.S. CDV isolates. The results suggest that CDV has also evolved in Japan, and further studies will be needed for an evaluation and possible improvement of the efficacies of current CDV vaccines. PMID:10449479

  6. Insights into nitrogen allocation and recycling from nitrogen elemental analysis and 15N isotope labelling in 14 genotypes of willow.

    PubMed

    Brereton, Nicholas J B; Pitre, Frederic E; Shield, Ian; Hanley, Steven J; Ray, Michael J; Murphy, Richard J; Karp, Angela

    2014-11-01

    Minimizing nitrogen (N) fertilization inputs during cultivation is essential for sustainable production of bioenergy and biofuels. The biomass crop willow (Salix spp.) is considered to have low N fertilizer requirements due to efficient recycling of nutrients during the perennial cycle. To investigate how successfully different willow genotypes assimilate and allocate N during growth, and remobilize and consequently recycle N before the onset of winter dormancy, N allocation and N remobilization (to and between different organs) were examined in 14 genotypes of a genetic family using elemental analysis and (15)N as a label. Cuttings were established in pots in April and sampled in June, August and at onset of senescence in October. Biomass yield of the trees correlated well with yields recorded in the field. Genotype-specific variation was observed for all traits measured and general trends spanning these sampling points were identified when trees were grouped by biomass yield. Nitrogen reserves in the cutting fuelled the entirety of the canopy establishment, yet earlier cessation of this dependency was linked to higher biomass yields. The stem was found to be the major N reserve by autumn, which constitutes a major source of N loss at harvest, typically every 2-3 years. These data contribute to understanding N remobilization in short rotation coppice willow and to the identification of traits that could potentially be selected for in breeding programmes to further improve the sustainability of biomass production.

  7. A genome-wide analysis of multidrug-resistant and extensively drug-resistant strains of Mycobacterium tuberculosis Beijing genotype.

    PubMed

    Wu, Wei; Zheng, Huajun; Zhang, Lu; Wen, Zilu; Zhang, Shulin; Pei, Hao; Yu, Guohua; Zhu, Yongqiang; Cui, Zhenling; Hu, Zhongyi; Wang, Honghai; Li, Yao

    2013-09-01

    The Beijing genotype of Mycobacterium tuberculosis (MTB) is one of the most successful MTB lineages that has disseminated in the world. In China, the rate of multidrug-resistant (MDR) tuberculosis is significantly higher than the global average rate, and the Beijing genotype strains take the largest share of MDR strains. To study the genetic basis of the epidemiological findings that Beijing genotype has often been associated with tuberculosis outbreaks and drug resistance, we determined the genome sequences of four clinical isolates: two extensively drug resistant (XDR1219, XDR1221) and two multidrug resistant (WX1, WX3), using whole-genome sequencing. A large number of individual and shared SNPs of the four Beijing strains were identified. Our isolates harbored almost all classic drug resistance-associated mutations. The mutations responsible for drug resistance in the two XDR strains were consistent with the clinical quantitative drug resistance levels. COG analysis revealed that Beijing strains have significantly higher abundances of the mutations responsible for cell wall/membrane/envelope biogenesis (COG M), secondary metabolites biosynthesis, transport and catabolism (COG Q), lipid transport and metabolism (COG I) and defense mechanisms (COG V). The shared mutated genes of the four studied Beijing strains were significantly overrepresented in three DNA repair pathways. Our analyses promote the understanding of the genome polymorphism of the Beijing family strains and provide the molecular genetic basis for their wide dissemination capacity and drug resistance.

  8. Hepatitis C virus genotype 3: Meta-analysis on sustained virologic response rates with currently available treatment options

    PubMed Central

    Ampuero, Javier; Reddy, K Rajender; Romero-Gomez, Manuel

    2016-01-01

    AIM: To address the therapeutic efficacy of various treatment regimens in genotype 3 selecting randomized clinical trials and prospective National Cohort Studies. METHODS: (1) PEG-INF-based therapy including sofosbuvir (SOF) + RBV for 12 wk vs SOF + RBV 24 wk; (2) SOF + RBV therapy 12 wk/16 wk vs 24 wk; and (3) the role of RBV in SOF + daclatasvir (DCV) and SOF + ledipasvir (LDV) combinations. This meta-analysis provides robust information with the intention of addressing treatment strategy for hepatitis C virus genotype 3. RESULTS: A combination treatment including SOF + RBV + PEG-IFN for 12 wk notes better SVR than with only SOF + RBV for 12 wk, although its association with more frequent adverse effects may be a limiting factor. Longer duration therapy with SOF + RBV (24 wk) has achieved higher SVR rates than shorter durations (12 or 16 wk). SOF + LDV are not an ideal treatment for genotype 3. CONCLUSION: Lastly, SOF + DCV combination is probably the best oral therapy option and the addition of RBV does not appear to be needed to increase SVR rates substantially. PMID:27298572

  9. Cysteine proteases of Trypanosoma (Megatrypanum) theileri: cathepsin L-like gene sequences as targets for phylogenetic analysis, genotyping diagnosis.

    PubMed

    Rodrigues, Adriana C; Garcia, Herakles A; Ortiz, Paola A; Cortez, Alane P; Martinkovic, Franjo; Paiva, Fernando; Batista, Jael S; Minervino, Antonio H; Campaner, Marta; Pral, Elizabeth M; Alfieri, Silvia C; Teixeira, Marta M G

    2010-09-01

    Although Trypanosomatheileri and allied trypanosomes are the most widespread trypanosomes in bovids little is known about proteolytic enzymes in these species. We have characterized genes encoding for cathepsin L-like (CATL) cysteine proteases from isolates of cattle, water buffalo and deer that largely diverged from homologues of other trypanosome species. Analysis of 78 CATL catalytic domain sequences from 22 T. theileri trypanosomes disclosed 6 genotypes tightly clustered together into the T. theileri clade. The CATL genes in these trypanosomes are organized in tandem arrays of approximately 1.7kb located in 2 chromosomal bands of 600-720kb. A diagnostic PCR assay targeting CATL sequences detected T. theileri of all genotypes from cattle, buffaloes and cervids and also from tabanid vectors. Expression of T. theileri cysteine proteases was demonstrated by proteolytic activity in gelatin gels and hydrolysis of Z-Phe-Arg-AMC substrate. Results from this work agree with previous data using ribosomal and spliced leader genes demonstrating that CATL gene sequences are useful for diagnosis, population genotyping and evolutionary studies of T. theileri trypanosomes.

  10. Genotypic analysis of β-tubulin in Onchocerca volvulus from communities and individuals showing poor parasitological response to ivermectin treatment.

    PubMed

    Osei-Atweneboana, Mike Y; Boakye, Daniel A; Awadzi, Kwablah; Gyapong, John O; Prichard, Roger K

    2012-12-01

    Ivermectin (IVM) has been in operational use for the control of onchocerciasis for two decades and remains the only drug of choice. To investigate the parasitological responses and genetic profile of Onchocerca volvulus, we carried out a 21 month epidemiological study to determine the response of the parasite to IVM in 10 Ghanaian endemic communities. Onchocerca nodules were surgically removed from patients in three IVM response categories (good, intermediate and poor) and one IVM naïve community. DNA from adult worms was analyzed to determine any association between genotype and IVM response phenotypic. Embryogramme analysis showed significantly higher reproductive activity in worms from poor response communities, which had up to 41% of females with live stretched microfilaria (mf) in utero, despite IVM treatment, compared with good response communities, which had no intra-uterine stretched mf. β-tubulin isotype 1 gene has been shown to be linked to IVM selection in O. volvulus and also known to be associated with IVM resistance in veterinary nematodes. We have genotyped the full length genomic DNA sequence of the β-tubulin gene from 127 adult worms obtained from the four community categories. We found SNPs at 24 sites over the entire 3696 bp. Eight of the SNPs occurred at significantly higher (p < 0.05) frequencies in the poor response communities compared with the good response communities and the IVM naïve community. Phenotypic and genotypic analyses show that IVM resistance has been selected and the genotype (1183GG/1188CC/1308TT/1545GG) was strongly associated with the resistance phenotype. Since the region in the β-tubulin gene where these four SNPs occur is within 362 bp, it is feasible to develop a genetic marker for the early detection of IVM resistance. PMID:24533268

  11. Kinetics of viral loads and genotypic analysis of elephant endotheliotropic herpesvirus-1 infection in captive Asian elephants (Elephas maximus).

    PubMed

    Stanton, Jeffrey J; Zong, Jian-Chao; Eng, Crystal; Howard, Lauren; Flanagan, Joe; Stevens, Martina; Schmitt, Dennis; Wiedner, Ellen; Graham, Danielle; Junge, Randall E; Weber, Martha A; Fischer, Martha; Mejia, Alicia; Tan, Jie; Latimer, Erin; Herron, Alan; Hayward, Gary S; Ling, Paul D

    2013-03-01

    Elephant endotheliotropic herpesviruses (EEHVs) can cause fatal hemorrhagic disease in juvenile Asian elephants (Elphas maximus); however, sporadic shedding of virus in trunk washes collected from healthy elephants also has been detected. Data regarding the relationship of viral loads in blood compared with trunk washes are lacking, and questions about whether elephants can undergo multiple infections with EEHVs have not been addressed previously. Real-time quantitative polymerase chain reaction was used to determine the kinetics of EEHV1 loads, and genotypic analysis was performed on EEHV1 DNA detected in various fluid samples obtained from five Asian elephants that survived detectable EEHV1 DNAemia on at least two separate occasions. In three elephants displaying clinical signs of illness, preclinical EEHV1 DNAemia was detectable, and peak whole-blood viral loads occurred 3-8 days after the onset of clinical signs. In two elephants with EEHV1 DNAemia that persisted for 7-21 days, no clinical signs of illness were observed. Detection of EEHV1 DNA in trunk washes peaked approximately 21 days after DNAemia, and viral genotypes detected during DNAemia matched those detected in subsequent trunk washes from the same elephant. In each of the five elephants, two distinct EEHV1 genotypes were identified in whole blood and trunk washes at different time points. In each case, these genotypes represented both an EEHV1A and an EEHV1B subtype. These data suggest that knowledge of viral loads could be useful for the management of elephants before or during clinical illness. Furthermore, sequential infection with both EEHV1 subtypes occurs in Asian elephants, suggesting that they do not elicit cross-protective sterilizing immunity. These data will be useful to individuals involved in the husbandry and clinical care of Asian elephants.

  12. Glutathione S-transferase M1 (GSTM1) null genotype and coronary artery disease risk: a meta-analysis

    PubMed Central

    Zhang, Zhen-Xian; Zhang, Ye

    2014-01-01

    Background: The Glutathione S-Transferase M1 (GSTM1) null genotype has been indicated to be correlated with coronary artery disease (CAD) susceptibility, but study results are still debatable. Thus, a meta-analysis was conducted. Materials and methods: Databases including PubMed, Embase, Web of Science, and Chinese National Knowledge Infrastructure (CNKI) were searched. Data were extracted and pooled odds ratios (OR) with 95% confidence intervals (CI) were calculated. Results: Twenty-six studies with 10595 cases and 13782 controls were included in this meta-analysis. The association between GSTM1 null genotype and CAD risk was significant (OR = 1.35; 95% CI, 1.09 - 1.67; P < 0.01). When stratified by ethnicity, the significantly elevated risk were observed in Caucasians (OR = 1.39; 95% CI, 1.07 - 1.81; P = 0.01) but not in Asians (OR = 1.27; 95% CI, 0.87 - 1.86; P = 0.22). No significantly increased myocardial infarction risk was observed (OR = 0.96; 95% CI, 0.78 - 1.18; P = 0.68). Subgroup analysis on the smoking status showed that the increased risk was found in smokers (OR = 1.66; 95% CI, 1.14 - 2.42; P < 0.01) but not in non-smokers (OR = 1.30; 95% CI, 1.74 - 2.28; P = 0.37). Conclusion: In conclusion, this meta-analysis suggested that GSTM1 null genotype was a risk factor for CAD, especially in Caucasians and smokers. PMID:25419371

  13. Identification and Complete Genome Sequence Analysis of a Genotype XIV Newcastle Disease Virus from Nigeria

    PubMed Central

    Shittu, Ismaila; Sharma, Poonam; Volkening, Jeremy D.; Solomon, Ponman; Sulaiman, Lanre K.; Joannis, Tony M.; Williams-Coplin, Dawn; Miller, Patti J.; Dimitrov, Kiril M.

    2016-01-01

    The first complete genome sequence of a strain of Newcastle disease virus (NDV) from genotype XIV is reported here. Strain duck/Nigeria/NG-695/KG.LOM.11-16/2009 was isolated from an apparently healthy domestic duck from a live bird market in Kogi State, Nigeria, in 2009. This strain is classified as a member of subgenotype XIVb of class II. PMID:26823576

  14. Comparative proteome analysis of metabolic changes by low phosphorus stress in two Brassica napus genotypes.

    PubMed

    Yao, Yinan; Sun, Haiyan; Xu, Fangsen; Zhang, Xuejiang; Liu, Shengyi

    2011-03-01

    In an attempt to determine the adaptation strategy to phosphorous (Pi) deficiency in oilseed rape, comparative proteome analyses were conducted to investigate the differences of metabolic changes in two oilseed rape genotypes with different tolerance to low phosphorus (LP). Generally in either roots or leaves, there existed few low phosphorus (LP)-induced proteins shared in the two lines. The LP-tolerant genotype 102 maintained higher Pi concentrations than LP-sensitive genotype 105 when growing hydroponically under the 5-μM phosphorus condition. In 102 we observed the downregulation of the proteins related to gene transcription, protein translation, carbon metabolism, and energy transfer in leaves and roots, and the downregulation of proteins related to leaf growth and root cellular organization. But the proteins related to the formation of lateral root were upregulated, such as the auxin-responsive family proteins in roots and the sucrose-phosphate synthase-like protein in roots and leaves. On the other hand, the LP-sensitive genotype 105 maintained the low level of Pi concentrations and suffered high oxidative pressure under the LP condition, and stress-shocking proteins were pronouncedly upregulated such as the proteins for signal transduction, gene transcription, secondary metabolism, universal stress family proteins, as well as the proteins involved in lipid oxygenation and the disease resistance in both leaves and roots. Although the leaf proteins for growth in 105 were downregulated, the protein expressions in roots related to glycolysis and tricarboxylic acid (TCA) cycle were enhanced to satisfy the requirement of organic acid secretion. PMID:21110039

  15. Phenotype-genotype analysis of CYP2C19 in Colombian mestizo individuals

    PubMed Central

    Isaza, Carlos; Henao, Julieta; Martínez, José H Isaza; Arias, Juan C Sepúlveda; Beltrán, Leonardo

    2007-01-01

    Background Omeprazole is metabolized by the hepatic cytochrome P450 (CYP) 2C19 enzyme to 5-hydroxyomeprazole. CYP2C19 exhibits genetic polymorphisms responsible for the presence of poor metabolizers (PMs), intermediate metabolizers (IMs) and extensive metabolizers (EMs). The defective mutations of the enzyme and their frequencies change between different ethnic groups; however, the polymorphism of the CYP2C19 gene has not been studied in Colombian mestizos. The aim of this study was to evaluate the genotype and phenotype status of CYP2C19 in Colombian mestizos, in order to contribute to the use of appropriate strategies of drug therapy for this population. Methods 189 subjects were genotyped using the multiplex SNaPshot technique and a subgroup of 44 individuals received 20 mg of omeprazole followed by blood collection at 3 hours to determine the omeprazole hydroxylation index by HPLC. Results 83.6%, 15.3% and 1.1% of the subjects were genotyped as EMs, IMs and PMs, respectively. The frequencies of the CYP2C29*1 and CYP2C19*2 alleles were 91.3% and 8.7% respectively whereas the *3, *4, *5, *6 and *8 alleles were not found. No discrepancies were found between the genotype and phenotype of CYP2C19. Conclusion The frequency of poor metabolizers (1.1%) in the Colombian mestizos included in this study is similar to that in Bolivian mestizos (1%) but lower than in Mexican-Americans (3.2%), West Mexicans (6%), Caucasians (5%) and African Americans (5.4%). The results of this study will be useful for drug dosage recommendations in Colombian mestizos. PMID:17623107

  16. An association analysis between OXT genotype and milk yield and flow in Italian Mediterranean river buffalo.

    PubMed

    Pauciullo, Alfredo; Cosenza, Gianfranco; Steri, Roberto; Coletta, Angelo; Jemma, Lazzaro; Feligini, Maria; Di Berardino, Dino; Macciotta, Nicolò P P; Ramunno, Luigi

    2012-05-01

    The aim of this study was to evaluate possible associations between three SNPs at the oxytocin locus (AM234538: g.28C>T; g.204A>G and g.1627G>T) and two productive traits, milk yield and milkability, in Italian Mediterranean river buffaloes. Effects of parity, calving season and month of production were also evaluated. A total of 41 980 test-day records belonging to 219 lactations of 163 buffalo cows were investigated. The allele call rate was 98·8% and the major allele frequency for all the investigated loci was 0·76. The OXT genotype was significantly associated with milk yield (P=0·029). The TT genotype showed an average daily milk yield approximately 1·7 kg higher than GT buffaloes. Such a difference represents about 23% more milk/d. A large dominance effect (-1·17±0·43 kg) was estimated, whereas the contribution of OXT genotype (r(2)(OXT)) to the total phenotypic variance in milk yield was equal to 0·06. The TT genotype showed higher values also for the milk flow, even though the estimated difference did not reach a level of statistical significance (P=0·07). Such an association, among the first reported for the oxytocin locus in ruminants, should be tested on a population scale and possible effects on milk composition traits should be evaluated in order to supply useful indications for the application of marker-assisted selection programmes in river buffaloes. PMID:22280971

  17. Synthesis of a select group of proteins by Neisseria gonorrhoeae in response to thermal stress.

    PubMed Central

    Woods, M L; Bonfiglioli, R; McGee, Z A; Georgopoulos, C

    1990-01-01

    We report the thermal conditions that induce the heat shock response in Neisseria gonorrhoeae. Under conditions of thermal stress, Neisseria gonorrhoeae synthesizes heat shock proteins (hsps), which differ quantitatively from conventionally studied gonococcal proteins. Gonococci accelerate the rate of synthesis of the hsps as early as 5 min after the appropriate stimulus is applied, with synthesis continuing for 30 min, as demonstrated by in vivo labeling experiments with L-[35S]methionine. Two of the gonococcal hsps are immunologically cross-reactive with the hsps of Escherichia coli, DnaK and GroEL, as demonstrated by Western blot (immunoblot) analysis. Ten hsps can be identified on two-dimensional autoradiograms of whole gonococci (total protein). Four hsps can be identified on two-dimensional autoradiograms of 1% N-lauroylsarcosine (sodium salt) (Sarkosyl)-insoluble membrane fractions. Two of the hsps from the 1% Sarkosyl-insoluble fraction are found exclusively in this fraction, suggesting that they are membrane proteins. The identification of this group of proteins will facilitate further study of the function of these proteins and provide insight into the possible role of hsps in disease pathogenesis. Images PMID:2106493

  18. Synthesis of a select group of proteins by Neisseria gonorrhoeae in response to thermal stress.

    PubMed

    Woods, M L; Bonfiglioli, R; McGee, Z A; Georgopoulos, C

    1990-03-01

    We report the thermal conditions that induce the heat shock response in Neisseria gonorrhoeae. Under conditions of thermal stress, Neisseria gonorrhoeae synthesizes heat shock proteins (hsps), which differ quantitatively from conventionally studied gonococcal proteins. Gonococci accelerate the rate of synthesis of the hsps as early as 5 min after the appropriate stimulus is applied, with synthesis continuing for 30 min, as demonstrated by in vivo labeling experiments with L-[35S]methionine. Two of the gonococcal hsps are immunologically cross-reactive with the hsps of Escherichia coli, DnaK and GroEL, as demonstrated by Western blot (immunoblot) analysis. Ten hsps can be identified on two-dimensional autoradiograms of whole gonococci (total protein). Four hsps can be identified on two-dimensional autoradiograms of 1% N-lauroylsarcosine (sodium salt) (Sarkosyl)-insoluble membrane fractions. Two of the hsps from the 1% Sarkosyl-insoluble fraction are found exclusively in this fraction, suggesting that they are membrane proteins. The identification of this group of proteins will facilitate further study of the function of these proteins and provide insight into the possible role of hsps in disease pathogenesis.

  19. Affordable hands-on DNA sequencing and genotyping: an exercise for teaching DNA analysis to undergraduates.

    PubMed

    Shah, Kushani; Thomas, Shelby; Stein, Arnold

    2013-01-01

    In this report, we describe a 5-week laboratory exercise for undergraduate biology and biochemistry students in which students learn to sequence DNA and to genotype their DNA for selected single nucleotide polymorphisms (SNPs). Students use miniaturized DNA sequencing gels that require approximately 8 min to run. The students perform G, A, T, C Sanger sequencing reactions. They prepare and run the gels, perform Southern blots (which require only 10 min), and detect sequencing ladders using a colorimetric detection system. Students enlarge their sequencing ladders from digital images of their small nylon membranes, and read the sequence manually. They compare their reads with the actual DNA sequence using BLAST2. After mastering the DNA sequencing system, students prepare their own DNA from a cheek swab, polymerase chain reaction-amplify a region of their DNA that encompasses a SNP of interest, and perform sequencing to determine their genotype at the SNP position. A family pedigree can also be constructed. The SNP chosen by the instructor was rs17822931, which is in the ABCC11 gene and is the determinant of human earwax type. Genotypes at the rs178229931 site vary in different ethnic populations.

  20. Genotyping of PCR-based polymorphisms and linkage-disequilibrium analysis at the NF1 locus

    SciTech Connect

    Purandare, S.M.; Viskochil, D.H.; Cawthon, R.

    1996-07-01

    Six polymorphism across the NF1 gene have been adapted for genotyping through application of PCR-based assays. Three exon-based polymorphisms - at positions 702, 2034, and 10647 in the NF1 cDNA - were genotyped by mutagenically separated PCR (MS-PCR). A fourth polymorphism, DV1.9, is an L1 insertion element in intron 30, and the other two polymorphisms, GXAlu and EVI-20, are short tandem repeats in intron 27b. All the polymorphisms were evaluated in a cohort of 110 CEPH individuals who previously had been analyzed by use of eight RFLPs at the NF1 locus. Pairwise linkage-disequilibrium analyses with the six PCR-based polymorphisms and their flanking markers demonstrated disequilibrium between all tested loci. Genotypes of the four diallelic polymorphisms (702, 2034, 10647, and DV1.9) were also evaluated in cohorts from the CEPH, African, and Japanese populations. The CEPH and Japanese cohorts showed similar heterozygosities and linkage-disequilibrium coefficients. The African cohort showed a higher degree of heterozygosity and lower linkage-disequilibrium values, compared with the CEPH and Japanese cohorts. 36 refs., 2 figs., 3 tabs.

  1. Genotype-phenotype analysis of 4q deletion syndrome: proposal of a critical region.

    PubMed

    Strehle, Eugen-Matthias; Yu, Linbo; Rosenfeld, Jill A; Donkervoort, Sandra; Zhou, Yulin; Chen, Tian-Jian; Martinez, Jose E; Fan, Yao-Shan; Barbouth, Deborah; Zhu, Hongbo; Vaglio, Alicia; Smith, Rosemarie; Stevens, Cathy A; Curry, Cynthia J; Ladda, Roger L; Fan, Zheng Jane; Fox, Joyce E; Martin, Judith A; Abdel-Hamid, Hoda Z; McCracken, Elizabeth A; McGillivray, Barbara C; Masser-Frye, Diane; Huang, Taosheng

    2012-09-01

    Chromosome 4q deletion syndrome (4q- syndrome) is a rare condition, with an estimated incidence of 1 in 100,000. Although variable, the clinical spectrum commonly includes craniofacial, developmental, digital, skeletal, and cardiac involvement. Data on the genotype-phenotype correlation within the 4q arm are limited. We present detailed clinical and genetic information by array CGH on 20 patients with 4q deletions. We identified a patient who has a ∼465 kb deletion (186,770,069-187,234,800, hg18 coordinates) in 4q35.1 with all clinical features for 4q deletion syndrome except for developmental delay, suggesting that this is a critical region for this condition and a specific gene responsible for orofacial clefts and congenital heart defects resides in this region. Since the patients with terminal deletions all had cleft palate, our results provide further evidence that a gene associated with clefts is located on the terminal segment of 4q. By comparing and contrasting our patients' genetic information and clinical features, we found significant genotype-phenotype correlations at a single gene level linking specific phenotypes to individual genes. Based on these data, we constructed a hypothetical partial phenotype-genotype map for chromosome 4q which includes BMP3, SEC31A, MAPK10, SPARCL1, DMP1, IBSP, PKD2, GRID2, PITX2, NEUROG2, ANK2, FGF2, HAND2, and DUX4 genes.

  2. Metal Binding Specificity of the MntABC Permease of Neisseria gonorrhoeae and Its Influence on Bacterial Growth and Interaction with Cervical Epithelial Cells▿ †

    PubMed Central

    Lim, Karen H. L.; Jones, Christopher E.; vanden Hoven, Rachel N.; Edwards, Jennifer L.; Falsetta, Megan L.; Apicella, Michael A.; Jennings, Michael P.; McEwan, Alastair G.

    2008-01-01

    mntABC from Neisseria gonorrhoeae encodes an ABC permease which includes a periplasmic divalent cation binding receptor protein of the cluster IX family, encoded by mntC. Analysis of an mntC mutant showed that growth of N. gonorrhoeae could be stimulated by addition of either manganese(II) or zinc(II) ions, suggesting that the MntABC system could transport both ions. In contrast, growth of the mntAB mutant in liquid culture was possible only when the medium was supplemented with an antioxidant such as mannitol, consistent with the view that ion transport via MntABC is essential for protection of N. gonorrhoeae against oxidative stress. Using recombinant MntC, we determined that MntC binds Zn2+ and Mn2+ with almost equal affinity (dissociation constant of ∼0.1 μM). Competition assays with the metallochromic zinc indicator 4-(2-pyridylazo)resorcinol showed that MntC binds Mn2+ and Zn2+ at the same binding site. Analysis of the N. gonorrhoeae genome showed that MntC is the only Mn/Zn metal binding receptor protein cluster IX in this bacterium, in contrast to the situation in many other bacteria which have systems with dedicated Mn and Zn binding proteins as part of distinctive ABC cassette permeases. Both the mntC and mntAB mutants had reduced intracellular survival in a human cervical epithelial cell model and showed reduced ability to form a biofilm. These data suggest that the MntABC transporter is of importance for survival of Neisseria gonorrhoeae in the human host. PMID:18426887

  3. Towards fully automated genotyping: use of an X linked recessive spastic paraplegia family to test alternative analysis methods.

    PubMed

    Kobayashi, H; Matise, T C; Perlin, M W; Marks, H G; Hoffman, E P

    1995-05-01

    Advances in dinucleotide-based genetic maps open possibilities for large scale genotyping at high resolution. The current rate-limiting steps in use of these dense maps is data interpretation (allele definition), data entry, and statistical calculations. We have recently reported automated allele identification methods. Here we show that a 10-cM framework map of the human X chromosome can be analyzed on two lanes of an automated sequencer per individual (10-12 loci per lane). We use this map and analysis strategy to generate allele data for an X-linked recessive spastic paraplegia family with a known PLP mutation. We analyzed 198 genotypes in a single gel and used the data to test three methods of data analysis: manual meiotic breakpoint mapping, automated concordance analysis, and whole chromosome multipoint linkage analysis. All methods pinpointed the correct location of the gene. We propose that multipoint exclusion mapping may permit valid inflation of LOD scores using the equation max LOD-(next best LOD). PMID:7759066

  4. Transcriptome Analysis of Cotton (Gossypium hirsutum L.) Genotypes That Are Susceptible, Resistant, and Hypersensitive to Reniform Nematode (Rotylenchulus reniformis)

    PubMed Central

    Li, Ruijuan; Rashotte, Aaron M.; Singh, Narendra K.; Lawrence, Kathy S.; Weaver, David B.; Locy, Robert D.

    2015-01-01

    Reniform nematode is a semi-endoparasitic nematode species causing significant yield loss in numerous crops, including cotton (Gossypium hirsutum L.). An RNA-sequencing analysis was conducted to measure transcript abundance in reniform nematode susceptible (DP90 & SG747), resistant (BARBREN-713), and hypersensitive (LONREN-1) genotypes of cotton (Gossypium hirsutum L.) with and without reniform nematode infestation. Over 90 million trimmed high quality reads were assembled into 84,711 and 80, 353 transcripts using the G. arboreum and the G. raimondii genomes as references. Many transcripts were significantly differentially expressed between the three different genotypes both prior to and during nematode pathogenesis, including transcripts corresponding to the gene ontology categories of cell wall, hormone metabolism and signaling, redox reactions, secondary metabolism, transcriptional regulation, stress responses, and signaling. Further analysis revealed that a number of these differentially expressed transcripts mapped to the G. raimondii and/or the G. arboreum genomes within 1 megabase of quantitative trait loci that had previously been linked to reniform nematode resistance. Several resistance genes encoding proteins known to be strongly linked to pathogen perception and resistance, including LRR-like and NBS-LRR domain-containing proteins, were among the differentially expressed transcripts mapping near these quantitative trait loci. Further investigation is required to confirm a role for these transcripts in reniform nematode susceptibility, hypersensitivity, and/or resistance. This study presents the first systemic investigation of reniform nematode resistance-associated genes using different genotypes of cotton. The candidate reniform nematode resistance-associated genes identified in this study can serve as the basis for further functional analysis and aid in further development of reniform a nematode resistant cotton germplasm. PMID:26571375

  5. Transcriptome Analysis of Sunflower Genotypes with Contrasting Oxidative Stress Tolerance Reveals Individual- and Combined- Biotic and Abiotic Stress Tolerance Mechanisms.

    PubMed

    Ramu, Vemanna S; Paramanantham, Anjugam; Ramegowda, Venkategowda; Mohan-Raju, Basavaiah; Udayakumar, Makarla; Senthil-Kumar, Muthappa

    2016-01-01

    In nature plants are often simultaneously challenged by different biotic and abiotic stresses. Although the mechanisms underlying plant responses against single stress have been studied considerably, plant tolerance mechanisms under combined stress is not understood. Also, the mechanism used to combat independently and sequentially occurring many number of biotic and abiotic stresses has also not systematically studied. From this context, in this study, we attempted to explore the shared response of sunflower plants to many independent stresses by using meta-analysis of publically available transcriptome data and transcript profiling by quantitative PCR. Further, we have also analyzed the possible role of the genes so identified in contributing to combined stress tolerance. Meta-analysis of transcriptomic data from many abiotic and biotic stresses indicated the common representation of oxidative stress responsive genes. Further, menadione-mediated oxidative stress in sunflower seedlings showed similar pattern of changes in the oxidative stress related genes. Based on this a large scale screening of 55 sunflower genotypes was performed under menadione stress and those contrasting in oxidative stress tolerance were identified. Further to confirm the role of genes identified in individual and combined stress tolerance the contrasting genotypes were individually and simultaneously challenged with few abiotic and biotic stresses. The tolerant hybrid showed reduced levels of stress damage both under combined stress and few independent stresses. Transcript profiling of the genes identified from meta-analysis in the tolerant hybrid also indicated that the selected genes were up-regulated under individual and combined stresses. Our results indicate that menadione-based screening can identify genotypes not only tolerant to multiple number of individual biotic and abiotic stresses, but also the combined stresses.

  6. Transcriptome Analysis of Cotton (Gossypium hirsutum L.) Genotypes That Are Susceptible, Resistant, and Hypersensitive to Reniform Nematode (Rotylenchulus reniformis).

    PubMed

    Li, Ruijuan; Rashotte, Aaron M; Singh, Narendra K; Lawrence, Kathy S; Weaver, David B; Locy, Robert D

    2015-01-01

    Reniform nematode is a semi-endoparasitic nematode species causing significant yield loss in numerous crops, including cotton (Gossypium hirsutum L.). An RNA-sequencing analysis was conducted to measure transcript abundance in reniform nematode susceptible (DP90 & SG747), resistant (BARBREN-713), and hypersensitive (LONREN-1) genotypes of cotton (Gossypium hirsutum L.) with and without reniform nematode infestation. Over 90 million trimmed high quality reads were assembled into 84,711 and 80, 353 transcripts using the G. arboreum and the G. raimondii genomes as references. Many transcripts were significantly differentially expressed between the three different genotypes both prior to and during nematode pathogenesis, including transcripts corresponding to the gene ontology categories of cell wall, hormone metabolism and signaling, redox reactions, secondary metabolism, transcriptional regulation, stress responses, and signaling. Further analysis revealed that a number of these differentially expressed transcripts mapped to the G. raimondii and/or the G. arboreum genomes within 1 megabase of quantitative trait loci that had previously been linked to reniform nematode resistance. Several resistance genes encoding proteins known to be strongly linked to pathogen perception and resistance, including LRR-like and NBS-LRR domain-containing proteins, were among the differentially expressed transcripts mapping near these quantitative trait loci. Further investigation is required to confirm a role for these transcripts in reniform nematode susceptibility, hypersensitivity, and/or resistance. This study presents the first systemic investigation of reniform nematode resistance-associated genes using different genotypes of cotton. The candidate reniform nematode resistance-associated genes identified in this study can serve as the basis for further functional analysis and aid in further development of reniform a nematode resistant cotton germplasm. PMID:26571375

  7. Transcriptome Analysis of Cotton (Gossypium hirsutum L.) Genotypes That Are Susceptible, Resistant, and Hypersensitive to Reniform Nematode (Rotylenchulus reniformis).

    PubMed

    Li, Ruijuan; Rashotte, Aaron M; Singh, Narendra K; Lawrence, Kathy S; Weaver, David B; Locy, Robert D

    2015-01-01

    Reniform nematode is a semi-endoparasitic nematode species causing significant yield loss in numerous crops, including cotton (Gossypium hirsutum L.). An RNA-sequencing analysis was conducted to measure transcript abundance in reniform nematode susceptible (DP90 & SG747), resistant (BARBREN-713), and hypersensitive (LONREN-1) genotypes of cotton (Gossypium hirsutum L.) with and without reniform nematode infestation. Over 90 million trimmed high quality reads were assembled into 84,711 and 80, 353 transcripts using the G. arboreum and the G. raimondii genomes as references. Many transcripts were significantly differentially expressed between the three different genotypes both prior to and during nematode pathogenesis, including transcripts corresponding to the gene ontology categories of cell wall, hormone metabolism and signaling, redox reactions, secondary metabolism, transcriptional regulation, stress responses, and signaling. Further analysis revealed that a number of these differentially expressed transcripts mapped to the G. raimondii and/or the G. arboreum genomes within 1 megabase of quantitative trait loci that had previously been linked to reniform nematode resistance. Several resistance genes encoding proteins known to be strongly linked to pathogen perception and resistance, including LRR-like and NBS-LRR domain-containing proteins, were among the differentially expressed transcripts mapping near these quantitative trait loci. Further investigation is required to confirm a role for these transcripts in reniform nematode susceptibility, hypersensitivity, and/or resistance. This study presents the first systemic investigation of reniform nematode resistance-associated genes using different genotypes of cotton. The candidate reniform nematode resistance-associated genes identified in this study can serve as the basis for further functional analysis and aid in further development of reniform a nematode resistant cotton germplasm.

  8. Transcriptome Analysis of Sunflower Genotypes with Contrasting Oxidative Stress Tolerance Reveals Individual- and Combined- Biotic and Abiotic Stress Tolerance Mechanisms

    PubMed Central

    Ramu, Vemanna S.; Paramanantham, Anjugam; Ramegowda, Venkategowda; Mohan-Raju, Basavaiah; Udayakumar, Makarla

    2016-01-01

    In nature plants are often simultaneously challenged by different biotic and abiotic stresses. Although the mechanisms underlying plant responses against single stress have been studied considerably, plant tolerance mechanisms under combined stress is not understood. Also, the mechanism used to combat independently and sequentially occurring many number of biotic and abiotic stresses has also not systematically studied. From this context, in this study, we attempted to explore the shared response of sunflower plants to many independent stresses by using meta-analysis of publically available transcriptome data and transcript profiling by quantitative PCR. Further, we have also analyzed the possible role of the genes so identified in contributing to combined stress tolerance. Meta-analysis of transcriptomic data from many abiotic and biotic stresses indicated the common representation of oxidative stress responsive genes. Further, menadione-mediated oxidative stress in sunflower seedlings showed similar pattern of changes in the oxidative stress related genes. Based on this a large scale screening of 55 sunflower genotypes was performed under menadione stress and those contrasting in oxidative stress tolerance were identified. Further to confirm the role of genes identified in individual and combined stress tolerance the contrasting genotypes were individually and simultaneously challenged with few abiotic and biotic stresses. The tolerant hybrid showed reduced levels of stress damage both under combined stress and few independent stresses. Transcript profiling of the genes identified from meta-analysis in the tolerant hybrid also indicated that the selected genes were up-regulated under individual and combined stresses. Our results indicate that menadione-based screening can identify genotypes not only tolerant to multiple number of individual biotic and abiotic stresses, but also the combined stresses. PMID:27314499

  9. Rapid genotyping of human rotavirus using SYBR green real-time reverse transcription-polymerase chain reaction with melting curve analysis

    PubMed Central

    Tong, Yupin; Lee, Bonita E; Pang, Xiaoli L

    2015-01-01

    AIM: To develop a real-time reverse transcription-polymerase chain reaction (RT-PCR) assay to genotype rotavirus (G and P) in Alberta from January 2012 to June 2013. METHODS: We developed and validated a different approach to perform rotavirus G and P genotyping using a two-step SYBR green RT-PCR (rt-gPCR) by selecting genotype-specific primers of published conventional RT nested PCR (cnRT-PCR) assay and optimizing the amplification conditions. cDNA was first synthesized from total RNA with SuperScript™ II reverse transcriptase kit followed by amplication step using monoplex SYBR green real-time PCR. After the PCR reaction, melting curve analysis was used to determine specific genotype. Sixteen samples previously genotyped using cnRT-PCR were tested using the new assay and the genotyping results were compared as sensitivity analysis. Assay specificity was evaluated by testing other gastroenteritis viruses with the new assay. The amplicon size of each available genotype was determined by gel-electrophoresis and DNA sequences were obtained using Sanger-sequencing method. After validation and optimization, the new assay was used to genotype 122 pediatric clinical stool samples previously tested positive for rotavirus using electron microscopy between January 2012 and June 2013. RESULTS: The new rt-gPCR assay was validated and optimized. The assay detected G1 to G4, G9, G12 and P[4] and P[8] that were available as positive controls in our laboratory. A single and clear peak of melting curve was generated for each of specific G and P genotypes with a Tm ranging from 80 °C to 82 °C. The sensitivity of rt-gPCR was comparable to cnRT-PCR with 100% correlation of the 16 samples with known G and P genotypes. No cross reaction was found with other gastroenteritis viruses. Using the new rt-gPCR assay, genotypes were obtained for 121 of the 122 pediatric clinical samples tested positive for rotavirus: G1P[8] (42.6%), G2P[4] (4.9%), G3P[8] (10.7%), G9P[8] (10.7%), G9P[4

  10. Antigenic analysis of divergent genotypes human Enterovirus 71 viruses by a panel of neutralizing monoclonal antibodies: current genotyping of EV71 does not reflect their antigenicity.

