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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. First Neisseria gonorrhoeae Genotyping Analysis in France: Identification of a Strain Cluster with Reduced Susceptibility to Ceftriaxone ▿

    PubMed Central

    Monfort, Laura; Caro, Valérie; Devaux, Zaelle; Delannoy, Anne-Sophie; Brisse, Sylvain; Sednaoui, Patrice

    2009-01-01

    Sexually transmitted infections are a major public health problem in France and other European countries. Particularly, surveillance data about Neisseria gonorrhoeae infections have clearly indicated an increase in the incidence of gonorrhoea in France in 2006. The French laboratories participated on voluntary basis in the RENAGO (Réseau National du Gonocoque) network and sent all of their collected strains to the National Reference Center for Neisseria gonorrhoeae. In this first French molecular epidemiological study, 93 isolates collected in 2006 and representative of the French gonorrhoea epidemiology were selected. Antibiotic susceptibility to six antibiotics was determined, and serotyping and N. gonorrhoeae multiantigen sequence typing (NG-MAST) were performed. NG-MAST identified 53 sequence types (STs), of which 13 STs contained 2 to 16 isolates. The major STs identified in France were previously described elsewhere. However, two newly described STs, ST1479 and ST1987, had only been found in France until now. ST1479 was characterized by a multiple-resistance phenotype, whereas ST1987 presented a susceptibility phenotype. Moreover, among the predominant French STs, ST225, which had already been described in many countries, comprised isolates (14/16) resistant to ciprofloxacin and with reduced susceptibility to ceftriaxone. Thus, the surveillance of resistance to antibiotics is a priority in order to adapt treatment and decrease the transmission of resistant strains. Of note, no predominant ST was identified among rectal isolates from men who have sex with men. PMID:19794054

  3. Influence of conserved and hypervariable genetic markers on genotyping circulating strains of Neisseria gonorrhoeae.

    PubMed

    Vidovic, Sinisa; Horsman, Greg B; Liao, Mingmin; Dillon, Jo-Anne R

    2011-01-01

    Presently there is no vaccine against Neisseria gonorrhoeae and therefore accurate information on gonococcal transmission plays a crucial role for interventions designed to limit the spread of infections caused by this microorganism. We evaluated the impact of two different categories of genetic markers, (i) concatenated sequences of 10 housekeeping genes and (ii) hypervariable porB DNA sequences, on the genetic relatedness and subsequently on genotyping analysis of this human pathogen. Eighty gonococcal isolates from Canada, China, the US, Argentina, Venezuela and Chile, collected over different times, were analyzed. Our results show that the choice of genetic marker had a profound effect on the interpretation of genotyping results associated with N. gonorrhoeae. The concatenated sequences of the housekeeping genes preserved the genetic relatedness of closely related isolates, enabling detection of the predominant strains circulating within a community (Saskatchewan, Canada) over an extended period of time. In contrast, a genetic marker based on antigen gene, porB, may lead to a failure to detect these predominant circulating strains. Based on the analysis of the DNA sequences of the 10 housekeeping genes, we identified two major clonal complexes, CC33 and CC22, which comprised STs from China, and Argentina as well as two STs from Canada. Several minor clonal complexes were observed among isolates from Saskatchewan. eBURST analysis suggested that the majority of the tested gonococcal isolates from Saskatchewan, Canada were endemic, with only a couple of genotypes introduced.

  4. Gonorrhoea

    PubMed Central

    2014-01-01

    Introduction In 2012, the diagnosis rates for gonorrhoea among adults aged 20 to 24 years in the UK were 249 per 100,000 for men and 140 per 100,000 for women. Resistance to one or more antimicrobial agent is reported in more than one quarter of isolates. Co-infection with Chlamydia trachomatis is reported in 10% to 40% of people with gonorrhoea in the US and UK. Methods and outcomes We conducted a systematic review and aimed to answer the following clinical questions: What are the effects of treatments for uncomplicated infections in men and non-pregnant women, and in pregnant women? What are the effects of treatments for disseminated gonococcal infection? What are the effects of dual treatment for gonorrhoea and chlamydia infection? We searched: Medline, Embase, The Cochrane Library, and other important databases up to September 2013 (Clinical Evidence reviews are updated periodically, please check our website for the most up-to-date version of this review). We included harms alerts from relevant organisations such as the US Food and Drug Administration (FDA) and the UK Medicines and Healthcare products Regulatory Agency (MHRA). Results We found 7 studies that met our inclusion criteria. We performed a GRADE evaluation of the quality of evidence for interventions. Conclusions In this systematic review we present information relating to the effectiveness and safety of the following interventions: antibiotic regimens (dual treatment, multiple dose, single dose). PMID:24559849

  5. Efficacy and safety of ceftriaxone for uncomplicated gonorrhoea: a meta-analysis of randomized controlled trials.

    PubMed

    Bai, Z-G; Bao, X-J; Cheng, W-D; Yang, K-H; Li, Y-P

    2012-02-01

    We conducted a systematic review and meta-analysis of ceftriaxone for treatment of uncomplicated gonorrhoea compared with four other antibiotics. Thirteen randomized controlled trials (RCTs) totalling treatment of 2557 patients with uncomplicated gonorrhoea were included. Statistically significant differences were observed in side-effects, which were increased after ceftriaxone 250 mg versus cefotaxime 500 mg (odds ratio [OR] 1.87; 95% confidence interval [CI] 1.14-3.08). Cure rates of ceftriaxone 250 mg were significantly better than cefixime 400 mg (OR 1.77; 95% CI 1.11-2.80) as was ceftriaxone 125 mg versus spectinomycin 2 g (OR 3.44; 95% CI 1.08-10.90). There was no statistically significant difference between ceftriaxone 250 mg and cefixime 800 mg in cure rates (OR 1.39; 95% CI 0.92-2.10) or adverse effects (OR 1.29, 95% CI 0.58-2.84) for treating uncomplicated gonorrhoea. The cure rate after ceftriaxone 250 mg was not significantly different from that after spectinomycin 2 g (OR 1.96; 95% CI 1.00-3.87). In conclusion, this meta-analysis revealed that 250 mg ceftriaxone had a higher efficacy than 400 mg cefixime for uncomplicated gonorrhoea. Also, ceftriaxone 125 mg is a better choice than spectinomycin 2 g for patients with uncomplicated gonorrhoea, but ceftriaxone had higher side-effect rates than cefotaxime. In the current era further randomized controlled clinical trials of ceftriaxone for uncomplicated gonorrhoea are warranted.

  6. 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...

  7. 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

  8. 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

  9. 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

  10. Functional analysis of NsrR, a nitric oxide sensing Rrf2 repressor in Neisseria gonorrhoeae

    PubMed Central

    Isabella, Vincent M.; Lapek, John D.; Kennedy, Edward M.; Clark, Virginia L.

    2008-01-01

    Nitric oxide has been shown to be an important component of the human immune response, and as such, it is important to understand how pathogenic organisms respond to its presence. In Neisseria gonorrhoeae, recent work has revealed that NsrR, an Rrf2-type transcriptional repressor, can sense NO and control the expression of genes responsible for NO metabolism. A highly pure extract of epitope tagged NsrR was isolated and mass spectroscopic analysis suggested that the protein contained a [2Fe-2S] cluster. NsrR/DNA interactions were thoroughly analyzed in vitro. Using EMSA analysis, NsrR::FLAG was shown to interact with predicted operators in the norB, aniA, and nsrR upstream regions with a Kd of 7 nM, 19 nM, and 35 nM respectively. DNase I footprint analysis was performed on the upstream regions of norB and nsrR, where NsrR was shown to protect the predicted 29 bp binding sites. The presence of exogenously added NO inhibited DNA binding by NsrR. Alanine substitution of C90, C97, or C103 in NsrR abrogated repression of norB::lacZ and inhibited DNA binding, consistent with their presumed role in coordination of a NO-sensitive Fe-S center required for DNA binding. PMID:19007408

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

    PubMed Central

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

    2016-01-01

    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 103 to 104 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

  12. 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.

  13. Transcriptional and Functional Analysis of the Neisseria gonorrhoeae Fur Regulon▿ †

    PubMed Central

    Jackson, Lydgia A.; Ducey, Thomas F.; Day, Michael W.; Zaitshik, Jeremy B.; Orvis, Joshua; Dyer, David W.