    PubMed

    Chen, Yixin; Li, Chuan; He, Delei; Cheng, Tong; Ge, Shengxiang; Shih, James Wai-Kuo; Zhao, Qinjian; Chen, Pei-Jer; Zhang, Jun; Xia, Ningshao

    2013-01-01

    In recent year, Enterovirus 71 (EV71)-associated hand, foot and mouth disease (HFMD) has become an important public health issue in China. EV71 has been classified into genotypes A, B1-B5 and C1-C5. With such genetic diversity, whether the convalescent or recovery antibody responses can cross-protect infections from other genotypes remains a question. Understanding of the antigenicity of such diverse genetic EV71 isolates is crucial for the EV71 vaccine development. Here, a total of 186 clones anti-EV71 MAbs was generated and characterized with Western blot and cell-based neutralization assay. Forty neutralizing anti-EV71 MAbs were further used to analyze the antigenic properties of 18 recent EV71 isolates representing seven genotypes in neutralization assay. We found that most neutralizing anti-EV71 MAbs are specific to conformational epitopes. We also classified the 40 neutralizing anti-EV71 MAbs into two classes according to their reactivity patterns with 18 EV71 isolates. Class I MAb can neutralize all isolates, suggesting conserved epitopes are present among EV71. Class II MAb includes four subclasses (IIa-IId) and neutralizes only subgroups of EV71 strains. Conversely, 18 EV71 strains were grouped into antigenic types 1 and four antigenic subtypes (2.1-2.4). These results suggest that the current genotyping of EV71 does not reflect their antigenicity which may be important in the selection of EV71 vaccine strains. This panel of neutralizing anti-EV71 MAbs may be useful for the recognition of emerging antigenic variants of EV71 and vaccine development.

  11. Genotype-phenotype matching analysis of 38 Lactococcus lactis strains using random forest methods

    PubMed Central

    2013-01-01

    Background Lactococcus lactis is used in dairy food fermentation and for the efficient production of industrially relevant enzymes. The genome content and different phenotypes have been determined for multiple L. lactis strains in order to understand intra-species genotype and phenotype diversity and annotate gene functions. In this study, we identified relations between gene presence and a collection of 207 phenotypes across 38 L. lactis strains of dairy and plant origin. Gene occurrence and phenotype data were used in an iterative gene selection procedure, based on the Random Forest algorithm, to identify genotype-phenotype relations. Results A total of 1388 gene-phenotype relations were found, of which some confirmed known gene-phenotype relations, such as the importance of arabinose utilization genes only for strains of plant origin. We also identified a gene cluster related to growth on melibiose, a plant disaccharide; this cluster is present only in melibiose-positive strains and can be used as a genetic marker in trait improvement. Additionally, several novel gene-phenotype relations were uncovered, for instance, genes related to arsenite resistance or arginine metabolism. Conclusions Our results indicate that genotype-phenotype matching by integrating large data sets provides the possibility to identify gene-phenotype relations, possibly improve gene function annotation and identified relations can be used for screening bacterial culture collections for desired phenotypes. In addition to all gene-phenotype relations, we also provide coherent phenotype data for 38 Lactococcus strains assessed in 207 different phenotyping experiments, which to our knowledge is the largest to date for the Lactococcus lactis species. PMID:23530958

  12. Meta-analysis of three diabetes population studies: association of inactive ALDH2 genotype with maternal inheritance of diabetes.

    PubMed

    Murata, C; Taniyama, M; Kuriyama, S; Muramatsu, T; Atsumi, Y; Matsuoka, K; Suzuki, Y

    2004-12-01

    To date, there have been three population studies that examined the association of mitochondrial aldehyde dehydrogenase 2 (ALDH2) genotype with inheritance of diabetes. Here, we summarize the results by meta-analysis. The study 1 consisted of 212 type 2 diabetics who did not have renal failure. The study 2 consisted of 73 type 2 diabetics who had renal failure. The study 3 consisted of 230 type 1 diabetics. In total, 515 subjects were examined for the association of ALDH2 genotype with inheritance of diabetes. Out of 515 subjects, 307 (60%) had active ALDH2 (ALDH2*1/ALDH2*1) and 208 (40%) had inactive ALDH2 (175 had ALDH2*1/ALDH2*2 and 33 had ALDH2*2/ALDH2*2). As for family history, 25 subjects (8.1%) in the active ALDH2 group had a diabetic mother, compared with 43 (20.6%) in the inactive ALDH2 group. Twenty-nine subjects (9.4%) in the active ALDH2 group had a diabetic father, compared with 14 (6.7%) in the inactive ALDH2 group. The percentage of diabetic mother was higher in the inactive ALDH2 group, the differences were statistically significant (P < 0.0001). We hence speculate that diabetic patients with inactive ALDH2 genotype may have underlying background of mitochondria etiology, thereby showing maternal trait of diabetes inheritance. In conclusion, meta-analysis using three diabetes population studies strongly confirmed the association between ALDH2 inactivity and maternal inheritance.

  13. Strain differentiation of Neisseria gonorrhoeae by reverse passive hemagglutination.

    PubMed Central

    Armstrong, A S; Mathias, J R; DeYoung, M I; Hirata, A A

    1979-01-01

    A reverse passive hemagglutination test that utilizes human erythrocytes coated with antibody to gonococci was developed to distinguish differences among 11 strains of Neisseria gonorrhoeae. Different rabbits were immunized with each strain of gonococcus. Antibody was purified by passing antiserum over an immunoadsorbent column containing homologous cell walls trapped in a cross-linked polyacrylamide gel. Antibody, after absorption with N. meningitidis, was used for coating 11 individual suspensions of erythrocytes, each with antibody to one gonococcal strain. The panel of coated erythrocytes was added to microtiter trays containing dilutions of homologous bacterial lysate and lysates from 10 heterologous strains. Agglutination titers were highest with homologous lysates, although cross-reactions occurred among some heterologous lysates. Lysates of nongonococcal Neisseria species and of other genera did not agglutinate coated erythrocytes. The reverse passive hemagglutination test can be a useful procedure to distinguish differences among strains of N. gonorrhoeae. Images PMID:110695

  14. Neisseria gonorrhoeae and humans perform an evolutionary LINE dance.

    PubMed

    Anderson, Mark T; Seifert, H Steven

    2011-05-01

    Horizontal gene transfer is an important mechanism for generating genetic diversity. As the number of sequenced genomes continues to increase, so do the examples of horizontal genetic exchange between both related and divergent organisms. Here we discuss the recent finding that certain strains of the human pathogen Neisseria gonorrhoeae have incorporated a small fragment of human DNA sequence into their genomes. The horizontally acquired sequence exhibits 98-100% nucleotide identity to a 685 bp portion of the highly repetitive retrotransposable element L1 and its presence in the gonococcal genome has been confirmed by multiple molecular techniques. The possibility of similar L1 horizontal gene transfer events having occurred in other bacteria based on genomic sequence evidence is explored. Potential mechanisms of how N. gonorrhoeae was able to acquire and maintain this human sequence are also discussed in addition to the evolutionary implications of such an event. PMID:22016852

  15. Quinolones for the Treatment of Neisseria Gonorrhoeae and Chlamydia Trachomatis

    PubMed Central

    1993-01-01

    The most commonly sexually transmitted bacteria are Neisseria gonorrhoeae and Chlamydia trachomatis. The quinolones ofloxacin and ciprofloxacin have been shown to have activity against both of these bacteria in vitro and in vivo. Ofloxacin is particularly well suited for the treatment of N. gonorrhoeae and C. trachomatis cervical infection, which can be considered the earliest manifestation of pelvic inflammatory disease (PID). Not only can ofloxacin be effectively used as a single agent, it is also useful in treating urinary tract infections caused by Enterobacteriaceae. Although it has moderate activity against anaerobes in general, ofloxacin does have activity against the anaerobes commonly isolated from female patients with soft tissue pelvic infections. Thus, ofloxacin has the potential for being utilized to treat early salpingitis. PMID:18475328

  16. Application of restriction fragment length polymorphism analysis to simple and rapid genotyping of bovine viral diarrhea virus strains isolated in Japan.

    PubMed

    Seki, Yoshihisa; Seimiya, Yukio M; Motokawa, Masato; Yaegashi, Gakuji; Nagai, Makoto; Hayashi, Michiko

    2008-04-01

    The E2 regions of 177 bovine viral diarrhea virus (BVDV) strains isolated in Japan between 1957 and 2006 were analyzed for genotyping. The strains were classified into 8 genotypes (1a, 1b, 1c, 1d, 1e, 1f, So and 2a) based on the phylogenetic analysis. The restriction fragment length polymorphism (RFLP) analysis of the RT-PCR products using 6 selected enzymes (Apo I, Mly I, BstAP I, Pvu II, Ear I, EcoR V) disclosed the cutting patterns classified into 11 groups (I-XI), each of that consisted of strains belonging to a single genotype. Namely, groups-I and -II were composed by genotype-1a strains, groups-III and -IV by 1b strains, and groups-V and -VI by 1c strains. Other groups-VII, -VIII, -IX, -X and -XI comprised genotypes-1d, -1e, -1f, -So and -2a strains, respectively. The results suggest that the RFLP analysis can simply and rapidly differentiate the 8 genotypes of BVDV strains.

  17. Data in support of comparative physiology and proteomic analysis of two wheat genotypes contrasting in drought tolerance.

    PubMed

    Faghani, Elham; Gharechahi, Javad; Komatsu, Setsuko; Mirzaei, Mehdi; Ali Khavarinejad, Ramzan; Najafi, Farzaneh; Karimi Farsad, Laleh; Salekdeh, Ghasem Hosseini

    2015-03-01

    Here, we present the data from a comparative physiology and proteomics approach used to analyze the response of two wheat genotypes (SERI M 82 (SE) and SW89.5193/kAu2 (SW)) with contrasting responses to drought stress. Proteomic analysis resulted in identification of 49 unique proteins with significant change in abundance (2-fold) under water shortage in roots and leaves. Gene ontology analysis of drought-responsive proteins (DRPs) suggested an induction of proteins related to cell wall biogenesis, ATP synthesis, photosynthesis, and carbohydrate/energy metabolism in leaves under stress condition. A large fraction of root proteins were identified to be involved in defense and oxidative stress response. In addition, a significant change was detected in proteins related to protein synthesis, ATP synthesis, and germin-like proteins in response to drought stress. A detailed analysis of this data may be obtained from Ref. [1]. PMID:26217700

  18. Gonorrhoea treatment failures to cefixime and azithromycin in England, 2010.

    PubMed

    Ison, C A; Hussey, J; Sankar, K N; Evans, J; Alexander, S

    2011-04-07

    Successful treatment of gonorrhoea is the mainstay of public health control. Cefixime and ceftriaxone, highly active third generation cephalosporins, are today the recommended first-line agents in most countries and azithromycin is a second-line agent. However, there is increasing evidence of decreasing susceptibility and emergence of therapeutic failures. In this report two cases of clinical failure to cefixime are described, one of which additionally shows failure to azithromycin and selection of a less susceptible strain during treatment.

  19. A re-audit of the management of gonorrhoea.

    PubMed

    Ekanayaka, Ratna; Challenor, Rachel

    2016-08-01

    A re-audit of the management of gonorrhoea was undertaken in 2014. Six out of nine auditable outcomes were met in the second audit (2014) compared with three out of eight in the first audit (2012). The new measures that were introduced following the original audit may have helped to improve outcomes. However, electronic patient records were introduced in December 2012. Documentation was much improved with the use of patient record templates and this has contributed considerably to the improved outcomes.

  20. Genetic analysis of the capsid gene of genotype GII.2 noroviruses.

    PubMed

    Iritani, Nobuhiro; Vennema, Harry; Siebenga, J Joukje; Siezen, Roland J; Renckens, Bernadet; Seto, Yoshiyuki; Kaida, Atsushi; Koopmans, Marion

    2008-08-01

    Noroviruses (NoVs) are considered to be a major cause of acute nonbacterial gastroenteritis in humans. The NoV genus is genetically diverse, and genotype GII.4 has been most commonly identified worldwide in recent years. In this study we analyzed the complete capsid gene of NoV strains belonging to the less prevalent genotype GII.2. We compared a total of 36 complete capsid sequences of GII.2 sequences obtained from the GenBank (n = 5) and from outbreaks or sporadic cases that occurred in The Netherlands (n = 10) and in Osaka City, Japan (n = 21), between 1976 and 2005. Alignment of all capsid sequences did not show fixation of amino acid substitutions over time as an indication for genetic drift. In contrast, when strains previously recognized as recombinants were excluded from the alignment, genetic drift was observed. Substitutions were found at five informative sites (two in the P1 subdomain and three in the P2 subdomain), segregating strains into five genetic groups (1994 to 1997, 1999 to 2000, 2001 to 2003, 2004, and 2005). Only one amino acid position changed consistently between each group (position 345). Homology modeling of the GII.2 capsid protein showed that the five amino acids were located on the surface of the capsid and close to each other at the interface of two monomers. The data suggest that these changes were induced by selective pressure, driving virus evolution. Remarkably, this was observed only for nonrecombinant genomes, suggesting differences in behavior with recombinant strains.

  1. [Genotype analysis of RhD-negative donors with immune antibodies].

    PubMed

    Zhang, Chun-Yan; Li, Ji-Hong; Zhao, Su-Zen; Liu, Jie

    2012-06-01

    In order to analyze the genotype of RhD-negative blood donors with immune antibodies in Harbin, the voluntary blood donors from 1 April 2008 to 30 september 2011 were detected serologically to determine the RhD-negative donors. The blood donors confirmed to be RhD negative were detected to screen the immune antibodies, the samples with immune antibodies were analyzed by PCR-SSP and DNA sequencing to detect RhD genotype. The results showed that the 12 cases of the immune antibodies (0.95%) were screened out from 1265 cases of RhD-negative donors, among which 9 cases showed anti-D-antibody, 3 cases showed anti-(D+C) antibody; 10 cases were RhD-negative, 2 cases were RHD 711D(el)C. It is concluded that RhD negative and RHD 711D(el)C are easy to be immunized to produce the immune antibodies; RhD-negative population, especially women should be highly aware of avoiding mis-transfusion of RhD-positive blood, and also avoiding multiple pregnancies resulting in newborn's hemolytic disease.

  2. Large-Scale Transcriptome Analysis of Two Sugarcane Genotypes Contrasting for Lignin Content.

    PubMed

    Vicentini, Renato; Bottcher, Alexandra; Brito, Michael Dos Santos; Dos Santos, Adriana Brombini; Creste, Silvana; Landell, Marcos Guimarães de Andrade; Cesarino, Igor; Mazzafera, Paulo

    2015-01-01

    Sugarcane is an important crop worldwide for sugar and first generation ethanol production. Recently, the residue of sugarcane mills, named bagasse, has been considered a promising lignocellulosic biomass to produce the second-generation ethanol. Lignin is a major factor limiting the use of bagasse and other plant lignocellulosic materials to produce second-generation ethanol. Lignin biosynthesis pathway is a complex network and changes in the expression of genes of this pathway have in general led to diverse and undesirable impacts on plant structure and physiology. Despite its economic importance, sugarcane genome was still not sequenced. In this study a high-throughput transcriptome evaluation of two sugarcane genotypes contrasting for lignin content was carried out. We generated a set of 85,151 transcripts of sugarcane using RNA-seq and de novo assembling. More than 2,000 transcripts showed differential expression between the genotypes, including several genes involved in the lignin biosynthetic pathway. This information can give valuable knowledge on the lignin biosynthesis and its interactions with other metabolic pathways in the complex sugarcane genome.

  3. Large-Scale Transcriptome Analysis of Two Sugarcane Genotypes Contrasting for Lignin Content

    PubMed Central

    Vicentini, Renato; Bottcher, Alexandra; Brito, Michael dos Santos; dos Santos, Adriana Brombini; Creste, Silvana; Landell, Marcos Guimarães de Andrade; Cesarino, Igor; Mazzafera, Paulo

    2015-01-01

    Sugarcane is an important crop worldwide for sugar and first generation ethanol production. Recently, the residue of sugarcane mills, named bagasse, has been considered a promising lignocellulosic biomass to produce the second-generation ethanol. Lignin is a major factor limiting the use of bagasse and other plant lignocellulosic materials to produce second-generation ethanol. Lignin biosynthesis pathway is a complex network and changes in the expression of genes of this pathway have in general led to diverse and undesirable impacts on plant structure and physiology. Despite its economic importance, sugarcane genome was still not sequenced. In this study a high-throughput transcriptome evaluation of two sugarcane genotypes contrasting for lignin content was carried out. We generated a set of 85,151 transcripts of sugarcane using RNA-seq and de novo assembling. More than 2,000 transcripts showed differential expression between the genotypes, including several genes involved in the lignin biosynthetic pathway. This information can give valuable knowledge on the lignin biosynthesis and its interactions with other metabolic pathways in the complex sugarcane genome. PMID:26241317

  4. [MOLECULAR MECHANISMS OF DRUG RESISTANCE NEISSERIA GONORRHOEAE HISTORY AND PROSPECTS].

    PubMed

    Bodoev, I N; Il'ina, E N

    2015-01-01

    Neisseria gonorrhoeae (gonococcus) is a strict human pathogen, which causes gonorrhea--an infectious disease, whose origin dates back to more than two thousand years. Due to the unique plasticity of the genetic material, these bacteria have acquired the capacity to adapt to the host immune system, cause repeated infections, as well as withstand antimicrobials. Since the introduction of antibiotics in 1930s, gonococcus has displayed its propensity to develop resistance to all clinically useful antibiotics. It is important to note that the known resistance determinants of N. gonorrhoeae were acquired through horizontal gene transfer, recombination and spontaneous mutagenesis, and may be located both in the chromosome and on the plasmid. After introduction of a new antimicrobial drug, gonococcus becomes resistant within two decades and replaces sensitive bacterial population. Currently Ceftriaxone is the last remaining antibiotic for first-line treatment of gonorrhea. However, the first gonococcus displaying high-level resistance to Ceftriaxone was isolated in Japan a few years ago. Therefore, in the near future, gonorrhea may become untreatable. In the present review, we discuss the chronology of the anti-gonorrhea drugs (antibiotics) replacement, the evolution of resistance mechanisms emergence and future perspectives of N. gonorrhoeae treatment.

  5. Phylogenetic analysis of Japanese encephalitis virus: envelope gene based analysis reveals a fifth genotype, geographic clustering, and multiple introductions of the virus into the Indian subcontinent.

    PubMed

    Uchil, P D; Satchidanandam, V

    2001-09-01

    We report the analysis of the complete nucleotide sequence for the Indian isolate (P20778; Genbank Accession number AF080251) of Japanese encephalitis virus (JEV). The phylogenetic tree topology obtained using thirteen complete genome sequences of JEV was reproduced with the envelope, NS1, NS3, and NS5 genes and revealed extensive divergence between the two Indian strains included. A more exhaustive analysis of JEV evolution using 107 envelope sequences available for isolates from different geographic locations worldwide revealed five distinct genotypes of JEV, displaying a minimum nucleotide divergence of 7% with high bootstrap support values. The tree also revealed overall clustering of strains based on geographic location, as well as multiple introductions of JEV into the Indian subcontinent. Nonsynonymous nucleotide divergence rates of the envelope gene estimated that the ancestor common to all JEV genotypes arose within the last three hundred years.

  6. De Novo Assembly and Transcriptome Analysis of Bulb Onion (Allium cepa L.) during Cold Acclimation Using Contrasting Genotypes.

    PubMed

    Han, Jeongsukhyeon; Thamilarasan, Senthil Kumar; Natarajan, Sathishkumar; Park, Jong-In; Chung, Mi-Young; Nou, Ill-Sup

    2016-01-01

    Bulb onion (Allium cepa) is the second most widely cultivated and consumed vegetable crop in the world. During winter, cold injury can limit the production of bulb onion. Genomic resources available for bulb onion are still very limited. To date, no studies on heritably durable cold and freezing tolerance have been carried out in bulb onion genotypes. We applied high-throughput sequencing technology to cold (2°C), freezing (-5 and -15°C), and control (25°C)-treated samples of cold tolerant (CT) and cold susceptible (CS) genotypes of A. cepa lines. A total of 452 million paired-end reads were de novo assembled into 54,047 genes with an average length of 1,331 bp. Based on similarity searches, these genes were aligned with entries in the public non-redundant (nr) database, as well as KEGG and COG database. Differentially expressed genes (DEGs) were identified using log10 values with the FPKM method. Among 5,167DEGs, 491 genes were differentially expressed at freezing temperature compared to the control temperature in both CT and CS libraries. The DEG results were validated with qRT-PCR. We performed GO and KEGG pathway enrichment analyses of all DEGs and iPath interactive analysis found 31 pathways including those related to metabolism of carbohydrate, nucleotide, energy, cofactors and vitamins, other amino acids and xenobiotics biodegradation. Furthermore, a large number of molecular markers were identified from the assembled genes, including simple sequence repeats (SSRs) 4,437 and SNP substitutions of transition and transversion types of CT and CS. Our study is the first to provide a transcriptome sequence resource for Allium spp. with regard to cold and freezing stress. We identified a large set of genes and determined their DEG profiles under cold and freezing conditions using two different genotypes. These data represent a valuable resource for genetic and genomic studies of Allium spp. PMID:27627679

  7. De Novo Assembly and Transcriptome Analysis of Bulb Onion (Allium cepa L.) during Cold Acclimation Using Contrasting Genotypes

    PubMed Central

    Natarajan, Sathishkumar; Park, Jong-In; Chung, Mi-Young; Nou, Ill-Sup

    2016-01-01

    Bulb onion (Allium cepa) is the second most widely cultivated and consumed vegetable crop in the world. During winter, cold injury can limit the production of bulb onion. Genomic resources available for bulb onion are still very limited. To date, no studies on heritably durable cold and freezing tolerance have been carried out in bulb onion genotypes. We applied high-throughput sequencing technology to cold (2°C), freezing (-5 and -15°C), and control (25°C)-treated samples of cold tolerant (CT) and cold susceptible (CS) genotypes of A. cepa lines. A total of 452 million paired-end reads were de novo assembled into 54,047 genes with an average length of 1,331 bp. Based on similarity searches, these genes were aligned with entries in the public non-redundant (nr) database, as well as KEGG and COG database. Differentially expressed genes (DEGs) were identified using log10 values with the FPKM method. Among 5,167DEGs, 491 genes were differentially expressed at freezing temperature compared to the control temperature in both CT and CS libraries. The DEG results were validated with qRT-PCR. We performed GO and KEGG pathway enrichment analyses of all DEGs and iPath interactive analysis found 31 pathways including those related to metabolism of carbohydrate, nucleotide, energy, cofactors and vitamins, other amino acids and xenobiotics biodegradation. Furthermore, a large number of molecular markers were identified from the assembled genes, including simple sequence repeats (SSRs) 4,437 and SNP substitutions of transition and transversion types of CT and CS. Our study is the first to provide a transcriptome sequence resource for Allium spp. with regard to cold and freezing stress. We identified a large set of genes and determined their DEG profiles under cold and freezing conditions using two different genotypes. These data represent a valuable resource for genetic and genomic studies of Allium spp. PMID:27627679

  8. De Novo Assembly and Transcriptome Analysis of Bulb Onion (Allium cepa L.) during Cold Acclimation Using Contrasting Genotypes.

    PubMed

    Han, Jeongsukhyeon; Thamilarasan, Senthil Kumar; Natarajan, Sathishkumar; Park, Jong-In; Chung, Mi-Young; Nou, Ill-Sup

    2016-01-01

    Bulb onion (Allium cepa) is the second most widely cultivated and consumed vegetable crop in the world. During winter, cold injury can limit the production of bulb onion. Genomic resources available for bulb onion are still very limited. To date, no studies on heritably durable cold and freezing tolerance have been carried out in bulb onion genotypes. We applied high-throughput sequencing technology to cold (2°C), freezing (-5 and -15°C), and control (25°C)-treated samples of cold tolerant (CT) and cold susceptible (CS) genotypes of A. cepa lines. A total of 452 million paired-end reads were de novo assembled into 54,047 genes with an average length of 1,331 bp. Based on similarity searches, these genes were aligned with entries in the public non-redundant (nr) database, as well as KEGG and COG database. Differentially expressed genes (DEGs) were identified using log10 values with the FPKM method. Among 5,167DEGs, 491 genes were differentially expressed at freezing temperature compared to the control temperature in both CT and CS libraries. The DEG results were validated with qRT-PCR. We performed GO and KEGG pathway enrichment analyses of all DEGs and iPath interactive analysis found 31 pathways including those related to metabolism of carbohydrate, nucleotide, energy, cofactors and vitamins, other amino acids and xenobiotics biodegradation. Furthermore, a large number of molecular markers were identified from the assembled genes, including simple sequence repeats (SSRs) 4,437 and SNP substitutions of transition and transversion types of CT and CS. Our study is the first to provide a transcriptome sequence resource for Allium spp. with regard to cold and freezing stress. We identified a large set of genes and determined their DEG profiles under cold and freezing conditions using two different genotypes. These data represent a valuable resource for genetic and genomic studies of Allium spp.

  9. Characterization of spliced leader genes of Trypanosoma ( Megatrypanum) theileri: phylogeographical analysis of Brazilian isolates from cattle supports spatial clustering of genotypes and parity with ribosomal markers.

    PubMed

    Rodrigues, A C; Garcia, H A; Batista, J S; Minervino, A H H; Góes-Cavalcante, G; Maia da Silva, F; Ferreira, R C; Campaner, M; Paiva, F; Teixeira, M M G

    2010-01-01

    Trypanosoma (Megatrypanum) theileri from cattle and trypanosomes of other artiodactyls form a clade of closely related species in analyses using ribosomal sequences. Analysis of polymorphic sequences of a larger number of trypanosomes from broader geographical origins is required to evaluate the clustering of isolates as suggested by previous studies. Here, we determined the sequences of the spliced leader (SL) genes of 21 isolates from cattle and 2 from water buffalo from distant regions of Brazil. Analysis of SL gene repeats revealed that the 5S rRNA gene is inserted within the intergenic region. Phylogeographical patterns inferred using SL sequences showed at least 5 major genotypes of T. theileri distributed in 2 strongly divergent lineages. Lineage TthI comprises genotypes IA and IB from buffalo and cattle, respectively, from the Southeast and Central regions, whereas genotype IC is restricted to cattle from the Southern region. Lineage TthII includes cattle genotypes IIA, which is restricted to the North and Northeast, and IIB, found in the Centre, West, North and Northeast. PCR-RFLP of SL genes revealed valuable markers for genotyping T. theileri. The results of this study emphasize the genetic complexity and corroborate the geographical structuring of T. theileri genotypes found in cattle.

  10. Comparative Transcriptome Analysis between Low- and High-Cadmium-Accumulating Genotypes of Pakchoi (Brassica chinensis L.) in Response to Cadmium Stress.

    PubMed

    Zhou, Qian; Guo, Jing-Jie; He, Chun-Tao; Shen, Chuang; Huang, Ying-Ying; Chen, Jing-Xin; Guo, Jian-Hua; Yuan, Jian-Gang; Yang, Zhong-Yi

    2016-06-21

    To reduce cadmium (Cd) pollution of food chains, screening and breeding of low-Cd-accumulating cultivars are the focus of much study. Two previously identified genotypes, a low-Cd-accumulating genotype (LAJK) and a high-Cd-accumulating genotype (HAJS) of pakchoi (Brassica chinesis L.), were stressed by Cd (12.5 μM) for 0 h (T0), 3 h (T3) and 24 h (T24). By comparative transcriptome analysis for root tissue, 3005 and 4343 differentially expressed genes (DEGs) were identified in LAJK at T3 (vs T0) and T24 (vs T3), respectively, whereas 8677 and 5081 DEGs were detected in HAJS. Gene expression pattern analysis suggested a delay of Cd responded transcriptional changes in LAJK compared to HAJS. DEG functional enrichments proposed genotype-specific biological processes coped with Cd stress. Cell wall biosynthesis and glutathione (GSH) metabolism were found to involve in Cd resistance in HAJS, whereas DNA repair and abscisic acid (ABA) signal transduction pathways played important roles in LAJK. Furthermore, the genes participating in Cd efflux such as PDR8 were overexpressed in LAJK, whereas those responsible for Cd transport such as YSL1 were more enhanced in HAJS, exhibiting different Cd transport processes between two genotypes. These novel findings should be useful for molecular assisted screening and breeding of low-Cd-accumulating genotypes for pakchoi. PMID:27228483

  11. Analysis of Behavioral and Emotional Problems in Children Highlights the Role of Genotype × Environment Interaction.

    PubMed

    Molenaar, Dylan; Middeldorp, Christel; van Beijsterveldt, Toos; Boomsma, Dorret I

    2015-01-01

    This study tested for Genotype × Environment (G × E) interaction on behavioral and emotional problems in children using new methods that do not require identification of candidate genes or environments, can distinguish between interaction with shared and unique environment, and are insensitive to scale effects. Parental ratings of problem behavior from 14,755 twin pairs (5.3 years, SD = 0.22) indicated G × E interaction on emotional liability, social isolation, aggression, attention problems, dependency, anxiety, and physical coordination. Environmental influences increased in children who were genetically more predisposed to problem behavior, with ~20% of the variance due to G × E interaction (8% for anxiety to 37% for attention problems). Ignoring G × E interaction does not greatly bias heritability estimates, but it does offer a comprehensive model of the etiology for childhood problems.

  12. Neisseria gonorrhoeae induces a tolerogenic phenotype in macrophages to modulate host immunity.

    PubMed

    Escobar, Alejandro; Candia, Enzo; Reyes-Cerpa, Sebastian; Villegas-Valdes, Bélgica; Neira, Tanya; Lopez, Mercedes; Maisey, Kevin; Tempio, Fabián; Ríos, Miguel; Acuña-Castillo, Claudio; Imarai, Mónica

    2013-01-01

    Neisseria gonorrhoeae is the etiological agent of gonorrhoea, which is a sexually transmitted disease widespread throughout the world. N. gonorrhoeae does not improve immune response in patients with reinfection, suggesting that gonococcus displays several mechanisms to evade immune response and survive in the host. N. gonorrhoeae is able to suppress the protective immune response at different levels, such as B and T lymphocytes and dendritic cells. In this study, we determined whether N. gonorrhoeae directly conditions the phenotype of RAW 264.7 murine macrophage cell line and its response. We established that gonococcus was effectively phagocytosed by the RAW 264.7 cells and upregulates production of immunoregulatory cytokines (IL-10 and TGF- β 1) but not the production of proinflammatory cytokine TNF- α , indicating that gonococcus induces a shift towards anti-inflammatory cytokine production. Moreover, N. gonorrhoeae did not induce significant upregulation of costimulatory CD86 and MHC class II molecules. We also showed that N. gonorrhoeae infected macrophage cell line fails to elicit proliferative CD4+ response. This implies that macrophage that can phagocytose gonococcus do not display proper antigen-presenting functions. These results indicate that N. gonorrhoeae induces a tolerogenic phenotype in antigen-presenting cells, which seems to be one of the mechanisms to induce evasion of immune response.

  13. Neisseria gonorrhoeae Induces a Tolerogenic Phenotype in Macrophages to Modulate Host Immunity

    PubMed Central

    Candia, Enzo; Reyes-Cerpa, Sebastian; Villegas-Valdes, Bélgica; Neira, Tanya; Lopez, Mercedes; Maisey, Kevin; Tempio, Fabián; Ríos, Miguel; Acuña-Castillo, Claudio; Imarai, Mónica

    2013-01-01

    Neisseria gonorrhoeae is the etiological agent of gonorrhoea, which is a sexually transmitted disease widespread throughout the world. N. gonorrhoeae does not improve immune response in patients with reinfection, suggesting that gonococcus displays several mechanisms to evade immune response and survive in the host. N. gonorrhoeae is able to suppress the protective immune response at different levels, such as B and T lymphocytes and dendritic cells. In this study, we determined whether N. gonorrhoeae directly conditions the phenotype of RAW 264.7 murine macrophage cell line and its response. We established that gonococcus was effectively phagocytosed by the RAW 264.7 cells and upregulates production of immunoregulatory cytokines (IL-10 and TGF-β1) but not the production of proinflammatory cytokine TNF-α, indicating that gonococcus induces a shift towards anti-inflammatory cytokine production. Moreover, N. gonorrhoeae did not induce significant upregulation of costimulatory CD86 and MHC class II molecules. We also showed that N. gonorrhoeae infected macrophage cell line fails to elicit proliferative CD4+ response. This implies that macrophage that can phagocytose gonococcus do not display proper antigen-presenting functions. These results indicate that N. gonorrhoeae induces a tolerogenic phenotype in antigen-presenting cells, which seems to be one of the mechanisms to induce evasion of immune response. PMID:24204097

  14. Antibacterial activity of resazurin-based compounds against Neisseria gonorrhoeae in vitro and in vivo.

    PubMed

    Schmitt, Deanna M; Connolly, Kristie L; Jerse, Ann E; Detrick, Melinda S; Horzempa, Joseph

    2016-10-01

    Neisseria gonorrhoeae is the cause of the second most common sexually transmitted bacterial infection, with ca. 80 million new cases of gonorrhoea reported annually. The recent emergence of clinical isolates resistant to the last monotherapy against this bacterium, the cephalosporins, illustrates the need for new antigonococcal agents. Here we have characterised a new group of antimicrobials based on the compound resazurin that exhibits robust activity against N. gonorrhoeae in vitro. Resazurin inhibits the growth of a broad range of N. gonorrhoeae isolates, including those resistant to multiple antibiotics. Furthermore, treatment of human endometrial cells infected with N. gonorrhoeae with resazurin significantly reduces the number of intracellular bacteria. Whilst resazurin exhibited potent in vitro antimicrobial activity, in vivo resazurin did not limit the colonisation of mice with N. gonorrhoeae following vaginal infection. The ineffectiveness of resazurin in vivo is likely due to its interaction with serum albumin, which completely diminishes its antimicrobial activity. However, treatment of mice with a resazurin analogue (resorufin pentyl ether) that maintains its antimicrobial activity in the presence of serum albumin approached a significant decrease in the percentage of mice vaginally colonised. This treatment also decreased vaginal colonisation by N. gonorrhoeae over time. Together, these data suggest that resazurin derivatives have potential for the treatment of gonorrhoea.

  15. Complete Genome Sequences of Neisseria gonorrhoeae with Coresistance to First-Line Antimicrobials.

    PubMed

    Bharat, Amrita; Martin, Irene; Demczuk, Walter; Allen, Vanessa; Haldane, David; Hoang, Linda; Mulvey, Michael R

    2016-01-01

    Neisseria gonorrhoeae strains with coresistance to the first-line antimicrobial treatments azithromycin and ceftriaxone are an emerging public health threat. Here, we present the complete genome sequences of three strains of N. gonorrhoeae, including one susceptible strain and two strains with coresistance to ceftriaxone and azithromycin. PMID:27609929

  16. Complete Genome Sequences of Neisseria gonorrhoeae with Coresistance to First-Line Antimicrobials

    PubMed Central

    Bharat, Amrita; Martin, Irene; Demczuk, Walter; Allen, Vanessa; Haldane, David; Hoang, Linda

    2016-01-01

    Neisseria gonorrhoeae strains with coresistance to the first-line antimicrobial treatments azithromycin and ceftriaxone are an emerging public health threat. Here, we present the complete genome sequences of three strains of N. gonorrhoeae, including one susceptible strain and two strains with coresistance to ceftriaxone and azithromycin. PMID:27609929

  17. Emergence of high level azithromycin-resistant Neisseria gonorrhoeae strain isolated in Argentina.

    PubMed

    Galarza, Patricia G; Alcalá, Belén; Salcedo, Celia; Canigia, Liliana Fernández; Buscemi, Luis; Pagano, Irene; Oviedo, Claudia; Vázquez, Julio A

    2009-12-01

    One Neisseria gonorrhoeae strains highly resistant to azithromycin AzHLR (MIC >2048 mg/L) was isolated in Argentina in 2001 and it has been characterized by N. gonorrhoeae multiantigen sequence typing (NG-MAST) as ST696, suggesting a different event to other isolates in Europe. Neither, mtrR mutations or presence of mef gene were detected.

  18. Antibacterial activity of resazurin-based compounds against Neisseria gonorrhoeae in vitro and in vivo.

    PubMed

    Schmitt, Deanna M; Connolly, Kristie L; Jerse, Ann E; Detrick, Melinda S; Horzempa, Joseph

    2016-10-01

    Neisseria gonorrhoeae is the cause of the second most common sexually transmitted bacterial infection, with ca. 80 million new cases of gonorrhoea reported annually. The recent emergence of clinical isolates resistant to the last monotherapy against this bacterium, the cephalosporins, illustrates the need for new antigonococcal agents. Here we have characterised a new group of antimicrobials based on the compound resazurin that exhibits robust activity against N. gonorrhoeae in vitro. Resazurin inhibits the growth of a broad range of N. gonorrhoeae isolates, including those resistant to multiple antibiotics. Furthermore, treatment of human endometrial cells infected with N. gonorrhoeae with resazurin significantly reduces the number of intracellular bacteria. Whilst resazurin exhibited potent in vitro antimicrobial activity, in vivo resazurin did not limit the colonisation of mice with N. gonorrhoeae following vaginal infection. The ineffectiveness of resazurin in vivo is likely due to its interaction with serum albumin, which completely diminishes its antimicrobial activity. However, treatment of mice with a resazurin analogue (resorufin pentyl ether) that maintains its antimicrobial activity in the presence of serum albumin approached a significant decrease in the percentage of mice vaginally colonised. This treatment also decreased vaginal colonisation by N. gonorrhoeae over time. Together, these data suggest that resazurin derivatives have potential for the treatment of gonorrhoea. PMID:27451856

  19. Analysis of hepatitis B virus genotyping and drug resistance gene mutations based on massively parallel sequencing.

    PubMed

    Han, Yingxin; Zhang, Yinxin; Mei, Yanhua; Wang, Yuqi; Liu, Tao; Guan, Yanfang; Tan, Deming; Liang, Yu; Yang, Ling; Yi, Xin

    2013-11-01

    Drug resistance to nucleoside analogs is a serious problem worldwide. Both drug resistance gene mutation detection and HBV genotyping are helpful for guiding clinical treatment. Total HBV DNA from 395 patients who were treated with single or multiple drugs including Lamivudine, Adefovir, Entecavir, Telbivudine, Tenofovir and Emtricitabine were sequenced using the HiSeq 2000 sequencing system and validated using the 3730 sequencing system. In addition, a mixed sample of HBV plasmid DNA was used to determine the cutoff value for HiSeq-sequencing, and 52 of the 395 samples were sequenced three times to evaluate the repeatability and stability of this technology. Of the 395 samples sequenced using both HiSeq and 3730 sequencing, the results from 346 were consistent, and the results from 49 were inconsistent. Among the 49 inconsistent results, 13 samples were detected as drug-resistance-positive using HiSeq but negative using 3730, and the other 36 samples showed a higher number of drug-resistance-positive gene mutations using HiSeq 2000 than using 3730. Gene mutations had an apparent frequency of 1% as assessed by the plasmid testing. Therefore, a 1% cutoff value was adopted. Furthermore, the experiment was repeated three times, and the same results were obtained in 49/52 samples using the HiSeq sequencing system. HiSeq sequencing can be used to analyze HBV gene mutations with high sensitivity, high fidelity, high throughput and automation and is a potential method for hepatitis B virus gene mutation detection and genotyping.

  20. Genotyping and Classification of Tunisian Strains of Avian Reovirus using RT-PCR and RFLP Analysis.

    PubMed

    Kort, Ymene Hellal; Bourogâa, Hager; Gribaa, Latifa; Hassen, Jihene; Ghram, Abdelgelil

    2015-03-01

    Since 1998, avian reovirus (ARV) infection has been detected in broiler and breeding chicken flocks in Tunisia. The genotype of avian reoviruses was established using simple and rapid approaches. Reverse transcription PCR (RT-PCR) on both sigma C (σC) and sigma B (σB)-encoding genes followed by restriction fragment length polymorphism (RFLP) analyses were used to better characterize Tunisian isolated strains. The RT-PCR amplified fragments of 738 and 540 bp for σC- and σB-encoding genes, respectively, of 15 ARV Tunisian strains. DNA fragments amplified from S 1133 vaccine and isolated strains were digested with different restrictions enzymes. RFLP on the σC gene indicated that the field isolates and the S 1133 vaccine strain have identical profiles when separately digested with TaqI, PstI, DdeI, and HincII. Considering the σB gene, RFLP profiles were identical with RsaI, BclI, DpnII, and NciI restriction enzymes for all the strains. However, using MseI and AciI enzymes, it was shown that all tested isolates could be clearly distinguished from the vaccine strain. ARV strains could be classified in groups with strong relatedness. Strain-typing based on cleavage site results are in agreement with ARV clustering based on nucleotide sequences of both the σC and σB genes. RT-PCR-RFLP provides a simple and a rapid approach for genotyping ARV isolates, especially when a large number of isolates are being studied. Additionally, this approach may also determine whether a new variant strain has been introduced into a flock or if a given virus strain is being spread from one flock to another. PMID:26292528

  1. Metabolic analysis of kiwifruit (Actinidia deliciosa) berries from extreme genotypes reveals hallmarks for fruit starch metabolism.

    PubMed

    Nardozza, Simona; Boldingh, Helen L; Osorio, Sonia; Höhne, Melanie; Wohlers, Mark; Gleave, Andrew P; MacRae, Elspeth A; Richardson, Annette C; Atkinson, Ross G; Sulpice, Ronan; Fernie, Alisdair R; Clearwater, Michael J

    2013-11-01

    Tomato, melon, grape, peach, and strawberry primarily accumulate soluble sugars during fruit development. In contrast, kiwifruit (Actinidia Lindl. spp.) and banana store a large amount of starch that is released as soluble sugars only after the fruit has reached maturity. By integrating metabolites measured by gas chromatography-mass spectrometry, enzyme activities measured by a robot-based platform, and transcript data sets during fruit development of Actinidia deliciosa genotypes contrasting in starch concentration and size, this study identified the metabolic changes occurring during kiwifruit development, including the metabolic hallmarks of starch accumulation and turnover. At cell division, a rise in glucose (Glc) concentration was associated with neutral invertase (NI) activity, and the decline of both Glc and NI activity defined the transition to the cell expansion and starch accumulation phase. The high transcript levels of β-amylase 9 (BAM9) during cell division, prior to net starch accumulation, and the correlation between sucrose phosphate synthase (SPS) activity and sucrose suggest the occurrence of sucrose cycling and starch turnover. ADP-Glc pyrophosphorylase (AGPase) is identified as a key enzyme for starch accumulation in kiwifruit berries, as high-starch genotypes had 2- to 5-fold higher AGPase activity, which was maintained over a longer period of time and was also associated with enhanced and extended transcription of the AGPase large subunit 4 (APL4). The data also revealed that SPS and galactinol might affect kiwifruit starch accumulation, and suggest that phloem unloading into kiwifruit is symplastic. These results are relevant to the genetic improvement of quality traits such as sweetness and sugar/acid balance in a range of fruit species.

  2. GST genotypes and lung cancer susceptibility in Asian populations with indoor air pollution exposures: a meta-analysis.

    PubMed

    Hosgood, H Dean; Berndt, Sonja I; Lan, Qing

    2007-01-01

    About half of the world's population is exposed to smoke from heating or cooking with coal, wood, or biomass. These exposures, and fumes from cooking oil use, have been associated with increased lung cancer risk. Glutathione S-transferases play an important role in the detoxification of a wide range of human carcinogens in these exposures. Functional polymorphisms have been identified in the GSTM1, GSTT1, and GSTP1 genes, which may alter the risk of lung cancer among individuals exposed to coal, wood, and biomass smoke, and cooking oil fumes. We performed a meta-analysis of 6 published studies (912 cases; 1063 controls) from regions in Asia where indoor air pollution makes a substantial contribution to lung cancer risk, and evaluated the association between the GSTM1 null, GSTT1 null, and GSTP1 105Val polymorphisms and lung cancer risk. Using a random effects model, we found that carriers of the GSTM1 null genotype had a borderline significant increased lung cancer risk (odds ratio (OR), 1.31; 95% confidence interval (CI), 0.95-1.79; p=0.10), which was particularly evident in the summary risk estimate for the four studies carried out in regions of Asia that use coal for heating and cooking (OR, 1.64; 95% CI, 1.25-2.14; p=0.0003). The GSTT1 null genotype was also associated with an increased lung cancer risk (OR, 1.49; 95% CI, 1.17-1.89; p=0.001), but no association was observed for the GSTP1 105Val allele. Previous meta- and pooled-analyses suggest at most a small association between the GSTM1 null genotype and lung cancer risk in populations where the vast majority of lung cancer is attributed to tobacco, and where indoor air pollution from domestic heating and cooking is much less than in developing Asian countries. Our results suggest that the GSTM1 null genotype may be associated with a more substantial risk of lung cancer in populations with coal exposure.

  3. Population genetics of Neisseria gonorrhoeae in a high-prevalence community using a hypervariable outer membrane porB and 13 slowly evolving housekeeping genes.

    PubMed

    Pérez-Losada, Marcos; Viscidi, Raphael P; Demma, James C; Zenilman, Jonathan; Crandall, Keith A

    2005-09-01

    Baltimore, Md., is an urban community with a high prevalence of Neisseria gonorrhoeae. Due to partially protective immune responses, introduction of new strains from other host populations, and exposure of N. gonorrhoeae to antibiotics, the phenotypic and genotypic characteristics of the circulating strains can fluctuate over time. Understanding the overall genetic diversity and population structure of N. gonorrhoeae is essential for informing public health interventions to eliminate this pathogen. We studied gonococci population genetics in Baltimore by analyzing a hypervariable and strongly selected outer membrane porB gene and 13 slowly evolving and presumably neutral housekeeping genes (abcZ, adk, aroE, fumC, gdh, glnA, gnd, pdhC, pgm, pilA, ppk, pyrD, and serC) in 204 isolates collected in 1991, 1996, and 2001 from male and female patients of two public sexually transmitted diseases clinics. Genetic diversity (), recombination (C), growth (g), population structure, and adaptive selection under codon-substitution and amino acid property models were estimated and compared between these two gene classes. Estimates of the F(ST) fixation index and the chi(2) test of sequence absolute frequencies revealed significant temporal substructuring for both gene types. Baltimore's N. gonorrhoeae populations have increased since 1991 as indicated by consistent positive values of g. Female patients showed similar or lower levels of and C than male patients. Within the MLST housekeeping genes, levels of and C ranged from 0.001-0.013 and 0.000-0.018, respectively. Overall recombination seems to be the dominant force driving evolution in these populations. All loci showed amino acid sites and physicochemical properties under adaptive (or positive-destabilizing) selection, rejecting the generally assumed hypothesis of stabilizing selection for these MLST genes. Within the porB gene, protein I B showed higher and C values than protein I A. Directional positive selection possibly

  4. Analysis of genetic variation in sorghum (Sorghum bicolor (L.) Moench) genotypes with various agronomical traits using SPAR methods.

    PubMed

    Satish, Lakkakula; Shilpha, Jayabalan; Pandian, Subramani; Rency, Arockiam Sagina; Rathinapriya, Periyasamy; Ceasar, Stanislaus Antony; Largia, Muthiah Joe Virgin; Kumar, Are Ashok; Ramesh, Manikandan

    2016-01-15

    Genetic variation among 45 genotypes of sorghum (Sorghum bicolor L.) representing seven subpopulations was assessed using three single primer amplification reaction (SPAR) methods viz., inter-simple sequence repeat (ISSR), random amplified polymorphic DNA (RAPD) and directed amplification of minisatellite-region DNA (DAMD). Totally 15 ISSR, 8 RAPD and 7 DAMD primers generated 263 amplification products, accounting for 84.6% polymorphism across all the genotypes. The Mantel's test of correlation revealed the best correlation between ISSR and cumulative data with a correlation coefficient (r) of 0.84. Assessment of population diversity indicated that the maximum intra population genetic diversity was recorded among high FeZn lines (HFL) having maximum values of Nei's genetic diversity (h) (0.244), Shannon information index (I) (0.368) and the percentage of polymorphic loci (Pp) (72.65%) while the corresponding lowest values of 0.074, 0.109 and 17.95% respectively were observed among the members of MDT subpopulation. The mean coefficient of gene differentiation (GST) and the gene flow (Nm) between populations were observed to be 0.396 and 0.7680 respectively. The analysis of molecular variance (AMOVA) suggested that maximum genetic variation exists within populations (95%) than among populations (5%). Thus the information obtained from this study could be utilized in sorghum breeding programmes for the development of varieties with improved nutrition and agronomic values in future.

  5. Genotype analysis of enterotoxin H-positive Staphylococcus aureus strains isolated from food samples in the Czech Republic.

    PubMed

    Růzicková, Vladislava; Karpísková, Renata; Pantůcek, Roman; Pospísilová, Markéta; Cerníková, Pavla; Doskar, Jirí

    2008-01-15

    Twenty-eight enterotoxin H-positive Staphylococcus aureus strains isolated from food samples collected in eleven districts of the Czech Republic between 2000 and 2005 were genotypically characterized by pulsed-field gel electrophoresis (PFGE) profiling, spa gene polymorphism analysis, enterobacterial repetitive intergenic consensus sequence-based PCR (ERIC-PCR) fingerprinting and prophage carriage detection. These strains accounted for about 21% of the food-derived, staphylococcal enterotoxin (SE)-positive isolates. One strain, detected in feta cheese, was implicated in a case of enterotoxinosis. Sixteen of the twenty-eight isolates carried the seh gene alone. The remaining twelve strains harbored the seh gene in combination with other enterotoxin genes, most often the seg and sei genes, followed by the sea, seb, sec and sed genes. Comparison of various genomic profiles resulted in the determination of twenty genotypes designated G-1 to G-20. Two new, to date not defined, spa types (t2000 and t2002) were identified in one strain isolated from raw meat and two strains obtained from prepacked pizza. Evidence has been given that the seh-positive S. aureus isolates from foodstuffs did not originate from a single source or a common ancestor.

  6. Identification of Novel Immunogenic Proteins of Neisseria gonorrhoeae by Phage Display

    PubMed Central

    Connor, Daniel O.; Zantow, Jonas; Hust, Michael; Bier, Frank F.; von Nickisch-Rosenegk, Markus

    2016-01-01

    Neisseria gonorrhoeae is one of the most prevalent sexually transmitted diseases worldwide with more than 100 million new infections per year. A lack of intense research over the last decades and increasing resistances to the recommended antibiotics call for a better understanding of gonococcal infection, fast diagnostics and therapeutic measures against N. gonorrhoeae. Therefore, the aim of this work was to identify novel immunogenic proteins as a first step to advance those unresolved problems. For the identification of immunogenic proteins, pHORF oligopeptide phage display libraries of the entire N. gonorrhoeae genome were constructed. Several immunogenic oligopeptides were identified using polyclonal rabbit antibodies against N. gonorrhoeae. Corresponding full-length proteins of the identified oligopeptides were expressed and their immunogenic character was verified by ELISA. The immunogenic character of six proteins was identified for the first time. Additional 13 proteins were verified as immunogenic proteins in N. gonorrhoeae. PMID:26859666

  7. An audit of pharyngeal gonorrhoea treatment in a public sexual health clinic in Adelaide, South Australia.

    PubMed

    Hustig, A; Bell, C; Waddell, R

    2013-05-01

    In recent times there have been changes to guidelines regarding the management of gonorrhoea, from both the Centers for Disease Control and Prevention in 2010 and the British Association for Sexual Health and HIV (BASHH) in 2011. Coinciding with their release we conducted a clinical audit of our treatment protocol for gonorrhoea. In 2010, local data on the minimum inhibitory concentrations for Neisseria gonorrhoeae indicated an increase in local isolates that were less sensitive to ceftriaxone (11.6% c.f. 5.3% in 2009). We have a long history of using 250 mg of ceftriaxone to treat all standard sites of gonorrhoea infection followed with tests of cure in all cases. In a retrospective clinical audit of an 11-year period from 2000 up to and including 2010 we identified six test-of-cure failures over 11 years after treating a total of 215 patients with pharyngeal gonorrhoea.

  8. Identification of Novel Immunogenic Proteins of Neisseria gonorrhoeae by Phage Display.

    PubMed

    Connor, Daniel O; Zantow, Jonas; Hust, Michael; Bier, Frank F; von Nickisch-Rosenegk, Markus

    2016-01-01

    Neisseria gonorrhoeae is one of the most prevalent sexually transmitted diseases worldwide with more than 100 million new infections per year. A lack of intense research over the last decades and increasing resistances to the recommended antibiotics call for a better understanding of gonococcal infection, fast diagnostics and therapeutic measures against N. gonorrhoeae. Therefore, the aim of this work was to identify novel immunogenic proteins as a first step to advance those unresolved problems. For the identification of immunogenic proteins, pHORF oligopeptide phage display libraries of the entire N. gonorrhoeae genome were constructed. Several immunogenic oligopeptides were identified using polyclonal rabbit antibodies against N. gonorrhoeae. Corresponding full-length proteins of the identified oligopeptides were expressed and their immunogenic character was verified by ELISA. The immunogenic character of six proteins was identified for the first time. Additional 13 proteins were verified as immunogenic proteins in N. gonorrhoeae.

  9. Identification of Novel Immunogenic Proteins of Neisseria gonorrhoeae by Phage Display.

    PubMed

    Connor, Daniel O; Zantow, Jonas; Hust, Michael; Bier, Frank F; von Nickisch-Rosenegk, Markus

    2016-01-01

    Neisseria gonorrhoeae is one of the most prevalent sexually transmitted diseases worldwide with more than 100 million new infections per year. A lack of intense research over the last decades and increasing resistances to the recommended antibiotics call for a better understanding of gonococcal infection, fast diagnostics and therapeutic measures against N. gonorrhoeae. Therefore, the aim of this work was to identify novel immunogenic proteins as a first step to advance those unresolved problems. For the identification of immunogenic proteins, pHORF oligopeptide phage display libraries of the entire N. gonorrhoeae genome were constructed. Several immunogenic oligopeptides were identified using polyclonal rabbit antibodies against N. gonorrhoeae. Corresponding full-length proteins of the identified oligopeptides were expressed and their immunogenic character was verified by ELISA. The immunogenic character of six proteins was identified for the first time. Additional 13 proteins were verified as immunogenic proteins in N. gonorrhoeae. PMID:26859666

  10. Pharyngeal Neisseria gonorrhoeae detection in oral-throat wash specimens of male patients with urethritis.

    PubMed

    Takahashi, Satoshi; Kurimura, Yuichiro; Hashimoto, Jiro; Takeyama, Koh; Koroku, Mikio; Tanda, Hitoshi; Nishimura, Masahiro; Tsukamoto, Taiji

    2008-12-01

    Detection of Neisseria gonorrhoeae and Chlamydia trachomatis in the pharynx has been highlighted in the prevention of the unexpected spread of sexually transmitted diseases. We tried to clarify the detection rate of Neisseria gonorrhoeae in the pharynx and the clinical relevance of oral-throat wash specimens to detect the organism in heterosexual men with gonococcal and nongonococcal urethritis. In our cohort of 79 male patients with urethritis, oral throat wash specimens were collected after they had gargled with normal saline for approximately 30 to 60 s. Positive pharyngeal N. gonorrhoeae was defined as a positive result on the strand displacement amplification test for the specimen from the oral-throat wash. N. gonorrhoeae was detected in the oral-throat wash specimens of 13 (31.7%) of the 41 male patients with gonococcal urethritis. Oral-throat wash with a nucleic acid amplification test can detect pharyngeal N. gonorrhoeae easily and efficiently.

  11. H-NS suppresses pilE intragenic transcription and antigenic variation in Neisseria gonorrhoeae.

    PubMed

    Masters, Thao L; Wachter, Shaun; Wachter, Jenny; Hill, Stuart A

    2016-01-01

    Initially, pilE transcription in Neisseria gonorrhoeae appeared to be complicated, yet it was eventually simplified into a model where integration host factor activates a single -35/ -10 promoter. However, with the advent of high-throughput RNA sequencing, numerous small pil-specific RNAs (sense as well as antisense) have been identified at the pilE locus as well as at various pilS loci. Using a combination of in vitro transcription, site-directed mutagenesis, Northern analysis and quantitative reverse transcriptase PCR (qRT-PCR) analysis, we have identified three additional non-canonical promoter elements within the pilE gene; two are located within the midgene region (one sense and one antisense), with the third, an antisense promoter, located immediately downstream of the pilE ORF. Using strand-specific qRT-PCR analysis, an inverse correlation exists between the level of antisense expression and the amount of sense message. By their nature, promoter sequences tend to be AT-rich. In Escherichia coli, the small DNA-binding protein H-NS binds to AT-rich sequences and inhibits intragenic transcription. In N. gonorrhoeae hns mutants, pilE antisense transcription was increased twofold, with a concomitant decrease in sense transcript levels. However, most noticeably in these mutants, the absence of H-NS protein caused pilE/pilS recombination to increase dramatically when compared with WT values. Consequently, H-NS protein suppresses pilE intragenic transcription as well as antigenic variation through the pilE/pilS recombination system.

  12. 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

  13. Analysis of the full-length genome of a hepatitis E virus isolate obtained from a wild boar in Japan that is classifiable into a novel genotype.

    PubMed

    Takahashi, Masaharu; Nishizawa, Tsutomu; Sato, Hiroyuki; Sato, Yukihiro; Jirintai; Nagashima, Shigeo; Okamoto, Hiroaki

    2011-04-01

    While performing a nationwide survey of hepatitis E virus (HEV) infection among 450 wild boars (Sus scrofa leucomystax) that had been captured in Japan between November 2005 and March 2010, we found 16 boars (3.6%) with ongoing HEV infection: 11 had genotype 3 HEV, four had genotype 4 HEV and the remaining boar was infected with HEV of an unrecognized genotype (designated wbJOY_06). The entire wbJOY_06 genome was sequenced and was found to comprise 7246 nt excluding the poly(A) tail. The wbJOY_06 isolate was highly divergent from known genotype 1-4 HEV isolates derived from humans, swine, wild boars, deer, mongoose and rabbits (n=145) by 22.6-27.7%, rat HEV isolates (n=2) by 46.0-46.2%, and avian HEV isolates (n=5) by 52.5-53.1% over the entire genome. A Simplot analysis revealed no significant recombination between the existing HEV strains of genotypes 1-4. Therefore, we propose that the wbJOY_06 isolate is the first member of a previously unidentified genotype.

  14. Pleiotropy and genotype by diet interaction: A multivariate genetic analysis of HDL-C subfractions

    SciTech Connect

    Mahaney, M.C.; Blangero, J.; Comuzzie, A.G.

    1994-09-01

    Reduced high density lipoprotein cholesterol (HDL-C) is a risk factor for cardiovascular disease in humans. Both major genes and major genotype by diet interaction have been reported for HDL-C, but the genetics of the HDL-C subfractions are less well known. In a baboon model for human atherosclerosis, we investigated the pleiotropic effects of genes on normal quantitative variation in three HDL-C subfractions (HDL{sub 1}-C, HDL{sub 2}-C, and HDL{sub 3}-C) in two dietary environments -- a basal diet and a 7 week high cholesterol, saturated fat (HCSF) diet. We analyzed data on serum HDL-C subfraction levels, quantified by gradient gel eletrophoresis, for 942 baboons (Papo hamadryas, sensu lato) from 17 pedigrees. We used multivariate maximum likelihood methods to simultaneously estimate phenotypic means, standard deviations, and heritabilities (h{sup 2}); effects of sex, age-by-sex, age{sup 2}-by-sex, percent subspecies admixture, and infant feeding modality; plus estimated significant h{sup 2} values for all three subfractions on both diets. When tested within dietary environments, we obtained significant genetic correlations between all three subfractions [i.e., P({rho}{sub G} = 0) < 0.001] and evidence of complete pleiotropy [i.e., P({vert_bar}{rho}{sub G}{vert_bar} = 1.0) > 0.1] between HDL{sub 1}-C and HDL{sub 3}-C ({rho}{sub G} = 0.81) on the basal diet. On the HCSF diet, only the genetic correlation between HDL{sub 1}-C and HDL{sub 3}-C ({rho}{sub g} = 0.61) was significant (p > 0.1). Complete pleiotropy was observed for each of the three subfractions between both diets. Given these results, we reject genotype by diet interaction for HDL{sub 1}-C, HDL{sub 2}-C or HDL{sub 3}-C; i.e., the same genes influence variation in each subfraction to the same degree on either diet. However, the apparent disruption of pleiotropy between HDL{sub 2}-C and the other two subfractions needs to be investigated further.

  15. Proteins that appear to be associated with pili in Neisseria gonorrhoeae.

    PubMed Central

    Muir, L L; Strugnell, R A; Davies, J K

    1988-01-01

    Pili of Neisseria gonorrhoeae are thought to be composed entirely of identical subunits, called pilin, that self-assemble in vitro. Previous pilus purification methods have relied on this latter point, and dissociation and reassociation of pilin subunits has yielded pilin preparations of high purity. Such a procedure could result in the loss of any pilus-associated proteins. We have developed a procedure for the isolation of intact native pili in a deoxycholate-urea buffer in which the pili are fractionated on the basis of size and hydrophobicity. Electron microscopy indicates that the pili are largely free from outer membrane vesicles and other cellular material. Electrophoretic analysis has shown that a number of proteins copurify with pilin. Antibodies to these proteins could be removed from an antiserum against whole piliated cells by absorption with piliated cells but not by absorption with nonpiliated cells. Hence, our results indicate that these proteins could be pilus associated. Images PMID:2898429

  16. The U.S. military's Neisseria gonorrhoeae resistance surveillance initiatives in selected populations of five countries.

    PubMed

    Tsai, Alice Y; Dueger, Erica; Macalino, Grace E; Montano, Silvia M; Tilley, Drake H; Mbuchi, Margaret; Wurapa, Eyako K; Saylors, Karen; Duplessis, Christopher C; Puplampu, Naiki; Garges, Eric C; McClelland, R Scott; Sanchez, Jose L

    2013-02-01

    Multi-drug resistant Neisseria gonorrhoeae (GC) threatens the successful treatment of gonorrhea. This report presents preliminary findings with regard to the prevalence of laboratory-confirmed GC and the extent of drug-resistance among sample populations in five countries. Between October 2010 and January 2013, 1,694 subjects (54% male; 45% female; 1% unknown) were enrolled and screened for the presence of laboratory-confirmed GC in the United States, Djibouti, Ghana, Kenya, and Peru. Overall, 108 (6%) of enrolled subjects tested positive for GC. Antimicrobial susceptibility testing results were available for 66 GC isolates. Resistance to at least three antibiotics was observed at each overseas site. All isolates tested in Ghana (n=6) were resistant to ciprofloxacin, penicillin, and tetracycline. In Djibouti, preliminary results suggested resistance to penicillin, tetracycline, ciprofloxacin, cefepime, and ceftriaxone. The small sample size and missing data prevent comparative analysis and limit the generalizability of these preliminary findings. PMID:23461308

  17. 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

  18. 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

  19. Genotypic analysis of Mycobacterium tuberculosis isolates from a Lisbon hospital in Portugal.

    PubMed

    Perdigão, João; Milho, Catarina; Carrilho, Lurdes; Brum, Laura; Portugal, Isabel

    2009-01-01

    Portugal has one of the highest tuberculosis notification rates of the European Union with Lisbon Health Region having an incidence rate well above the national average. The present study analyses the transmission, drug susceptibility and characteristics of a study population from a Central Lisbon's Hospital. One hundred and thirty -two Mycobacterium tuberculosis clinical isolates were previously tested for drug susceptibility to first -line drugs. The multidrug (MDR) resistance rate was found to be 3.0%, while 13.6% of the isolates were resistant to one or more first -line drugs. HIV serology was available for 98 patients, 26 (26.5%) were positive. Genotyping was performed by MIRU -VNTR and 53 (40.2%) out of the 132 isolates were found to be distributed through 17 MIRU -VNTR clusters of two or more isolates. Lisboa strains accounted for 25.8% of all strains. We conclude that transmission of resistant and susceptible Mycobacterium tuberculosis strains is occurring, with special concern for Lisboa strains.

  20. 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.

  1. 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

  2. SSR genotyping.

    PubMed

    Mason, Annaliese S

    2015-01-01

    SSR genotyping involves the use of simple sequence repeats (SSRs) as DNA markers. SSRs, also called microsatellites, are a type of repetitive DNA sequence ubiquitous in most plant genomes. SSRs contain repeats of a motif sequence 1-6 bp in length. Due to this structure SSRs frequently undergo mutations, mainly due to DNA polymerase errors, which involve the addition or subtraction of a repeat unit. Hence, SSR sequences are highly polymorphic and may be readily used for detection of allelic variation within populations. SSRs are present within both genic and nongenic regions and are occasionally transcribed, and hence may be identified in expressed sequence tags (ESTs) as well as more commonly in nongenic DNA sequences. SSR genotyping involves the design of DNA-based primers to amplify SSR sequences from extracted genomic DNA, followed by amplification of the SSR repeat region using polymerase chain reaction, and subsequent visualization of the resulting DNA products, usually using gel electrophoresis. These procedures are described in this chapter. SSRs have been one of the most favored molecular markers for plant genotyping in the last 20 years due to their high levels of polymorphism, wide distribution across most plant genomes, and ease of use and will continue to be a useful tool in many species for years to come.

  3. Clostridium botulinum Group I Strain Genotyping by 15-Locus Multilocus Variable-Number Tandem-Repeat Analysis ▿ †

    PubMed Central

    Fillo, Silvia; Giordani, Francesco; Anniballi, Fabrizio; Gorgé, Olivier; Ramisse, Vincent; Vergnaud, Gilles; Riehm, Julia M.; Scholz, Holger C.; Splettstoesser, Wolf D.; Kieboom, Jasper; Olsen, Jaran-Strand; Fenicia, Lucia; Lista, Florigio

    2011-01-01

    Clostridium botulinum is a taxonomic designation that encompasses a broad variety of spore-forming, Gram-positive bacteria producing the botulinum neurotoxin (BoNT). C. botulinum is the etiologic agent of botulism, a rare but severe neuroparalytic disease. Fine-resolution genetic characterization of C. botulinum isolates of any BoNT type is relevant for both epidemiological studies and forensic microbiology. A 10-locus multiple-locus variable-number tandem-repeat analysis (MLVA) was previously applied to isolates of C. botulinum type A. The present study includes five additional loci designed to better address proteolytic B and F serotypes. We investigated 79 C. botulinum group I strains isolated from human and food samples in several European countries, including types A (28), B (36), AB (4), and F (11) strains, and 5 nontoxic Clostridium sporogenes. Additional data were deduced from in silico analysis of 10 available fully sequenced genomes. This 15-locus MLVA (MLVA-15) scheme identified 86 distinct genotypes that clustered consistently with the results of amplified fragment length polymorphism (AFLP) and MLVA genotyping in previous reports. An MLVA-7 scheme, a subset of the MLVA-15, performed on a lab-on-a-chip device using a nonfluorescent subset of primers, is also proposed as a first-line assay. The phylogenetic grouping obtained with the MLVA-7 does not differ significantly from that generated by the MLVA-15. To our knowledge, this report is the first to analyze genetic variability among all of the C. botulinum group I serotypes by MLVA. Our data provide new insights into the genetic variability of group I C. botulinum isolates worldwide and demonstrate that this group is genetically highly diverse. PMID:22012011

  4. Clostridium botulinum group I strain genotyping by 15-locus multilocus variable-number tandem-repeat analysis.

    PubMed

    Fillo, Silvia; Giordani, Francesco; Anniballi, Fabrizio; Gorgé, Olivier; Ramisse, Vincent; Vergnaud, Gilles; Riehm, Julia M; Scholz, Holger C; Splettstoesser, Wolf D; Kieboom, Jasper; Olsen, Jaran-Strand; Fenicia, Lucia; Lista, Florigio

    2011-12-01

    Clostridium botulinum is a taxonomic designation that encompasses a broad variety of spore-forming, Gram-positive bacteria producing the botulinum neurotoxin (BoNT). C. botulinum is the etiologic agent of botulism, a rare but severe neuroparalytic disease. Fine-resolution genetic characterization of C. botulinum isolates of any BoNT type is relevant for both epidemiological studies and forensic microbiology. A 10-locus multiple-locus variable-number tandem-repeat analysis (MLVA) was previously applied to isolates of C. botulinum type A. The present study includes five additional loci designed to better address proteolytic B and F serotypes. We investigated 79 C. botulinum group I strains isolated from human and food samples in several European countries, including types A (28), B (36), AB (4), and F (11) strains, and 5 nontoxic Clostridium sporogenes. Additional data were deduced from in silico analysis of 10 available fully sequenced genomes. This 15-locus MLVA (MLVA-15) scheme identified 86 distinct genotypes that clustered consistently with the results of amplified fragment length polymorphism (AFLP) and MLVA genotyping in previous reports. An MLVA-7 scheme, a subset of the MLVA-15, performed on a lab-on-a-chip device using a nonfluorescent subset of primers, is also proposed as a first-line assay. The phylogenetic grouping obtained with the MLVA-7 does not differ significantly from that generated by the MLVA-15. To our knowledge, this report is the first to analyze genetic variability among all of the C. botulinum group I serotypes by MLVA. Our data provide new insights into the genetic variability of group I C. botulinum isolates worldwide and demonstrate that this group is genetically highly diverse.

  5. New cryptosporidium genotypes in HIV-infected persons.

    PubMed Central

    Pieniazek, N. J.; Bornay-Llinares, F. J.; Slemenda, S. B.; da Silva, A. J.; Moura, I. N.; Arrowood, M. J.; Ditrich, O.; Addiss, D. G.

    1999-01-01

    Using DNA sequencing and phylogenetic analysis, we identified four distinct Cryptosporidium genotypes in HIV-infected patients: genotype 1 (human), genotype 2 (bovine) Cryptosporidium parvum, a genotype identical to C. felis, and one identical to a Cryptosporidium sp. isolate from a dog. This is the first identification of human infection with the latter two genotypes. PMID:10341184

  6. Genetic signatures of Mycobacterium tuberculosis Nonthaburi genotype revealed by whole genome analysis of isolates from tuberculous meningitis patients in Thailand

    PubMed Central

    Coker, Olabisi Oluwabukola; Ngamphiw, Chumpol; Tongsima, Sissades; Regmi, Sanjib Mani; Clark, Taane G.; Ong, Rick Twee Hee; Teo, Yik-Ying; Prammananan, Therdsak; Palittapongarnpim, Prasit

    2016-01-01

    Genome sequencing plays a key role in understanding the genetic diversity of Mycobacterium tuberculosis (M.tb). The genotype-specific character of M. tb contributes to tuberculosis severity and emergence of drug resistance. Strains of M. tb complex can be classified into seven lineages. The Nonthaburi (NB) genotype, belonging to the Indo-Oceanic lineage (lineage 1), has a unique spoligotype and IS6110-RFLP pattern but has not previously undergone a detailed whole genome analysis. In addition, there is not much information available on the whole genome analysis of M. tb isolates from tuberculous meningitis (TBM) patients in public databases. Isolates CSF3053, 46-5069 and 43-13838 of NB genotype were obtained from the cerebrospinal fluids of TBM Thai patients in Siriraj Hospital, Bangkok. The whole genomes were subjected to high throughput sequencing. The sequence data of each isolate were assembled into draft genome. The sequences were also aligned to reference genome, to determine genomic variations. Single nucleotide polymorphisms (SNPs) were obtained and grouped according to the functions of the genes containing them. They were compared with SNPs from 1,601 genomes, representing the seven lineages of M. tb complex, to determine the uniqueness of NB genotype. Susceptibility to first-line, second-line and other antituberculosis drugs were determined and related to the SNPs previously reported in drug-resistant related genes. The assembled genomes have an average size of 4,364,461 bp, 4,154 genes, 48 RNAs and 64 pseudogenes. A 500 base pairs deletion, which includes ppe50, was found in all isolates. RD239, specific for members of Indo Oceanic lineage, and RD147c were identified. A total of 2,202 SNPs were common to the isolates and used to classify the NB strains as members of sublineage 1.2.1. Compared with 1,601 genomes from the seven lineages of M. tb complex, mutation G2342203C was found novel to the isolates in this study. Three mutations (T28910C, C1180580T

  7. Direct Analysis in Real Time by Mass Spectrometric Technique for Determining the Variation in Metabolite Profiles of Cinnamomum tamala Nees and Eberm Genotypes

    PubMed Central

    Singh, Vineeta; Gupta, Atul Kumar; Singh, S. P.; Kumar, Anil

    2012-01-01

    Cinnamomum tamala Nees & Eberm. is an important traditional medicinal plant, mentioned in various ancient literatures such as Ayurveda. Several of its medicinal properties have recently been proved. To characterize diversity in terms of metabolite profiles of Cinnamomum tamala Nees and Eberm genotypes, a newly emerging mass spectral ionization technique direct time in real time (DART) is very helpful. The DART ion source has been used to analyze an extremely wide range of phytochemicals present in leaves of Cinnamomum tamala. Ten genotypes were assessed for the presence of different phytochemicals. Phytochemical analysis showed the presence of mainly terpenes and phenols. These constituents vary in the different genotypes of Cinnamomum tamala. Principal component analysis has also been employed to analyze the DART data of these Cinnamomum genotypes. The result shows that the genotype of Cinnamomum tamala could be differentiated using DART MS data. The active components present in Cinnamomum tamala may be contributing significantly to high amount of antioxidant property of leaves and, in turn, conditional effects for diabetic patients. PMID:22701361

  8. Identification of unbalanced genome copy number abnormalities in patients with multiple myeloma by single-nucleotide polymorphism genotyping microarray analysis.

    PubMed

    Kamada, Yuhei; Sakata-Yanagimoto, Mamiko; Sanada, Masashi; Sato-Otsubo, Aiko; Enami, Terukazu; Suzukawa, Kazumi; Kurita, Naoki; Nishikii, Hidekazu; Yokoyama, Yasuhisa; Okoshi, Yasushi; Hasegawa, Yuichi; Ogawa, Seishi; Chiba, Shigeru

    2012-10-01

    Single-nucleotide polymorphism genotyping microarray (SNP array) analysis provides detailed information on chromosomal copy number aberrations. To obtain detailed information on genomic abnormalities related to pathogenesis or prognosis of multiple myeloma (MM), we performed 250K SNP array analysis in 39 MM patients and 11 cell lines. We identified an accumulation of deletions and uniparental disomies at 22q12.1. Among the hyperdiploid MM cases, chromosomal imbalance at this locus was associated with poor prognosis. On sequencing, we also found a mutation in the seizure-related 6 homolog (mouse)-like (SEZ6L) gene located at ch.22q12.1 in an MM cell line, NOP1. We further found isolated deletions in 17 genes, five of which are known tumor suppressor genes. Of these, deletion of protein tyrosine phosphatase, receptor type D (PTPRD) was found in three samples, including two patients. Consistent with previous reports, non-hyperdiploid MM, deletion of 13q (del13q) and gain of 1q in non-hyperdiploid MMs were predictive of poor prognosis (p = 0.039, p = 0.049, and p = 0.013, respectively). However, our analysis revealed that unless accompanied by gain of 1q, the prognosis of non-hyperdiploid MM was as good as that of hyperdiploid MM. Thus, SNP array analysis provides significant information useful to understanding the pathogenesis and prognosis of MM.