    2010-01-01

    To ensure survival in the host, bacteria have evolved strategies to acquire the essential element iron. In Neisseria gonorrhoeae, the ferric uptake regulator Fur regulates metabolism through transcriptional control of iron-responsive genes by binding conserved Fur box (FB) sequences in promoters during iron-replete growth. Our previous studies showed that Fur also controls the transcription of secondary regulators that may, in turn, control pathways important to pathogenesis, indicating an indirect role for Fur in controlling these downstream genes. To better define the iron-regulated cascade of transcriptional control, we combined three global strategies—temporal transcriptome analysis, genomewide in silico FB prediction, and Fur titration assays (FURTA)—to detect genomic regions able to bind Fur in vivo. The majority of the 300 iron-repressed genes were predicted to be of unknown function, followed by genes involved in iron metabolism, cell communication, and intermediary metabolism. The 107 iron-induced genes encoded hypothetical proteins or energy metabolism functions. We found 28 predicted FBs in FURTA-positive clones in the promoters and within the open reading frames of iron-repressed genes. We found lower levels of conservation at critical thymidine residues involved in Fur binding in the FB sequence logos of FURTA-positive clones with intragenic FBs than in the sequence logos generated from FURTA-positive promoter regions. In electrophoretic mobility shift assay studies, intragenic FBs bound Fur with a lower affinity than intergenic FBs. Our findings further indicate that transcription under iron stress is indirectly controlled by Fur through 12 potential secondary regulators. PMID:19854902

  14. 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.

  15. 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.

  16. Identifying opportunities for sexually transmitted infection prevention: analysis of critical points in the care pathways of patients with gonorrhoea.

    PubMed

    Abu-Rajab, K; Scoular, A; Church, S; Connell, J; Winter, A; Hart, G

    2009-03-01

    We applied the principles of Hazard Analysis and Critical Control Points (HACCP) to systematically analyse the care pathway of patients diagnosed with gonorrhoea to identify potential intervention opportunities for preventive action. Data were collected on individuals with culture-positive gonococcal infection during 27 February 2003 to 08 January 2004. Qualitative data were gathered within individual semi-structured interviews. Two hundred and twenty-three gonorrhoea patient episodes were evaluated. The median interval between presentation and treatment was significantly longer in females and men having sex with men (MSM), compared with heterosexual men (P = 0.002). Females were significantly more likely to be in regular relationships at the timepoint of perceived infection acquisition than heterosexuals or MSM (P < 0.0001). Four major themes emerged from the interviews: life-stage and infection risk, determinants of risk perception around sexual encounters, attitudes to preventing re-infection and condom use. These informed three potential 'critical control points': health-related attitudes/behaviours preceding infection; access to appropriate care and optimizing health promotion to prevent further infection.

  17. 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

  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. Deep sequencing-based analysis of the anaerobic stimulon in Neisseria gonorrhoeae

    PubMed Central

    2011-01-01

    Background Maintenance of an anaerobic denitrification system in the obligate human pathogen, Neisseria gonorrhoeae, suggests that an anaerobic lifestyle may be important during the course of infection. Furthermore, mounting evidence suggests that reduction of host-produced nitric oxide has several immunomodulary effects on the host. However, at this point there have been no studies analyzing the complete gonococcal transcriptome response to anaerobiosis. Here we performed deep sequencing to compare the gonococcal transcriptomes of aerobically and anaerobically grown cells. Using the information derived from this sequencing, we discuss the implications of the robust transcriptional response to anaerobic growth. Results We determined that 198 chromosomal genes were differentially expressed (~10% of the genome) in response to anaerobic conditions. We also observed a large induction of genes encoded within the cryptic plasmid, pJD1. Validation of RNA-seq data using translational-lacZ fusions or RT-PCR demonstrated the RNA-seq results to be very reproducible. Surprisingly, many genes of prophage origin were induced anaerobically, as well as several transcriptional regulators previously unknown to be involved in anaerobic growth. We also confirmed expression and regulation of a small RNA, likely a functional equivalent of fnrS in the Enterobacteriaceae family. We also determined that many genes found to be responsive to anaerobiosis have also been shown to be responsive to iron and/or oxidative stress. Conclusions Gonococci will be subject to many forms of environmental stress, including oxygen-limitation, during the course of infection. Here we determined that the anaerobic stimulon in gonococci was larger than previous studies would suggest. Many new targets for future research have been uncovered, and the results derived from this study may have helped to elucidate factors or mechanisms of virulence that may have otherwise been overlooked. PMID:21251255

  20. 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}.

  1. A systematic review and meta-analysis of the prevalence of chlamydia, gonorrhoea and syphilis in incarcerated persons.

    PubMed

    Kouyoumdjian, F G; Leto, D; John, S; Henein, H; Bondy, S

    2012-04-01

    Communicable diseases are common in people who are incarcerated. We aimed to define the prevalence of chlamydia, gonorrhoea and syphilis in people who are incarcerated and to identify subgroups with the highest risk of infection. We searched for prevalence studies of chlamydia, gonorrhoea or syphilis in incarcerated populations. Pooled estimates were generated, and meta-regression was conducted. Random effects models yielded pooled prevalence estimates of 5.75% (95% confidence interval [CI] 5.01, 6.48) and 12.31% (95% CI 10.61, 14.01) for chlamydia in men and women, 1.4% (95% CI 1.09, 1.70) and 5.73% (4.76, 6.69) for gonorrhoea in men and women, and 2.45% (95% CI 2.08, 2.82) and 6.10% (95% CI 4.75, 7.46) for syphilis in men and women, respectively. Each infection was associated with female gender in meta-regression models. Chlamydia, gonorrhoea and syphilis are highly prevalent in these populations. Primary and secondary prevention efforts could improve individual and population health.

  2. 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

  3. [Neisseria gonorrhoeae infections].

    PubMed

    Furuya, Ryusaburo; Tanaka, Masatoshi

    2009-01-01

    Neisseria gonorrhoeae infections are common bacterial sexually transmitted diseases. Men will usually experience lower urinary tract symptons attributed to urethritis, epididymitis, proctitis, or prostatitis, with associated mucopurulent urethral discharge. Many women are asymptomatic. But, occasionally, they have symptons of vaginal and pelvic discomfort of dysuria, and these infections can lead to pelvic inflammatory disease. Recentry, high prevalence of Neisseria gonorrhoeae isolates resistant to antimicrobial agents is a serious problem in the treatment of gonorrhea. For example, in Fukuoka city, Japan, the proportion of the isolates resistant to ciprofloxacin (CPFX) were 73.4% in 2006 and it was still so high. The proportion of the isolates resistant to tetracycline (TC) was 38.5% in 2006 and that of isolates resistant to penicillin G (PCG) was 17.5%. Owing to this high prevalence of antimicrobial-resistant Neisseria gonorrhoeae in Japan, the clinical efficacy rates of oral antimicrobial agents have become lower. So, as first-line therapy for gonococcal infections, only three parenteral regimens of single doses of ceftriaxone, cefodizime or spectinomycin are recommended by the Japanese Society for Sexually Transmitted Diseases. In the circumstances, we studied in vitro activity of combinations of oral agents such as, beta-lactam and azithromycin, fluoroquinolone and azithromycin, or beta-lactam and fluoroquinolone against Neisseria gonorrhoeae. The cefixime+azithromycin combination demonstrated greater synergy than other combinations.

  4. Worldwide Susceptibility Rates of Neisseria gonorrhoeae Isolates to Cefixime and Cefpodoxime: A Systematic Review and Meta-Analysis

    PubMed Central

    Yu, Rui-xing; Yin, Yueping; Wang, Guan-qun; Chen, Shao-chun; Zheng, Bing-jie; Dai, Xiu-qin; Han, Yan; Li, Qi; Zhang, Guo-yi; Chen, Xiangsheng

    2014-01-01

    Background Neisseria gonorrhoeae (NG) infection is a serious public health problem. The third-generation extended-spectrum cephalosporins (ESCs) have been used as the first-line treatment for NG infection for almost three decades. However, in recent years, treatment failures with the oral third-generation ESCs have been reported worldwide. This study aimed to estimate worldwide susceptibility rates of NG to cefixime and cefpodoxime by analyzing data from all relevant published studies. Methodology/principal findings Two researchers independently searched five databases to identify studies on susceptibilities of NG to cefixime and cefpodoxime published between January 1, 1984 and October 15, 2012. A fixed-effect model was used to perform group analysis, and a χ2 test was employed to make subgroup comparison. Publication bias was assessed with the Begg rank correlation test. The pooled susceptibility rate of NG isolates to cefixime was 99.8% (95% CI: 99.7%–99.8%). The cefixime susceptibility rate of NG isolates from men was significantly lower than that from patients without information of gender or from men and women; the susceptibility rate of NG isolates from Asia was significantly lower than that from other continents; and the susceptibility rate of NG isolates collected before or during 2003 was significantly higher than that after 2003. The pooled susceptibility rate of NG isolates to cefpodoxime was 92.8% (95% CI: 89.0%–95.3%), which was lower than that to cefixime (92.8% vs. 99.8%, χ2 = 951.809, P<0.01). Conclusions The susceptibility rate of NG isolates to cefixime varied with the gender of patients and geographical location from which NG isolates were collected, and declined with time. The reported lower susceptibility rate of NG isolates to cefixime and associated treatment failures, as well as the emergence of NG strains with cephalosporin resistance call for the more effective control of NG infection and the development of new antibiotics. PMID

  5. [Analysis of syphilis and gonorrhoea cases, based on data from the National STD Centre, Department of Dermatology and Venerology, Semmelweis University (2005-2008)].

    PubMed

    Pónyai, Katinka; Marschalkó, Márta; Schöffler, Mária; Ostorházi, Eszter; Rozgonyi, Ferenc; Várkonyi, Viktória; Kárpáti, Sarolta

    2009-09-20

    The STD Department of Semmelweis University Budapest is the National Centre of Hungary, which is responsible for screening and care of sexually transmitted diseases (STD), including syphilis and gonorrhoea. 42,114 patients attended the STD Department and 25,362 anonymous screening (HIV: 12,337, syphilis: 13,025) were done between January 2005 and December 2008. During this period 600 syphilitic and 339 gonorrhoea infections were diagnosed. The obligatory HIV screening of patients with sexually transmitted infections (STI) resulted positive result in 47 cases, and 63 patients infected with HIV acquired new syphilitic or gonorrhoea infection. Contact tracing was successful in around 400 syphilis cases, and 150-200 gonorrhoea cases per year. We present our statistical data in order to call attention to the resurgence of syphilis and gonorrhoea and the importance of STD co-infections.

  6. Antigenic diversity of the serotype antigen complex of Neisseria gonorrhoeae: analysis by an indirect enzyme-linked immunoassay.

    PubMed Central

    Johnston, K H

    1980-01-01

    An indirect enzyme-linked immunoassay (ELISA) has been developed to analyze the antigenic profile of the outer membrane serotype complex of Neisseria gonorrhoeae. Antisera raised in rabbits to serotype-specific vesicles (SSV) reacted primarily with homologous SSV; however, there was significant cross-reactivity (less than 50%) with heterologous SSV. N. meningitidis SSV cross-reacted with all antigonococcal SSV but at a lower degree (less than 20%). Preimmune sera did not cross-react significantly with all antigonoccoccal SSV. The sensitivity of the ELISA was enhanced when the integral SSV proteins 1a and 2 were used as adsorbed antigen. Heterologous anti-SSV cross-reacted slightly, having ELISA values less than 15% of the homologous reaction. Antisera prepared by immunoabsorbent affinity columns were highly specific. Homologous affinity anti-SSV reacted only with proteins 1a and 2. The reaction of immune sera was inhibited by homologous proteins 1a and 2; lipopolysaccharide and proteins 1a and 2 isolated from heterologous serotypes did not inhibit the reaction. The reaction of affinity-purified antisera could be inhibited only by homologous protein 1a. By the use of affinity-purified antisera, a specific and highly sensitive ELISA was developed to analyze the antigenic profile of strains of N. gonorrhoeae. PMID:6769815

  7. 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

  8. 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

  9. Conjugative Plasmids of Neisseria gonorrhoeae

    PubMed Central

    Pachulec, Emilia; van der Does, Chris

    2010-01-01

    Many clinical isolates of the human pathogen Neisseria gonorrhoeae contain conjugative plasmids. The host range of these plasmids is limited to Neisseria species, but presence of a tetracycline (tetM) determinant inserted in several of these plasmids is an important cause of the rapid spread of tetracycline resistance. Previously plasmids with different backbones (Dutch and American type backbones) and with and without different tetM determinants (Dutch and American type tetM determinants) have been identified. Within the isolates tested, all plasmids with American or Dutch type tetM determinants contained a Dutch type plasmid backbone. This demonstrated that tetM determinants should not be used to differentiate between conjugal plasmid backbones. The nucleotide sequences of conjugative plasmids with Dutch type plasmid backbones either not containing the tetM determinant (pEP5233) or containing Dutch (pEP5289) or American (pEP5050) type tetM determinants were determined. Analysis of the backbone sequences showed that they belong to a novel IncP1 subfamily divergent from the IncP1α, β, γ, δ and ε subfamilies. The tetM determinants were inserted in a genetic load region found in all these plasmids. Insertion was accompanied by the insertion of a gene with an unknown function, and rearrangement of a toxin/antitoxin gene cluster. The genetic load region contains two toxin/antitoxins of the Zeta/Epsilon toxin/antitoxin family previously only found in Gram positive organisms and the virulence associated protein D of the VapD/VapX toxin/antitoxin family. Remarkably, presence of VapX of pJD1, a small cryptic neisserial plasmid, in the acceptor strain strongly increased the conjugation efficiency, suggesting that it functions as an antitoxin for the conjugative plasmid. The presence of the toxin and antitoxin on different plasmids might explain why the host range of this IncP1 plasmid is limited to Neisseria species. The isolated plasmids conjugated efficiently between

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

    PubMed Central

    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. PMID:25483706

  11. 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

  12. Adaptability and genotypic stability of Coffea arabica genotypes based on REML/BLUP analysis in Rio de Janeiro State, Brazil.

    PubMed

    Rodrigues, W P; Vieira, H D; Barbosa, D H S G; Souza Filho, G R; Candido, L S

    2013-07-15

    Biannuality in coffee culture causes temporal variability in plant productivity. Consequently, it is essential to evaluate genotypes during various crop years to ensure selection of productive and stable genotypes. We evaluated the effectiveness of simultaneous selection of coffee genotypes along harvests, based on productivity, stability, and adaptability, via mixed models, for indication of varieties suitable for Rio de Janeiro State. We evaluated 25 genotypes during 4 crop seasons (2009-2012), in a randomized block design with 5 replications. The ranking of genotypes was obtained on the basis of the adaptability and temporal stability methods (harmonic average of genetic values, relative performance of genetic values, and harmonic mean of the relative performance of the genetic values), obtained via restricted maximum likelihood/best linear unbiased procedure analysis. The selection accuracy (0.8717), associated with the high magnitude of mean heritability, indicate good reliability and prospects for success in the indication of agronomically superior genotypes. There was little variation in the ordering of genotypes among the environments, indicating low influence of harvests in the performance of the genotypes. Five of the 25 genotypes were superior and could be recommended for planting in the northwestern region of Rio de Janeiro State, due to high predicted productivity and stability. We recommend that these methodologies for evaluation of productivity, stability, and adaptability be included in the selection criteria for recommendation of genotypes for commercial plantings.

  13. Comparative analysis of African swine fever virus genotypes and serogroups.

    PubMed

    Malogolovkin, Alexander; Burmakina, Galina; Titov, Ilya; Sereda, Alexey; Gogin, Andrey; Baryshnikova, Elena; Kolbasov, Denis

    2015-02-01

    African swine fever virus (ASFV) causes highly lethal hemorrhagic disease among pigs, and ASFV's extreme antigenic diversity hinders vaccine development. We show that p72 ASFV phylogenetic analysis does not accurately define ASFV hemadsorption inhibition assay serogroups. Thus, conventional ASFV genotyping cannot discriminate between viruses of different virulence or predict efficacy of a specific ASFV vaccine.

  14. Diagnosis, treatment and prevention of gonorrhoea.

    PubMed

    Turner, Rosemarie; Brown, Leonie; Davidson, Clare; Roberts, Colin

    Neisseria gonorrhoeae is a Gram-negative bacteria responsible for the sexually transmitted infection gonorrhoea, which is increasingly common in the UK. Drug-resistant strains of the bacteria have emerged, which is making gonorrhoea difficult to treat. Therefore, preventing infection is important. This article identifies people at increased risk of contracting the infection, and explores how nurses can offer testing and treatment as well as helping to prevent infection through education and health promotion.

  15. 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.

  16. In vitro activity of tigecycline alone and antimicrobial combinations against clinical Neisseria gonorrhoeae isolates.