  9. Evaluation of a Susceptibility Gene for Schizophrenia: Genotype Based Meta-Analysis of RGS4 Polymorphisms from Thirteen Independent Samples

    PubMed Central

    Talkowski, Michael E.; Seltman, Howard; Bassett, Anne S.; Brzustowicz, Linda M.; Chen, Xiangning; Chowdari, Kodavali V.; Collier, David A.; Cordeiro, Quirino; Corvin, Aiden P.; Deshpande, Smita N.; Egan, Michael F.; Gill, Michael; Kendler, Kenneth S.; Kirov, George; Heston, Leonard L.; Levitt, Pat; Lewis, David A.; Li, Tao; Mirnics, Karoly; Morris, Derek W.; Norton, Nadine; O’Donovan, Michael C.; Owen, Michael J.; Richard, Christian; Semwal, Prachi; Sobell, Janet L.; Clair, David St; Straub, Richard E.; Thelma, B.K.; Vallada, Homero; Weinberger, Daniel R.; Williams, Nigel M.; Wood, Joel; Zhang, Feng; Devlin, Bernie; Nimgaonkar, Vishwajit L.

    2011-01-01

    Background Associations between schizophrenia (SCZ) and polymorphisms at the regulator of G-protein signaling 4 (RGS4) gene have been reported (single nucleotide polymorphisms [SNPs] 1, 4, 7, and 18). Yet, similar to other SCZ candidate genes, studies have been inconsistent with respect to the associated alleles. Methods In an effort to resolve the role for RGS4 in SCZ susceptibility, we undertook a genotype-based meta-analysis using both published and unpublished family-based and case-control samples (total n = 13,807). Results The family-based dataset consisted of 10 samples (2160 families). Significant associations with individual SNPs/haplotypes were not observed. In contrast, global analysis revealed significant transmission distortion (p = .0009). Specifically, analyses suggested overtransmission of two common haplotypes that account for the vast majority of all haplotypes. Separate analyses of 3486 cases and 3755 control samples (eight samples) detected a significant association with SNP 4 (p = .01). Individual haplotype analyses were not significant, but evaluation of test statistics from individual samples suggested significant associations. Conclusions Our collaborative meta-analysis represents one of the largest SCZ association studies to date. No individual risk factor arose from our analyses, but interpretation of these results is not straightforward. Our analyses suggest risk due to at least two common haplotypes in the presence of heterogeneity. Similar analysis for other putative susceptibility genes is warranted. PMID:16631129

  10. Genotypic and phenotypic analysis of S. mutans isolated from dental biofilms formed in vivo under high cariogenic conditions.

    PubMed

    Arthur, Rodrigo Alex; Cury, Altair Antoninha Del Bel; Graner, Renata Oliveira Mattos; Rosalen, Pedro Luiz; Vale, Gláuber Campos; Paes Leme, Adriana Franco; Cury, Jaime Aparecido; Tabchoury, Cínthia Pereira Machado

    2011-01-01

    The oral cavity harbors several Streptococcus mutans genotypes, which could present distinct virulence properties. However, little is known about the diversity and virulence traits of S. mutans genotypes isolated in vivo under controlled conditions of high cariogenic challenge. This study evaluated the genotypic diversity of S. mutans isolated from dental biofilms formed in vivo under sucrose exposure, as well as their acidogenicity and aciduricity. To form biofilms, subjects rinsed their mouths with distilled water or sucrose solution 8 times/day for 3 days. S. mutans collected from saliva and biofilms were genotyped by arbitrarily-primed PCR. Genotypes identified in the biofilms were evaluated regarding their ability to lower the suspension pH through glycolysis and their acid susceptibility and F-ATPase activity. Most subjects harbored only one genotype in saliva, which was detected in almost all biofilm samples at high proportions. Genotypes isolated only in the presence of sucrose had higher acidogenicity than those isolated only in the presence of water. Genotypes from biofilms formed with sucrose were more aciduric after 30 and 60 min of incubation at pH 2.8 and 5.0, respectively. The present results suggest that biofilms formed under high cariogenic conditions may harbor more aciduric and acidogenic S. mutans genotypes.

  11. Pili-Induced Clustering of N. gonorrhoeae Bacteria.

    PubMed

    Taktikos, Johannes; Lin, Yen Ting; Stark, Holger; Biais, Nicolas; Zaburdaev, Vasily

    2015-01-01

    Type IV pili (Tfp) are prokaryotic retractable appendages known to mediate surface attachment, motility, and subsequent clustering of cells. Tfp are the main means of motility for Neisseria gonorrhoeae, the causative agent of gonorrhea. Tfp are also involved in formation of the microcolonies, which play a crucial role in the progression of the disease. While motility of individual cells is relatively well understood, little is known about the dynamics of N. gonorrhoeae aggregation. We investigate how individual N. gonorrhoeae cells, initially uniformly dispersed on flat plastic or glass surfaces, agglomerate into spherical microcolonies within hours. We quantify the clustering process by measuring the area fraction covered by the cells, number of cell aggregates, and their average size as a function of time. We observe that the microcolonies are also able to move but their mobility rapidly vanishes as the size of the colony increases. After a certain critical size they become immobile. We propose a simple theoretical model which assumes a pili-pili interaction of cells as the main clustering mechanism. Numerical simulations of the model quantitatively reproduce the experimental data on clustering and thus suggest that the agglomeration process can be entirely explained by the Tfp-mediated interactions. In agreement with this hypothesis mutants lacking pili are not able to form colonies. Moreover, cells with deficient quorum sensing mechanism show similar aggregation as the wild-type bacteria. Therefore, our results demonstrate that pili provide an essential mechanism for colony formation, while additional chemical cues, for example quorum sensing, might be of secondary importance. PMID:26355966

  12. HPV prevalence and genotypes in different histological subtypes of cervical adenocarcinoma, a worldwide analysis of 760 cases.

    PubMed

    Pirog, Edyta C; Lloveras, Belen; Molijn, Anco; Tous, Sara; Guimerà, Núria; Alejo, Maria; Clavero, Omar; Klaustermeier, Joellen; Jenkins, David; Quint, Wim Gv; Xavier Bosch, Francesc; Alemany, Laia; de Sanjosé, Silvia

    2014-12-01

    The goal of our study was to provide comprehensive data on the worldwide human papillomavirus (HPV) genotype distribution in patients with invasive cervical adenocarcinoma in correlation with histologic tumor subtypes, geographical location, patients' age, and duration of sample storage. Paraffin-embedded samples of 760 cervical adenocarcinoma cases were collected worldwide. A three-level pathology review of cases was performed to obtain consensus histologic diagnoses and 682 cases were determined to be eligible for further analysis. HPV DNA detection and genotyping was performed using SPF-10/DEIA/LiPA(25) system (version 1). Classic cervical adenocarcinoma accounted for 83.1% of cases, while rare histological variants accounted for a few percent of cases individually. HPV positivity varied significantly between the different histologic tumor subtypes. Classic cervical adenocarcinoma showed high HPV positivity (71.8%), while other adenocarcinoma types had significantly lower HPV prevalence (endometrioid 27.3%, serous 25%, clear cell 20%, not otherwise specified 13.9%, and minimal deviation 8.3%). In all, 91.8% of HPV-positive tumors showed the presence of a single viral type and in 7% of cases multiple viral types were detected. Three HPV genotypes, HPV 16, 18, and 45, dominated in all adenocarcinomas and together accounted for 94.1% of HPV-positive tumors. HPV16 was the most common and found in 50.9% of HPV-positive cases, followed by HPV18 (31.6%) and HPV45 (11.6%). HPV prevalence varied depending on geographical region, patient age, and sample storage time. Tumors from older patients and tumor samples with longer storage time showed lower HPV prevalence. Our results indicate that HPV vaccines may prevent up to 82.5% (HPV16/18) and up to 95.3% (9-valent vaccine) of HPV-positive cervical adenocarcinomas, mostly the classic type. HPV testing and vaccination will not provide full coverage for a very small subset of classical adenocarcinomas and most of the rare

  13. Comparison of Antimicrobial Susceptibility of Urogenital Neisseria gonorrhoeae Isolates Obtained from Women and Men

    PubMed Central

    Kidd, Sarah; Moore, Page C.; Kirkcaldy, Robert D.; Philip, Susan S.; Wiesenfeld, Harold C.; Papp, John R.; Kerndt, Peter R.; Venkatasubramanian, Lalitha; Ghanem, Khalil G.; Hook, Edward W.

    2015-01-01

    Background The United States’ (US) system for gonococcal antimicrobial susceptibility surveillance monitors trends exclusively among men with urethral infection, the population from whom the yield of gonococcal culture is highest. Little is known about the susceptibility of female urogenital isolates, and it is unclear whether gonococcal susceptibility among men who report sex exclusively with women (MSW) is representative of susceptibility among women. Methods Using isolates collected during a recent treatment trial in five US cities, we performed a secondary analysis to compare antimicrobial susceptibilities of N. gonorrhoeae urogenital isolates obtained from women, MSW, and men who have sex with men (MSM). Pre-treatment isolates were collected from trial participants; minimum inhibitory concentrations (MICs) were determined by agar dilution. Geometric mean MICs were adjusted for geographic location using general linear models. Results Susceptibility data for urogenital isolates from 56 women, 252 MSW, and 170 MSM were studied. The adjusted geometric mean ceftriaxone MIC was similar among women (0.0067 μg/ml, 95% CI 0.0049–0.0092 μg/ml) and MSW (0.0060 μg/ml, 95% CI 0.0053–0.0066 μg/ml). In contrast, the adjusted geometric mean ceftriaxone MIC was higher among MSM (0.0098 μg/ml, 95% CI 0.0082–0.0119 μg/ml) than MSW. This same pattern was observed for other antimicrobials, including cefixime and azithromycin Conclusions Ceftriaxone, cefixime, and azithromycin MICs were higher among MSM than MSW, but were similar among women and MSW. These findings suggest that gonococcal antimicrobial susceptibility surveillance based on urethral isolates from MSW may adequately represent susceptibility of urogenital N. gonorrhoeae in women. PMID:26165435

  14. Prevalence of Hepatitis C Virus Genotypes Among Patients in Countries of the Eastern Mediterranean Regional Office of WHO (EMRO): A Systematic Review and Meta-Analysis

    PubMed Central

    Sadeghi, Farzin; Salehi-Vaziri, Mostafa; Almasi-Hashiani, Amir; Gholami-Fesharaki, Mohammad; Pakzad, Reza; Alavian, Seyed Moayed

    2016-01-01

    Context Hepatitis C virus (HCV) infection is a major global public health issue. The Eastern Mediterranean regional office (EMRO) of the world health organization (WHO) seems to have one of the highest prevalence rates worldwide, with at least 21.3 million HCV-infected patients. Objectives The aim of the present study was to review systematically all epidemiological data related to the prevalence of HCV genotypes in infected patients in EMRO countries. Data Sources A systematic search was conducted of peer-reviewed journals indexed in electronic databases (PubMed, Scopus, ISI, PakMediNet, and IMEMR, and Persian-specific databases including SID, Iran Medex, and MagIran). Study Selection A systematic search was performed with temporal limits (papers published between January 2000 up to June 2015), regarding the prevalence and distribution of HCV genotypes in EMRO countries. Data Extraction The prevalence rates of HCV genotypes were pooled by metan command in Stata 14. Statistical heterogeneity was explored using the I-square at the 5% significance level. Publication bias was assessed, graphically and statistically, by funnel plot and Begg and Egger tests. Results A total of 563 records were identified through the electronic search. Of these records, 134 studies comprising 67681 HCV-infected individuals were included in the meta-analysis. In Iran, subtype 1a was the predominant subtype with a rate of 42% (95% CI, 39 - 46), followed by subtype 3a, 35% (95% CI, 31 - 38). In Pakistan, Subtype 3a was the most common subtype with a rate of 56% (95% CI, 49 - 62), followed by subtype 3b, 10% (95% CI, 7 - 12). In Saudi Arabia and Egypt, genotype 4 was the most prevalent genotype with a rate of 65% (95% CI, 59 - 72) and 69% (95% CI, 36 - 100) respectively. In Tunisia and Morocco, subtype 1b was the most common subtype with a rate of 69% (95% CI, 50 - 88) and 32% (95% CI, 7 - 56) respectively. Conclusions The genotype distribution of HCV takes diverse patterns in EMRO countries

  15. [Antimicrobal resistance of Neisseria gonorrhoeae strains in Hungary].

    PubMed

    Nemes-Nikodém, Éva; Brunner, Alexandra; Tóth, Béla; Tóth, Veronika; Bánvölgyi, András; Ostorházi, Eszter

    2015-02-01

    Bevezetés: A Neisseria gonorrhoeae-infekciók kezelésére kiadott európai ajánlás elsősorban a nyugat-európai adatok alapján készült, és nem egyértelműen használható a magyarországi helyzet ismeretében. Célkitűzés: A szerzők 2011. január és 2014. június közötti időszakban a Semmelweis Egyetem, Bőr-, Nemikórtani és Bőronkológiai Klinika Országos Szexuális Úton Terjedő Betegségek Centrumában izolált Neisseria gonorrhoeae törzsek rezisztenciaadatait összevetették az izolált törzsek molekuláris tipizálási eredményeivel, azzal a céllal, hogy pontos adatokat kapjanak a hazánkban előforduló Neisseria gonorrhoeae törzsek antimikrobiális rezisztenciájáról. Módszer: Az antibiotikumrezisztencia-meghatározás minimális inhibitorkoncentráció-méréssel, a szekvenciameghatározás a Neisseria gonorrhoeae Multi Antigen Sequence Typing módszerrel történt. Eredmények: A jelenleg terápiának ajánlott széles spektrumú cefalosporinok elleni rezisztencia ritka, az utóbbi években az azithromycinrezisztencia előfordulása viszont rohamosan növekedett. Következtetések: Az új terápiás irányelvek készítésekor figyelembe kell venni, hogy a gyakran fertőzést okozó molekuláris típusba sorolható törzsek között kiemelkedően magas az azithromycinrezisztensek aránya. Orv. Hetil., 2015, 156(6), 226–229.

  16. Production of Neisseria gonorrhoeae pili (fimbriae) in Pseudomonas aeruginosa.

    PubMed Central

    Hoyne, P A; Haas, R; Meyer, T F; Davies, J K; Elleman, T C

    1992-01-01

    Pseudomonas aeruginosa K/2PfS, when transformed with an expression plasmid harboring the pilin gene (pilE1) of Neisseria gonorrhoeae MS11, was able to express and assemble gonococcal pilin monomers into surface-associated pili, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and immunoelectron microscopy. Concomitant with the expression of gonococcal pili in P. aeruginosa was the virtual loss of production of P. aeruginosa K/2PfS pili normally associated with the host cell. Images PMID:1358873

  17. Dense cultures of Neisseria gonorrhoeae in liquid medium.

    PubMed

    Brookes, R; Hedén, C G

    1967-03-01

    Cultivation of Neisseria gonorrhoeae was effected in a conical glass culture vessel surrounded by a constant-temperature water jacket, and with facilities for stirring, aeration, and pH measurement and control. With the use of an aerated peptone-based medium, containing polypropylene glycol to prevent foam build-up, the yields obtained over the pH range from 5.8 to 7.4 were determined. The greatest yield was obtained at pH 6.4 when the dry weight was 1.5 g/liter. At pH 7.2 to 7.6, lysis was extensive.

  18. Genome Wide Analysis of Flowering Time Trait in Multiple Environments via High-Throughput Genotyping Technique in Brassica napus L.

    PubMed Central

    Dalton-Morgan, Jessica; Batley, Jacqueline; Yu, Longjiang; Meng, Jinling; Li, Maoteng

    2015-01-01

    The prediction of the flowering time (FT) trait in Brassica napus based on genome-wide markers and the detection of underlying genetic factors is important not only for oilseed producers around the world but also for the other crop industry in the rotation system in China. In previous studies the low density and mixture of biomarkers used obstructed genomic selection in B. napus and comprehensive mapping of FT related loci. In this study, a high-density genome-wide SNP set was genotyped from a double-haploid population of B. napus. We first performed genomic prediction of FT traits in B. napus using SNPs across the genome under ten environments of three geographic regions via eight existing genomic predictive models. The results showed that all the models achieved comparably high accuracies, verifying the feasibility of genomic prediction in B. napus. Next, we performed a large-scale mapping of FT related loci among three regions, and found 437 associated SNPs, some of which represented known FT genes, such as AP1 and PHYE. The genes tagged by the associated SNPs were enriched in biological processes involved in the formation of flowers. Epistasis analysis showed that significant interactions were found between detected loci, even among some known FT related genes. All the results showed that our large scale and high-density genotype data are of great practical and scientific values for B. napus. To our best knowledge, this is the first evaluation of genomic selection models in B. napus based on a high-density SNP dataset and large-scale mapping of FT loci. PMID:25790019

  19. Genome wide analysis of flowering time trait in multiple environments via high-throughput genotyping technique in Brassica napus L.

    PubMed

    Li, Lun; Long, Yan; Zhang, Libin; Dalton-Morgan, Jessica; Batley, Jacqueline; Yu, Longjiang; Meng, Jinling; Li, Maoteng

    2015-01-01

    The prediction of the flowering time (FT) trait in Brassica napus based on genome-wide markers and the detection of underlying genetic factors is important not only for oilseed producers around the world but also for the other crop industry in the rotation system in China. In previous studies the low density and mixture of biomarkers used obstructed genomic selection in B. napus and comprehensive mapping of FT related loci. In this study, a high-density genome-wide SNP set was genotyped from a double-haploid population of B. napus. We first performed genomic prediction of FT traits in B. napus using SNPs across the genome under ten environments of three geographic regions via eight existing genomic predictive models. The results showed that all the models achieved comparably high accuracies, verifying the feasibility of genomic prediction in B. napus. Next, we performed a large-scale mapping of FT related loci among three regions, and found 437 associated SNPs, some of which represented known FT genes, such as AP1 and PHYE. The genes tagged by the associated SNPs were enriched in biological processes involved in the formation of flowers. Epistasis analysis showed that significant interactions were found between detected loci, even among some known FT related genes. All the results showed that our large scale and high-density genotype data are of great practical and scientific values for B. napus. To our best knowledge, this is the first evaluation of genomic selection models in B. napus based on a high-density SNP dataset and large-scale mapping of FT loci.

  20. QTL analysis of the spring wheat "Chapio" identifies stable stripe rust resistance despite inter-continental genotype × environment interactions.

    PubMed

    Yang, E-N; Rosewarne, G M; Herrera-Foessel, S A; Huerta-Espino, J; Tang, Z-X; Sun, C-F; Ren, Z-L; Singh, R P

    2013-07-01

    Chapio is a spring wheat developed by CIMMYT in Mexico by a breeding program that focused on multigenic resistances to leaf rust and stripe rust. A population consisting of 277 recombinant inbred lines (RILs) was developed by crossing Chapio with Avocet. The RILs were genotyped with DArT markers (137 randomly selected RILs) and bulked segregant analysis conducted to supplement the map with informative SSR markers. The final map consisted of 264 markers. Phenotyping against stripe rust was conducted for three seasons in Toluca, Mexico and at three sites over two seasons (total of four environments) in Sichuan Province, China. Significant loci across the two inter-continental regions included Lr34/Yr18 on 7DS, Sr2/Yr30 on 3BS, and a QTL on 3D. There were significant genotype × environment interactions with resistance gene Yr31 on 2BS being effective in most of the Toluca environments; however, a late incursion of a virulent pathotype in 2009 rendered this gene ineffective. This locus also had no effect in China. Conversely, a 5BL locus was only effective in the Chinese environments. There were also complex additive interactions. In the Mexican environments, Yr31 suppressed the additive effect of Yr30 and the 3D locus, but not of Lr34/Yr18, while in China, the 3D and 5BL loci were generally not additive with each other, but were additive when combined with other loci. These results indicate the importance of maintaining diverse, multi-genic resistances as Chapio had stable inter-continental resistance despite the fact that there were QTLs that were not effective in either one or the other region.

  1. Distinct defensin profiles in Neisseria gonorrhoeae and Chlamydia trachomatis urethritis reveal novel epithelial cell-neutrophil interactions.

    PubMed

    Porter, Edith; Yang, Huixia; Yavagal, Sujata; Preza, Gloria C; Murillo, Omar; Lima, Heriberto; Greene, Sheila; Mahoozi, Laily; Klein-Patel, Marcia; Diamond, Gill; Gulati, Sunita; Ganz, Tomas; Rice, Peter A; Quayle, Alison J

    2005-08-01

    Defensins are key participants in mucosal innate defense. The varied antimicrobial activity and differential distribution of defensins at mucosal sites indicate that peptide repertoires are tailored to site-specific innate defense requirements. Nonetheless, few studies have investigated changes in peptide profiles and function after in vivo pathogen challenge. Here, we determined defensin profiles in urethral secretions of healthy men and men with Chlamydia trachomatis- and Neisseria gonorrhoeae-mediated urethritis by immunoblotting for the epithelial defensins HBD1, HBD2, and HD5 and the neutrophil defensins HNP1 to -3 (HNP1-3). HBD1 was not detectable in secretions, and HBD2 was only induced in a small proportion of the urethritis patients; however, HD5 and HNP1-3 were increased in C. trachomatis infection and significantly elevated in N. gonorrhoeae infection. When HNP1-3 levels were low, HD5 appeared mostly as the propeptide; however, when HNP1-3 levels were >10 microg/ml, HD5 was proteolytically processed, suggesting neutrophil proteases might contribute to HD5 processing. HD5 and HNP1-3 were bactericidal against C. trachomatis and N. gonorrhoeae, but HD5 activity was dependent upon N-terminal processing of the peptide. In vitro proteolysis of proHD5 by neutrophil proteases and analysis of urethral secretions by surface-enhanced laser desorption ionization substantiated that neutrophils contribute the key convertases for proHD5 in the urethra during these infections. This contrasts with the small intestine, where Paneth cells secrete both proHD5 and its processing enzyme, trypsin. In conclusion, we describe a unique defensin expression repertoire in response to inflammatory sexually transmitted infections and a novel host defense mechanism wherein epithelial cells collaborate with neutrophils to establish an antimicrobial barrier during infection. PMID:16040996

  2. Neisseria gonorrhoeae infection protects human endocervical epithelial cells from apoptosis via expression of host antiapoptotic proteins.

    PubMed

    Follows, S A; Murlidharan, J; Massari, P; Wetzler, L M; Genco, C A

    2009-09-01

    Several microbial pathogens can modulate the host apoptotic response to infection, which may contribute to immune evasion. Various studies have reported that infection with the sexually transmitted disease pathogen Neisseria gonorrhoeae can either inhibit or induce apoptosis. N. gonorrhoeae infection initiates at the mucosal epithelium, and in women, cells from the ectocervix and endocervix are among the first host cells encountered by this pathogen. In this study, we defined the antiapoptotic effect of N. gonorrhoeae infection in human endocervical epithelial cells (End/E6E7 cells). We first established that N. gonorrhoeae strain FA1090B failed to induce cell death in End/E6E7 cells. Subsequently, we demonstrated that stimulation with N. gonorrhoeae protected these cells from staurosporine (STS)-induced apoptosis. Importantly, only End/E6E7 cells incubated with live bacteria and in direct association with N. gonorrhoeae were protected from STS-induced apoptosis, while heat-killed and antibiotic-killed bacteria failed to induce protection. Stimulation of End/E6E7 cells with live N. gonorrhoeae induced NF-kappaB activation and resulted in increased gene expression of the NF-kappaB-regulated antiapoptotic genes bfl-1, cIAP-2, and c-FLIP. Furthermore, cIAP-2 protein levels also increased in End/E6E7 cells incubated with gonococci. Collectively, our results indicate that the antiapoptotic effect of N. gonorrhoeae in human endocervical epithelial cells results from live infection via expression of host antiapoptotic proteins. Securing an intracellular niche through the inhibition of apoptosis may be an important mechanism utilized by N. gonorrhoeae for microbial survival and immune evasion in cervical epithelial cells. PMID:19546192

  3. High-resolution melting curve analysis for genotyping of common SNP in MTHFR gene using fixed-cell suspension.

    PubMed

    Sinthuwiwat, Thivaratana; Poowasanpetch, Phanasit; Wongngamrungroj, Angsana; Promso, Somying; Auewarakul, Chirayu; Mooney, Sean; Tocharoentanaphol, Chintana

    2008-01-01

    Genetic variation in MTHFR might explain the interindividual differences in both therapeutic and toxic responses to the treatment of cancer and rheumatoid arthritis with methotrexate, and can be involved in the sensitivity of developing diseases like cancer and congenital anomalies. We investigated the common sequence variation, C677T, in the MTHFR gene in fixed-cell specimens archived after chromosomal analysis using a novel gene scanning method based on post PCR analysis of high-resolution melting curves (HRM). These fixed specimens were stored after routine chromosomal analysis for 1 year at -20 degrees C in a 3:1 methanol:acetic acid solution. The method revealed a distinct pattern between homozygous and heterozygous alleles. Sensitivity and specificity of the HRM based method were comparable to that obtained by a hybridization probe. While the success rate for genotyping of a common SNP in MTHFR was similar to the hybridization probe approach, the HRM based method was more cost-effective and had a shorter turnaround time. PMID:18725286

  4. HIV-1 sensitivity to zidovudine: a consensus culture technique validated by genotypic analysis of the reverse transcriptase.

    PubMed

    Brun-Vézinet, F; Ingrand, D; Deforges, L; Gochi, K; Ferchal, F; Schmitt, M P; Jung, M; Masquelier, B; Aubert, J; Buffet-Janvresse, C

    1992-05-01

    In order to select and standardize a reliable assay for the analysis of sensitivity of HIV isolates to AZT, we have compared two culture methods. The first assay (Cell-Associated Isolate Sensitivity Assay: CAISA) quantified AZT-resistant HIV isolates by end-point dilution cultures of peripheral blood mononuclear cells (PBMCs) in the presence of various concentrations of AZT. In the second assay (Cell-Free Isolate Sensitivity Assay: CFISA), following a conventional isolation of HIV, dilutions of infected cell-free supernatants were cultivated with fresh normal donor PBMCs in the presence of increasing concentrations of AZT. Samples from 64 untreated and AZT-treated patients were studied by CAISA (41), CFISA (43) or both assays (20). The CFISA, which allows the determination of titration parameters with respect to various kinetics patterns of viral replication was selected, and some of the CFISA phenotypically characterized isolates were further studied by nucleotide sequence analysis of the reverse transcriptase gene. CFISA showed that isolates from untreated patients were susceptible to AZT while the frequency of resistance increased with the duration of therapy. Genotypic analysis of CFISA-resistant isolates exhibited mutations at crucial positions, particularly at residue 215. We consider CFISA as a consensus culture technique for longitudinal studies of isolates from patients receiving AZT or other analogs of nucleosides.

  5. Analysis of gene expression and proteomic profiles of clonal genotypes from Theobroma cacao subjected to soil flooding.

    PubMed

    Bertolde, Fabiana Z; Almeida, Alex-Alan F; Pirovani, Carlos P

    2014-01-01

    Soil flooding causes changes in gene transcription, synthesis and degradation of proteins and cell metabolism. The main objective of this study was to understand the biological events of Theobroma cacao during soil flooding-induced stress, using the analyses of gene expression and activity of key enzymes involved in fermentation, as well as the identification of differentially expressed proteins by mass spectrometry in two contrasting genotypes for flooding tolerance (tolerant - TSA-792 and susceptible - TSH-774). Soil anoxia caused by flooding has led to changes in the expression pattern of genes associated with the biosynthesis of alcohol dehydrogenase (ADH), pyruvate decarboxylase (PDC) and lactate dehydrogenase (LDH) in leaves and roots of the two evaluated genotypes. Significant differences were observed between the enzyme activities of the two genotypes. Leaves and roots of the TSA-792 genotype showed higher ADH activity as compared to the TSH-774 genotype, whereas the activities of PDC and LDH have varied over the 96 h of soil flooding, being higher for TSA-792 genotype, at the initial stage, and TSH-774 genotype, at the final stage. Some of the identified proteins are those typical of the anaerobic metabolism-involved in glycolysis and alcoholic fermentation-and different proteins associated with photosynthesis, protein metabolism and oxidative stress. The ability to maintain glycolysis and induce fermentation was observed to play an important role in anoxia tolerance in cacao and may also serve to distinguish tolerant and susceptible genotypes in relation to this stressor.

  6. Analysis of Gene Expression and Proteomic Profiles of Clonal Genotypes from Theobroma cacao Subjected to Soil Flooding

    PubMed Central

    Bertolde, Fabiana Z.; Almeida, Alex-Alan F.; Pirovani, Carlos P.

    2014-01-01

    Soil flooding causes changes in gene transcription, synthesis and degradation of proteins and cell metabolism. The main objective of this study was to understand the biological events of Theobroma cacao during soil flooding-induced stress, using the analyses of gene expression and activity of key enzymes involved in fermentation, as well as the identification of differentially expressed proteins by mass spectrometry in two contrasting genotypes for flooding tolerance (tolerant - TSA-792 and susceptible - TSH-774). Soil anoxia caused by flooding has led to changes in the expression pattern of genes associated with the biosynthesis of alcohol dehydrogenase (ADH), pyruvate decarboxylase (PDC) and lactate dehydrogenase (LDH) in leaves and roots of the two evaluated genotypes. Significant differences were observed between the enzyme activities of the two genotypes. Leaves and roots of the TSA-792 genotype showed higher ADH activity as compared to the TSH-774 genotype, whereas the activities of PDC and LDH have varied over the 96 h of soil flooding, being higher for TSA-792 genotype, at the initial stage, and TSH-774 genotype, at the final stage. Some of the identified proteins are those typical of the anaerobic metabolism-involved in glycolysis and alcoholic fermentation-and different proteins associated with photosynthesis, protein metabolism and oxidative stress. The ability to maintain glycolysis and induce fermentation was observed to play an important role in anoxia tolerance in cacao and may also serve to distinguish tolerant and susceptible genotypes in relation to this stressor. PMID:25289700

  7. HBV Genotypic Variability in Cuba

    PubMed Central

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

    2015-01-01

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

  8. HBV genotypic variability in Cuba.

    PubMed

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

    2015-01-01

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

  9. Rapid detection of gyrA and parC mutations in fluoroquinolone-resistant Neisseria gonorrhoeae by denaturing high-performance liquid chromatography.

    PubMed

    Shigemura, Katsumi; Shirakawa, Toshiro; Okada, Hiroshi; Tanaka, Kazushi; Udaka, Tohru; Kamidono, Sadao; Arakawa, Soichi; Gotoh, Akinobu

    2004-12-01

    The detection of DNA sequence variation is fundamental to the identification of the genomic basis of phenotypic variability. Denaturing high-performance liquid chromatography (DHPLC) is a novel technique that is used to detect mutations in human DNA. This is the first report that this technique is used as a tool to detect mutations in genes encoding fluoroquinolone resistance in Neisseria gonorrhoeae. Eighty-one strains of N. gonorrhoeae were used in this study. Genomic DNA from each strain was subjected to PCR amplification of 225 bp in gyrA and 166 bp in parC spanning the fluoroquinolone-resistance determining regions (QRDRs). After we performed DNA sequencing of these amplicons and identification of mutations in the QRDRs, DHPLC was undertaken to investigate whether its results correlate the distinctive chromatogram with their DNA mutations pattern. The profilings detected by DHPLC completely corresponded to the results of the DNA sequencing in mutation patters in gyrA and parC genes. They resulted in the following amino acid substitutions: Ser-91Phe, Asp-95Gly, and Asp-95Asn in gyrA; and Gly-85Asp, Asp-86Asn, Ser-87Arg, and Ser-88Pro in parC, respectively. These mutations existed alone or as combinations, and we identified five mutations patterns in gyrA and six in parC including wild-type. These mutations and their patterns could be rapidly and reproducibly identified from the PCR products using DHPLC, producing specific peak patterns that correlate with genotypes. This novel detection system facilitates the detection of resistance alleles, providing a rapid (5 min per sample), economic (96 sample per run), and reliable technique for characterizing fluoroquinolone resistance in N. gonorrhoeae. PMID:15488283

  10. Rapid spread of Neisseria gonorrhoeae ciprofloxacin resistance due to a newly introduced resistant strain in Nuuk, Greenland, 2012–2015: a community-based prospective cohort study

    PubMed Central

    Pedersen, Michael Lynge; Poulsen, Peter; Berthelsen, Lene; Nørgaard, Christina; Hoffmann, Steen; Jensen, Jørgen Skov

    2016-01-01

    Objectives To determine the antimicrobial susceptibility and genotype distribution of Neisseria gonorrhoeae strains isolated from a cohort of patients in Nuuk, Greenland in order to assess the risk of rapid spread in the event of introduction of new strains. Methods Gonococcal isolates (n=102) obtained from a prospective cohort study of ciprofloxacin resistance were collected between March 2012 and February 2013. Etest minimal inhibitory concentrations (MICs) were determined for ciprofloxacin, azithromycin, ceftriaxone, penicillin, tetracycline, spectinomycin and gentamicin. All isolates were subjected to molecular typing using N. gonorrhoeae multiantigen sequence typing (NG-MAST). After the introduction of a ciprofloxacin-resistant strain in early 2014, an additional 18 isolates were characterised. Results During the study period, all 102 isolates were fully susceptible to ciprofloxacin (≤0.03 mg/L), azithromycin, spectinomycin, gentamicin and ceftriaxone. 10 different NG-MAST types circulated in Nuuk but 7 were found as single isolates, and 3 of the 7 belonged to 1 of the 3 major genogroups (G210, G9816 and G9817) together comprising 96% of the 102 isolates. ST210 accounted for 55% of the 102 strains. The newly introduced ciprofloxacin resistant strain belonged to ST2400 and dominated the population with 59% resistant strains within 6 months after its introduction. All G2400 strains had MICs≥2 mg/L. Conclusions Introduction of a ciprofloxacin-resistant strain into a very homogeneous N. gonorrhoeae population led to an explosive spread of the resistant clone, probably as a result of large sexual networks suggested by the strain homogeneity. Careful surveillance of antimicrobial susceptibility is essential to avoid widespread treatment failure in closed populations. PMID:27577587

  11. The obligate human pathogen, Neisseria gonorrhoeae, is polyploid.

    PubMed

    Tobiason, Deborah M; Seifert, H Steven

    2006-06-01

    We show using several methodologies that the Gram-negative, diplococcal-bacterium Neisseria gonorrhoeae has more than one complete genome copy per cell. Gene dosage measurements demonstrated that only a single replication initiation event per chromosome occurs per round of cell division, and that there is a single origin of replication. The region containing the origin does not encode any genes previously associated with bacterial origins of replication. Quantitative PCR results showed that there are on average three genome copies per coccal cell unit. These findings allow a model for gonococcal DNA replication and cell division to be proposed, in which a minimum of two chromosomal copies exist per coccal unit within a monococcal or diplococcal cell, and these chromosomes replicate in unison to produce four chromosomal copies during cell division. Immune evasion via antigenic variation is an important mechanism that allows these organisms to continually infect a high risk population of people. We propose that polyploidy may be necessary for the high frequency gene conversion system that mediates pilin antigenic variation and the propagation of N. gonorrhoeae within its human hosts.

  12. Immunological basis of serum resistance of Neisseria gonorrhoeae.

    PubMed

    Schneider, H; Griffiss, J M; Williams, G D; Pier, G B

    1982-01-01

    The immunological basis for resistance of certain strains of Neisseria gonorrhoeae to the bactericidal action of normal human serum was studied by investigating the potential role of factors which are known to interfere with each of the sequential steps that result in immune lysis of Gram-negative bacteria. Strains of N. gonorrhoeae were characterized as serum-sensitive (sers) or serum-resistant (serr) on the basis of their sensitivity to lysis by the sera of six normal individuals. Neither intrinsic resistance to the lytic action of activated human complement nor inaccessibility of the cell membrane to C5b accounted for serr. Outer membrane lipopolysaccharide (LPS) was the target antigen for lytic antibody in normal human sera. The gross chemical composition and molecular size of the LPS of the strains were heterogeneous and no consistent patterns of differences between those extracted from serr and from sers strains were found. Neither IgA nor IgG 'blocking' antibody in normal human serum was responsible for serr. We conclude that serr results from the absence from the LPS of the strains of antigenic loci for the lytic antibody in most normal human sera, or, expressed as a function of the host, the absence from the sera of most normal humans of lytic antibody directed against LPS antigenic loci for immune lysis.