    PubMed

    Lee, Hyukmin; Kim, Hyunsoo; Seo, Young Hee; Yong, Dongeun; Jeong, Seok Hoon; Lee, Kyungwon; Chong, Younsop

    2017-02-01

    In this study, we determined the in vitro activity of various combinations of antimicrobial agents against 54 Neisseria gonorrhoeae isolates. The combined activity of ceftriaxone (CRO) and azithromycin (AZM), CRO and doxycycline (DOX), CRO and spectinomycin (SPT), cefixime (CFX) and AZM, CFX and DOX, and CFX and SPT was determined using a checkerboard method. The fractional inhibitory concentration index (FICI) values for all combinations were either additive or indifferent, and no synergistic or antagonistic effects were found. The FICI comparison in each combination did not show any difference according to the N.gonorrhoeae-resistant phenotypes and genotypic characteristics, including penicillinase-producing N. gonorrhoeae, tetracycline-resistant N. gonorrhoeae, stratified MIC of all antibiotics, and N. gonorrhoeae multiantigen sequence typing. MIC50 and MIC90 of tigecycline by agar dilution were 0.5 mg/L and 0.5 mg/L, respectively, which were lower than that of tetracycline and DOX. Additive/indifference results could suggest that combinations that include CRO may be used safely without a significant likelihood of generating resistance.

  17. 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

  18. [Incidence of Neisseria gonorrhoeae infections in Belgium: trends 2000-2006].

    PubMed

    Defraye, A; Crucitti, T; Ducoffre, G; Mak, R; Sasse, A

    2009-01-01

    In Belgium, three registration systems collect epidemiological information on N. gonorrhoeae infections. The descriptive analysis of the data presented in this article allows describing the epidemiology of N. gonorrhoeae infections in Belgium in terms of trends in time, describing the characteristics of the patients, and providing information on resistance to antibiotics. The results on the incidence of N. gonorrhoeae infections show an important increase since the year 2000, and this increase is even more pronounced between 2005 and 2006. The majority of the patients reside in big cities, mainly in the district of Antwerp and in the Brussels-Capital region. Among the N. gonorrhoeae specimens that were sent to the reference laboratory, the proportion of specimens resistant to ciprofloxacine increases each year; this proportion reaches 61.4% in 2006. The increase in the incidence of N. gonorrhoeae infections and in antimicrobial resistance is also observed in other European countries. The increase in incidence may be partly related to the important increase of resistance to ciprofloxacine. It is very important to continue the surveillance of antimicrobial resistance, to adapt treatment in function of the recent evolutions and to inform physicians at a regular basis. The results show that homo- and bisexual men are most at risk for N. gonorrhoeae infections. The prevention campaigns for sexually transmitted infections and screening policy have to be reinforced, particularly among homo- and bisexual men.

  19. Characterization of the Neisseria gonorrhoeae Iron and Fur Regulatory Network

    PubMed Central

    Yu, Chunxiao; McClure, Ryan; Daou, Nadine

    2016-01-01

    ABSTRACT The Neisseria gonorrhoeae ferric uptake regulator (Fur) protein controls expression of iron homeostasis genes in response to intracellular iron levels. In this study, using transcriptome sequencing (RNA-seq) analysis of an N. gonorrhoeae fur strain, we defined the gonococcal Fur and iron regulons and characterized Fur-controlled expression of an ArsR-like DNA binding protein. We observed that 158 genes (8% of the genome) showed differential expression in response to iron in an N. gonorrhoeae wild-type or fur strain, while 54 genes exhibited differential expression in response to Fur. The Fur regulon was extended to additional regulators, including NrrF and 13 other small RNAs (sRNAs), and two transcriptional factors. One transcriptional factor, coding for an ArsR-like regulator (ArsR), exhibited increased expression under iron-replete conditions in the wild-type strain but showed decreased expression across iron conditions in the fur strain, an effect that was reversed in a fur-complemented strain. Fur was shown to bind to the promoter region of the arsR gene downstream of a predicted σ70 promoter region. Electrophoretic mobility shift assay (EMSA) analysis confirmed binding of the ArsR protein to the norB promoter region, and sequence analysis identified two additional putative targets, NGO1411 and NGO1646. A gonococcal arsR strain demonstrated decreased survival in human endocervical epithelial cells compared to that of the wild-type and arsR-complemented strains, suggesting that the ArsR regulon includes genes required for survival in host cells. Collectively, these results demonstrate that the N. gonorrhoeae Fur functions as a global regulatory protein to repress or activate expression of a large repertoire of genes, including additional transcriptional regulatory proteins. IMPORTANCE Gene regulation in bacteria in response to environmental stimuli, including iron, is of paramount importance to both bacterial replication and, in the case of pathogenic

  20. 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.

  1. Nucleic Acid Amplification Testing for Neisseria gonorrhoeae

    PubMed Central

    Whiley, David M.; Tapsall, John W.; Sloots, Theo P.

    2006-01-01

    Nucleic acid amplification tests (NAATs) for the detection of Neisseria gonorrhoeae became available in the early 1990s. Although offering several advantages over traditional detection methods, N. gonorrhoeae NAATs do have some limitations. These include cost, risk of carryover contamination, inhibition, and inability to provide antibiotic resistance data. In addition, there are sequence-related limitations that are unique to N. gonorrhoeae NAATs. In particular, false-positive results are a major consideration. These primarily stem from the frequent horizontal genetic exchange occurring within the Neisseria genus, leading to commensal Neisseria species acquiring N. gonorrhoeae genes. Furthermore, some N. gonorrhoeae subtypes may lack specific sequences targeted by a particular NAAT. Therefore, NAAT false-negative results because of sequence variation may occur in some gonococcal populations. Overall, the N. gonorrhoeae species continues to present a considerable challenge for molecular diagnostics. The need to evaluate N. gonorrhoeae NAATs before their use in any new patient population and to educate physicians on the limitations of these tests is emphasized in this review. PMID:16436629

  2. Genotypic analysis of Mucor from the platypus in Australia.

    PubMed

    Connolly, J H; Stodart, B J; Ash, G J

    2010-01-01

    Mucor amphibiorum is the only pathogen known to cause significant morbidity and mortality in the free-living platypus (Ornithorhynchus anatinus) in Tasmania. Infection has also been reported in free-ranging cane toads (Bufo marinus) and green tree frogs (Litoria caerulea) from mainland Australia but has not been confirmed in platypuses from the mainland. To date, there has been little genotyping specifically conducted on M. amphibiorum. A collection of 21 Mucor isolates representing isolates from the platypus, frogs and toads, and environmental samples were obtained for genotypic analysis. Internal transcribed spacer (ITS) region sequencing and GenBank comparison confirmed the identity of most of the isolates. Representative isolates from infected platypuses formed a clade containing the reference isolates of M. amphibiorum from the Centraal Bureau voor Schimmelcultures repository. The M. amphibiorum isolates showed a close sequence identity with Mucor indicus and consisted of two haplotypes, differentiated by single nucleotide polymorphisms within the ITS1 and ITS2 regions. With the exception of isolate 96-4049, all isolates from platypuses were in one haplotype. Multilocus fingerprinting via the use of intersimple sequence repeats polymerase chain reaction identified 19 genotypes. Two major clusters were evident: 1) M. amphibiorum and Mucor racemosus; and 2) Mucor circinelloides, Mucor ramosissimus, and Mucor fragilis. Seven M. amphibiorum isolates from platypuses were present in two subclusters, with isolate 96-4053 appearing genetically distinct from all other isolates. Isolates classified as M. circinelloides by sequence analysis formed a separate subcluster, distinct from other Mucor spp. The combination of sequencing and multilocus fingerprinting has the potential to provide the tools for rapid identification of M. amphibiorum. Data presented on the diversity of the pathogen and further work in linking genetic diversity to functional diversity will provide

  3. 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.

  4. ACPA: automated cluster plot analysis of genotype data.

    PubMed

    Schillert, Arne; Schwarz, Daniel F; Vens, Maren; Szymczak, Silke; König, Inke R; Ziegler, Andreas

    2009-12-15

    Genome-wide association studies have become standard in genetic epidemiology. Analyzing hundreds of thousands of markers simultaneously imposes some challenges for statisticians. One issue is the problem of multiplicity, which has been compared with the search for the needle in a haystack. To reduce the number of false-positive findings, a number of quality filters such as exclusion of single-nucleotide polymorphisms (SNPs) with a high missing fraction are employed. Another filter is exclusion of SNPs for which the calling algorithm had difficulties in assigning the genotypes. The only way to do this is the visual inspection of the cluster plots, also termed signal intensity plots, but this approach is often neglected. We developed an algorithm ACPA (automated cluster plot analysis), which performs this task automatically for autosomal SNPs. It is based on counting samples that lie too close to the cluster of a different genotype; SNPs are excluded when a certain threshold is exceeded. We evaluated ACPA using 1,000 randomly selected quality controlled SNPs from the Framingham Heart Study data that were provided for the Genetic Analysis Workshop 16. We compared the decision of ACPA with the decision made by two independent readers. We achieved a sensitivity of 88% (95% CI: 81%-93%) and a specificity of 86% (95% CI: 83%-89%). In a screening setting in which one aims at not losing any good SNP, we achieved 99% (95% CI: 98%-100%) specificity and still detected every second low-quality SNP.