  13. The obligate human pathogen, Neisseria gonorrhoeae, is polyploid.

    PubMed

    Tobiason, Deborah M; Seifert, H Steven

    2006-06-01

    We show using several methodologies that the Gram-negative, diplococcal-bacterium Neisseria gonorrhoeae has more than one complete genome copy per cell. Gene dosage measurements demonstrated that only a single replication initiation event per chromosome occurs per round of cell division, and that there is a single origin of replication. The region containing the origin does not encode any genes previously associated with bacterial origins of replication. Quantitative PCR results showed that there are on average three genome copies per coccal cell unit. These findings allow a model for gonococcal DNA replication and cell division to be proposed, in which a minimum of two chromosomal copies exist per coccal unit within a monococcal or diplococcal cell, and these chromosomes replicate in unison to produce four chromosomal copies during cell division. Immune evasion via antigenic variation is an important mechanism that allows these organisms to continually infect a high risk population of people. We propose that polyploidy may be necessary for the high frequency gene conversion system that mediates pilin antigenic variation and the propagation of N. gonorrhoeae within its human hosts. PMID:16719561

  14. Emerging resistance in Neisseria meningitidis and Neisseria gonorrhoeae.

    PubMed

    Stefanelli, Paola

    2011-02-01

    The value of monitoring antimicrobial resistance is particularly significant for Neisseria meningitidis and Neisseria gonorrhoeae diseases, even if it is for different reasons. Although there is no global alert for the spread of resistant meningococcal strains, the emergence of resistance is correlated to the outcome of treatment and the successful prophylaxis of close contacts. Few cases of resistance among meningococci have been recorded worldwide; it remains unclear what intriguing mechanism is responsible for maintaining resistance in these cases in the absence of significant antibiotic selective pressure, as in the case of penicillin; on the contrary, although rifampicin is the antibiotic of choice in the prophylaxis of close contacts, there is a very low rate of resistance. The emergence of multidrug-resistant N. gonorrhoeae is a great challenge in controlling gonorrhea as one of the main sexually transmitted bacterial diseases. International surveillance programs permit the monitoring of the susceptibility of the pathogen and allow the revision of the standardized treatment regimen when the situation changes. PMID:21342071

  15. Identification of alleles and genotypes of beta-casein with DNA sequencing analysis in Chinese Holstein cow.

    PubMed

    Dai, Ronghua; Fang, Yu; Zhao, Wenjing; Liu, Shuyun; Ding, Jinmei; Xu, Ke; Yang, Lingyu; He, Chuan; Ding, Fangmei; Meng, He

    2016-08-01

    The study reported in this Regional Research Communication aimed to analyse the genetic polymorphisms of β-casein in Chinese Holstein cows. β-casein has received considerable research interest in the dairy industry and animal breeding in recent years as a source not only of high quality protein, but also of bioactive peptides that may be linked to health effects. Morever, the polymorphic nature of β-casein and its association with milk production traits, composition, and quality also attracted several efforts in evaluating the allelic distribution of β-casein locus as a potential dairy trait marker. However, few data on beta-casein variants are available for the Chinese Holstein cow. In the present paper, one hundred and thirty three Holstein cows were included in the analysis. Results revealed the presence of 5 variants (A1, A2, A3, B and I), preponderance of the genotype A1A2 (0·353) and superiorities of A1/A2 alleles (0·432 and 0·459, respectively) in the population. Sequence analysis of β-casein gene in the cows showed four nucleotide changes in exon 7. Our study can provide reference and guidance for selection for superior milk for industrial applications and crossbreeding and genetic improvement programmes.

  16. Identification of alleles and genotypes of beta-casein with DNA sequencing analysis in Chinese Holstein cow.

    PubMed

    Dai, Ronghua; Fang, Yu; Zhao, Wenjing; Liu, Shuyun; Ding, Jinmei; Xu, Ke; Yang, Lingyu; He, Chuan; Ding, Fangmei; Meng, He

    2016-08-01

    The study reported in this Regional Research Communication aimed to analyse the genetic polymorphisms of β-casein in Chinese Holstein cows. β-casein has received considerable research interest in the dairy industry and animal breeding in recent years as a source not only of high quality protein, but also of bioactive peptides that may be linked to health effects. Morever, the polymorphic nature of β-casein and its association with milk production traits, composition, and quality also attracted several efforts in evaluating the allelic distribution of β-casein locus as a potential dairy trait marker. However, few data on beta-casein variants are available for the Chinese Holstein cow. In the present paper, one hundred and thirty three Holstein cows were included in the analysis. Results revealed the presence of 5 variants (A1, A2, A3, B and I), preponderance of the genotype A1A2 (0·353) and superiorities of A1/A2 alleles (0·432 and 0·459, respectively) in the population. Sequence analysis of β-casein gene in the cows showed four nucleotide changes in exon 7. Our study can provide reference and guidance for selection for superior milk for industrial applications and crossbreeding and genetic improvement programmes. PMID:27600965

  17. Latent Class Analysis of Antisocial Behavior: Interaction of Serotonin Transporter Genotype and Maltreatment

    ERIC Educational Resources Information Center

    Li, James J.; Lee, Steve S.

    2010-01-01

    To improve understanding about genetic and environmental influences on antisocial behavior (ASB), we tested the association of the 44-base pair polymorphism of the serotonin transporter gene (5-HTTLPR) and maltreatment using latent class analysis in 2,488 boys and girls from Wave 1 of the National Longitudinal Study of Adolescent Health. In boys,…

  18. ApoE Genotype and Alzheimer's Disease in Adults with Down Syndrome: Meta-Analysis.

    ERIC Educational Resources Information Center

    Prashner, V. P.; Chowdhury, T. A.; Rowe, B. R.; Bain, S. C.

    1997-01-01

    ApoE gene polymorphism was examined in 100 adults with Down syndrome with and without dementia (Alzheimer's disease) and 346 control subjects. Additionally, a meta analysis of studies (total N=480 subjects) was performed. Results indicated a similar incidence of the gene across groups but subjects with the allele tended to an earlier onset of…

  19. 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

  20. Antimicrobial susceptibility and penicillin-binding protein 1 and 2 mutations in Neisseria gonorrhoeae isolated from male urethritis in Sapporo, Japan.

    PubMed

    Takahashi, Satoshi; Kurimura, Yuichiro; Hashimoto, Jiro; Uehara, Teruhisa; Hiyama, Yoshiki; Iwasawa, Akihiko; Nishimura, Masahiro; Sunaoshi, Kenichi; Takeda, Koichi; Suzuki, Nobukazu; Tsukamoto, Taiji

    2013-02-01

    The spread of antimicrobial-resistant Neisseria gonorrhoeae worldwide is a critical issue in the control of sexually transmitted infections. The purpose of this study was to clarify recent trends in the susceptibility of N. gonorrhoeae to various antimicrobial agents and to compare these data with our previous data. Minimum inhibitory concentrations (MICs) of various antimicrobial agents were determined in N. gonorrhoeae strains clinically isolated from male gonococcal urethritis. In addition, amino acid sequencing of penicillin-binding protein (PBP) 2, encoded by the penA gene, was analyzed so that genetic analysis of mosaic PBP 2 could clarify the susceptibility of the strains to cefixime and other cephalosporins. The susceptibility rate for ceftriaxone, cefodizime, and spectinomycin, agents whose use is recommended by the guideline of the Japanese Society of Sexually Transmitted Infections (JSSTI), was 100 %. The susceptibility rates of the strains to penicillin G and ciprofloxacin were lower than those in previous reports. Mosaic PBP 2 structures were detected in 51.9 % of the strains and the MICs of the strains with the mosaic PBP 2 to cefixime were much higher than those of the strains without the mosaic PBP 2. In the clinical situation, the treatment regimen recommended by the JSSTI remains appropriate; however, the susceptibility to cephalosporins should be intensively surveyed because strains with mosaic PBP 2 were commonly detected.

  1. Genotypic tropism testing in proviral DNA to guide maraviroc initiation in aviremic subjects: 48-week analysis of the PROTEST study

    PubMed Central

    Garcia, Federico; Poveda, Eva; Jesús Pérez-Elías, Maria; Hernández Quero, José; Àngels Ribas, Maria; Martínez-Madrid, Onofre J; Flores, Juan; Crespo, Manel; Gutiérrez, Félix; García-Deltoro, Miguel; Imaz, Arkaitz; Ocampo, Antonio; Artero, Arturo; Blanco, Francisco; Bernal, Enrique; Pasquau, Juan; Mínguez-Gallego, Carlos; Pérez, Núria; Aiestarán, Aintzane; Paredes, Roger

    2014-01-01

    Introduction In a previous interim 24-week virological safety analysis of the PROTEST study [1], initiation of Maraviroc (MVC) plus 2 nucleoside reverse-transcriptase inhibitors (NRTIs) in aviremic subjects based on genotypic tropism testing of proviral HIV-1 DNA was associated with low rates of virological failure. Here we present the final 48-week analysis of the study. Methods PROTEST was a phase 4, prospective, single-arm clinical trial (ID: NCT01378910) carried on in 24 HIV care centres in Spain. Maraviroc-naïve HIV-1-positive adults with HIV-1 RNA (VL) <50 c/mL on stable ART during the previous 6 months, requiring an ART change due to toxicity, with no antiretroviral resistance to the ART started, and R5 HIV by proviral DNA genotypic tropism testing (defined as a G2P FPR >10% in a singleton), initiated MVC with 2 NRTIs and were followed for 48 weeks. Virological failure was defined as two consecutive VL>50 c/mL. Recent adherence was calculated as: (# pills taken/# pills prescribed during the previous week)*100. Results Tropism results were available from 141/175 (80.6%) subjects screened: 87/141 (60%) were R5 and 74/87 (85%) were finally included in the study. Their median age was 48 years, 16% were women, 31% were MSM, 36% had CDC category C at study entry, 62% were HCV+ and 10% were HBV+. Median CD4+ counts were 616 cells/mm3 at screening, and median nadir CD4+ counts were 143 cells/mm3. Previous ART included PIs in 46 (62%) subjects, NNRTIs in 27 (36%) and integrase inhibitors (INIs) in 1 (2%). The main reasons for treatment change were dyslipidemia (42%), gastrointestinal symptoms (22%), and liver toxicity (15%). MVC was given alongside TDF/FTC in 40 (54%) subjects, ABC/3TC in 30 (40%), AZT/3TC in 2 (3%) and ABC/TDF in 2 (3%). Sixty-two (84%) subjects maintained VL<50 c/mL through week 48, whereas 12 (16%) discontinued treatment: two (3%) withdrew informed consent, one (1%) had a R5→X4 shift in HIV tropism between the screening and baseline visits, one

  2. Comparative genetic analysis of trichome-less and normal pod genotypes of Mucuna pruriens (Fabaceae).

    PubMed

    Dhawan, S S; Rai, G K; Darokar, M P; Lal, R K; Misra, H O; Khanuja, S P S

    2011-01-01

    Velvet bean (Mucuna pruriens) seeds contain the catecholic amino acid L-DoPA (L-3,4-dihydroxyphenylalanine), which is a neurotransmitter precursor and used for the treatment of Parkinson's disease and mental disorders. The great demand for L-DoPA is largely met by the pharmaceutical industry through extraction of the compound from wild populations of this plant; commercial exploitation of this compound is hampered because of its limited availability. The trichomes present on the pods can cause severe itching, blisters and dermatitis, discouraging cultivation. We screened genetic stocks of velvet bean for the trichome-less trait, along with high seed yield and L-DoPA content. The highest yielding trichome-less elite strain was selected and indentified on the basis of a PCR-based DNA fingerprinting method (RAPD), using deca-nucleotide primers. A genetic similarity index matrix was obtained through multivariant analysis using Nei and Li's coefficient. The similarity coefficients were used to generate a tree for cluster analysis using the UPGMA method. Analysis of amplification spectra of 408 bands obtained with 56 primers allowed us to distinguish a trichome-less elite strain of M. pruriens. PMID:21968621

  3. Source tracking of an anthrax outbreak in northeastern China using complete genome analysis and MLVA genotyping.

    PubMed

    Li, S; An, X; Huang, Y; Pei, G; Cao, D; Mi, Z; Gu, Z; Zhao, X; Li, J; Gu, G; Tong, Y

    2015-01-01

    Anthrax is caused by Bacillus anthracis, an etiological agent behind zoonotic diseases worldwide. B. anthracis is also one of the most dangerous bioterrorism agents. An anthrax outbreak took place in Liaoning Province in northeastern China in August 2012. It resulted in seven human infections and dozens of dead cows. One B. anthracis strain, named Han, was isolated from a dead cow. This strain showed minor pathogenicity in mice and was suspected to be derived from the locally administered vaccine strain, Vac. In order to determine if the Han isolate was derived from the vaccine strain Vac and to track the source of the anthrax outbreak and, so, exclude the possibility of terrorism attack, a complete genome sequencing of these two B. anthracis strains was conducted. With the genome sequencing data, canonical single nucleotide polymorphism (SNP) analysis and whole-genome SNP screening were performed. The results indicate that the Han strain was markedly different from the Vac strain. Further analysis by multiple-locus variable-number tandem repeat analysis (MLVA) showed that Han clustered with previously reported Chinese strains. The result of MLVA15 confirmed that the Han strain is a naturally occurring isolate instead of an engineered agent deliberately distributed by terrorists or other parties. In conclusion, our method used in this study not only facilitates epidemiological studies but also made it easier to distinguish naturally occurring outbreaks from intentionally released pathogens.

  4. Reconstructing CNV genotypes using segregation analysis: combining pedigree information with CNV assay

    PubMed Central

    2010-01-01

    Background Repeated blocks of genome sequence have been shown to be associated with genetic diversity and disease risk in humans, and with phenotypic diversity in model organisms and domestic animals. Reliable tests are desirable to determine whether individuals are carriers of copy number variants associated with disease risk in humans and livestock, or associated with economically important traits in livestock. In some cases, copy number variants affect the phenotype through a dosage effect but in other cases, allele combinations have non-additive effects. In the latter cases, it has been difficult to develop tests because assays typically return an estimate of the sum of the copy number counts on the maternally and paternally inherited chromosome segments, and this sum does not uniquely determine the allele configuration. In this study, we show that there is an old solution to this new problem: segregation analysis, which has been used for many years to infer alleles in pedigreed populations. Methods Segregation analysis was used to estimate copy number alleles from assay data on simulated half-sib sheep populations. Copy number variation at the Agouti locus, known to be responsible for the recessive self-colour black phenotype, was used as a model for the simulation and an appropriate penetrance function was derived. The precision with which carriers and non-carriers of the undesirable single copy allele could be identified, was used to evaluate the method for various family sizes, assay strategies and assay accuracies. Results Using relationship data and segregation analysis, the probabilities of carrying the copy number alleles responsible for black or white fleece were estimated with much greater precision than by analyzing assay results for animals individually. The proportion of lambs correctly identified as non-carriers of the undesirable allele increased from 7% when the lambs were analysed alone to 80% when the lambs were analysed in half-sib families

  5. Cefixime-resistant Neisseria gonorrhoeae in the UK: a time to reflect on practice and recommendations.

    PubMed

    Forsyth, S; Penney, P; Rooney, G

    2011-05-01

    A 40-year-old man who has sex with men (MSM) with urethral gonorrhoea failed to respond to treatment with 400 mg cefixime orally. Laboratory isolation of the post-treatment strain showed a minimum inhibitory concentration of ≥0.25 mg/L, which is a level of tolerance to cefixime that has not been previously documented in the UK. This case illustrates the importance of assessing all patients after treatment for gonorrhoea so that treatment failure and antibiotic resistance can be identified. It is vital that gonorrhoea culture continues to be attempted from all infected individuals to enable accurate diagnosis and antibiotic sensitivities. We also recommend that laboratories test for cefixime sensitivity routinely, given that it is one of the most commonly prescribed treatments for gonorrhoea.

  6. Sequence and phylogenetic analysis of virulent Newcastle disease virus isolates from Pakistan during 2009–2013 reveals circulation of new sub genotype

    SciTech Connect

    Siddique, Naila; Naeem, Khalid; Abbas, Muhammad Athar; Ali Malik, Akbar; Rashid, Farooq; Rafique, Saba; Ghafar, Abdul; Rehman, Abdul

    2013-09-15

    Despite observing the standard bio-security measures at commercial poultry farms and extensive use of Newcastle disease vaccines, a new genotype VII-f of Newcastle disease virus (NDV) got introduced in Pakistan during 2011. In this regard 300 ND outbreaks recorded so far have resulted into huge losses of approximately USD 200 million during 2011–2013. A total of 33 NDV isolates recovered during 2009–2013 throughout Pakistan were characterized biologically and phylogenetically. The phylogenetic analysis revealed a new velogenic sub genotype VII-f circulating in commercial and domestic poultry along with the earlier reported sub genotype VII-b. Partial sequencing of Fusion gene revealed two types of cleavage site motifs; lentogenic {sup 112}GRQGRL{sup 117} and velogenic {sup 112}RRQKRF{sup 117} along with some point mutations indicative of genetic diversity. We report here a new sub genotype of virulent NDV circulating in commercial and backyard poultry in Pakistan and provide evidence for the possible genetic diversity which may be causing new NDV out breaks. - Highlights: • The first report of isolation of new genotype VII-f of virulent Newcastle disease virus (NDV) in Pakistan. • We report the partial Fusion gene sequences of new genotype VII-f of virulent NDV from Pakistan. • We report the phylogenetic relationship of new NDV strains with reported NDV strains. • Provide outbreak history of new virulent NDV strain in commercial and backyard poultry in Pakistan. • We provide possible evidence for the role of backyard poultry in NDV outbreaks.

  7. High-throughput method for determination of apolipoprotein E genotypes with use of restriction digestion analysis by microplate array diagonal gel electrophoresis.

    PubMed

    Bolla, M K; Haddad, L; Humphries, S E; Winder, A F; Day, I N

    1995-11-01

    Molecular epidemiological research has identified the association of a common apolipoprotein E (apo E) isoform (E4 as opposed to E3), with risk both of coronary artery disease and of Alzheimer dementia. In addition, the role of apo E genotype (usually E2/E2) in Type III hyperlipidemia is well known. However, both for diagnostic and research purposes, apo E genotyping is cumbersome. The preferred approach is electrophoretic sizing of restriction digestion fragments, enabling simultaneous analysis of the two codons (112 and 158) that represent the six common genotypes (E2/E2; E2/E3; E2/E4; E3/E3; E3/E4; E4/E4). However, the consequent demands of high-yield PCR, high-resolution, high-throughput electrophoresis, and sufficient detection sensitivity have left shortfalls in published protocols. In conjunction with a high-throughput electrophoresis system we described recently, microplate array diagonal gel electrophoresis (MADGE), we have constructed extensively optimized, simplified protocols for DNA isolation from mouthwash samples for PCR setup and high-yield PCR, for restriction digestion, and for subsequent MADGE gel image analysis. The integral system enables one worker to readily undertake apo E genotyping of as many as hundreds of DNA samples per day, without special equipment.

  8. Incidence of gonorrhoea diagnosed in GUM clinics in South Thames (west) region

    PubMed Central

    Hickman, M.; Judd, A.; Maguire, H.; Hay, P.; Charlett, A.; Catchpole, M.; Nayagam, A.; Renton, A.

    1999-01-01

    OBJECTIVES: To describe the incidence of gonorrhoea diagnosed in genitourinary medicine (GUM) clinics in South Thames (West) between 1995 and 1996, and how it changed among population subgroups. SETTINGS AND SUBJECTS: Cases of uncomplicated and complicated gonorrhoea diagnosed at 13 GUM clinics in the former South Thames West (STW) Regional Health Authority that reported disaggregate data to the South Thames GUM Clinic Collaborative STD Surveillance Scheme. METHODS: Annual incidence rates (per 100,000) of gonorrhoea diagnoses by sex, age group, ethnic group, area of residence, and year were calculated. Poisson regression models were used to calculate risk ratios (RR) to describe the key differences in the variation of gonorrhoea cases by these variables. Relative differences in the incidence of diagnosed gonorrhoea between 1995 and 1996 were investigated by including an interaction between year and the other variables (age group, sex, ethnic group, region) and testing whether any were significant using a likelihood ratio test. RESULTS: Area of residence, sex, age group, and ethnic group were key predictors of the rates of diagnosed gonorrhoea. The risk ratio for gonorrhoea (after adjustment for the other variables) was: 13 times higher among blacks than the white population; twice as high in inner London compared with outer London; and three times lower in the "shire" region compared with outer London. The rate of diagnosed gonorrhoea was significantly higher in the black population in the shire region than the inner London white population. The rate of gonorrhoea diagnosed by GUM clinics from 1995 to 1996 almost doubled in the white population aged 15-44 years, from 16 cases per 100,000 to 30 cases per 100,000 (adjusted RR 2.0, 95% CI 1.6 to 2.4), whereas increased rates in the black and Asian/other ethnic groups were not statistically significant (adjusted RR 1.1, 95% CI 0.9 to 1.4; and 1.4, 95% CI 0.7 to 2.7 respectively). CONCLUSION: The observed increase in

  9. Efficacy of a Novel Tricyclic Topoisomerase Inhibitor in a Murine Model of Neisseria gonorrhoeae Infection.

    PubMed

    Savage, Victoria J; Charrier, Cédric; Salisbury, Anne-Marie; Box, Helen; Chaffer-Malam, Nathan; Huxley, Anthony; Kirk, Ralph; Noonan, Gary M; Mohmed, Sarfraz; Craighead, Mark W; Ratcliffe, Andrew J; Best, Stuart A; Stokes, Neil R

    2016-09-01

    There is an urgent need for new antibiotics to treat multidrug-resistant Neisseria gonorrhoeae In this report, the microbiology, in vivo pharmacokinetics, and efficacy of REDX05931, a representative novel tricyclic topoisomerase inhibitor, were evaluated. REDX05931 demonstrated high oral bioavailability in mice and reduced N. gonorrhoeae infection after a single dose in a mouse model of gonorrhea. These data support the potential of this series of small molecules as a new treatment for drug-resistant gonorrheal infections. PMID:27324777

  10. Penicillinase-producing plasmid types in Neisseria gonorrhoeae clinical isolates from Australia.

    PubMed

    Whiley, David; Trembizki, Ella; Buckley, Cameron; Freeman, Kevin; Lawrence, Andrew; Limnios, Athena; Pearson, Julie; Smith, Helen; Stevens, Kerrie; Lahra, Monica M

    2014-12-01

    Penicillinase-producing Neisseria gonorrhoeae (PPNG) carrying the blaTEM-135 gene is of particular concern, as it is considered a stepping stone toward resistance to extended-spectrum cephalosporins. Here, we sought to characterize plasmid types and the occurrence of the blaTEM-135 gene for N. gonorrhoeae clinical isolates from Australia. We found that blaTEM-135 was prevalent in Australian PPNG and was detected on all three major plasmid types.

  11. An insight into the drug resistance profile & mechanism of drug resistance in Neisseria gonorrhoeae.

    PubMed

    Patel, Achchhe Lal; Chaudhry, Uma; Sachdev, Divya; Sachdeva, Poonam Nagpal; Bala, Manju; Saluja, Daman

    2011-10-01

    Among the aetiological agents of treatable sexually transmitted diseases (STDs), Neissseria gonorrhoeae is considered to be most important because of emerging antibiotic resistant strains that compromise the effectiveness of treatment of the disease - gonorrhoea. In most of the developing countries, treatment of gonorrhoea relies mainly on syndromic management rather than the aetiological based therapy. Gonococcal infections are usually treated with single-dose therapy with an agent found to cure > 95 per cent of cases. Unfortunately during the last few decades, N. gonorrhoeae has developed resistance not only to less expensive antimicrobials such as sulphonamides, penicillin and tetracyclines but also to fluoroquinolones. The resistance trend of N. gonorrhoeae towards these antimicrobials can be categorised into pre-quinolone, quinolone and post-quinolone era. Among the antimicrobials available so far, only the third-generation cephalosporins could be safely recommended as first-line therapy for gonorrhoea globally. However, resistance to oral third-generation cephalosporins has also started emerging in some countries. Therefore, it has become imperative to initiate sustained national and international efforts to reduce infection and misuse of antibiotics so as to prevent further emergence and spread of antimicrobial resistance. It is necessary not only to monitor drug resistance and optimise treatment regimens, but also to gain insight into how gonococcus develops drug resistance. Knowledge of mechanism of resistance would help us to devise methods to prevent the occurrence of drug resistance against existing and new drugs. Such studies could also help in finding out new drug targets in N. gonorrhoeae and also a possibility of identification of new drugs for treating gonorrhoea. PMID:22089602

  12. Association of susceptible genotypes to periodontal disease with the clinical outcome and tooth survival after non-surgical periodontal therapy: A systematic review and meta-analysis

    PubMed Central

    Doufexi, Aikaterini-Ellisavet; Kalogirou, Fotini

    2016-01-01

    Background The real clinical utility of genetic testing is the prognostic value of genetic factors in the clinical outcome of periodontal treatment and the tooth survival. A meta-analysis was undertaken to estimate the effect of a susceptible genotype to periodontitis on the clinical outcomes of non-surgical periodontal therapy and the tooth survival. Material and Methods A systematic search of MEDLINE-Pubmed, Cochrane Library and Scopus was performed. Additionally, a hand search was done in three journals. No specific language restriction was applied. Two reviewers screened independently titles and abstracts or full text copies. Quality assessment of all the included studies was held. Results Initial screening of electronic databases resulted in 283 articles. Ten studies met the inclusion criteria, nine of them examined the clinical outcome, while the other one investigated the tooth survival in susceptible individuals after non-surgical periodontal therapy. Eight of included studies were selected for the meta-analysis. IL-1 positive genotypes increase the risk of tooth loss, while no association found between the bleeding on probing (BOP), clinical attachment loss (CAL) and plaque index (PI) with the genotype status. Probing pocket depth (PPD) reduction in the first three months and in long-term results found to have a significant association with the genotype. Conclusions There is no difference in the clinical measurements after non-surgical periodontal treatment, apart from PPD. More publications are needed to identify a cause-effect relationship. Key words:Periodontal disease, periodontitis, periodontal therapy, clinical outcome, tooth loss, susceptibility, polymorphism, genotype, meta-analysis, systematic review. PMID:26595831

  13. Genomic Epidemiology and Molecular Resistance Mechanisms of Azithromycin-Resistant Neisseria gonorrhoeae in Canada from 1997 to 2014.

    PubMed

    Demczuk, Walter; Martin, Irene; Peterson, Shelley; Bharat, Amrita; Van Domselaar, Gary; Graham, Morag; Lefebvre, Brigitte; Allen, Vanessa; Hoang, Linda; Tyrrell, Greg; Horsman, Greg; Wylie, John; Haldane, David; Archibald, Chris; Wong, Tom; Unemo, Magnus; Mulvey, Michael R

    2016-05-01

    The emergence of Neisseria gonorrhoeae strains with decreased susceptibility to cephalosporins and azithromycin (AZM) resistance (AZM(r)) represents a public health threat of untreatable gonorrhea infections. Genomic epidemiology through whole-genome sequencing was used to describe the emergence, dissemination, and spread of AZM(r) strains. The genomes of 213 AZM(r) and 23 AZM-susceptible N. gonorrhoeae isolates collected in Canada from 1989 to 2014 were sequenced. Core single nucleotide polymorphism (SNP) phylogenomic analysis resolved 246 isolates into 13 lineages. High-level AZM(r) (MICs ≥ 256 μg/ml) was found in 5 phylogenetically diverse isolates, all of which possessed the A2059G mutation (Escherichia coli numbering) in all four 23S rRNA alleles. One isolate with high-level AZM(r) collected in 2009 concurrently had decreased susceptibility to ceftriaxone (MIC = 0.125 μg/ml). An increase in the number of 23S rRNA alleles with the C2611T mutations (E. coli numbering) conferred low to moderate levels of AZM(r) (MICs = 2 to 4 and 8 to 32 μg/ml, respectively). Low-level AZM(r) was also associated with mtrR promoter mutations, including the -35A deletion and the presence of Neisseria meningitidis-like sequences. Geographic and temporal phylogenetic clustering indicates that emergent AZM(r) strains arise independently and can then rapidly expand clonally in a region through local sexual networks. PMID:26935729

  14. Non-cytotoxic nanomaterials enhance antimicrobial activities of cefmetazole against multidrug-resistant Neisseria gonorrhoeae.

    PubMed

    Li, Lan-Hui; Yen, Muh-Yong; Ho, Chao-Chi; Wu, Ping; Wang, Chien-Chun; Maurya, Pawan Kumar; Chen, Pai-Shan; Chen, Wei; Hsieh, Wan-Yu; Chen, Huei-Wen

    2013-01-01

    The emergence and spread of antibiotic-resistant Neisseria gonorrhoeae has led to difficulties in treating patients, and novel strategies to prevent and treat this infection are urgently needed. Here, we examined 21 different nanomaterials for their potential activity against N. gonorrhoeae (ATCC 49226). Silver nanoparticles (Ag NPs, 120 nm) showed the greatest potency for reducing N. gonorrhoeae colony formation (MIC: 12.5 µg/ml) and possessed the dominant influence on the antibacterial activity with their properties of the nanoparticles within a concentration range that did not induce cytotoxicity in human fibroblasts or epithelial cells. Electron microscopy revealed that the Ag NPs significantly reduced bacterial cell membrane integrity. Furthermore, the use of clinical isolates of multidrug-resistant N. gonorrhoeae showed that combined treatment with 120 nm Ag NPs and cefmetazole produced additive effects. This is the first report to screen the effectiveness of nanomaterials against N. gonorrhoeae, and our results indicate that 120 nm Ag NPs deliver low levels of toxicity to human epithelial cells and could be used as an adjuvant with antibiotic therapy, either for topical use or as a coating for biomaterials, to prevent or treat multidrug-resistant N. gonorrhoeae. PMID:23705013

  15. Multivariate analysis of backcross progeny of Passiflora L. (Passifloraceae) for pre-breeding genotype selection.

    PubMed

    Melo, C A F; Souza, M M; Sousa, A G R; Viana, A P; Santos, E A

    2015-01-01

    The Ward-MLM procedure was used to evaluate genetic variation in four backcross progenies and in their parents, hybrid F1 HD13 and donor parent Passiflora sublanceolata. Sixteen quantitative descriptors and five qualitative characteristics of relevance to ornamental flower production were assessed. Using the pseudo-F and pseudo-T² criteria, we identified four groups among these plants in two evaluation periods. In both evaluations, the BC1 plants showed greater dissimilarity to their recurrent parent, but showed high genetic similarity with the P. sublanceolata parent. The first two canonical variables produced by the Ward-MLM procedure accounted for over 90% of the variation in both evaluation periods, enabling the representation of diversity through two-dimensional graphics. Groups II and IV were formed in the first assessment period. Groups I and IV formed in the second period and showed plants with selection potential. We found that it was essential to use both qualitative and quantitative variables for this analysis. Assessments of quantitative descriptors indicate that the selection of BC1 plants can be performed in any of the four progenies. Because of the similarities observed for some floral descriptors between BC1 and the P. sublanceolata parent, a second generation backcross was not recommended. However, the selection of BC1 plants for evaluation and direct use as an ornamental cultivar, or as a resource in other breeding programs, can be recommended. PMID:26634503

  16. Latent Class Analysis of Antisocial Behavior: Interaction of Serotonin Transporter Genotype and Maltreatment

    PubMed Central

    Li, James J.

    2010-01-01

    To improve understanding about genetic and environmental influences on antisocial behavior (ASB), we tested the association of the 44-base pair polymorphism of the serotonin transporter gene (5-HTTLPR) and maltreatment using latent class analysis in 2,488 boys and girls from Wave 1 of the National Longitudinal Study of Adolescent Health. In boys, ASB was defined by three classes (Exclusive Covert, Mixed Covert and Overt, and No Problems) whereas in girls, ASB was defined by two classes (Exclusive Covert, No Problems). In boys, 5-HTTLPR and maltreatment were not significantly related to ASB. However, in girls, maltreatment, but not 5-HTTLPR, was significantly associated with ASB. A significant interaction between 5-HTTLPR and maltreatment was also observed, where maltreated girls homozygous for the short allele were 12 times more likely to be classified in the Exclusive Covert group than in the No Problems group. Structural differences in the latent structure of ASB at Wave 2 and Wave 3 prevented repeat LCA modeling. However, using counts of ASB, 5-HTTLPR, maltreatment, and its interaction were unrelated to overt and covert ASB at Wave 2 and only maltreatment was related to covert ASB at Wave 3. We discuss these findings within the context of sex differences in ASB and relevant models of gene-environment interplay across developmental periods. PMID:20405199

  17. Genetic diversity analysis among male and female Jojoba genotypes employing gene targeted molecular markers, start codon targeted (SCoT) polymorphism and CAAT box-derived polymorphism (CBDP) markers.

    PubMed

    Heikrujam, Monika; Kumar, Jatin; Agrawal, Veena

    2015-09-01

    To detect genetic variations among different Simmondsia chinensis genotypes, two gene targeted markers, start codon targeted (SCoT) polymorphism and CAAT box-derived polymorphism (CBDP) were employed in terms of their informativeness and efficiency in analyzing genetic relationships among different genotypes. A total of 15 SCoT and 17 CBDP primers detected genetic polymorphism among 39 Jojoba genotypes (22 females and 17 males). Comparatively, CBDP markers proved to be more effective than SCoT markers in terms of percentage polymorphism as the former detecting an average of 53.4% and the latter as 49.4%. The Polymorphic information content (PIC) value and marker index (MI) of CBPD were 0.43 and 1.10, respectively which were higher than those of SCoT where the respective values of PIC and MI were 0.38 and 1.09. While comparing male and female genotype populations, the former showed higher variation in respect of polymorphic percentage and PIC, MI and Rp values over female populations. Nei's diversity (h) and Shannon index (I) were calculated for each genotype and found that the genotype "MS F" (in both markers) was highly diverse and genotypes "Q104 F" (SCoT) and "82-18 F" (CBDP) were least diverse among the female genotype populations. Among male genotypes, "32 M" (CBDP) and "MS M" (SCoT) revealed highest h and I values while "58-5 M" (both markers) was the least diverse. Jaccard's similarity co-efficient of SCoT markers ranged from 0.733 to 0.922 in female genotypes and 0.941 to 0.746 in male genotype population. Likewise, CBDP data analysis also revealed similarity ranging from 0.751 to 0.958 within female genotypes and 0.754 to 0.976 within male genotype populations thereby, indicating genetically diverse Jojoba population. Employing the NTSYS (Numerical taxonomy and multivariate analysis system) Version 2.1 software, both the markers generated dendrograms which revealed that all the Jojoba genotypes were clustered into two major groups, one group consisting of

  18. Genetic diversity analysis among male and female Jojoba genotypes employing gene targeted molecular markers, start codon targeted (SCoT) polymorphism and CAAT box-derived polymorphism (CBDP) markers

    PubMed Central

    Heikrujam, Monika; Kumar, Jatin; Agrawal, Veena

    2015-01-01

    To detect genetic variations among different Simmondsia chinensis genotypes, two gene targeted markers, start codon targeted (SCoT) polymorphism and CAAT box-derived polymorphism (CBDP) were employed in terms of their informativeness and efficiency in analyzing genetic relationships among different genotypes. A total of 15 SCoT and 17 CBDP primers detected genetic polymorphism among 39 Jojoba genotypes (22 females and 17 males). Comparatively, CBDP markers proved to be more effective than SCoT markers in terms of percentage polymorphism as the former detecting an average of 53.4% and the latter as 49.4%. The Polymorphic information content (PIC) value and marker index (MI) of CBPD were 0.43 and 1.10, respectively which were higher than those of SCoT where the respective values of PIC and MI were 0.38 and 1.09. While comparing male and female genotype populations, the former showed higher variation in respect of polymorphic percentage and PIC, MI and Rp values over female populations. Nei's diversity (h) and Shannon index (I) were calculated for each genotype and found that the genotype “MS F” (in both markers) was highly diverse and genotypes “Q104 F” (SCoT) and “82–18 F” (CBDP) were least diverse among the female genotype populations. Among male genotypes, “32 M” (CBDP) and “MS M” (SCoT) revealed highest h and I values while “58-5 M” (both markers) was the least diverse. Jaccard's similarity co-efficient of SCoT markers ranged from 0.733 to 0.922 in female genotypes and 0.941 to 0.746 in male genotype population. Likewise, CBDP data analysis also revealed similarity ranging from 0.751 to 0.958 within female genotypes and 0.754 to 0.976 within male genotype populations thereby, indicating genetically diverse Jojoba population. Employing the NTSYS (Numerical taxonomy and multivariate analysis system) Version 2.1 software, both the markers generated dendrograms which revealed that all the Jojoba genotypes were clustered into two major groups

  19. Bioinformatics analysis of hepatitis C virus genotype 2a-induced human hepatocellular carcinoma in Huh7 cells

    PubMed Central

    Xu, Ping; Wu, Meiying; Chen, Hui; Xu, Junchi; Wu, Minjuan; Li, Ming; Qian, Feng; Xu, Junhua

    2016-01-01

    Hepatocellular carcinoma (HCC) is a liver cancer that could be induced by hepatitis C virus genotype 2a Japanese fulminant hepatitis-1 (JFH-1) strain. The aim of this study was to investigate the molecular mechanisms of HCC. The microarray data GSE20948 includes 14 JFH-1- and 14 mock (equal volume of medium [control])-infected Huh7 samples. The data were downloaded from the Gene Expression Omnibus. After data processing, soft cluster analyses were performed to identify co-regulated genes with similar temporal expression patterns. Functional and pathway enrichment analyses, as well as functional annotation analysis, were performed. Subsequently, combined networks of protein–protein interaction network, microRNA regulatory network, and transcriptional regulatory network were constructed. Hub nodes, modules, and five clusters of co-regulated genes were also identified. In total, 173 up and 207 down co-regulated genes were separately identified in JFH-1-infected Huh7 cells compared with those of control cells. Functional enrichment analysis indicated that up co-regulated genes were related to skeletal system morphogenesis and neuron differentiation and down co-regulated genes were related to steroid/cholesterol/sterol metabolisms. Hub genes (such as IRF1, GBP1, ICAM1, Foxa1, DHCR7, HMGCS2, and MSMO1) were identified. Transcription factors IRF1 and Foxa1 were the targets of miR-130a, miR-17-5p, and miR-20a. PPARGC1A was targeted by miR-29 family, and MSMO1 was the target of miR-23 family. Hub nodes (such as IRF1, GBP1, ICAM1, Foxa1, DHCR7, HMGCS2, and MSMO1) and microRNAs might be used as candidate biomarkers of JFH-1-infected HCC. PMID:26811688

  20. Bioinformatics analysis of hepatitis C virus genotype 2a-induced human hepatocellular carcinoma in Huh7 cells.

    PubMed

    Xu, Ping; Wu, Meiying; Chen, Hui; Xu, Junchi; Wu, Minjuan; Li, Ming; Qian, Feng; Xu, Junhua

    2016-01-01

    Hepatocellular carcinoma (HCC) is a liver cancer that could be induced by hepatitis C virus genotype 2a Japanese fulminant hepatitis-1 (JFH-1) strain. The aim of this study was to investigate the molecular mechanisms of HCC. The microarray data GSE20948 includes 14 JFH-1- and 14 mock (equal volume of medium [control])-infected Huh7 samples. The data were downloaded from the Gene Expression Omnibus. After data processing, soft cluster analyses were performed to identify co-regulated genes with similar temporal expression patterns. Functional and pathway enrichment analyses, as well as functional annotation analysis, were performed. Subsequently, combined networks of protein-protein interaction network, microRNA regulatory network, and transcriptional regulatory network were constructed. Hub nodes, modules, and five clusters of co-regulated genes were also identified. In total, 173 up and 207 down co-regulated genes were separately identified in JFH-1-infected Huh7 cells compared with those of control cells. Functional enrichment analysis indicated that up co-regulated genes were related to skeletal system morphogenesis and neuron differentiation and down co-regulated genes were related to steroid/cholesterol/sterol metabolisms. Hub genes (such as IRF1, GBP1, ICAM1, Foxa1, DHCR7, HMGCS2, and MSMO1) were identified. Transcription factors IRF1 and Foxa1 were the targets of miR-130a, miR-17-5p, and miR-20a. PPARGC1A was targeted by miR-29 family, and MSMO1 was the target of miR-23 family. Hub nodes (such as IRF1, GBP1, ICAM1, Foxa1, DHCR7, HMGCS2, and MSMO1) and microRNAs might be used as candidate biomarkers of JFH-1-infected HCC. PMID:26811688

  1. Understanding of HLA-conferred susceptibility to chronic hepatitis B infection requires HLA genotyping-based association analysis

    PubMed Central

    Nishida, Nao; Ohashi, Jun; Khor, Seik-Soon; Sugiyama, Masaya; Tsuchiura, Takayo; Sawai, Hiromi; Hino, Keisuke; Honda, Masao; Kaneko, Shuichi; Yatsuhashi, Hiroshi; Yokosuka, Osamu; Koike, Kazuhiko; Kurosaki, Masayuki; Izumi, Namiki; Korenaga, Masaaki; Kang, Jong-Hon; Tanaka, Eiji; Taketomi, Akinobu; Eguchi, Yuichiro; Sakamoto, Naoya; Yamamoto, Kazuhide; Tamori, Akihiro; Sakaida, Isao; Hige, Shuhei; Itoh, Yoshito; Mochida, Satoshi; Mita, Eiji; Takikawa, Yasuhiro; Ide, Tatsuya; Hiasa, Yoichi; Kojima, Hiroto; Yamamoto, Ken; Nakamura, Minoru; Saji, Hiroh; Sasazuki, Takehiko; Kanto, Tatsuya; Tokunaga, Katsushi; Mizokami, Masashi

    2016-01-01

    Associations of variants located in the HLA class II region with chronic hepatitis B (CHB) infection have been identified in Asian populations. Here, HLA imputation method was applied to determine HLA alleles using genome-wide SNP typing data of 1,975 Japanese individuals (1,033 HBV patients and 942 healthy controls). Together with data of an additional 1,481 Japanese healthy controls, association tests of six HLA loci including HLA-A, C, B, DRB1, DQB1, and DPB1, were performed. Although the strongest association was detected at a SNP located in the HLA-DP locus in a SNP-based GWAS using data from the 1,975 Japanese individuals, HLA genotyping-based analysis identified DQB1*06:01 as having the strongest association, showing a greater association with CHB susceptibility (OR = 1.76, P = 6.57 × 10−18) than any one of five HLA-DPB1 alleles that were previously reported as CHB susceptibility alleles. Moreover, HLA haplotype analysis showed that, among the five previously reported HLA-DPB1 susceptibility and protective alleles, the association of two DPB1 alleles (DPB1*09:01, and *04:01) had come from linkage disequilibrium with HLA-DR-DQ haplotypes, DRB1*15:02-DQB1*06:01 and DRB1*13:02-DQB1*06:04, respectively. The present study showed an example that SNP-based GWAS does not necessarily detect the primary susceptibility locus in the HLA region. PMID:27091392

  2. Epidemiology of HPV Genotypes among HIV Positive Women in Kenya: A Systematic Review and Meta-Analysis

    PubMed Central

    Menon, Sonia; Wusiman, Aibibula; Boily, Marie Claude; Kariisa, Mbabazi; Mabeya, Hillary; Luchters, Stanley; Forland, Frode; Rossi, Rodolfo; Callens, Steven; vanden Broeck, Davy

    2016-01-01

    Background There is a scarcity of data on the distribution of human papillomavirus (HPV) genotypes in the HIV positive population and in invasive cervical cancer (ICC) in Kenya. This may be different from genotypes found in abnormal cytology. Yet, with the advent of preventive HPV vaccines that target HPV 16 and 18, and the nonavalent vaccine targeting 90% of all ICC cases, such HPV genotype distribution data are indispensable for predicting the impact of vaccination and HPV screening on prevention. Even with a successful vaccination program, vaccinated women will still require screening to detect those who will develop ICC from other High risk (HR) HPV genotypes not prevented by current vaccines. The aim of this review is to report on the prevalence of pHR/HR HPV types and multiple pHR/HR HPV genotypes in Kenya among HIV positive women with normal, abnormal cytology and ICC. Methods PUBMED, EMBASE, SCOPUS, and PROQUEST were searched for articles on HPV infection up to August 2nd 2016. Search terms were HIV, HPV, Cervical Cancer, Incidence or Prevalence, and Kenya. Results The 13 studies included yielded a total of 2116 HIV-infected women, of which 89 had ICC. The overall prevalence of pHR/HR HPV genotypes among HIV-infected women was 64% (95%CI: 50%-77%). There was a borderline significant difference in the prevalence of pHR/HR HPV genotypes between Female Sex workers (FSW) compared to non-FSW in women with both normal and abnormal cytology. Multiple pHR/HR HPV genotypes were highly prominent in both normal cytology/HSIL and ICC. The most prevalent HR HPV genotypes in women with abnormal cytology were HPV 16 with 26%, (95%CI: 23.0%-30.0%) followed by HPV 35 and 52, with 21% (95%CI: 18%-25%) and 18% (95%CI: 15%-21%), respectively. In women with ICC, the most prevalent HPV genotypes were HPV 16 (37%; 95%CI: 28%-47%) and HPV 18 (24%; 95%CI: 16%-33%). Conclusion HPV 16/18 gains prominence as the severity of cervical disease increases, with HPV 16/18 accounting for 61

  3. Application of Microsatellite Loci for Molecular Identification of Elite Genotypes, Analysis of Clonality, and Genetic Diversity in Aspen Populus tremula L. (Salicaceae)

    PubMed Central

    Politov, Dmitry V.; Belokon, Maryana M.; Belokon, Yuri S.; Polyakova, Tatyana A.; Shatokhina, Anna V.; Mudrik, Elena A.; Azarova, Anna B.; Filippov, Mikhail V.; Shestibratov, Konstantin A.

    2015-01-01

    Testing systems for molecular identification of micropropagated elite aspen (Populus tremula L.) genotypes were developed on the base on microsatellite (SSR) loci. Out of 33 tested microsatellite loci, 14 were selected due to sustainable PCR amplification and substantial variability in elite clones of aspen aimed for establishment of fast-rotated forest plantations. All eight tested clones had different multilocus genotypes. Among 114 trees from three reference native stands located near the established plantations, 80 haplotypes were identified while some repeated genotypes were attributed to natural clones which appeared as a result of sprouting. The selected set of SSR markers showed reliable individual identification with low probability of appearance of identical aspen genotypes (a minimum of 4.8 · 10−10 and 1 × 10−4 for unrelated and related individuals, resp.). Case studies demonstrating practical applications of the test system are described including analysis of clonal structure and levels of genetic diversity in three natural aspen stands growing in the regions where plantations made of elite clones were established. PMID:26823661

  4. Application of Microsatellite Loci for Molecular Identification of Elite Genotypes, Analysis of Clonality, and Genetic Diversity in Aspen Populus tremula L. (Salicaceae).

    PubMed

    Politov, Dmitry V; Belokon, Maryana M; Belokon, Yuri S; Polyakova, Tatyana A; Shatokhina, Anna V; Mudrik, Elena A; Azarova, Anna B; Filippov, Mikhail V; Shestibratov, Konstantin A

    2015-01-01

    Testing systems for molecular identification of micropropagated elite aspen (Populus tremula L.) genotypes were developed on the base on microsatellite (SSR) loci. Out of 33 tested microsatellite loci, 14 were selected due to sustainable PCR amplification and substantial variability in elite clones of aspen aimed for establishment of fast-rotated forest plantations. All eight tested clones had different multilocus genotypes. Among 114 trees from three reference native stands located near the established plantations, 80 haplotypes were identified while some repeated genotypes were attributed to natural clones which appeared as a result of sprouting. The selected set of SSR markers showed reliable individual identification with low probability of appearance of identical aspen genotypes (a minimum of 4.8 · 10(-10) and 1 × 10(-4) for unrelated and related individuals, resp.). Case studies demonstrating practical applications of the test system are described including analysis of clonal structure and levels of genetic diversity in three natural aspen stands growing in the regions where plantations made of elite clones were established.

  5. ACTN3 Genotype, Athletic Status, and Life Course Physical Capability: Meta-Analysis of the Published Literature and Findings from Nine Studies

    PubMed Central

    Alfred, Tamuno; Ben-Shlomo, Yoav; Cooper, Rachel; Hardy, Rebecca; Cooper, Cyrus; Deary, Ian J; Gunnell, David; Harris, Sarah E; Kumari, Meena; Martin, Richard M; Moran, Colin N; Pitsiladis, Yannis P; Ring, Susan M; Sayer, Avan Aihie; Smith, George Davey; Starr, John M; Kuh, Diana; Day, Ian NM

    2011-01-01

    The ACTN3 R577X (rs1815739) genotype has been associated with athletic status and muscle phenotypes, although not consistently. Our objective was to conduct a meta-analysis of the published literature on athletic status and investigate its associations with physical capability in several new population-based studies. Relevant data were extracted from studies in the literature, comparing genotype frequencies between controls and sprint/power and endurance athletes. For life course physical capability, data were used from two studies of adolescents and seven studies in the Healthy Ageing across the Life Course (HALCyon) collaborative research program, involving individuals aged between 53 and 90+ years. We found evidence from the published literature to support the hypothesis that in Europeans the RR genotype is more common among sprint/power athletes compared with their controls. There is currently no evidence that the X allele is advantageous to endurance athleticism. We found no association between R577X and grip strength (P = 0.09, n = 7,672 in males; P = 0.90, n = 7,839 in females), standing balance, timed get up and go, or chair rises in our studies of physical capability. The ACTN3 R577X genotype is associated with sprint/power athletic status in Europeans, but does not appear to be associated with objective measures of physical capability in the general population. Hum Mutat 32:1–11, 2011. © 2011 Wiley-Liss, Inc. PMID:21542061

  6. Potential Therapy for Neisseria Gonorrhoeae Infections With Human Chorionic Gonadotropin.

    PubMed

    Rao, C V

    2015-12-01

    The scientific evidence suggests that Neisseria gonorrhoeae (NG) infects human fallopian tubes by molecular mimicry in which pathogens act like a ligand to bind to epithelial cell surface human chorionic gonadotrophin (hCG)/luteinizing hormone (LH) receptors. The hCG-like molecule has been identified as ribosomal protein L12 in NG coat surface. Human fallopian tube epithelial cells have been shown to contain functional hCG/LH receptors. As previously shown in human fallopian tube organ and cell culture studies, cellular invasion and infection can be prevented by exposing the cells to excess hCG, which would outnumber and outcompete NG for receptor binding. Based on these data, we suggest testing hCG in clinical trials on infected women.

  7. Diagnosis and treatment of chlamydia and gonorrhoea in general practice in England 2000–2011: a population-based study using data from the UK Clinical Practice Research Datalink

    PubMed Central

    Wetten, Sally; Mohammed, Hamish; Yung, Mandy; Mercer, Catherine H; Cassell, Jackie A; Hughes, Gwenda

    2015-01-01

    Objectives To determine the relative contribution of general practices (GPs) to the diagnosis of chlamydia and gonorrhoea in England and whether treatment complied with national guidelines. Design Analysis of longitudinal electronic health records in the Clinical Practice Research Datalink (CPRD) and national sexually transmitted infection (STI) surveillance databases, England, 2000–2011. Setting GPs, and community and specialist STI services. Participants Patients diagnosed with chlamydia (n=1 386 169) and gonorrhoea (n=232 720) at CPRD GPs, and community and specialist STI Services from 2000–2011. Main outcome measures Numbers and rates of chlamydia and gonorrhoea diagnoses; percentages of patients diagnosed by GPs relative to other services; percentage of GP patients treated and antimicrobials used; percentage of GP patients referred. Results The diagnosis rate (95% CI) per 100 000 population of chlamydia in GP increased from 22.8 (22.4–23.2) in 2000 to 29.3 (28.8–29.7) in 2011 (p<0.001), while the proportion treated increased from 59.5% to 78.4% (p=0.001). Over 90% were prescribed a recommended antimicrobial. Over the same period, the diagnosis rate (95% CI) per 100 000 population of gonorrhoea in GP ranged between 3.2 (3–3.3) and 2.4 (2.2–2.5; p=0.607), and the proportion treated ranged between 32.7% and 53.6% (p=0.262). Despite being discontinued as a recommended therapy for gonorrhoea in 2005, ciprofloxacin accounted for 42% of prescriptions in 2007 and 20% in 2011. Over the study period, GPs diagnosed between 9% and 16% of chlamydia cases and between 6% and 9% of gonorrhoea cases in England. Conclusions GP makes an important contribution to the diagnosis and treatment of bacterial STIs in England. While most patients diagnosed with chlamydia were managed appropriately, many of those treated for gonorrhoea received antimicrobials no longer recommended for use. Given the global threat of antimicrobial resistance, GPs should remain

  8. Phylodynamic analysis of porcine circovirus type 2 reveals global waves of emerging genotypes and the circulation of recombinant forms.

    PubMed

    Franzo, Giovanni; Cortey, Marti; Segalés, Joaquim; Hughes, Joseph; Drigo, Michele

    2016-07-01

    Since the first description of Porcine circovirus type 2 (PCV2), four genotypes (PCV2a, PCV2b, PCV2c and PCV2d) have been recognized and three of them have been shown to exhibit worldwide distribution. Here, the population dynamics of PCV2 has been reconstructed over time and the factors that have shaped its evolution determined. The results obtained confirm that PCV2 originated approximately at the beginning of the 20th century. The most recent common ancestor of genotypes PCV2a, PCV2b, PCV2c and PCV2d circulated in the 1950s, 1980s, 1960s and 1950s, respectively, and the population sizes of the individual genotypes remained low until the mid 90s, coinciding with the identification of PCV2 as a major pathogen of the pig industry. The population dynamics of PCV2 have been characterized by the appearance of periodic waves of distinct genotypes that, after an initial rise, spread following major swine commercial routes and were then superseded by subsequent emerging genotypes. Various recombinant forms displayed comparable population dynamics and spreading routes to those of major genotypes, suggesting that recombinant strains are able to compete with parental ones. The capsid gene is subjected to immune selection and evasion of the host immune response seems to be a major force for the emergence and spread of new genotypes. In contrast, the evolution of other genes appears to be constrained by the particular genomic organization of PCV2. In summary, obtained results suggest that changes in farming strategies, international trade, host population immunity, recombination and the constraints imposed by genome organization have all played a major role in the evolutionary dynamics of PCV2. PMID:27114187

  9. Immunologic classification of Neisseria gonorrhoeae with micro-immunofluorescence.

    PubMed

    Wang, S P; Holmes, K K; Knapp, J S; Ott, S; Kyzer, D D

    1977-09-01

    A reproducible immunologic classification of Neisseria gonorrhoeae strains has been achieved by the micro-immunofluorescence (Micro-IF)3 method by using formalinized whole organisms as test antigens and mouse antisera prepared by i.v. immunization with the whole organisms as antibody. Immunologic differences among Neisseria species were also distinct in this test system. Immunologic differences among gonococcal strains were not influenced by gonococcal colony type. Classification of gonococci was facilitated by use of antisera absorbed with an antigenically unique gonococcus strain. Of 180 gonococcal strains, 175 could be classified into three immunotypes: A, B, and C. Each type was further divided into subtypes designated A1, A2, A3, B1, B2, B3, C1, and C2. Minor antigenic differences still exist within each subtype. The two gonococcal isolates from each of 17 pairs of sexual contacts fell into the same subtype. Seventy-one of 73 isolates which required arginine, hypoxanthine, and uracil for growth (Arg-Hyx-Ura-) and seven of 107 other auxotypes belonged to subtypes A2 and A3. Marked geographical differences in distribution of gonococcal immunotypes were observed among those available for testing. Subtypes A2 and A3 were predominant in Seattle whereas types B and C were predominant in Southeast Asia. The only Arg-Hyx-Ura- isolates not belonging to subtypes A2 or A3 were the only two that were serum sensitive. This Micro-IF immunotyping appears potentially useful for future immunologic, epidemiologic, and genetic studies of N. gonorrhoeae. PMID:408415

  10. Resolving the Effects of Maternal and Offspring Genotype on Dyadic Outcomes in Genome Wide Complex Trait Analysis (“M-GCTA”)

    PubMed Central

    Pourcain, Beate St.; Smith, George Davey; York, Timothy P.; Evans, David M.

    2014-01-01

    Genome wide complex trait analysis (GCTA) is extended to include environmental effects of the maternal genotype on offspring phenotype (“maternal effects”, M-GCTA). The model includes parameters for the direct effects of the offspring genotype, maternal effects and the covariance between direct and maternal effects. Analysis of simulated data, conducted in OpenMx, confirmed that model parameters could be recovered by full information maximum likelihood (FIML) and evaluated the biases that arise in conventional GCTA when indirect genetic effects are ignored. Estimates derived from FIML in OpenMx showed very close agreement to those obtained by restricted maximum likelihood using the published algorithm for GCTA. The method was also applied to illustrative perinatal phenotypes from ∼4,000 mother-offspring pairs from the Avon Longitudinal Study of Parents and Children. The relative merits of extended GCTA in contrast to quantitative genetic approaches based on analyzing the phenotypic covariance structure of kinships are considered. PMID:25060210

  11. Genotyping of velvet antlers for identification of country of origin using mitochondrial DNA and fluorescence melting curve analysis with locked nucleic acid probes.

    PubMed

    Ahn, Jeong Jin; Kim, Youngjoo; Hong, Ji Young; Kim, Gi Won; Hwang, Seung Yong

    2016-07-01

    Velvet antlers are used medicinally in Asia and possess various therapeutic effects. Prices are set according to the country of origin, which is unidentifiable to the naked eye, and therefore counterfeiting is prevalent. Additionally, antlers of the Canadian elk, which can generate chronic wasting disease, are prevalently smuggled and distributed in the market. Thus, a method for identifying the country of origin of velvet antlers was developed, using polymorphisms in mitochondrial DNA, fluorescence melting curve analysis and analysis of locked nucleic acids (LNA). This combined method is capable of identifying five genotypes of velvet antlers in a single experiment using two probes. It also has advantages in multiplexing, simplicity and efficiency in genotyping, when compared to real-time PCR or microarrays. The developed method can be used to improve identification rates in the velvet antler market and, by extension, research based on polymorphisms in DNA sequences.

  12. Multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) using multiplex PCR and multicolor capillary electrophoresis: application to the genotyping of Brucella species.

    PubMed

    Garofolo, Giuliano

    2015-01-01

    The multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) is a genetic typing method based on the evaluation of the number of repeated sequences in multiple selected loci of microbial DNA. Although several MLVA typing panels have been proposed for brucellae, the 16-loci panel is recognized as the standard genotyping method, also used for the Brucella international online database. This chapter describes a high-throughput MLVA-16 protocol using multiplex PCRs and multicolor capillary electrophoresis.

  13. Novel Genotyping and Quantitative Analysis of Neuraminidase Inhibitor Resistance-Associated Mutations in Influenza A Viruses by Single-Nucleotide Polymorphism Analysis▿§

    PubMed Central

    Duan, Susu; Boltz, David A.; Li, Jiang; Oshansky, Christine M.; Marjuki, Henju; Barman, Subrata; Webby, Richard J.; Webster, Robert G.; Govorkova, Elena A.

    2011-01-01

    Neuraminidase (NA) inhibitors are among the first line of defense against influenza virus infection. With the increased worldwide use of the drugs, antiviral susceptibility surveillance is increasingly important for effective clinical management and for public health epidemiology. Effective monitoring requires effective resistance detection methods. We have developed and validated a novel genotyping method for rapid detection of established NA inhibitor resistance markers in influenza viruses by single nucleotide polymorphism (SNP) analysis. The multi- or monoplex SNP analysis based on single nucleotide extension assays was developed to detect NA mutations H275Y and I223R/V in pandemic H1N1 viruses, H275Y in seasonal H1N1 viruses, E119V and R292K in seasonal H3N2 viruses, and H275Y and N295S in H5N1 viruses. The SNP analysis demonstrated high sensitivity for low-content NA amplicons (0.1 to 1 ng/μl) and showed 100% accordant results against a panel of defined clinical isolates. The monoplex assays for the H275Y NA mutation allowed precise and accurate quantification of the proportions of wild-type and mutant genotypes in virus mixtures (5% to 10% discrimination), with results comparable to those of pyrosequencing. The SNP analysis revealed the lower growth fitness of an H275Y mutant compared to the wild-type pandemic H1N1 virus by quantitatively genotyping progeny viruses grown in normal human bronchial epithelial cells. This novel method offers high-throughput screening capacity, relatively low costs, and the wide availability of the necessary equipment, and thus it could provide a much-needed approach for genotypic screening of NA inhibitor resistance in influenza viruses. PMID:21730113

  14. Identification of genes associated with nitrogen-use efficiency by genome-wide transcriptional analysis of two soybean genotypes

    PubMed Central

    2011-01-01

    Background Soybean is a valuable crop that provides protein and oil. Soybean requires a large amount of nitrogen (N) to accumulate high levels of N in the seed. The yield and protein content of soybean seeds are directly affected by the N-use efficiency (NUE) of the plant, and improvements in NUE will improve yields and quality of soybean products. Genetic engineering is one of the approaches to improve NUE, but at present, it is hampered by the lack of information on genes associated with NUE. Solexa sequencing is a new method for estimating gene expression in the transcription level. Here, the expression profiles were analyzed between two soybean varieties in N-limited conditions to identify genes related to NUE. Results Two soybean genotypes were grown under N-limited conditions; a low-N-tolerant variety (No.116) and a low-N-sensitive variety (No.84-70). The shoots and roots of soybeans were used for sequencing. Eight libraries were generated for analysis: 2 genotypes × 2 tissues (roots and shoots) × 2 time periods [short-term (0.5 to 12 h) and long-term (3 to 12 d) responses] and compared the transcriptomes by high-throughput tag-sequencing analysis. 5,739,999, 5,846,807, 5,731,901, 5,970,775, 5,476,878, 5,900,343, 5,930,716, and 5,862,642 clean tags were obtained for the eight libraries: L1, 116-shoot short-term; L2 84-70-shoot short-term; L3 116-shoot long-term; L4 84-70-shoot long-term; L5 116-root short-term; L6 84-70-root short-term; L7 116-root long-term;L8 84-70-root long-term; these corresponded to 224,154, 162,415, 191,994, 181,792, 204,639, 206,998, 233,839 and 257,077 distinct tags, respectively. The clean tags were mapped to the reference sequences for annotation of expressed genes. Many genes showed substantial differences in expression among the libraries. In total, 3,231genes involved in twenty-two metabolic and signal transduction pathways were up- or down-regulated. Twenty-four genes were randomly selected and confirmed their expression

  15. A simple and rapid genotyping assay for simultaneous detection of two ADRB2 allelic variants using fluorescence resonance energy transfer probes and melting curve analysis.

    PubMed

    Sábato, M Fernanda; Irani, Anne-Marie; Bukaveckas, Bonny L; Schwartz, Lawrence B; Wilkinson, David S; Ferreira-Gonzalez, Andrea

    2008-05-01

    Allelic variants at codons 16 and 27 of the beta(2)-adrenergic receptor gene (ADRB2) have shown clinical and pharmacological implications in asthma, hypertension, ischemic heart failure, diabetes, obesity, and cystic fibrosis. We have developed a simultaneous genotyping assay for the c.46A>G and c.79C>G allelic variants using hybridization probes and melting curve analysis. The assay was optimized on a panel of 30 DNA samples of known ADRB2 genotype as determined by sequencing with 100% concordance between the two techniques. Melting temperature (Tm) ranges for the different genotypes were obtained using data from three independent experiments. Single peaks for p.Arg16Arg (Tm = 57.76 degrees C +/- 0.10 degrees C) and p.Gly16Gly (Tm = 66.73 degrees C +/- 0.18 degrees C) and two melting peaks for p.Arg16Gly were obtained. Similarly, single peaks for p.Gln27Gln (Tm = 53.98 degrees C +/- 0.19 degrees C) and p.Glu27Glu (Tm = 64.93 degrees C +/- 0.16 degrees C) and two peaks for p.Gln27Glu were detected. Independent operators easily assigned genotypes in a sample set of 385 asthmatic patients. Haplotype and allele frequencies were in concordance with previously published data: Arg allele frequencies in children/adults were 0.34/0.30 in Caucasians and 0.45/0.52 in African Americans, and Gln allele frequencies were 0.58/0.52 in Caucasians and 0.82/0.84 in African Americans. Thus, the ADRB2 genotyping assay represents a highly reliable and rapid technique for routine clinical use in the simultaneous detection of ADRB2 variants.

  16. A Simple and Rapid Genotyping Assay for Simultaneous Detection of Two ADRB2 Allelic Variants Using Fluorescence Resonance Energy Transfer Probes and Melting Curve Analysis

    PubMed Central

    Sábato, M. Fernanda; Irani, Anne-Marie; Bukaveckas, Bonny L.; Schwartz, Lawrence B.; Wilkinson, David S.; Ferreira-Gonzalez, Andrea

    2008-01-01

    Allelic variants at codons 16 and 27 of the β2-adrenergic receptor gene (ADRB2) have shown clinical and pharmacological implications in asthma, hypertension, ischemic heart failure, diabetes, obesity, and cystic fibrosis. We have developed a simultaneous genotyping assay for the c.46A>G and c.79C>G allelic variants using hybridization probes and melting curve analysis. The assay was optimized on a panel of 30 DNA samples of known ADRB2 genotype as determined by sequencing with 100% concordance between the two techniques. Melting temperature (Tm) ranges for the different genotypes were obtained using data from three independent experiments. Single peaks for p.Arg16Arg (Tm = 57.76°C ± 0.10°C) and p.Gly16Gly (Tm = 66.73°C ± 0.18°C) and two melting peaks for p.Arg16Gly were obtained. Similarly, single peaks for p.Gln27Gln (Tm = 53.98°C ± 0.19°C) and p.Glu27Glu (Tm = 64.93°C ± 0.16°C) and two peaks for p.Gln27Glu were detected. Independent operators easily assigned genotypes in a sample set of 385 asthmatic patients. Haplotype and allele frequencies were in concordance with previously published data: Arg allele frequencies in children/adults were 0.34/0.30 in Caucasians and 0.45/0.52 in African Americans, and Gln allele frequencies were 0.58/0.52 in Caucasians and 0.82/0.84 in African Americans. Thus, the ADRB2 genotyping assay represents a highly reliable and rapid technique for routine clinical use in the simultaneous detection of ADRB2 variants. PMID:18440968

  17. COMT Val(158) Met genotype is associated with reward learning: a replication study and meta-analysis.

    PubMed

    Corral-Frías, N S; Pizzagalli, D A; Carré, J M; Michalski, L J; Nikolova, Y S; Perlis, R H; Fagerness, J; Lee, M R; Conley, E Drabant; Lancaster, T M; Haddad, S; Wolf, A; Smoller, J W; Hariri, A R; Bogdan, R

    2016-06-01

    Identifying mechanisms through which individual differences in reward learning emerge offers an opportunity to understand both a fundamental form of adaptive responding as well as etiological pathways through which aberrant reward learning may contribute to maladaptive behaviors and psychopathology. One candidate mechanism through which individual differences in reward learning may emerge is variability in dopaminergic reinforcement signaling. A common functional polymorphism within the catechol-O-methyl transferase gene (COMT; rs4680, Val(158) Met) has been linked to reward learning, where homozygosity for the Met allele (linked to heightened prefrontal dopamine function and decreased dopamine synthesis in the midbrain) has been associated with relatively increased reward learning. Here, we used a probabilistic reward learning task to asses response bias, a behavioral form of reward learning, across three separate samples that were combined for analyses (age: 21.80 ± 3.95; n = 392; 268 female; European-American: n = 208). We replicate prior reports that COMT rs4680 Met allele homozygosity is associated with increased reward learning in European-American participants (β = 0.20, t = 2.75, P < 0.01; ΔR(2) = 0.04). Moreover, a meta-analysis of 4 studies, including the current one, confirmed the association between COMT rs4680 genotype and reward learning (95% CI -0.11 to -0.03; z = 3.2; P < 0.01). These results suggest that variability in dopamine signaling associated with COMT rs4680 influences individual differences in reward which may potentially contribute to psychopathology characterized by reward dysfunction. PMID:27138112

  18. 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.

  19. A comprehensive transcriptome analysis of silique development and dehiscence in Arabidopsis and Brassica integrating genotypic, interspecies and developmental comparisons

    PubMed Central

    Jaradat, Masrur R; Ruegger, Max; Bowling, Andrew; Butler, Holly; Cutler, Adrian J

    2014-01-01

    Asynchronous flowering of Brassica napus (canola) leads to seeds and siliques at varying stages of maturity as harvest approaches. This range of maturation can result in premature silique dehiscence (pod shattering), resulting in yield losses, which may be worsened by environmental stresses. Therefore, a goal for canola crop improvement is to reduce shattering in order to maximize yield. We performed a comprehensive transcriptome analysis on the dehiscence zone (DZ) and valve of Arabidopsis and Brassica siliques in shatter resistant and sensitive genotypes at several developmental stages. Among known Arabidopsis dehiscence genes, we confirmed that homologs of SHP1/2, FUL, ADPG1, NST1/3 and IND were associated with shattering in B. juncea and B. napus. We noted a correlation between reduced pectin degradation genes and shatter-resistance. Tension between lignified and non-lignified cells in the silique DZ plays a major role in dehiscence. Light microscopy revealed a smaller non-lignified separation layer in relatively shatter-resistant B. juncea relative to B. napus and this corresponded to increased expression of peroxidases involved in monolignol polymerization. Sustained repression of auxin biosynthesis, transport and signaling in B. juncea relative to B. napus may cause differences in dehiscence zone structure and cell wall constituents. Tension on the dehiscence zone is a consequence of shrinkage and loss of flexibility in the valves, which is caused by senescence and desiccation. Reduced shattering was generally associated with upregulation of ABA signaling and down-regulation of ethylene and jasmonate signaling, corresponding to more pronounced stress responses and reduced senescence and photosynthesis. Overall, we identified 124 cell wall related genes and 103 transcription factors potentially involved in silique dehiscence. PMID:25523176

  20. A comprehensive transcriptome analysis of silique development and dehiscence in Arabidopsis and Brassica integrating genotypic, interspecies and developmental comparisons.

    PubMed

    Jaradat, Masrur R; Ruegger, Max; Bowling, Andrew; Butler, Holly; Cutler, Adrian J

    2014-01-01

    Asynchronous flowering of Brassica napus (canola) leads to seeds and siliques at varying stages of maturity as harvest approaches. This range of maturation can result in premature silique dehiscence (pod shattering), resulting in yield losses, which may be worsened by environmental stresses. Therefore, a goal for canola crop improvement is to reduce shattering in order to maximize yield. We performed a comprehensive transcriptome analysis on the dehiscence zone (DZ) and valve of Arabidopsis and Brassica siliques in shatter resistant and sensitive genotypes at several developmental stages. Among known Arabidopsis dehiscence genes, we confirmed that homologs of SHP1/2, FUL, ADPG1, NST1/3 and IND were associated with shattering in B. juncea and B. napus. We noted a correlation between reduced pectin degradation genes and shatter-resistance. Tension between lignified and non-lignified cells in the silique DZ plays a major role in dehiscence. Light microscopy revealed a smaller non-lignified separation layer in relatively shatter-resistant B. juncea relative to B. napus and this corresponded to increased expression of peroxidases involved in monolignol polymerization. Sustained repression of auxin biosynthesis, transport and signaling in B. juncea relative to B. napus may cause differences in dehiscence zone structure and cell wall constituents. Tension on the dehiscence zone is a consequence of shrinkage and loss of flexibility in the valves, which is caused by senescence and desiccation. Reduced shattering was generally associated with upregulation of ABA signaling and down-regulation of ethylene and jasmonate signaling, corresponding to more pronounced stress responses and reduced senescence and photosynthesis. Overall, we identified 124 cell wall related genes and 103 transcription factors potentially involved in silique dehiscence. PMID:25523176

  1. People of the British Isles: preliminary analysis of genotypes and surnames in a UK-control population

    PubMed Central

    Winney, Bruce; Boumertit, Abdelhamid; Day, Tammy; Davison, Dan; Echeta, Chikodi; Evseeva, Irina; Hutnik, Katarzyna; Leslie, Stephen; Nicodemus, Kristin; Royrvik, Ellen C; Tonks, Susan; Yang, Xiaofeng; Cheshire, James; Longley, Paul; Mateos, Pablo; Groom, Alexandra; Relton, Caroline; Bishop, D Tim; Black, Kathryn; Northwood, Emma; Parkinson, Louise; Frayling, Timothy M; Steele, Anna; Sampson, Julian R; King, Turi; Dixon, Ron; Middleton, Derek; Jennings, Barbara; Bowden, Rory; Donnelly, Peter; Bodmer, Walter

    2012-01-01

    There is a great deal of interest in a fine-scale population structure in the UK, both as a signature of historical immigration events and because of the effect population structure may have on disease association studies. Although population structure appears to have a minor impact on the current generation of genome-wide association studies, it is likely to have a significant part in the next generation of studies designed to search for rare variants. A powerful way of detecting such structure is to control and document carefully the provenance of the samples involved. In this study, we describe the collection of a cohort of rural UK samples (The People of the British Isles), aimed at providing a well-characterised UK-control population that can be used as a resource by the research community, as well as providing a fine-scale genetic information on the British population. So far, some 4000 samples have been collected, the majority of which fit the criteria of coming from a rural area and having all four grandparents from approximately the same area. Analysis of the first 3865 samples that have been geocoded indicates that 75% have a mean distance between grandparental places of birth of 37.3 km, and that about 70% of grandparental places of birth can be classed as rural. Preliminary genotyping of 1057 samples demonstrates the value of these samples for investigating a fine-scale population structure within the UK, and shows how this can be enhanced by the use of surnames. PMID:21829225

  2. 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

  3. COMT Val(158) Met genotype is associated with reward learning: a replication study and meta-analysis.

    PubMed

    Corral-Frías, N S; Pizzagalli, D A; Carré, J M; Michalski, L J; Nikolova, Y S; Perlis, R H; Fagerness, J; Lee, M R; Conley, E Drabant; Lancaster, T M; Haddad, S; Wolf, A; Smoller, J W; Hariri, A R; Bogdan, R

    2016-06-01

    Identifying mechanisms through which individual differences in reward learning emerge offers an opportunity to understand both a fundamental form of adaptive responding as well as etiological pathways through which aberrant reward learning may contribute to maladaptive behaviors and psychopathology. One candidate mechanism through which individual differences in reward learning may emerge is variability in dopaminergic reinforcement signaling. A common functional polymorphism within the catechol-O-methyl transferase gene (COMT; rs4680, Val(158) Met) has been linked to reward learning, where homozygosity for the Met allele (linked to heightened prefrontal dopamine function and decreased dopamine synthesis in the midbrain) has been associated with relatively increased reward learning. Here, we used a probabilistic reward learning task to asses response bias, a behavioral form of reward learning, across three separate samples that were combined for analyses (age: 21.80 ± 3.95; n = 392; 268 female; European-American: n = 208). We replicate prior reports that COMT rs4680 Met allele homozygosity is associated with increased reward learning in European-American participants (β = 0.20, t = 2.75, P < 0.01; ΔR(2) = 0.04). Moreover, a meta-analysis of 4 studies, including the current one, confirmed the association between COMT rs4680 genotype and reward learning (95% CI -0.11 to -0.03; z = 3.2; P < 0.01). These results suggest that variability in dopamine signaling associated with COMT rs4680 influences individual differences in reward which may potentially contribute to psychopathology characterized by reward dysfunction.

  4. The Genotype-Tissue Expression (GTEx) Project: Linking Clinical Data with Molecular Analysis to Advance Personalized Medicine

    PubMed Central

    Keen, Judy C.; Moore, Helen M.

    2015-01-01

    Evaluation of how genetic mutations or variability can directly affect phenotypic outcomes, the development of disease, or determination of a tailored treatment protocol is fundamental to advancing personalized medicine. To understand how a genotype affects gene expression and specific phenotypic traits, as well as the correlative and causative associations between such, the Genotype-Tissue Expression (GTEx) Project was initiated The GTEx collection of biospecimens and associated clinical data links extensive clinical data with genotype and gene expression data to provide a wealth of data and resources to study the underlying genetics of normal physiology. These data will help inform personalized medicine through the identification of normal variation that does not contribute to disease. Additionally, these data can lead to insights into how gene variation affects pharmacodynamics and individualized responses to therapy. PMID:25809799

  5. Successful Combination of Nucleic Acid Amplification Test Diagnostics and Targeted Deferred Neisseria gonorrhoeae Culture

    PubMed Central

    Wind, Carolien M.; de Vries, Henry J. C.; Schim van der Loeff, Maarten F.; Unemo, Magnus

    2015-01-01

    Nucleic acid amplification tests (NAATs) are recommended for the diagnosis of N. gonorrhoeae infections because of their superior sensitivity. Increasing NAAT use causes a decline in crucial antimicrobial resistance (AMR) surveillance data, which rely on culture. We analyzed the suitability of the ESwab system for NAAT diagnostics and deferred targeted N. gonorrhoeae culture to allow selective and efficient culture based on NAAT results. We included patients visiting the STI Clinic Amsterdam, The Netherlands, in 2013. Patient characteristics and urogenital and rectal samples for direct N. gonorrhoeae culture, standard NAAT, and ESwab were collected. Standard NAAT and NAAT on ESwab samples were performed using the Aptima Combo 2 assay for N. gonorrhoeae and C. trachomatis. Two deferred N. gonorrhoeae cultures were performed on NAAT-positive ESwab samples after storage at 4°C for 1 to 3 days. We included 2,452 samples from 1,893 patients. In the standard NAAT, 107 samples were N. gonorrhoeae positive and 284 were C. trachomatis positive. The sensitivities of NAAT on ESwab samples were 83% (95% confidence interval [CI], 75 to 90%) and 87% (95% CI, 82 to 90%), respectively. ESwab samples were available for 98 of the gonorrhea-positive samples. Of these, 82% were positive in direct culture and 69% and 56% were positive in the 1st and 2nd deferred cultures, respectively (median storage times, 27 and 48 h, respectively). Deferred culture was more often successful in urogenital samples or when the patient had symptoms at the sampling site. Deferred N. gonorrhoeae culture of stored ESwab samples is feasible and enables AMR surveillance. To limit the loss in NAAT sensitivity, we recommend obtaining separate samples for NAAT and deferred culture. PMID:25832300

  6. Molecular Assay for Detection of Genetic Markers Associated with Decreased Susceptibility to Cephalosporins in Neisseria gonorrhoeae

    PubMed Central

    Peterson, S. W.; Martin, I.; Demczuk, W.; Bharat, A.; Hoang, L.; Wylie, J.; Allen, V.; Lefebvre, B.; Tyrrell, G.; Horsman, G.; Haldane, D.; Garceau, R.; Wong, T.