  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 Helicobacter pylori genotypes in clinical gastric wash samples.

    PubMed

    Miyamoto, Shuichi; Watanabe, Yoshiyuki; Oikawa, Ritsuko; Ono, Shoko; Mabe, Katsuhiro; Kudo, Takahiko; Yamamoto, Hiroyuki; Itoh, Fumio; Kato, Mototsugu; Sakamoto, Naoya

    2016-08-01

    Helicobacter pylori is a key factor in the development of gastric cancer; indeed, clearance of H. pylori helps prevent gastric cancer. However, the relationship between gastric cancer and the abundance and diversity of H. pylori genotypes in the stomach remains unknown. Here, we present, for the first time, a quantitative analysis of H. pylori genotypes in gastric washes. A method was first developed to assess diversity and abundance by pyrosequencing and analysis of single nucleotide polymorphisms in 23S ribosomal RNA (rRNA), a gene associated with clarithromycin resistance. This method was then validated using arbitrarily mixed plasmids carrying 23S rRNA with single nucleotide polymorphisms. Multiple strains were detected in many of 34 clinical samples, with frequency 24.3 ± 24.2 and 26.3 ± 33.8 % for the A2143G and A2144G strains, respectively. Importantly, results obtained from gastric washes were similar to those obtained from biopsy samples. The method provides opportunities to investigate drug resistance in H. pylori and assess potential biomarkers of gastric cancer risk, and should thus be validated in large-scale clinical trials.

  7. 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

  8. Characterization of the ftsZ cell division gene of Neisseria gonorrhoeae: expression in Escherichia coli and N. gonorrhoeae.

    PubMed

    Salimnia, H; Radia, A; Bernatchez, S; Beveridge, T J; Dillon, J R

    2000-01-01

    We cloned the cell division gene ftsZ of the gram-negative coccus Neisseria gonorrhoeae (Ng) strain CH811, characterized it genetically and phenotypically, and studied its localization in N. gonorrhoeae and Escherichia coli (Ec). The 1,179-bp ORF of ftsZ(Ng) encodes a protein with a predicted molecular mass of 41.5 kDa. Protein sequence alignments indicate that FtsZ(Ng) is similar to other FtsZ proteins and contains the conserved GTP binding motif. FtsZ homologues were identified in several N. gonorrhoeae strains and in Neisseria lactamica, Neisseria sicca, Neisseria polysaccharae and Neisseria cinerea either by Western blot or by PCR-Southern blot analysis. Attempts to inactivate the ftsZ(Ng) on the chromosome failed, indicating that it is essential for gonococcal growth. FtsZ(Ng) was synthesized in an in vitro transcription/translation system and was shown to be 43 kDa, the same size as in Western blots. Expression of the ftsZ(Ng) gene from nongonococcal promoters resulted in a filamentous phenotype in E. coli. Under controlled expression, the FtsZ(Ng)-GFP fusion protein localized at the mid-cell division site in E. coli. E. coli expressing high levels of the FtsZ(Ng)-GFP fusion protein formed filaments and exhibited different fluorescent structures including helices, spiral tubules extending from pole to pole, and regularly spaced dots or bands that did not localize at the middle of the cell. Expression of the FtsZ(Ng)-GFP fusion protein in N. gonorrhoeae resulted in abnormal cell division as shown by electron microscopy. FtsZ(Ng)-GFP fusions were also expressed in a gonococcal background using a unique shuttle vector.

  9. Stable shuttle vectors for Neisseria gonorrhoeae, Haemophilus spp. and other bacteria based on a single origin of replication.

    PubMed

    Pagotto, F J; Salimnia, H; Totten, P A; Dillon, J R

    2000-02-22

    An origin of replication (ori) was obtained from a naturally occurring beta-lactamase-producing plasmid isolated from Neisseria gonorrhoeae and used to construct shuttle vectors capable of replicating in N. gonorrhoeae, Haemophilus ducreyi, Haemophilus influenzae and Escherichia coli. Using the gonococcal proAB genes, we complemented proline-requiring N. gonorrhoeae F62 and E. coli HB101 in trans. The first demonstration of the expression of the green fluorescent protein (GFP) in either N. gonorrhoeae or H. ducreyi was shown using this vector, indicating that GFP may be a useful tool in the analysis of these organisms. This is the first report of a gonococcal vector based on a broad host range, genetically defined ori, and should facilitate the molecular analysis of gonococcal and Haemophilus genes.

  10. Biplot analysis of phenotypic stability in upland cotton genotypes in Mato Grosso.

    PubMed

    Farias, F J C; Carvalho, L P; Silva Filho, J L; Teodoro, P E

    2016-05-20

    Seed cotton yield is a trait governed by multiple genes that cause changes in the performance of genotypes depending on the cultivation environment. Breeding programs examine the genotype x environment interaction (GE) using precise statistical methods, such as AMMI (additive main effects and multiplicative interaction) and GGE biplot (genotype main effects + genotype x environment interaction). The AMMI method combines the analysis of variance and principal components, to adjust the main effects (genotypes and environments) and the effects of GE interaction, respectively. The GGE biplot groups the genotype additive effect together with the multiplicative effect of the GE interaction, and submits both of these to the principal components analysis. The aim of this study was to investigate the association between the AMMI and GGE biplot methods and select cotton genotypes that simultaneously showed high productivity of seed cotton and stability in Mato Grosso environments. Trials were conducted with cotton cultivars in eight environments across Mato Grosso State in the 2008/2009 crop season. The experiment used a randomized block design with 16 genotypes and four replicates per genotype x environment combination. Data for seeds cotton productivity were analyzed by AMMI and GGE biplot methods. Both methods were concordant in the discrimination of environments and genotypes for phenotypic stability. The genotypes BRS ARAÇÁ and LD 05 CV had high seed cotton productivity and phenotypic stability, and could be grown in all environments across Mato Grosso State.

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

    PubMed

    Fu, Yong-Bi

    2014-03-13

    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.

  12. Genotyping and phylogenetic analysis of Acanthamoeba isolates associated with keratitis.

    PubMed

    Risler, Arnaud; Coupat-Goutaland, Bénédicte; Pélandakis, Michel

    2013-11-01

    We examined a partial SSU-rDNA sequence from 20 Acanthamoeba isolates associated with keratitis infections. The phylogenetic tree inferred from this partial sequence allowed to assign isolates to genotypes. Among the 20 isolates examined, 16 were found to be of the T4 genotype, 2 were T3, 1 was a T5, and 1 was a T2, confirming the predominance of T4 in infections. However, the study highlighted other genotypes more rarely associated with infections, particularly the T2 genotype. Our study is the second one to detect that this genotype is associated with keratitis. Additionally, the phylogenetic analyses showed five main emerging clusters, T4/T3/T11, T2/T6, T10/T12/T14, T13/T16, and T7/T8/T9/T17, regularly obtained whichever method was used. A similar branching pattern was found when the full rDNA sequence was investigated.

  13. Molecular analyses of TEM genes and their corresponding penicillinase-producing Neisseria gonorrhoeae isolates in Bangkok, Thailand.

    PubMed

    Nakayama, Shu-Ichi; Tribuddharat, Chanwit; Prombhul, Sasiprapa; Shimuta, Ken; Srifuengfung, Somporn; Unemo, Magnus; Ohnishi, Makoto

    2012-02-01

    Neisseria gonorrhoeae is a major public health problem globally, especially because the bacterium has developed resistance to most antimicrobials introduced for first-line treatment of gonorrhea. In the present study, 96 N. gonorrhoeae isolates with high-level resistance to penicillin from 121 clinical isolates in Thailand were examined to investigate changes related to their plasmid-mediated penicillin resistance and their molecular epidemiological relationships. A β-lactamase (TEM) gene variant, bla(TEM-135), that may be a precursor in the transitional stage of a traditional bla(TEM-1) gene into an extended-spectrum β-lactamase (ESBL), possibly causing high resistance to all extended-spectrum cephalosporins in N. gonorrhoeae, was identified. Clonal analysis using multilocus sequence typing (MLST) and N. gonorrhoeae multiantigen sequence typing (NG-MAST) revealed the existence of a sexual network among patients from Japan and Thailand. Molecular analysis of the bla(TEM-135) gene showed that the emergence of this allele might not be a rare genetic event and that the allele has evolved in different plasmid backgrounds, which results possibly indicate that it is selected due to antimicrobial pressure. The presence of the bla(TEM-135) allele in the penicillinase-producing N. gonorrhoeae population may call for monitoring for the possible emergence of ESBL-producing N. gonorrhoeae in the future. This study identified a bla(TEM) variant (bla(TEM-135)) that is a possible intermediate precursor for an ESBL, which warrants international awareness.

  14. NGMASTER: in silico multi-antigen sequence typing for Neisseria gonorrhoeae

    PubMed Central

    Gonçalves da Silva, Anders; Dyet, Kristin; Williamson, Deborah A.; Stinear, Timothy P.; Howden, Benjamin P.; Seemann, Torsten

    2016-01-01

    Whole-genome sequencing (WGS) provides the highest resolution analysis for comparison of bacterial isolates in public health microbiology. However, although increasingly being used routinely for some pathogens such as Listeria monocytogenes and Salmonella enterica, the use of WGS is still limited for other organisms, such as Neisseria gonorrhoeae. Multi-antigen sequence typing (NG-MAST) is the most widely performed typing method for epidemiological surveillance of gonorrhoea. Here, we present NGMASTER, a command-line software tool for performing in silico NG-MAST on assembled genome data. NGMASTER rapidly and accurately determined the NG-MAST of 630 assembled genomes, facilitating comparisons between WGS and previously published gonorrhoea epidemiological studies. The source code and user documentation are available at https://github.com/MDU-PHL/ngmaster. PMID:28348871

  15. 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.

  16. Genotype and Phenotype Analysis in Pediatric Patients with Cystinuria

    PubMed Central

    2017-01-01

    Cystinuria is an inherited disorder characterized by defective renal reabsorption of cystine and dibasic amino acids leading to nephrolithiasis. This study was conducted to analyze the genotypes and phenotypes of pediatric patients with cystinuria. Eight children from Seoul National University Hospital and Asan Medical Center presenting with cystinuria from January 2003 to June 2016 were retrospectively analyzed. Mutational studies were performed by direct sequencing. Two of the 8 were male and 6 were female. The median ages at onset and diagnosis were 1.5 (range, 0.3–13.6) and 2.6 (range, 0.7–16.7) years, respectively. The median followed up was 7.7 (range, 3.4–14.0) years. Mutational analyses were performed in 7 patients and revealed biallelic SLC3A1 mutations (AA genotype) in 4 patients, a single heterozygous SLC3A1 mutation (A- genotype) in 1 patient, biallelic SLC7A9 mutations (BB genotype) in 1 patient, and a single heterozygous SLC7A9 mutation (B- genotype) in 1 patient. Two of the mutations were novel. No genotype-phenotype correlations were observed, except for earlier onset age in patients with non-AA genotypes than in patients with the AA genotype. All patients suffered from recurrent attacks of symptomatic nephrolithiasis, which lead to urologic interventions. At the last follow-up, 3 patients had a mild-to-moderate degree of renal dysfunction. This is the first study of genotypic and phenotypic analyses of patients with cystinuria in Korea. PMID:28049243

  17. Neisseria Species Identification Assay for the Confirmation of Neisseria gonorrhoeae-Positive Results of the COBAS Amplicor PCR▿

    PubMed Central

    Mangold, Kathy A.; Regner, MaryAnn; Tajuddin, Mohammed; Tajuddin, Aamair M.; Jennings, Lawrence; Du, Hongyan; Kaul, Karen L.

    2007-01-01

    Screening assays for Neisseria gonorrhoeae exhibit low positive predictive values, particularly in low-prevalence populations. A new real-time PCR assay that detects and identifies individual Neisseria spp. using melt curve analysis was compared to two previously published supplementary assays. NsppID, a 16S rRNA real-time PCR/melt curve assay developed to distinguish N. gonorrhoeae from other Neisseria spp., was compared to real-time PCR assays targeting genes reportedly specific for N. gonorrhoeae, the cppB gene and the porA pseudogene. A total of 408 clinical specimens (324 female endocervical swabs and 84 male urine or urogenital swab specimens) were screened using the COBAS Amplicor assay for Chlamydia trachomatis and N. gonorrhoeae (CT/NG) (Roche Diagnostics, Indianapolis, IN) followed by confirmatory testing via real-time PCR. The NsppID assay detected Neisseria spp. in 150/181 COBAS-positive specimens (82%), including six dual infections, and identified N. gonorrhoeae in 102 (56%) specimens. Sixty-nine of 181 (38%) specimens were positive for N. gonorrhoeae by porA pseudogene, and 115/181 (64%) were positive for cppB. However, cppB was also positive in 15% of COBAS-negative specimens, more than either NsppID (4%) or porA pseudogene (2%) assays. The porA pseudogene assay had the highest specificity for both genders but the lowest sensitivity, especially in female specimens. NsppID had a slightly lower specificity but greater sensitivity and overall accuracy. The least desirable confirmatory assay was cppB, due to poor specificity. The NsppID assay is an accurate confirmatory assay for N. gonorrhoeae detection. In addition, the NsppID assay can identify the non-N. gonorrhoeae species responsible for the majority of false-positive results from the COBAS Amplicor CT/NG assay. PMID:17360838

  18. Pharmacogenomics of platinum-based chemotherapy response in NSCLC: a genotyping study and a pooled analysis

    PubMed Central

    Chen, Juan; Wang, Zhan; Zou, Ting; Cui, Jiajia; Yin, Jiye; Zheng, Wei; Jiang, Wuzhong; Zhou, Honghao; Liu, Zhaoqian

    2016-01-01

    Published data showed inconsistent results about associations of extensively studied polymorphisms with platinum-based chemotherapy response. Our study aimed to provide reliable conclusions of these associations by detecting genotypes of the SNPs in a larger sample size and summarizing a comprehensive pooled analysis. 13 SNPs in 8 genes were genotyped in 1024 NSCLC patients by SequenomMassARRAY. 39 published studies and our study were included in meta-analysis. Patients with GA or GG genotypes of XRCC1 G1196 had better response than AA genotype carriers (Genotyping study: OR = 0.72, 95%CI: 0.53-0.96, P = 0.028; Meta-analysis: OR = 0.74, 95%CI: 0.62-0.89, P = 0.001). Patients carrying CT or TT genotypes of XRCC1 C580T could be more sensitive to platinum-based chemotherapy compared to patients with CC genotype (OR = 0.54, 95%CI: 0.37-0.80, P = 0.002). CC genotype of XRCC3 C18067T carriers showed more resistance to platinum-based chemotherapy when compared to those with CT or TT genotypes (OR = 0.69, 95%CI: 0.52-0.91, P = 0.009). Our study indicated that XRCC1 G1196A/C580T and XRCC3 C18067T should be paid attention for personalized platinum-based chemotherapy in NSCLC patients. PMID:27248474

  19. High resolution melt analysis (HRMA); a viable alternative to agarose gel electrophoresis for mouse genotyping.

    PubMed

    Thomsen, Nicole; Ali, Radiya G; Ahmed, Jehangir N; Arkell, Ruth M

    2012-01-01

    Most mouse genetics laboratories maintain mouse strains that require genotyping in order to identify the genetically modified animals. The plethora of mutagenesis strategies and publicly available mouse alleles means that any one laboratory may maintain alleles with random or targeted insertions of orthologous or unrelated sequences as well as random or targeted deletions and point mutants. Many experiments require that different strains be cross bred conferring the need to genotype progeny at more than one locus. In contrast to the range of new technologies for mouse mutagenesis, genotyping methods have remained relatively static with alleles typically discriminated by agarose gel electrophoresis of PCR products. This requires a large amount of researcher time. Additionally it is susceptible to contamination of future genotyping experiments because it requires that tubes containing PCR products be opened for analysis. Progress has been made with the genotyping of mouse point mutants because a range of new high-throughput techniques have been developed for the detection of Single Nucleotide Polymorphisms. Some of these techniques are suitable for genotyping point mutants but do not detect insertion or deletion alleles. Ideally, mouse genetics laboratories would use a single, high-throughput platform that enables closed-tube analysis to genotype the entire range of possible insertion and deletion alleles and point mutants. Here we show that High Resolution Melt Analysis meets these criteria, it is suitable for closed-tube genotyping of all allele types and current genotyping assays can be converted to this technology with little or no effort.