    2015-01-01

    The incidence of antimicrobial-resistant Neisseria gonorrhoeae continues to rise in Canada; however, antimicrobial resistance data are lacking for approximately 70% of gonorrhea infections that are diagnosed directly from clinical specimens by nucleic acid amplification tests (NAATs). We developed a molecular assay for surveillance use to detect mutations in genes associated with decreased susceptibility to cephalosporins that can be applied to both culture isolates and clinical samples. Real-time PCR assays were developed to detect single nucleotide polymorphisms (SNPs) in ponA, mtrR, penA, porB, and one N. gonorrhoeae-specific marker (porA). We tested the real-time PCR assay with 252 gonococcal isolates, 50 nongonococcal isolates, 24 N. gonorrhoeae-negative NAAT specimens, and 34 N. gonorrhoeae-positive NAAT specimens. Twenty-four of the N. gonorrhoeae-positive NAAT specimens had matched culture isolates. Assay results were confirmed by comparison with whole-genome sequencing data. For 252 N. gonorrhoeae strains, the agreement between the DNA sequence and real-time PCR was 100% for porA, ponA, and penA, 99.6% for mtrR, and 95.2% for porB. The presence of ≥2 SNPs correlated with decreased susceptibility to ceftriaxone (sensitivities of >98%) and cefixime (sensitivities of >96%). Of 24 NAAT specimens with matched cultures, the agreement between the DNA sequence and real-time PCR was 100% for porB, 95.8% for ponA and mtrR, and 91.7% for penA. We demonstrated the utility of a real-time PCR assay for sensitive detection of known markers for the decreased susceptibility to cephalosporins in N. gonorrhoeae. Preliminary results with clinical NAAT specimens were also promising, as they correlated well with bacterial culture results. PMID:25878350

  7. Antibiotic-Resistant Neisseria gonorrhoeae Spread Faster with More Treatment, Not More Sexual Partners

    PubMed Central

    Bonhoeffer, Sebastian; Low, Nicola; Althaus, Christian L.

    2016-01-01

    The sexually transmitted bacterium Neisseria gonorrhoeae has developed resistance to all antibiotic classes that have been used for treatment and strains resistant to multiple antibiotic classes have evolved. In many countries, there is only one antibiotic remaining for empirical N. gonorrhoeae treatment, and antibiotic management to counteract resistance spread is urgently needed. Understanding dynamics and drivers of resistance spread can provide an improved rationale for antibiotic management. In our study, we first used antibiotic resistance surveillance data to estimate the rates at which antibiotic-resistant N. gonorrhoeae spread in two host populations, heterosexual men (HetM) and men who have sex with men (MSM). We found higher rates of spread for MSM (0.86 to 2.38 y−1, mean doubling time: 6 months) compared to HetM (0.24 to 0.86 y−1, mean doubling time: 16 months). We then developed a dynamic transmission model to reproduce the observed dynamics of N. gonorrhoeae transmission in populations of heterosexual men and women (HMW) and MSM. We parameterized the model using sexual behavior data and calibrated it to N. gonorrhoeae prevalence and incidence data. In the model, antibiotic-resistant N. gonorrhoeae spread with a median rate of 0.88 y−1 in HMW and 3.12 y−1 in MSM. These rates correspond to median doubling times of 9 (HMW) and 3 (MSM) months. Assuming no fitness costs, the model shows the difference in the host population’s treatment rate rather than the difference in the number of sexual partners explains the differential spread of resistance. As higher treatment rates result in faster spread of antibiotic resistance, treatment recommendations for N. gonorrhoeae should carefully balance prevention of infection and avoidance of resistance spread. PMID:27196299

  8. Molecular Assay for Detection of Genetic Markers Associated with Decreased Susceptibility to Cephalosporins in Neisseria gonorrhoeae.

    PubMed

    Peterson, S W; Martin, I; Demczuk, W; Bharat, A; Hoang, L; Wylie, J; Allen, V; Lefebvre, B; Tyrrell, G; Horsman, G; Haldane, D; Garceau, R; Wong, T; Mulvey, M R

    2015-07-01

    The incidence of antimicrobial-resistant Neisseria gonorrhoeae continues to rise in Canada; however, antimicrobial resistance data are lacking for approximately 70% of gonorrhea infections that are diagnosed directly from clinical specimens by nucleic acid amplification tests (NAATs). We developed a molecular assay for surveillance use to detect mutations in genes associated with decreased susceptibility to cephalosporins that can be applied to both culture isolates and clinical samples. Real-time PCR assays were developed to detect single nucleotide polymorphisms (SNPs) in ponA, mtrR, penA, porB, and one N. gonorrhoeae-specific marker (porA). We tested the real-time PCR assay with 252 gonococcal isolates, 50 nongonococcal isolates, 24 N. gonorrhoeae-negative NAAT specimens, and 34 N. gonorrhoeae-positive NAAT specimens. Twenty-four of the N. gonorrhoeae-positive NAAT specimens had matched culture isolates. Assay results were confirmed by comparison with whole-genome sequencing data. For 252 N. gonorrhoeae strains, the agreement between the DNA sequence and real-time PCR was 100% for porA, ponA, and penA, 99.6% for mtrR, and 95.2% for porB. The presence of ≥2 SNPs correlated with decreased susceptibility to ceftriaxone (sensitivities of >98%) and cefixime (sensitivities of >96%). Of 24 NAAT specimens with matched cultures, the agreement between the DNA sequence and real-time PCR was 100% for porB, 95.8% for ponA and mtrR, and 91.7% for penA. We demonstrated the utility of a real-time PCR assay for sensitive detection of known markers for the decreased susceptibility to cephalosporins in N. gonorrhoeae. Preliminary results with clinical NAAT specimens were also promising, as they correlated well with bacterial culture results.

  9. Antibiotic-Resistant Neisseria gonorrhoeae Spread Faster with More Treatment, Not More Sexual Partners.

    PubMed

    Fingerhuth, Stephanie M; Bonhoeffer, Sebastian; Low, Nicola; Althaus, Christian L

    2016-05-01

    The sexually transmitted bacterium Neisseria gonorrhoeae has developed resistance to all antibiotic classes that have been used for treatment and strains resistant to multiple antibiotic classes have evolved. In many countries, there is only one antibiotic remaining for empirical N. gonorrhoeae treatment, and antibiotic management to counteract resistance spread is urgently needed. Understanding dynamics and drivers of resistance spread can provide an improved rationale for antibiotic management. In our study, we first used antibiotic resistance surveillance data to estimate the rates at which antibiotic-resistant N. gonorrhoeae spread in two host populations, heterosexual men (HetM) and men who have sex with men (MSM). We found higher rates of spread for MSM (0.86 to 2.38 y-1, mean doubling time: 6 months) compared to HetM (0.24 to 0.86 y-1, mean doubling time: 16 months). We then developed a dynamic transmission model to reproduce the observed dynamics of N. gonorrhoeae transmission in populations of heterosexual men and women (HMW) and MSM. We parameterized the model using sexual behavior data and calibrated it to N. gonorrhoeae prevalence and incidence data. In the model, antibiotic-resistant N. gonorrhoeae spread with a median rate of 0.88 y-1 in HMW and 3.12 y-1 in MSM. These rates correspond to median doubling times of 9 (HMW) and 3 (MSM) months. Assuming no fitness costs, the model shows the difference in the host population's treatment rate rather than the difference in the number of sexual partners explains the differential spread of resistance. As higher treatment rates result in faster spread of antibiotic resistance, treatment recommendations for N. gonorrhoeae should carefully balance prevention of infection and avoidance of resistance spread.

  10. Antibiotic-Resistant Neisseria gonorrhoeae Spread Faster with More Treatment, Not More Sexual Partners.

    PubMed

    Fingerhuth, Stephanie M; Bonhoeffer, Sebastian; Low, Nicola; Althaus, Christian L

    2016-05-01

    The sexually transmitted bacterium Neisseria gonorrhoeae has developed resistance to all antibiotic classes that have been used for treatment and strains resistant to multiple antibiotic classes have evolved. In many countries, there is only one antibiotic remaining for empirical N. gonorrhoeae treatment, and antibiotic management to counteract resistance spread is urgently needed. Understanding dynamics and drivers of resistance spread can provide an improved rationale for antibiotic management. In our study, we first used antibiotic resistance surveillance data to estimate the rates at which antibiotic-resistant N. gonorrhoeae spread in two host populations, heterosexual men (HetM) and men who have sex with men (MSM). We found higher rates of spread for MSM (0.86 to 2.38 y-1, mean doubling time: 6 months) compared to HetM (0.24 to 0.86 y-1, mean doubling time: 16 months). We then developed a dynamic transmission model to reproduce the observed dynamics of N. gonorrhoeae transmission in populations of heterosexual men and women (HMW) and MSM. We parameterized the model using sexual behavior data and calibrated it to N. gonorrhoeae prevalence and incidence data. In the model, antibiotic-resistant N. gonorrhoeae spread with a median rate of 0.88 y-1 in HMW and 3.12 y-1 in MSM. These rates correspond to median doubling times of 9 (HMW) and 3 (MSM) months. Assuming no fitness costs, the model shows the difference in the host population's treatment rate rather than the difference in the number of sexual partners explains the differential spread of resistance. As higher treatment rates result in faster spread of antibiotic resistance, treatment recommendations for N. gonorrhoeae should carefully balance prevention of infection and avoidance of resistance spread. PMID:27196299

  11. Genotype-guided versus standard vitamin K antagonist dosing algorithms in patients initiating anticoagulation. A systematic review and meta-analysis.

    PubMed

    Belley-Cote, Emilie P; Hanif, Hasib; D'Aragon, Frederick; Eikelboom, John W; Anderson, Jeffrey L; Borgman, Mark; Jonas, Daniel E; Kimmel, Stephen E; Manolopoulos, Vangelis G; Baranova, Ekaterina; Maitland-van der Zee, Anke H; Pirmohamed, Munir; Whitlock, Richard P

    2015-10-01

    Variability in vitamin K antagonist (VKA) dosing is partially explained by genetic polymorphisms. We performed a meta-analysis to determine whether genotype-guided VKA dosing algorithms decrease a composite of death, thromboembolic events and major bleeding (primary outcome) and improve time in therapeutic range (TTR). We searched MEDLINE, EMBASE, CENTRAL, trial registries and conference proceedings for randomised trials comparing genotype-guided and standard (non genotype-guided) VKA dosing algorithms in adults initiating anticoagulation. Data were pooled using a random effects model. Of the 12 included studies (3,217 patients), six reported all components of the primary outcome of mortality, thromboembolic events and major bleeding (2,223 patients, 87 events). Our meta-analysis found no significant difference between groups for the primary outcome (relative risk 0.85, 95% confidence interval [CI] 0.54-1.34; heterogeneity Χ(²)=4.46, p=0.35, I(²)=10%). Based on 10 studies (2,767 patients), TTR was significantly higher in the genotype-guided group (mean difference (MD) 4.31%; 95% CI 0.35, 8.26; heterogeneity Χ(²)=43.31, p<0.001, I(²)=79%). Pre-specified exploratory analyses demonstrated that TTR was significantly higher when genotype-guided dosing was compared with fixed VKA dosing (6 trials, 997 patients: MD 8.41%; 95% CI 3.50,13.31; heterogeneity Χ(²)=15.18, p=0.01, I(²)=67%) but not when compared with clinical algorithm-guided dosing (4 trials, 1,770 patients: MD -0.29%; 95% CI -2.48,1.90; heterogeneity Χ(²)=1.53, p=0.68, I(²)=0%; p for interaction=0.002). In conclusion, genotype-guided compared with standard VKA dosing algorithms were not found to decrease a composite of death, thromboembolism and major bleeding, but did result in improved TTR. An improvement in TTR was observed in comparison with fixed VKA dosing algorithms, but not with clinical algorithms.

  12. Genotype-to-phenotype analysis: Search for clinical characteristics of a missense change in the GABA{sub A}-{beta}1 receptor gene

    SciTech Connect

    Sobell, J.L.; Sigurdson, D.C.; Sommer, S.S.

    1996-02-16

    Genotype-to-phenotype analysis reverses the classical approach to genetic disease in which an unknown genotype is sought for a known phenotype. This paper provides an example of genotype-to-phenotype analysis for the possible psychiatric effects of a missense mutation (H396Q) at a highly conserved residue of the {Beta}1 subunit gene of the gamma aminobutyric acid type A receptor. DNA samples from 1,507 Caucasians of Western European descent were screened, and 10 heterozygotes for H396Q were identified. These individuals were matched to homozygous normal individuals by age, gender, and length of available medical records. The complete medical records of these 20 individuals were reviewed blindly by two psychiatrists (D.C.S., L.L.H.) to assess psychiatric symptomatology, with an emphasis on anxiety and related disorders. However, no association was found between this missense change at a conserved amino acid and a dominant neuropsychiatric disease phenotype. Thus, this missense change may be neutral or only mildly deleterious, may only cause recessive disease in rare individuals, or may interact epistatically with some other gene(s). 17 refs.

  13. Associations between FTO genotype and total energy and macronutrient intake in adults: a systematic review and meta-analysis.

    PubMed

    Livingstone, K M; Celis-Morales, C; Lara, J; Ashor, A W; Lovegrove, J A; Martinez, J A; Saris, W H; Gibney, M; Manios, Y; Traczyk, I; Drevon, C A; Daniel, H; Gibney, E R; Brennan, L; Bouwman, J; Grimaldi, K A; Mathers, J C

    2015-08-01

    Risk variants of fat mass and obesity-associated (FTO) gene have been associated with increased obesity. However, the evidence for associations between FTO genotype and macronutrient intake has not been reviewed systematically. Our aim was to evaluate the potential associations between FTO genotype and intakes of total energy, fat, carbohydrate and protein. We undertook a systematic literature search in OVID MEDLINE, Scopus, EMBASE and Cochrane of associations between macronutrient intake and FTO genotype in adults. Beta coefficients and confidence intervals (CIs) were used for per allele comparisons. Random-effect models assessed the pooled effect sizes. We identified 56 eligible studies reporting on 213,173 adults. For each copy of the FTO risk allele, individuals reported 6.46 kcal day(-1) (95% CI: 10.76, 2.16) lower total energy intake (P = 0.003). Total fat (P = 0.028) and protein (P = 0.006), but not carbohydrate intakes, were higher in those carrying the FTO risk allele. After adjustment for body weight, total energy intakes remained significantly lower in individuals with the FTO risk genotype (P = 0.028). The FTO risk allele is associated with a lower reported total energy intake and with altered patterns of macronutrient intake. Although significant, these differences are small and further research is needed to determine whether the associations are independent of dietary misreporting.

  14. Analysis of single nucleotide polymorphism via genotyping-by-sequencing in the gall midge Mayetiola Destructor (Hessian Fly)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genotyping-by-sequencing (GBS) is a recently developed technology that has been used to identify DNA markers and map genes for specific traits in many organisms. The gall midge Mayetiola destructor, commonly known as Hessian fly, is a global destructive pest of wheat. In this study, we identified ...

  15. Draft Genome Sequence of Neisseria gonorrhoeae Strain NG_869 with Penicillin, Tetracycline and Ciprofloxacin Resistance Determinants Isolated from Malaysia.

    PubMed

    Ang, Geik Yong; Yu, Choo Yee; Yong, Delicia Ann; Cheong, Yuet Meng; Yin, Wai-Fong; Chan, Kok-Gan

    2016-06-01

    Gonorrhea is a sexually transmitted infection caused by Neisseria gonorrhoeae and the increasing reports of multidrug-resistant gonococcal isolates are a global public health care concern. Herein, we report the genome sequence of N. gonorrhoeae strain NG_869 isolated from Malaysia which may provide insights into the drug resistance determinants in gonococcal bacteria. PMID:27570316

  16. Neisseria gonorrhoeae with high-level resistance to azithromycin: case report of the first isolate identified in the United States.

    PubMed

    Katz, Alan R; Komeya, Alan Y; Soge, Olusegun O; Kiaha, Mandy I; Lee, Maria Veneranda C; Wasserman, Glenn M; Maningas, Eloisa V; Whelen, A Christian; Kirkcaldy, Robert D; Shapiro, Steven J; Bolan, Gail A; Holmes, King K

    2012-03-01

    We report on the first Neisseria gonorrhoeae isolate in the United States identified with high-level resistance to azithromycin. This report discusses the epidemiologic case investigation, the molecular studies of resistance-associated mutations and N. gonorrhoeae multiantigen sequence typing, and challenges posed by emerging gonococcal antimicrobial resistance. PMID:22184617

  17. Public health measures to control the spread of antimicrobial resistance in Neisseria gonorrhoeae in men who have sex with men.

    PubMed

    Xiridou, M; Soetens, L C; Koedijk, F D H; VAN DER Sande, M A B; Wallinga, J

    2015-06-01

    Gonorrhoea is one of the most common sexually transmitted infections. The control of gonorrhoea is extremely challenging because of the repeated development of resistance to the antibiotics used for its treatment. We explored different strategies to control the spread of antimicrobial resistance and prevent increases in gonorrhoea prevalence. We used a mathematical model that describes gonorrhoea transmission among men who have sex with men and distinguishes gonorrhoea strains sensitive or resistant to three antibiotics. We investigated the impact of combination therapy, switching first-line antibiotics according to resistance thresholds, and other control efforts (reduced sexual risk behaviour, increased treatment rate). Combination therapy can delay the spread of resistance better than using the 5% resistance threshold. Increased treatment rates, expected to enhance gonorrhoea control, may reduce gonorrhoea prevalence only in the short term, but could lead to more resistance and higher prevalence in the long term. Re-treatment of resistant cases with alternative antibiotics can substantially delay the spread of resistance. In conclusion, combination therapy and re-treatment of resistant cases with alternative antibiotics could be the most effective strategies to prevent increases in gonorrhoea prevalence due to antimicrobial resistance.

  18. The significant association of Taq1A genotypes in DRD2/ANKK1 with smoking cessation in a large-scale meta-analysis of Caucasian populations

    PubMed Central

    Ma, Y; Wang, M; Yuan, W; Su, K; Li, M D

    2015-01-01

    Although a number of studies have analyzed the relation between the DRD2/ANKK1 gene Taq1A polymorphism and smoking cessation, the results remain controversial. The primary objective of the present study was to determine whether this variant indeed has any effect on smoking cessation. The A1-dominant model that considers A1/* (*=A1 or A2) and A2/A2 as two genotypes and compares their frequencies in current and former smokers was applied. A total of 22 studies with 11 075 subjects were included in the meta-analyses. Considering the potential influence of between-study heterogeneity, we conducted stratified meta-analyses with the Comprehensive Meta-Analysis statistical software (version 2.0). Results based on either cross-sectional or longitudinal studies consistently showed a statistically significant association between Taq1A A1/* genotypes and smoking cessation. Further, a more significant association of the variant with smoking cessation was detected when both types of studies were combined. However, there was marginal evidence of heterogeneity among studies (I2=33.9% P=0.06). By excluding other ethnicities and subjects with cancer, the meta-analysis on the basis of 9487 Caucasians demonstrated that Taq1A A1/* genotypes indeed were significantly associated with smoking cessation under both the fixed- and random-effects models (pooled OR 1.22; 95% CI 1.11–1.34; P=3.9 × 10−5 for both models). No evidence of between-study heterogeneity or publication bias was observed. Thus, we conclude that the polymorphism of Taq1A has an important role in the process of abstaining from smoking, and smokers carrying A2/A2 genotype have a higher likelihood of smoking cessation than those who carry A1/A1 or A1/A2. PMID:26624925

  19. Genotyping of Brucella melitensis strains from dromedary camels (Camelus dromedarius) from the United Arab Emirates with multiple-locus variable-number tandem repeat analysis.

    PubMed

    Gyuranecz, Miklós; Wernery, Ulli; Kreizinger, Zsuzsa; Juhász, Judit; Felde, Orsolya; Nagy, Péter

    2016-04-15

    Camel brucellosis is a widespread zoonotic disease in camel-rearing countries caused by Brucella melitensis and Brucella abortus. The aim of this study was the first genetic analysis of B. melitensis strains isolated from dromedary camels (Camelus dromedarius) using multiple-locus variable-number tandem repeat analysis (MLVA). MLVA 16 and its MLVA 8 and MLVA11 subsets were used to determine the genotypes of 15 B. melitensis isolates from dromedary camels (11 strains) and other host species (4 strains) from the United Arab Emirates and the results were then compared to B. melitensis MLVA genotypes from other parts of the world. Five, including two novel genotypes were identified with MLVA 8. MLVA 16 further discriminated these five genotypes to ten variants. The eleven camel isolates clustered into four main genetic groups within the East-Mediterranean and African clades and this clustering correlated with the geographic origin of the hosts (United Arab Emirates, Kingdom of Saudi Arabia and Sudan) and the date of their isolation. The camel strains were also genetically related to strains isolated from wild and domestic ruminants from their close habitat or from other parts of the world. Although limited number of strains were analysed, based on our data imported animals from foreign countries, local small ruminants and wildlife species are hypothesized to be the main sources of camel brucellosis in the United Arab Emirates. MLVA was successfully applied to determine the epidemiological links between the different camel B. melitensis infections in the United Arab Emirates and it can be a beneficial tool in future disease control programs. PMID:27016751

  20. Analysis of HBV genotype, drug resistant mutations, and pre-core/basal core promoter mutations in Korean patients with acute hepatitis B.

    PubMed

    Lee, Jong Ho; Hong, Sun Pyo; Jang, Eun Sun; Park, Sang Jong; Hwang, Seong Gyu; Kang, Sook-Kyoung; Jeong, Sook-Hyang

    2015-06-01

    Acute hepatitis B, caused by hepatitis B virus (HBV) strains with drug resistant mutations or pre-core/basal core promoter (PC/BCP) mutations, is a public health concern, because this infection is often associated with poor disease outcome or difficulty in therapeutic choice. The HBV genotype, the prevalence of drug resistant mutations, and PC/BCP mutations in Korean patients with acute hepatitis B were studied. From 2006 to 2008, 36 patients with acute hepatitis B were enrolled prospectively in four general hospitals. Among them, 20 showed detectable HBV DNA (median value was 4.8 log copies/mL). HBV genotyping and analysis of HBV mutations that conferred resistance against lamivudine, adefovir, or entecavir and of PC/BCP mutations were performed using highly sensitive restriction fragment mass polymorphism (RFMP) analysis. All 20 patients were infected with HBV genotype C, which causes almost all cases of chronic hepatitis B in Korea. No patient showed mutations that conferred resistance against lamivudine (L180M, M204V/I), adefovir (A181T, N236S), or entecavir (I169M, A184T/V, S202I/G, M250V/I/L). However, four patients had BCP mutations, and two had PC mutations. Platelet counts were significantly lower in the four patients with PC/BCP mutations compared to those with wild type. In this study, all acute hepatitis B patients had genotype C HBV strains with no drug resistant mutations. However, 20% showed PC/BCP mutations. This highlights the need for further study on the significance of PC/BCP mutations.

  1. Genotyping of Brucella melitensis strains from dromedary camels (Camelus dromedarius) from the United Arab Emirates with multiple-locus variable-number tandem repeat analysis.

    PubMed

    Gyuranecz, Miklós; Wernery, Ulli; Kreizinger, Zsuzsa; Juhász, Judit; Felde, Orsolya; Nagy, Péter

    2016-04-15

    Camel brucellosis is a widespread zoonotic disease in camel-rearing countries caused by Brucella melitensis and Brucella abortus. The aim of this study was the first genetic analysis of B. melitensis strains isolated from dromedary camels (Camelus dromedarius) using multiple-locus variable-number tandem repeat analysis (MLVA). MLVA 16 and its MLVA 8 and MLVA11 subsets were used to determine the genotypes of 15 B. melitensis isolates from dromedary camels (11 strains) and other host species (4 strains) from the United Arab Emirates and the results were then compared to B. melitensis MLVA genotypes from other parts of the world. Five, including two novel genotypes were identified with MLVA 8. MLVA 16 further discriminated these five genotypes to ten variants. The eleven camel isolates clustered into four main genetic groups within the East-Mediterranean and African clades and this clustering correlated with the geographic origin of the hosts (United Arab Emirates, Kingdom of Saudi Arabia and Sudan) and the date of their isolation. The camel strains were also genetically related to strains isolated from wild and domestic ruminants from their close habitat or from other parts of the world. Although limited number of strains were analysed, based on our data imported animals from foreign countries, local small ruminants and wildlife species are hypothesized to be the main sources of camel brucellosis in the United Arab Emirates. MLVA was successfully applied to determine the epidemiological links between the different camel B. melitensis infections in the United Arab Emirates and it can be a beneficial tool in future disease control programs.

  2. Species-specificity of Neisseria gonorrhoeae infection: do human complement regulators contribute?

    PubMed

    Ngampasutadol, Jutamas; Tran, Connie; Gulati, Sunita; Blom, Anna M; Jerse, E Ann; Ram, Sanjay; Rice, Peter A

    2008-12-30

    Neisseria gonorrhoeae is the causative agent of gonorrhea, a disease restricted to humans. Complement forms a key arm of the innate immune system that combats gonococcal infections. N. gonorrhoeae uses its outer membrane porin (Por) molecules to bind complement down-regulatory proteins, C4b-binding protein (C4BP) and factor H (fH), to evade killing by human complement. In addition, sialylation of gonococcal lipooligosaccharide (LOS) also enables N. gonorrhoeae to bind fH. Strains of N. gonorrhoeae that resist killing by human serum complement are killed by serum from rodent, lagomorph and primate species, which cannot be readily infected experimentally with this organism and whose C4BP and/or fH molecules do not bind toN. gonorrhoeae. Serum resistance of gonococci is restored in these sera by human C4BP and/or fH. Direct binding specificity of human and chimpanzee C4BP and human fH to gonococci may explain, in part, species-specific restriction of natural gonococcal infection and address why Por1B, but not Por1A containing gonococcal strains, have been successful in experimental chimpanzee infection. Our findings may help to improve animal models for gonorrhea while also having implications in the choice of complement sources to evaluate neisserial vaccine candidates.

  3. Antibiotic resistance in Neisseria gonorrhoeae: origin, evolution, and lessons learned for the future.

    PubMed

    Unemo, Magnus; Shafer, William M

    2011-08-01

    The strict human pathogen Neisseria gonorrhoeae has caused gonorrhea for thousands of years, and currently gonorrhea is the second most prevalent bacterial sexually transmitted infection worldwide. Given the ancient nature of N. gonorrhoeae and its unique obligate relationship with humankind over the millennia, its remarkable ability to adapt to the host immune system and cause repeated infections, and its propensity to develop resistance to all clinically useful antibiotics, the gonococcus is an ideal pathogen on which to study the evolution of bacterial pathogenesis, including antimicrobial resistance, over the long term and within the host during infection. Recently, the first gonococcus displaying high-level resistance to ceftriaxone, identified in Japan, was characterized in detail. Ceftriaxone is the last remaining option for empirical first-line treatment, and N. gonorrhoeae now seems to be evolving into a true "superbug." In the near future, gonorrhea may become untreatable in certain circumstances. Herein, the history of antibiotics used for treatment of gonorrhea, the evolution of resistance emergence in N. gonorrhoeae, the linkage between resistance and biological fitness of N. gonorrhoeae, lessons learned, and future perspectives are reviewed and discussed.

  4. In vitro potency and combination testing of antimicrobial agents against Neisseria gonorrhoeae.

    PubMed

    Bharat, Amrita; Martin, Irene; Zhanel, George G; Mulvey, Michael R

    2016-03-01

    Antimicrobial resistant Neisseria gonorrhoeae is a major concern to public health due to decreased susceptibility to frontline antimicrobials. To find agents that are active against N. gonorrhoeae, we tested antimicrobials alone or in combination by Etest gradient strips. The potencies (as assessed by minimum inhibitory concentrations) of twenty-five antimicrobials were evaluated against nine reference strains of N. gonorrhoeae (WHO F, G, K, L, M, N, O, P and ATCC 49226). Potency was greatest for netilmicin, quinupristin-dalfopristin, ceftriaxone, ertapenem and piperacillin-tazobactam. Combinations of azithromycin, moxifloxacin, or gentamicin with ceftriaxone, doripenem, or aztreonam were tested against reference isolates and the fractional inhibitory concentration index (FICI) was calculated. All nine combinations resulted in indifference (>0.5 FICI ≤ 4). Combinations with FICI < 1 were further evaluated in nine clinical isolates which supported the finding of indifference. No antagonism was observed in any of the combinations tested. This is the first report in which the six combinations of azithromycin, moxifloxacin or gentamcin in combination with doripenem or aztreonam were tested in N. gonorrhoeae. These data on antimicrobials with higher potency and combinations that did not show antagonism can help to guide larger scale susceptibility studies for antimicrobial resistant N. gonorrhoeae.

  5. Antimicrobial susceptibility/resistance and molecular epidemiological characteristics of Neisseria gonorrhoeae in 2009 in Belarus.

    PubMed

    Glazkova, Slavyana; Golparian, Daniel; Titov, Leonid; Pankratova, Nataliya; Suhabokava, Nataliya; Shimanskaya, Irina; Domeika, Marius; Unemo, Magnus

    2011-08-01

    Increased antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a global concern, and ultimately gonorrhoea may become untreatable. Nonetheless, AMR data from East-Europe are scarce beyond Russia, and no AMR data or other characteristics of gonococci have been reported from Belarus for more than 20 years. The aim was to describe the prevalence of AMR, and report molecular epidemiological characteristics of gonococci circulating in 2009 in Belarus. In a sample of 80 isolates, resistance prevalences to antimicrobials used for gonorrhoea treatment in Belarus were: Ceftriaxone 0%, spectinomycin 0%, azithromycin 17.3%, tetracycline 25.9%, ciprofloxacin 34.6% and erythromycin 59.2%. The isolates displayed no penA mosaic alleles, 38 porB gene sequences and 35 N. gonorrhoeae multiantigen sequence types, of which 20 have not been described before worldwide. Due to the high levels of antimicrobial resistance, only ceftriaxone and spectinomycin can be recommended for empirical treatment of gonorrhoea in Belarus according to WHO recommendations. Continuous gonococcal AMR surveillance in Eastern Europe is crucial. This is now initiated in Belarus using WHO protocols.

  6. Spatiotemporal Analysis of Cryptosporidium Species/Genotypes and Relationships with Other Zoonotic Pathogens in Surface Water from Mixed-Use Watersheds

    PubMed Central

    Wilkes, Graham; Ruecker, Norma J.; Neumann, Norman F.; Gannon, Victor P. J.; Jokinen, Cassandra; Sunohara, Mark; Topp, Edward; Pintar, Katarina D. M.; Edge, Thomas A.

    2013-01-01

    Nearly 690 raw surface water samples were collected during a 6-year period from multiple watersheds in the South Nation River basin, Ontario, Canada. Cryptosporidium oocysts in water samples were enumerated, sequenced, and genotyped by detailed phylogenetic analysis. The resulting species and genotypes were assigned to broad, known host and human infection risk classes. Wildlife/unknown, livestock, avian, and human host classes occurred in 21, 13, 3, and <1% of sampled surface waters, respectively. Cryptosporidium andersoni was the most commonly detected livestock species, while muskrat I and II genotypes were the most dominant wildlife genotypes. The presence of Giardia spp., Salmonella spp., Campylobacter spp., and Escherichia coli O157:H7 was evaluated in all water samples. The greatest significant odds ratios (odds of pathogen presence when host class is present/odds of pathogen presence when host class is absent) for Giardia spp., Campylobacter spp., and Salmonella spp. in water were associated, respectively, with livestock (odds ratio of 3.1), avian (4.3), and livestock (9.3) host classes. Classification and regression tree analyses (CART) were used to group generalized host and human infection risk classes on the basis of a broad range of environmental and land use variables while tracking cooccurrence of zoonotic pathogens in these groupings. The occurrence of livestock-associated Cryptosporidium was most strongly related to agricultural water pollution in the fall (conditions also associated with elevated odds ratios of other zoonotic pathogens occurring in water in relation to all sampling conditions), whereas wildlife/unknown sources of Cryptosporidium were geospatially associated with smaller watercourses where urban/rural development was relatively lower. Conditions that support wildlife may not necessarily increase overall human infection risks associated with Cryptosporidium since most Cryptosporidium genotypes classed as wildlife in this study (e

  7. Comparative Analysis of Muscle Transcriptome between Pig Genotypes Identifies Genes and Regulatory Mechanisms Associated to Growth, Fatness and Metabolism

    PubMed Central

    Ayuso, Miriam; Fernández, Almudena; Núñez, Yolanda; Benítez, Rita; Isabel, Beatriz; Barragán, Carmen; Fernández, Ana Isabel; Rey, Ana Isabel; Medrano, Juan F.; Cánovas, Ángela; González-Bulnes, Antonio; López-Bote, Clemente; Ovilo, Cristina

    2015-01-01

    Iberian ham production includes both purebred (IB) and Duroc-crossbred (IBxDU) Iberian pigs, which show important differences in meat quality and production traits, such as muscle growth and fatness. This experiment was conducted to investigate gene expression differences, transcriptional regulation and genetic polymorphisms that could be associated with the observed phenotypic differences between IB and IBxDU pigs. Nine IB and 10 IBxDU pigs were slaughtered at birth. Morphometric measures and blood samples were obtained and samples from Biceps femoris muscle were employed for compositional and transcriptome analysis by RNA-Seq technology. Phenotypic differences were evident at this early age, including greater body size and weight in IBxDU and greater Biceps femoris intramuscular fat and plasma cholesterol content in IB newborns. We detected 149 differentially expressed genes between IB and IBxDU neonates (p < 0.01 and Fold-Change > 1. 5). Several were related to adipose and muscle tissues development (DLK1, FGF21 or UBC). The functional interpretation of the transcriptomic differences revealed enrichment of functions and pathways related to lipid metabolism in IB and to cellular and muscle growth in IBxDU pigs. Protein catabolism, cholesterol biosynthesis and immune system were functions enriched in both genotypes. We identified transcription factors potentially affecting the observed gene expression differences. Some of them have known functions on adipogenesis (CEBPA, EGRs), lipid metabolism (PPARGC1B) and myogenesis (FOXOs, MEF2D, MYOD1), which suggest a key role in the meat quality differences existing between IB and IBxDU hams. We also identified several polymorphisms showing differential segregation between IB and IBxDU pigs. Among them, non-synonymous variants were detected in several transcription factors as PPARGC1B and TRIM63 genes, which could be associated to altered gene function. Taken together, these results provide information about candidate

  8. Fur-mediated activation of gene transcription in the human pathogen Neisseria gonorrhoeae.

    PubMed

    Yu, Chunxiao; Genco, Caroline Attardo

    2012-04-01

    It is well established that the ferric uptake regulatory protein (Fur) functions as a transcriptional repressor in diverse microorganisms. Recent studies demonstrated that Fur also functions as a transcriptional activator. In this study we defined Fur-mediated activation of gene transcription in the sexually transmitted disease pathogen Neisseria gonorrhoeae. Analysis of 37 genes which were previously determined to be iron induced and which contained putative Fur boxes revealed that only 30 of these genes exhibited reduced transcription in a gonococcal fur mutant strain. Fur-mediated activation was established by examining binding of Fur to the putative promoter regions of 16 Fur-activated genes with variable binding affinities observed. Only ∼50% of the newly identified Fur-regulated genes bound Fur in vitro, suggesting that additional regulatory circuits exist which may function through a Fur-mediated indirect mechanism. The gonococcal Fur-activated genes displayed variable transcription patterns in a fur mutant strain, which correlated with the position of the Fur box in each (promoter) region. These results suggest that Fur-mediated direct transcriptional activation is fulfilled by multiple mechanisms involving either competing with a repressor or recruiting RNA polymerase. Collectively, our studies have established that gonococcal Fur functions as an activator of gene transcription through both direct and indirect mechanisms. PMID:22287521

  9. Fur-Mediated Activation of Gene Transcription in the Human Pathogen Neisseria gonorrhoeae

    PubMed Central

    Yu, Chunxiao

    2012-01-01

    It is well established that the ferric uptake regulatory protein (Fur) functions as a transcriptional repressor in diverse microorganisms. Recent studies demonstrated that Fur also functions as a transcriptional activator. In this study we defined Fur-mediated activation of gene transcription in the sexually transmitted disease pathogen Neisseria gonorrhoeae. Analysis of 37 genes which were previously determined to be iron induced and which contained putative Fur boxes revealed that only 30 of these genes exhibited reduced transcription in a gonococcal fur mutant strain. Fur-mediated activation was established by examining binding of Fur to the putative promoter regions of 16 Fur-activated genes with variable binding affinities observed. Only ∼50% of the newly identified Fur-regulated genes bound Fur in vitro, suggesting that additional regulatory circuits exist which may function through a Fur-mediated indirect mechanism. The gonococcal Fur-activated genes displayed variable transcription patterns in a fur mutant strain, which correlated with the position of the Fur box in each (promoter) region. These results suggest that Fur-mediated direct transcriptional activation is fulfilled by multiple mechanisms involving either competing with a repressor or recruiting RNA polymerase. Collectively, our studies have established that gonococcal Fur functions as an activator of gene transcription through both direct and indirect mechanisms. PMID:22287521

  10. Identification and Comparative Analysis of MicroRNAs Associated with Low-N Tolerance in Rice Genotypes

    PubMed Central

    Nischal, Lata; Mohsin, Mohd; Khan, Ishrat; Kardam, Hemant; Wadhwa, Asha; Abrol, Yash Pal; Iqbal, Muhammad; Ahmad, Altaf

    2012-01-01

    Background Nitrogen [N] is a critical limiting nutrient for plants and has to be exogenously supplied to many crops, to achieve high yield with significant economic and environmental costs, specifically for rice. Development of low-input nitrogen sustainable crop is necessary for sustainable agriculture. Identification of regulatory elements associated with low-N tolerance is imperative for formulating innovative approaches for developing low-N tolerant crop plants, using gene manipulation. MicroRNAs (miRNAs) are known to play crucial roles in the modulation of gene expression in plants under various environmental conditions. Methodology/Principal Findings MiRNAs associated with low-N tolerance have not been identified so far. In this study, we investigated microarray-based miRNA expression in low-N tolerant and low-N sensitive rice genotypes under low N condition. Expressions of 32 miRNAs differed significantly in the two genotypes. Of these 32 miRNAs, expressions of nine miRNAs were further validated experimentally in leaves as well as in roots. Of these differentially expressed miRNAs, six miRNAs (miR156, miR164, miR528, miR820, miR821 and miR1318) were reported in leaves and four (miR164, miR167, miR168 and miR528) in roots. Target genes of all the 32 miRNAs were predicted, which encode transcription factors, and proteins associated with metabolic processes or stress responses. Expression levels of some of the corresponding miRNA targets were analysed and found to be significantly higher in low N-tolerant genotype than low-N sensitive genotype. These findings suggested that miRNAs played an important role in low-N tolerance in rice. Conclusions/Significance Genome-wide differences in expression of miRNA in low N-tolerant and low N-sensitive rice genotypes were reported. This provides a platform for selection as well as manipulation of genotypes for better N utilization efficiency. PMID:23227161

  11. Recorded gonorrhoea rates in Denmark, 1900–2010: the impact of clinical testing activity and laboratory diagnostic procedures

    PubMed Central

    Lind, Inga; Hoffmann, Steen

    2015-01-01

    Objectives Assessment of the relations between recorded gonorrhoea rates and clinical testing activity and disposable diagnostic tests. Methods In Denmark, two sources of information on the epidemiology of gonorrhoea are available: (1) a mandatory clinical notification system (since 1867) comprising summary information about geographic distribution, season, age group and gender; in 1994, more detailed anonymous individualised epidemiological information was included; (2) a voluntary countrywide laboratory surveillance system for culture-confirmed cases (since 1957) comprising information about patient's age and gender, infected anatomical sites and medical setting attended. Results Both surveillance systems showed marked simultaneous changes in gonorrhoea rates, although periodically considerable under-reporting or under-diagnosing was demonstrated. The annual incidence of notified cases peaked in 1919 (474/100 000), in 1944 (583/100 000) and in 1972 (344/100 000). Since 1995, the incidence has been at a low endemic level (1.5–10/100 000) and the total male/female incidence ratios were from 3 to 7 times higher than previously recorded. Among approximately 2 million persons tested during 1974–1988 78 213 men and 63 143 women with culture-confirmed gonorrhoea were identified. During this period, pharyngeal sampling was performed in 36% of men and 25% of women with gonorrhoea; pharyngeal gonorrhoea was found in 10% and 16%, respectively; 40% and 30% of these patients had no concomitant urogenital gonorrhoea. Among men with gonorrhoea, 34% were sampled from the rectum; 9% had rectal gonorrhoea, among whom the rectum was the only infected site in 67%. Conclusions Crucial factors for case finding are clinical sampling tradition and appropriate laboratory diagnostic facilities. When case finding is insufficient, a reservoir of asymptomatic rectal or pharyngeal gonorrhoea remains unrecognised. PMID:26621510

  12. Comparative analysis of caffeoylquinic acids and lignans in roots and seeds among various burdock (Arctium lappa) genotypes with high antioxidant activity.