  20. HRAS mutation analysis in Costello syndrome: genotype and phenotype correlation.

    PubMed

    Gripp, Karen W; Lin, Angela E; Stabley, Deborah L; Nicholson, Linda; Scott, Charles I; Doyle, Daniel; Aoki, Yoko; Matsubara, Yoichi; Zackai, Elaine H; Lapunzina, Pablo; Gonzalez-Meneses, Antonio; Holbrook, Jennifer; Agresta, Cynthia A; Gonzalez, Iris L; Sol-Church, Katia

    2006-01-01

    Costello syndrome is a rare condition comprising mental retardation, distinctive facial appearance, cardiovascular abnormalities (typically pulmonic stenosis, hypertrophic cardiomyopathy, and/or atrial tachycardia), tumor predisposition, and skin and musculoskeletal abnormalities. Recently mutations in HRAS were identified in 12 Japanese and Italian patients with clinical information available on 7 of the Japanese patients. To expand the molecular delineation of Costello syndrome, we performed mutation analysis in 34 North American and 6 European (total 40) patients with Costello syndrome, and detected missense mutations in HRAS in 33 (82.5%) patients. All mutations affected either codon 12 or 13 of the protein product, with G12S occurring in 30 (90.9%) patients of the mutation-positive cases. In two patients, we found a mutation resulting in an alanine substitution in position 12 (G12A), and in one patient, we detected a novel mutation (G13C). Five different HRAS mutations have now been reported in Costello syndrome, however genotype-phenotype correlation remains incomplete.

  1. Factors associated with travel to non-local genitourinary medicine clinics for gonorrhoea: an analysis of patients diagnosed in London, 2009-10.

    PubMed

    Le Polain de Waroux, Olivier; Hughes, Gwenda; Maguire, Helen; Crook, Paul D

    2014-03-01

    We analysed factors associated with travelling to non-local genitourinary medicine clinics for gonorrhoea care in London. We used surveillance data on London residents attending genitourinary medicine clinics in 2009-10 and calculated distances between patients' areas of residence and both the nearest genitourinary medicine clinic and the clinic attended. Non-local clinics were attended by 5408 (46.7%) patients. Men having sex with men attended non-local services more than heterosexuals (OR 3.83, p < 0.001). Among heterosexual men, black Africans and black Caribbeans were more likely, and South Asians less likely, to attend non-local services compared to whites (OR [95%CI] 1.33 [1.04-1.72], 1.36 [1.11-1.67] and 0.46 [0.31-0.70] respectively). Similar associations, although not statistically significant, were found in women. People were more likely to attend local services if their local clinic provided walk-in and young people's services, weekend consultations and long opening hours. These findings could help design services meeting local population needs and facilitate prompt and equitable access to care.

  2. Small transcriptome analysis indicates that the enzyme RppH influences both the quality and quantity of sRNAs in Neisseria gonorrhoeae

    PubMed Central

    Wachter, Jenny; Hill, Stuart A.

    2015-01-01

    Prokaryotic mRNA turnover can be initiated by the removal of pyrophosphate from the 5′ end of a transcript using the RNA pyrophosphohydrolase enzyme RppH. Following the initial dephosphorylation step, RNaseE then degrades the message into small oligonucleotide segments. This study assessed the small RNA transcriptome of Neisseria gonorrhoeae strain MS11 in two genetic backgrounds; using wild type cells as well as cells carrying a rppH insertional mutation. It was found that the presence of the RppH enzyme affected both the quantity and length of small RNAs (sRNAs) in various chromosomal locations and involved sense transcripts (seRNAs), transcripts originating from the opposite strand (asRNAs) as well as inter-genic-derived RNAs (IGRs). In comparing the two transcriptomes, we found that not all small RNAs were expressed in both genetic backgrounds, suggesting that RppH apparently targets only a subset of transcripts. Overall, this study shows that small RNAs can be detected from the majority of genes within the chromosome, as well as from inter-genic regions, and that more sRNA transcripts are detected in the absence of the RppH enzyme. PMID:25688066

  3. 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.

  4. Seasonal variation in gonorrhoea incidence among men who have sex with men.

    PubMed

    Li, Bin; Bi, Peng; Chow, Eric P F; Donovan, Basil; McNulty, Anna; Ward, Alison; Bell, Charlotte; Fairley, Christopher K

    2016-10-07

    Background: After reviewing urethral gonorrhoea cases among men who have sex with men (MSM) at the South Australia Specialist Sexual Health (SASSH) in Adelaide, Australia, we noticed peaks of gonorrhoea among MSM occurred predominantly in the first quarter of the year (January-March). The aim of this study was to formally test this hypothesis against data from a similar period at three sexual health services, one each in Adelaide, Melbourne and Sydney. Methods: This study was a retrospective analysis of computerised records at the three Australian sexual health services. Potential risk factors for urethral gonorrhoea among MSM were also reviewed at the SASSH. Results: More peaks of gonorrhoea cases were observed in the first quarter of the year in Adelaide and Sydney and in the second and fourth quarter in Melbourne. Factors independently associated with urethral gonorrhoea at the SASSH were being a young MSM, especially those aged 25-29 (odds ratio (OR) 2.66, 95% confidence interval (CI): 2.00-3.54), having more than one sexual partner (OR 1.71, 95% CI: 1.43-2.04), having had sex interstate and overseas (OR 1.52, 95% CI: 1.06-2.17), and presenting in the first quarter (OR 1.30, 95% CI: 1.10-1.55). Conclusion: Our data suggest that gonorrhoea among MSM occurs in a seasonal pattern, particularly late summer into early autumn. This has implications for the provision of health services over the year and for the timing of health promotion activities.

  5. 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

  6. 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.

  7. Screening of eight Eucalypt genotypes (Eucalyptus sp.) for water deficit tolerance using multivariate cluster analysis.

    PubMed

    Cha-Um, S; Somsueb, S; Samphumphuang, T; Kirdmanee, C

    2014-06-01

    The present study evaluated eight genotypes of river red gum (Eucalyptus camaldulensis Dehnh.) and a hybrid (E. camaldulensis × E. urophylla) for mannitol-induced water deficit (WD) under photoautotrophic conditions using multivariate cluster analysis. Shoot height, plant dry weight, and chlorophyll a content in hybrid genotypes, 58H2 and 27A2, were maintained when exposed to 200 mM mannitol for 14 days. In addition, the diminution of photosynthetic abilities, i.e. maximum quantum yield of PSII, photon yield of PSII, photochemical quenching, and net photosynthetic rate, under WD was minimal in hybrid genotypes compared to that in selection clones of E. camaldulensis. Under WD condition, there was greater accumulation of proline in all genotypes. A positive relationship was observed between physiological and morphological attributes under WD stress. Using Ward's cluster analysis, hybrid genotypes-H4, 58H2, and 27A2-were classified as water deficit tolerant.

  8. 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

    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.

  9. 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

  10. Determination of hepatitis C virus genotypes in the United States by cleavase fragment length polymorphism analysis.

    PubMed Central

    Marshall, D J; Heisler, L M; Lyamichev, V; Murvine, C; Olive, D M; Ehrlich, G D; Neri, B P; de Arruda, M

    1997-01-01

    We describe the application of a new DNA-scanning method, which has been termed Cleavase Fragment Length Polymorphism (CFLP; Third Wave Technologies, Inc., Madison, Wis.), for the determination of the genotype of hepatitis C virus (HCV). CFLP analysis results in the generation of structural fingerprints that allow discrimination of different DNA sequences. We analyzed 251-bp cDNA products generated by reverse transcription-PCR of the well-conserved 5'-noncoding region of HCV. We determined the genotypes of 87 samples by DNA sequencing and found isolates representing 98% of the types typically encountered in the United States, i.e., types 1a, 1b, 2a/c, 2b, 3a, and 4. Blinded CFLP analysis of these samples was 100% concordant with DNA sequencing results, such that closely related genotypes yielded patterns with strong familial resemblance whereas more divergent sequences yielded patterns with pronounced dissimilarities. In each case, the aggregate pattern was indicative of genotypic grouping, while finer changes suggested subgenotypic differences. We also assessed the reproducibility of CFLP analysis in HCV genotyping by analyzing three distinct isolates belonging to a single subtype. These three isolates yielded indistinguishable CFLP patterns, as did replicate analysis of a single isolate. This study demonstrates the suitability of this technology for HCV genotyping and suggests that it may provide a low-cost, high-throughput alternative to DNA sequencing or other, more costly or cumbersome genotyping approaches. PMID:9399512