    PubMed

    Liu, Jingyi; Cai, Yi-Zhong; Wong, Ricky Ngok Shun; Lee, Calvin Kai-Fai; Tang, Sydney Chi Wai; Sze, Stephen Cho Wing; Tong, Yao; Zhang, Yanbo

    2012-04-25

    Caffeoylquinic acids and lignans in the crude extracts of both roots and seeds from different burdock ( Arctium lappa L.) genotypes were simultaneously characterized and systematically compared by LC-MS and matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry (MALDI-QIT-TOF MS), and their antioxidant activities were also investigated. A total of 14 lignans were identified in burdock seeds and 12 caffeoylquinic acids in burdock roots. High levels of caffeoylquinic acids were also detected in burdock seeds, but only trace amounts of lignans were found in burdock roots. Burdock seeds contained higher concentrations of lignans and caffeoylquinic acids than burdock roots. Quantitative analysis of caffeoylquinic acids and lignans in roots and seeds of various burdock genotypes was reported for the first time. Great variations in contents of both individual and total phenolic compounds as well as antioxidant activities were found among different genotypes. Burdock as a root vegetable or medicinal plants possessed considerably stronger antioxidant activity than common vegetables and fruits.

  13. Ticagrelor versus genotype-driven antiplatelet therapy for secondary prevention after acute coronary syndrome: a cost-effectiveness analysis

    PubMed Central

    Crespin, Daniel J.; Federspiel, Jerome J.; Biddle, Andrea K.; Jonas, Daniel E.; Rossi, Joseph S.

    2012-01-01

    Background Clopidogrel’s effectiveness is likely reduced significantly for prevention of thrombotic events after acute coronary syndrome (ACS) in patients exhibiting a decreased ability to metabolize clopidogrel into its active form. A genetic mutation responsible for this reduced effectiveness is detectable by genotyping. Ticagrelor is not dependent on gene-based metabolic activation and demonstrated greater clinical efficacy than clopidogrel in a recent secondary prevention trial. In 2011, clopidogrel will lose its patent protection and likely will be substantially less expensive than ticagrelor. Objective To determine the cost-effectiveness of ticagrelor compared with a genotype-driven selection of antiplatelet agents. Methods A hybrid decision tree/Markov model was used to estimate the 5-year medical costs (in 2009 US$) and outcomes for a cohort of ACS patients enrolled in Medicare receiving either genotype-driven or ticagrelor-only treatment. Outcomes included life years and quality-adjusted life years (QALYs) gained. Data comparing the clinical performance of ticagrelor and clopidogrel were derived from the Platelet Inhibition and Patient Outcomes trial. Results The incremental cost-effectiveness ratio (ICER) for universal ticagrelor was $10,059 per QALY compared to genotype-driven treatment, and was most sensitive to the price of ticagrelor and the hazard ratio for death for ticagrelor compared with clopidogrel. The ICER remained below $50,000 per QALY until a monthly ticagrelor price of $693 or a 0.93 hazard ratio for death for ticagrelor relative to clopidogrel. In probabilistic analyses, universal ticagrelor was below $50,000 per QALY in 97.7% of simulations. Conclusion Prescribing ticagrelor universally increases quality-adjusted life years for ACS patients at a cost below a typically accepted threshold. PMID:21669373

  14. Comparative Transcriptome Analysis of Resistant and Susceptible Common Bean Genotypes in Response to Soybean Cyst Nematode Infection.

    PubMed

    Jain, Shalu; Chittem, Kishore; Brueggeman, Robert; Osorno, Juan M; Richards, Jonathan; Nelson, Berlin D

    2016-01-01

    Soybean cyst nematode (SCN; Heterodera glycines Ichinohe) reproduces on the roots of common bean (Phaseolus vulgaris L.) and can cause reductions in plant growth and seed yield. The molecular changes in common bean roots caused by SCN infection are unknown. Identification of genetic factors associated with SCN resistance could help in development of improved bean varieties with high SCN resistance. Gene expression profiling was conducted on common bean roots infected by SCN HG type 0 using next generation RNA sequencing technology. Two pinto bean genotypes, PI533561 and GTS-900, resistant and susceptible to SCN infection, respectively, were used as RNA sources eight days post inoculation. Total reads generated ranged between ~ 3.2 and 5.7 million per library and were mapped to the common bean reference genome. Approximately 70-90% of filtered RNA-seq reads uniquely mapped to the reference genome. In the inoculated roots of resistant genotype PI533561, a total of 353 genes were differentially expressed with 154 up-regulated genes and 199 down-regulated genes when compared to the transcriptome of non- inoculated roots. On the other hand, 990 genes were differentially expressed in SCN-inoculated roots of susceptible genotype GTS-900 with 406 up-regulated and 584 down-regulated genes when compared to non-inoculated roots. Genes encoding nucleotide-binding site leucine-rich repeat resistance (NLR) proteins, WRKY transcription factors, pathogenesis-related (PR) proteins and heat shock proteins involved in diverse biological processes were differentially expressed in both resistant and susceptible genotypes. Overall, suppression of the photosystem was observed in both the responses. Furthermore, RNA-seq results were validated through quantitative real time PCR. This is the first report describing genes/transcripts involved in SCN-common bean interaction and the results will have important implications for further characterization of SCN resistance genes in common bean

  15. Comparative Transcriptome Analysis of Resistant and Susceptible Common Bean Genotypes in Response to Soybean Cyst Nematode Infection

    PubMed Central

    Jain, Shalu; Chittem, Kishore; Brueggeman, Robert; Osorno, Juan M.; Richards, Jonathan; Nelson, Berlin D.

    2016-01-01

    Soybean cyst nematode (SCN; Heterodera glycines Ichinohe) reproduces on the roots of common bean (Phaseolus vulgaris L.) and can cause reductions in plant growth and seed yield. The molecular changes in common bean roots caused by SCN infection are unknown. Identification of genetic factors associated with SCN resistance could help in development of improved bean varieties with high SCN resistance. Gene expression profiling was conducted on common bean roots infected by SCN HG type 0 using next generation RNA sequencing technology. Two pinto bean genotypes, PI533561 and GTS-900, resistant and susceptible to SCN infection, respectively, were used as RNA sources eight days post inoculation. Total reads generated ranged between ~ 3.2 and 5.7 million per library and were mapped to the common bean reference genome. Approximately 70–90% of filtered RNA-seq reads uniquely mapped to the reference genome. In the inoculated roots of resistant genotype PI533561, a total of 353 genes were differentially expressed with 154 up-regulated genes and 199 down-regulated genes when compared to the transcriptome of non- inoculated roots. On the other hand, 990 genes were differentially expressed in SCN-inoculated roots of susceptible genotype GTS-900 with 406 up-regulated and 584 down-regulated genes when compared to non-inoculated roots. Genes encoding nucleotide-binding site leucine-rich repeat resistance (NLR) proteins, WRKY transcription factors, pathogenesis-related (PR) proteins and heat shock proteins involved in diverse biological processes were differentially expressed in both resistant and susceptible genotypes. Overall, suppression of the photosystem was observed in both the responses. Furthermore, RNA-seq results were validated through quantitative real time PCR. This is the first report describing genes/transcripts involved in SCN-common bean interaction and the results will have important implications for further characterization of SCN resistance genes in common bean

  16. Crystal structure of the open state of the Neisseria gonorrhoeae MtrE outer membrane channel.

    PubMed

    Lei, Hsiang-Ting; Chou, Tsung-Han; Su, Chih-Chia; Bolla, Jani Reddy; Kumar, Nitin; Radhakrishnan, Abhijith; Long, Feng; Delmar, Jared A; Do, Sylvia V; Rajashankar, Kanagalaghatta R; Shafer, William M; Yu, Edward W

    2014-01-01

    Active efflux of antimicrobial agents is one of the most important strategies used by bacteria to defend against antimicrobial factors present in their environment. Mediating many cases of antibiotic resistance are transmembrane efflux pumps, composed of one or more proteins. The Neisseria gonorrhoeae MtrCDE tripartite multidrug efflux pump, belonging to the hydrophobic and amphiphilic efflux resistance-nodulation-cell division (HAE-RND) family, spans both the inner and outer membranes of N. gonorrhoeae and confers resistance to a variety of antibiotics and toxic compounds. We here describe the crystal structure of N. gonorrhoeae MtrE, the outer membrane component of the MtrCDE tripartite multidrug efflux system. This trimeric MtrE channel forms a vertical tunnel extending down contiguously from the outer membrane surface to the periplasmic end, indicating that our structure of MtrE depicts an open conformational state of this channel.

  17. An evaluation of Neisseria gonorrhoeae antimicrobial susceptibility testing in the UK.

    PubMed

    Jain, Anu; Cole, Michelle J; Planche, Tim; Ison, Catherine A

    2014-11-01

    The only method currently available to perform Neisseria gonorrhoeae antimicrobial susceptibility testing (Ng-AST) requires a viable organism obtained by culture. Reports of in vitro resistance to extended-spectrum cephalosporins, the treatment of choice for gonorrhoea, coupled with increasing gonorrhoea diagnoses is worrying. The aim of this study was to identify various methodologies employed by the UK microbiology laboratories to perform Ng-AST. Of the 118 laboratories that responded, 114 offered Ng-AST; the majority (82.5%, 94/114) of the laboratories used British Society for Antimicrobial Chemotherapy methodology for Ng-AST. The other main findings were infrequent use of quality control procedures and inconsistent susceptibility testing of the antibiotics used routinely for treatment.

  18. Crystal structure of the open state of the Neisseria gonorrhoeae MtrE outer membrane channel.

    PubMed

    Lei, Hsiang-Ting; Chou, Tsung-Han; Su, Chih-Chia; Bolla, Jani Reddy; Kumar, Nitin; Radhakrishnan, Abhijith; Long, Feng; Delmar, Jared A; Do, Sylvia V; Rajashankar, Kanagalaghatta R; Shafer, William M; Yu, Edward W

    2014-01-01

    Active efflux of antimicrobial agents is one of the most important strategies used by bacteria to defend against antimicrobial factors present in their environment. Mediating many cases of antibiotic resistance are transmembrane efflux pumps, composed of one or more proteins. The Neisseria gonorrhoeae MtrCDE tripartite multidrug efflux pump, belonging to the hydrophobic and amphiphilic efflux resistance-nodulation-cell division (HAE-RND) family, spans both the inner and outer membranes of N. gonorrhoeae and confers resistance to a variety of antibiotics and toxic compounds. We here describe the crystal structure of N. gonorrhoeae MtrE, the outer membrane component of the MtrCDE tripartite multidrug efflux system. This trimeric MtrE channel forms a vertical tunnel extending down contiguously from the outer membrane surface to the periplasmic end, indicating that our structure of MtrE depicts an open conformational state of this channel. PMID:24901251

  19. Whole-genome phylogenomic heterogeneity of Neisseria gonorrhoeae isolates with decreased cephalosporin susceptibility collected in Canada between 1989 and 2013.

    PubMed

    Demczuk, Walter; Lynch, Tarah; Martin, Irene; Van Domselaar, Gary; Graham, Morag; Bharat, Amrita; Allen, Vanessa; Hoang, Linda; Lefebvre, Brigitte; Tyrrell, Greg; Horsman, Greg; Haldane, David; Garceau, Richard; Wylie, John; Wong, Tom; Mulvey, Michael R

    2015-01-01

    A large-scale, whole-genome comparison of Canadian Neisseria gonorrhoeae isolates with high-level cephalosporin MICs was used to demonstrate a genomic epidemiology approach to investigate strain relatedness and dynamics. Although current typing methods have been very successful in tracing short-chain transmission of gonorrheal disease, investigating the temporal evolutionary relationships and geographical dissemination of highly clonal lineages requires enhanced resolution only available through whole-genome sequencing (WGS). Phylogenomic cluster analysis grouped 169 Canadian strains into 12 distinct clades. While some N. gonorrhoeae multiantigen sequence types (NG-MAST) agreed with specific phylogenomic clades or subclades, other sequence types (ST) and closely related groups of ST were widely distributed among clades. Decreased susceptibility to extended-spectrum cephalosporins (ESC-DS) emerged among a group of diverse strains in Canada during the 1990s with a variety of nonmosaic penA alleles, followed in 2000/2001 with the penA mosaic X allele and then in 2007 with ST1407 strains with the penA mosaic XXXIV allele. Five genetically distinct ESC-DS lineages were associated with penA mosaic X, XXXV, and XXXIV alleles and nonmosaic XII and XIII alleles. ESC-DS with coresistance to azithromycin was observed in 5 strains with 23S rRNA C2599T or A2143G mutations. As the costs associated with WGS decline and analysis tools are streamlined, WGS can provide a more thorough understanding of strain dynamics, facilitate epidemiological studies to better resolve social networks, and improve surveillance to optimize treatment for gonorrheal infections.

  20. Transcriptional landscape and essential genes of Neisseria gonorrhoeae

    PubMed Central

    Remmele, Christian W.; Xian, Yibo; Albrecht, Marco; Faulstich, Michaela; Fraunholz, Martin; Heinrichs, Elisabeth; Dittrich, Marcus T.; Müller, Tobias; Reinhardt, Richard; Rudel, Thomas

    2014-01-01

    The WHO has recently classified Neisseria gonorrhoeae as a super-bacterium due to the rapid spread of antibiotic resistant derivatives and an overall dramatic increase in infection incidences. Genome sequencing has identified potential genes, however, little is known about the transcriptional organization and the presence of non-coding RNAs in gonococci. We performed RNA sequencing to define the transcriptome and the transcriptional start sites of all gonococcal genes and operons. Numerous new transcripts including 253 potentially non-coding RNAs transcribed from intergenic regions or antisense to coding genes were identified. Strikingly, strong antisense transcription was detected for the phase-variable opa genes coding for a family of adhesins and invasins in pathogenic Neisseria, that may have regulatory functions. Based on the defined transcriptional start sites, promoter motifs were identified. We further generated and sequenced a high density Tn5 transposon library to predict a core of 827 gonococcal essential genes, 133 of which have no known function. Our combined RNA-Seq and Tn-Seq approach establishes a detailed map of gonococcal genes and defines the first core set of essential gonococcal genes. PMID:25143534

  1. Ethylenediaminetetraacetic acid-sensitive antiphagocytic activity of Neisseria gonorrhoeae.

    PubMed Central

    Rosenthal, R S; Fulbright, R S; Eads, M E; Sawyer, W D

    1977-01-01

    Colonial types of Neisseria gonorrhoeae were examined for the presence of pilus-independent antiphagocytic activity. Type 3 and depiliated type 1 gonococci had a shearing- and protease-resistant antiphagocytic activity that was eliminated by treatment with ethylenediaminetetraacetic acid (EDTA) and that was not present on type 4 bacteria. Incubation of EDTA-treated bacteria 37 degrees C for 90 min resulted in fas prevented by antibiotics that block the final assembly of cell wall macromolecules that depend on the C55-isoprenoid carrier for export. These include both lipopolysaccharide and peptidoglycan. Restoration was, however, unaffected by drugs that interfere with the synthesis of peptidoglycan, but not that of lipopolysaccharide, and by inhibitors of protein synthesis. These data suggested that gonococci have an antiphagocytic mechanism in addition to the previously described determinant (presumably pili) that was removed by blending or by treatment with proteases. Of the two antiphagocytic activities, type 1 had both, type 3 had only the EDTA-sensitive component, and type 4 had neither. PMID:404246

  2. [Nationwide antimicrobial susceptibility survey of Neisseria gonorrhoeae isolates in Japan].

    PubMed

    Tanaka, Masatoshi; Shimojima, Masahiro; Saika, Takeshi; Iyoda, Takako; Ikeda, Fumiaki; Kanayama, Akiko; Kobayashi, Intetsu

    2011-07-01

    In a nationwide antimicrobial susceptibility survey of 494 Nesseria gonorrhoeae isolates collected from February 2008 to December 2009 in 3 regions of Japan, 112 (22.7%) were collected from western Japan (Kinki, Chugoku, Shikoku, and Kyushu), 277 (56.1%) from mid-eastern Japan (Kanto), and 105 (21.1%) from eastern Japan (Tokai, Hokuriku, Koushinetsu, Tohoku, and Hokkaido). Resistance to ciprofloxacin (CPFX) was 72.8%, to penicillin G (PCG) 19.8%, and to tetracycline (TC) 18.2%. Intermediate resistance to CPFX was 1.8%, to PCG 73.7%, and to TC 43.7%. These results indicate that both types of resistance to the 3 agents were very high. Intermediate resistance to cefixime (CFIX) was 38.1% and to cefozidim (CDZM) 13.4%. Resistance to CFIX was only 0.4% and to CDZM 0%. Susceptibility to azithromycin was 96.6%, to ceftriaxone 99.8%, and to spectinomycin 100%. No significant difference in resistance was seen to different antimicrobial agent classes tested in the 3 regions, although intermediate resistance to CFIX in western Japan was significantly higher than in mid-eastern Japan.

  3. Concomitant infection with Neisseria gonorrhoeae and Chlamydia trachomatis in pregnancy.

    PubMed

    Christmas, J T; Wendel, G D; Bawdon, R E; Farris, R; Cartwright, G; Little, B B

    1989-09-01

    Gonorrhea is an important marker for endocervical chlamydial infections in nonpregnant women. Concomitant infection rates as high as 50% have been reported. There are few data on concomitant infection rates in pregnant patients. The purpose of this study was to examine the prevalence of endocervical chlamydial infections in pregnant women with gonorrhea. Patients with cervical cultures positive for Neisseria gonorrhoeae at their initial prenatal visit had endocervical specimens for Chlamydia trachomatis culture obtained before anti-gonorrheal therapy. Control patients were selected at random from the same prenatal population. The prevalence of C trachomatis in patients with gonorrhea was significantly greater than that in the control population (46 versus 5%; P less than .001). Patients with gonorrhea were younger, less often married, and more often black than the control population, but these demographic differences did not account for the large difference in the chlamydial prevalence. Erythromycin 500 mg four times daily provided an excellent cure rate without intolerable side effects. Pregnant patients being evaluated or treated for gonorrhea should also be considered at high risk for concomitant cervical chlamydial infection.

  4. Prevalence of Chlamydia trachomatis and Neisseria gonorrhoeae and repeat infection among pregnant urban adolescents

    PubMed Central

    Berggren, Erica K.; Patchen, Loral

    2013-01-01

    BACKGROUND Negative maternal and fetal consequences associated with Chlamydia trachomatis and Neisseria gonorrhoeae during pregnancy make diagnosis essential. The CDC recommends routine screening for sexually transmitted infections at the first prenatal visit, and third trimester repeat screening, specifically for C. trachomatis, is recommended for women under age 25 or at increased infection risk. The impact of repeat screening on diagnosis during pregnancy is not well documented among adolescents. METHODS A prospective cohort of 125 pregnant adolescents with at least one prenatal screen for C. trachomatis and N. gonorrhoeae was analyzed. All participants received prenatal care and delivered at one urban teaching hospital in Washington, DC. Screening results were documented for both sexually transmitted infections. Descriptive and univariate analyses were performed to describe disease prevalence. RESULTS Thirty-one percent of pregnant adolescents were diagnosed with either C. trachomatis or N. gonorrhoeae infection during pregnancy. Of the 75% (95/125) of patients who had more than one screening test, 11% (10/95) had a re-infection, and 7% (7/95) had a new infection on repeat testing. Nine percent (9/95) had recurrent C. trachomatis, while 4% (4/95) had a new diagnosis. Three percent (3/95) had recurrent N. gonorrhoeae, while 4% (4/95) had a new diagnosis. Some experienced co-infection at either initial or repeat testing. CONCLUSIONS Screening for C. trachomatis and N. gonorrhoeae is recommended during pregnancy. In this sample of pregnant adolescents, the overall high incidence and recurrence of C. trachomatis and N. gonorrhoeae support CDC screening and re-screening recommendations, regardless of initial test results. PMID:20938375

  5. Azithromycin Resistance and Its Mechanism in Neisseria gonorrhoeae Strains in Hyogo, Japan

    PubMed Central

    Osawa, Kayo; Miura, Makiko; Tanaka, Kazushi; Arakawa, Soichi; Shirakawa, Toshiro; Fujisawa, Masato

    2015-01-01

    Therapeutic options are limited for Neisseria gonorrhoeae infection, especially for oral drugs. The purpose of this study was to investigate the susceptibility of N. gonorrhoeae to oral azithromycin (AZM) and the correlation between AZM resistance-related gene mutations and MIC. We examined the AZM MICs of clinical strains of N. gonorrhoeae, sequenced the peptidyltransferase loop in domain V of 23S rRNA, and investigated the statistical correlation between AZM MIC and the presence and number of the mutations. Among 59 N. gonorrhoeae strains, our statistical data showed that a deletion mutation was seen significantly more often in the higher-MIC group (0.5 μg/ml or higher) (35/37; 94.6%) than in the lower-MIC group (0.25 μg/ml or less) (4/22; 18.2%) (P < 0.0001). However, a mutation of codon 40 (Ala→Asp) in the mtrR gene (helix-turn-helix) was seen significantly more often in the lower-MIC group (12/22; 54.5%) (P < 0.0001). In N. gonorrhoeae multiantigen sequence typing (NG-MAST) analyses, ST4777 was representative of the lower-MIC group and ST1407, ST6798, and ST6800 were representative of the higher-MIC group. NG-MAST type 1407 was detected as the most prevalent type in AZM-resistant or -intermediate strains, as previously described. In conclusion, a deletion mutation in the mtrR promoter region may be a significant indicator for higher MIC (0.5 μg/ml or higher). ST4777 was often seen in the lower-MIC group, and ST1407, ST6798, and ST6800 were characteristic of the higher-MIC group. Further research with a greater number of strains would help elucidate the mechanism of AZM resistance in N. gonorrhoeae infection. PMID:25712352

  6. Exploring the Benefits of Molecular Testing for Gonorrhoea Antibiotic Resistance Surveillance in Remote Settings

    PubMed Central

    Hui, Ben B.; Ryder, Nathan; Su, Jiunn-Yih; Ward, James; Chen, Marcus Y.; Donovan, Basil; Fairley, Christopher K.; Guy, Rebecca J.; Lahra, Monica M.; Law, Mathew G.; Whiley, David M.; Regan, David G.

    2015-01-01

    Background Surveillance for gonorrhoea antimicrobial resistance (AMR) is compromised by a move away from culture-based testing in favour of more convenient nucleic acid amplification test (NAAT) tests. We assessed the potential benefit of a molecular resistance test in terms of the timeliness of detection of gonorrhoea AMR. Methods and Findings An individual-based mathematical model was developed to describe the transmission of gonorrhoea in a remote Indigenous population in Australia. We estimated the impact of the molecular test on the time delay between first importation and the first confirmation that the prevalence of gonorrhoea AMR (resistance proportion) has breached the WHO-recommended 5% threshold (when a change in antibiotic should occur). In the remote setting evaluated in this study, the model predicts that when culture is the only available means of testing for AMR, the breach will only be detected when the actual prevalence of AMR in the population has already reached 8 – 18%, with an associated delay of ~43 – 69 months between first importation and detection. With the addition of a molecular resistance test, the number of samples for which AMR can be determined increases facilitating earlier detection at a lower resistance proportion. For the best case scenario, where AMR can be determined for all diagnostic samples, the alert would be triggered at least 8 months earlier than using culture alone and the resistance proportion will have only slightly exceeded the 5% notification threshold. Conclusions Molecular tests have the potential to provide more timely warning of the emergence of gonorrhoea AMR. This in turn will facilitate earlier treatment switching and more targeted treatment, which has the potential to reduce the population impact of gonorrhoea AMR. PMID:26181042

  7. Azithromycin resistance and its mechanism in Neisseria gonorrhoeae strains in Hyogo, Japan.

    PubMed

    Shigemura, Katsumi; Osawa, Kayo; Miura, Makiko; Tanaka, Kazushi; Arakawa, Soichi; Shirakawa, Toshiro; Fujisawa, Masato

    2015-05-01

    Therapeutic options are limited for Neisseria gonorrhoeae infection, especially for oral drugs. The purpose of this study was to investigate the susceptibility of N. gonorrhoeae to oral azithromycin (AZM) and the correlation between AZM resistance-related gene mutations and MIC. We examined the AZM MICs of clinical strains of N. gonorrhoeae, sequenced the peptidyltransferase loop in domain V of 23S rRNA, and investigated the statistical correlation between AZM MIC and the presence and number of the mutations. Among 59 N. gonorrhoeae strains, our statistical data showed that a deletion mutation was seen significantly more often in the higher-MIC group (0.5 μg/ml or higher) (35/37; 94.6%) than in the lower-MIC group (0.25 μg/ml or less) (4/22; 18.2%) (P < 0.0001). However, a mutation of codon 40 (Ala → Asp) in the mtrR gene (helix-turn-helix) was seen significantly more often in the lower-MIC group (12/22; 54.5%) (P < 0.0001). In N. gonorrhoeae multiantigen sequence typing (NG-MAST) analyses, ST4777 was representative of the lower-MIC group and ST1407, ST6798, and ST6800 were representative of the higher-MIC group. NG-MAST type 1407 was detected as the most prevalent type in AZM-resistant or -intermediate strains, as previously described. In conclusion, a deletion mutation in the mtrR promoter region may be a significant indicator for higher MIC (0.5 μg/ml or higher). ST4777 was often seen in the lower-MIC group, and ST1407, ST6798, and ST6800 were characteristic of the higher-MIC group. Further research with a greater number of strains would help elucidate the mechanism of AZM resistance in N. gonorrhoeae infection.

  8. Azithromycin resistance and its mechanism in Neisseria gonorrhoeae strains in Hyogo, Japan.

    PubMed

    Shigemura, Katsumi; Osawa, Kayo; Miura, Makiko; Tanaka, Kazushi; Arakawa, Soichi; Shirakawa, Toshiro; Fujisawa, Masato

    2015-05-01

    Therapeutic options are limited for Neisseria gonorrhoeae infection, especially for oral drugs. The purpose of this study was to investigate the susceptibility of N. gonorrhoeae to oral azithromycin (AZM) and the correlation between AZM resistance-related gene mutations and MIC. We examined the AZM MICs of clinical strains of N. gonorrhoeae, sequenced the peptidyltransferase loop in domain V of 23S rRNA, and investigated the statistical correlation between AZM MIC and the presence and number of the mutations. Among 59 N. gonorrhoeae strains, our statistical data showed that a deletion mutation was seen significantly more often in the higher-MIC group (0.5 μg/ml or higher) (35/37; 94.6%) than in the lower-MIC group (0.25 μg/ml or less) (4/22; 18.2%) (P < 0.0001). However, a mutation of codon 40 (Ala → Asp) in the mtrR gene (helix-turn-helix) was seen significantly more often in the lower-MIC group (12/22; 54.5%) (P < 0.0001). In N. gonorrhoeae multiantigen sequence typing (NG-MAST) analyses, ST4777 was representative of the lower-MIC group and ST1407, ST6798, and ST6800 were representative of the higher-MIC group. NG-MAST type 1407 was detected as the most prevalent type in AZM-resistant or -intermediate strains, as previously described. In conclusion, a deletion mutation in the mtrR promoter region may be a significant indicator for higher MIC (0.5 μg/ml or higher). ST4777 was often seen in the lower-MIC group, and ST1407, ST6798, and ST6800 were characteristic of the higher-MIC group. Further research with a greater number of strains would help elucidate the mechanism of AZM resistance in N. gonorrhoeae infection. PMID:25712352

  9. Genotyping of Salmonella enterica serovar Typhimurium isolates by multilocus variable number of tandem repeat high-resolution melting analysis (MLV-HRMA).

    PubMed

    Keeratipibul, Suwimon; Silamat, Panusanun; Phraephaisarn, Chirapiphat; Srisitthinam, Daranee; Takahashi, Hajime; Chaturongkasumrit, Yuphakhun; Vesaratchavest, Mongkol

    2015-01-01

    Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) is one of the most important virulent foodborne pathogens in industrialized countries. The ability to type bacterial strains is essential for surveillance, investigation of outbreaks, and epidemiological studies. Multilocus variable number tandem repeat combined with high-resolution melting analysis (MLV-HRMA) is a fast, cost-efficient, and easy sample genotyping method. In this study, MLV-HRMA and multilocus variable number tandem repeat analysis (MLVA) were used to differentiate between the allelic variants in 5 tandem repeat (TR) loci in 117 Salmonella Typhimurium isolates derived from various farms, slaughterhouses, market, and humans in Thailand. Both MLV-HRMA and MLVA analyses resulted in the identification of a total of 43 different genotypes, but slight differences were observed in cluster analysis results between the 2 methods. The unweighted pair-group method with arithmetic mean-based cluster analysis showed the same core clades; some small differences in the placement of sister-clades and subgrouping were observed due to the inability to reliably type the polymorphic STTR3 locus in the MLV-HRMA. The results of this study show that the MLV-HRMA, following the selection of suitable TR loci, is a relatively reliable and rapid screening method capable of differentiating between Salmonella Typhimurium isolates on the basis of allelic diversity at TR loci. As such, MLV-HRMA can be potentially used to investigate and track sources of contamination in order to effectively control Salmonella contamination in the food supply chain. PMID:25457374

  10. Characterization of an ntrX Mutant of Neisseria gonorrhoeae Reveals a Response Regulator That Controls Expression of Respiratory Enzymes in Oxidase-Positive Proteobacteria

    PubMed Central

    Atack, John M.; Srikhanta, Yogitha N.; Djoko, Karrera Y.; Welch, Jessica P.; Hasri, Norain H. M.; Steichen, Christopher T.; Vanden Hoven, Rachel N.; Grimmond, Sean M.; Othman, Dk Seti Maimonah Pg; Kappler, Ulrike; Apicella, Michael A.; Jennings, Michael P.; Edwards, Jennifer L.

    2013-01-01

    NtrYX is a sensor-histidine kinase/response regulator two-component system that has had limited characterization in a small number of Alphaproteobacteria. Phylogenetic analysis of the response regulator NtrX showed that this two-component system is extensively distributed across the bacterial domain, and it is present in a variety of Betaproteobacteria, including the human pathogen Neisseria gonorrhoeae. Microarray analysis revealed that the expression of several components of the respiratory chain was reduced in an N. gonorrhoeae ntrX mutant compared to that in the isogenic wild-type (WT) strain 1291. These included the cytochrome c oxidase subunit (ccoP), nitrite reductase (aniA), and nitric oxide reductase (norB). Enzyme activity assays showed decreased cytochrome oxidase and nitrite reductase activities in the ntrX mutant, consistent with microarray data. N. gonorrhoeae ntrX mutants had reduced capacity to survive inside primary cervical cells compared to the wild type, and although they retained the ability to form a biofilm, they exhibited reduced survival within the biofilm compared to wild-type cells, as indicated by LIVE/DEAD staining. Analyses of an ntrX mutant in a representative alphaproteobacterium, Rhodobacter capsulatus, showed that cytochrome oxidase activity was also reduced compared to that in the wild-type strain SB1003. Taken together, these data provide evidence that the NtrYX two-component system may be a key regulator in the expression of respiratory enzymes and, in particular, cytochrome c oxidase, across a wide range of proteobacteria, including a variety of bacterial pathogens. PMID:23564168

  11. The treatment of pharyngeal gonorrhoea with a single oral dose of cefixime.

    PubMed

    McMillan, A; Young, H

    2007-04-01

    An audit of the efficacy treatment of pharyngeal gonorrhoea with a single oral dose of 400 mg of cefixime was undertaken; 83% of patients also received a single oral dose of azithromycin to treat possible concurrent anogenital chlamydial infection. Of the 54 patients studied, only one of 45 patients who attended for at least one test of cure had a positive culture seven days after treatment; the possibility of reinfection could not have been excluded. Two tests of cure were obtained from 18 patients. Cefixime, therefore, seems to be effective in the treatment of pharyngeal gonorrhoea.

  12. Multicenter Investigation of Gepotidacin (GSK2140944) Agar Dilution Quality Control Determinations for Neisseria gonorrhoeae ATCC 49226.

    PubMed

    Jones, Ronald N; Fedler, Kelley A; Scangarella-Oman, Nicole E; Ross, James E; Flamm, Robert K

    2016-07-01

    Gepotidacin, a novel triazaacenaphthylene antibacterial agent, is the first in a new class of type IIA topoisomerase inhibitors with activity against many biothreat and conventional pathogens, including Neisseria gonorrhoeae To assist ongoing clinical studies of gepotidacin to treat gonorrhea, a multilaboratory quality assurance investigation determined the reference organism (N. gonorrhoeae ATCC 49226) quality control MIC range to be 0.25 to 1 μg/ml (88.8% of gepotidacin MIC results at the 0.5 μg/ml mode). PMID:27161642

  13. Induction of HIV-1 long terminal repeat-mediated transcription by Neisseria gonorrhoeae.

    PubMed

    Chen, Adrienne; Boulton, Ian C; Pongoski, Jodi; Cochrane, Alan; Gray-Owen, Scott D

    2003-03-01

    Gonorrhoea enhances the transmission of HIV through increased viral shedding and the increased probability of seroconversion among previously HIV-negative individuals. However, the mechanism(s) underlying these influences remain poorly understood. We demonstrated that exposure to Neisseria gonorrhoeae induces the nuclear factor kappa B-dependent transcription from the HIV-1 long terminal repeat in derivatives of the Jurkat CD4 T cell line. These data suggest that gonococcal infection directly impacts HIV-1 transmission through the localized stimulation of viral expression. PMID:12598784

  14. Comparison of Ahlstrom grade 226, Munktell TFN, and Whatman 903 filter papers for dried blood spot specimen collection and subsequent HIV-1 load and drug resistance genotyping analysis.

    PubMed

    Rottinghaus, Erin; Bile, Ebi; Modukanele, Mosetsanagape; Maruping, Maruping; Mine, Madisa; Nkengasong, John; Yang, Chunfu

    2013-01-01

    Dried blood spots (DBS) collected onto filter paper have eased the difficulty of blood collection in resource-limited settings. Currently, Whatman 903 (W-903) filter paper is the only filter paper that has been used for HIV load and HIV drug resistance (HIVDR) testing. We therefore evaluated two additional commercially available filter papers, Ahlstrom grade 226 (A-226) and Munktell TFN (M-TFN), for viral load (VL) testing and HIVDR genotyping using W-903 filter paper as a comparison group. DBS specimens were generated from 344 adult patients on antiretroviral therapy (ART) in Botswana. The VL was measured with NucliSENS EasyQ HIV-1 v2.0, and genotyping was performed for those specimens with a detectable VL (≥ 2.90 log(10) copies/ml) using an in-house method. Bland-A