  11. 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

  12. Chlamydia trachomatis and Neisseria gonorrhoeae Infections Among Men and Women Entering California Prisons

    PubMed Central

    Bernstein, Kyle T.; Chow, Joan M.; Ruiz, Juan; Schachter, Julius; Horowitz, Evalyn; Bunnell, Rebecca; Bolan, Gail

    2006-01-01

    Objective. We estimated the prevalence of Chlamydia trachomatis and Neisseria gonorrhoeae infection among newly arriving inmates at 6 California prisons. Methods. In this cross-sectional study in 1999, urine specimens collected from 698 men aged 18 to 25 years and 572 women aged 18 years or older were tested at intake for C trachomatis and N gonorrhoeae using ligase chain reaction. An analysis of demographic and arrest-related correlates of C trachomatis and N gonorrhoeae infection was performed. Results. The overall C trachomatis prevalence was 9.9% (95% CI=7.8%, 12.3%) among men aged 18 to 25 years, 8.9% (95% CI = 2.9%, 22.1%) among women aged 18 to 25 years, and 3.3% (95% CI=2.0%, 5.1%) among women overall. Three N gonorrhoeae cases were detected with an overall prevalence of 0.24% (95% CI=0.05%, 0.69%). Conclusions. The prevalence of C trachomatis infection at entry to California prisons, especially among young female and male inmates, was high, which supports routine screening at entry into prison. In addition, screening in a jail setting where most detainees are incarcerated before entry into the prison setting may provide an excellent earlier opportunity to identify these infections and treat disease to prevent complications and burden of infection in this high-risk population. PMID:17008584

  13. The Neisseria gonorrhoeae biofilm matrix contains DNA, and an endogenous nuclease controls its incorporation.

    PubMed

    Steichen, Christopher T; Cho, Christine; Shao, Jian Q; Apicella, Michael A

    2011-04-01

    Neisseria gonorrhoeae has been shown to produce biofilms both in experimental flow chambers and in the human host. Our laboratory has shown that extracellular DNA is an essential component of the gonococcal matrix. We have also identified a gene in N. gonorrhoeae, which we designated nuc. This gene has homology with the staphylococcus-secreted thermonuclease. Our laboratory has characterized nuc through phenotypic analysis of a nuc deletion mutant. Biofilms grown with this strain are significantly thicker and of greater biomass than the N. gonorrhoeae 1291 parent strain. Confocal microscopy indicates that the increased size of the mutant biofilms appears to be due to elevated amounts of extracellular DNA in the biofilm matrix. Chromosomal complementation of the nuc mutation restored the wild-type biofilm phenotype. In addition, we have cloned and expressed the Nuc protein in Escherichia coli, and our data indicate that it has the ability to digest multiple forms of DNA and is a thermonuclease. The ability of Nuc to digest DNA also extends to its ability to disrupt established gonococcal biofilms through degradation of the DNA in the biofilm matrix. Our studies indicate that the N. gonorrhoeae biofilm contains DNA and that the Nuc protein appears to play a role in biofilm formation and remodeling.

  14. 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

  15. Analyzing Neisseria gonorrhoeae Pilin Antigenic Variation Using 454 Sequencing Technology

    PubMed Central

    Rotman, Ella; Webber, David M.

    2016-01-01

    ABSTRACT Many pathogens use homologous recombination to vary surface antigens in order to avoid immune surveillance. Neisseria gonorrhoeae, the bacterium responsible for the sexually transmitted infection gonorrhea, achieves this in part by changing the sequence of the major subunit of the type IV pilus in a process termed pilin antigenic variation (Av). The N. gonorrhoeae chromosome contains one expression locus (pilE) and many promoterless, partial-coding silent copies (pilS) that act as reservoirs for variant pilin information. Pilin Av occurs by high-frequency gene conversion reactions, which transfer pilS sequences into the pilE locus. We have developed a 454 sequencing-based assay to analyze the frequency and characteristics of pilin Av that allows a more robust analysis of pilin Av than previous assays. We used this assay to analyze mutations and conditions previously shown to affect pilin Av, confirming many but not all of the previously reported phenotypes. We show that mutations or conditions that cause growth defects can result in Av phenotypes when analyzed by phase variation-based assays. Adapting the 454 sequencing to analyze pilin Av demonstrates the utility of this technology to analyze any diversity generation system that uses recombination to develop biological diversity. IMPORTANCE Measuring and analyzing complex recombination-based systems constitute a major barrier to understanding the mechanisms used to generate diversity. We have analyzed the contributions of many gonococcal mutations or conditions to the process of pilin antigenic variation. PMID:27381912

  16. Molecular analysis of HBV genotypes and subgenotypes in the Central-East region of Tunisia

    PubMed Central

    2010-01-01

    Background In Tunisia, country of intermediate endemicity for Hepatitis B virus (HBV) infection, most molecular studies on the virus have been carried out in the North of the country and little is known about other regions. The aim of this study was to determine HBV genotype and subgenotypes in Central-East Tunisia. A total of 217 HBs antigen positive patients were enrolled and determination of genotype was investigated in 130 patients with detectable HBV DNA. HBV genotyping methods were: PCR-RFLP on the pre-S region, a PCR using type-specific primers in the S region (TSP-PCR) and partial sequencing in the pre-S region. Results Three genotypes (D, B and A) were detected by the PCR-RFLP method and two (D and A) with the TSP-PCR method, the concordance between the two methods was 93%. Sequencing and phylogenetic analysis of 32 strains, retrieved the same genotype (D and A) for samples with concordant results and genotype D for samples with discordant results. The sequences of discordant genotypes had a restriction site in the pre-S gene which led to erroneous result by the PCR-RFLP method. Thus, prevalence of genotype D and A was 96% and 4%, respectively. Phylogenetic analysis showed the predominance of two subgenotypes D1 (55%) and D7 (41%). Only one strain clustered with D3 subgenotype (3%). Conclusions Predominance of subgenotype D7 appears to occur in northern regions of Africa with transition to subgenotype D1 in the East of the continent. HBV genetic variability may lead to wrong results in rapid genotyping methods and sequence analysis is needed to clarify atypical results. PMID:21050489

  17. Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry for the identification of Neisseria gonorrhoeae.

    PubMed

    Buchanan, R; Ball, D; Dolphin, H; Dave, J

    2016-09-01

    Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) was compared with the API NH biochemical method for the identification of Neisseria gonorrhoeae in routine clinical samples. A retrospective review of laboratory records for 1090 isolates for which both biochemical and MALDI-TOF MS identifications were available was performed. Cases of discrepant results were examined in detail for evidence supportive of a particular organism identification. Of 1090 isolates, 1082 were identified as N. gonorrhoeae by API NH. MALDI-TOF MS successfully identified 984 (91%) of these after one analysis, rising to 1081 (99.9%) after two analyses, with a positive predictive value of 99.3%. For those isolates requiring a repeat analysis, failure to generate an identifiable proteomic signature was the reason in 76% of cases, with alternative initial identifications accounting for the remaining 24%. MALDI-TOF MS identified eight isolates as N. gonorrhoeae that were not identified as such by API NH-examination of these discrepant results suggested that the MALDI-TOF MS identification may be the more reliable. MALDI-TOF MS is at least as accurate and reliable a method of identifying N. gonorrhoeae as API NH. We propose that MALDI-TOF MS could potentially be used as a single method for N. gonorrhoeae identification in routine cases by laboratories with access to this technology.

  18. Rapid and efficient zebrafish genotyping using PCR with high-resolution melt analysis.

    PubMed

    Xing, Lingyan; Quist, Tyler S; Stevenson, Tamara J; Dahlem, Timothy J; Bonkowsky, Joshua L

    2014-02-05

    Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA). This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts. Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

  19. Biplot analysis of strawberry genotypes recommended for the State of Espírito Santo.

    PubMed

    Costa, A F; Teodoro, P E; Bhering, L L; Leal, N R; Tardin, F D; Daher, R F

    2016-08-26

    Most strawberry genotypes grown commercially in Brazil originate from breeding programs in the United States, and are therefore not adapted to the various soil and climatic conditions found in Brazil. Thus, quantifying the magnitude of genotype x environment (GE) interactions serves as a primary means for increasing average Brazilian strawberry yields, and helps provide specific recommendations for farmers on which genotypes meet high yield and phenotypic stability thresholds. The aim of this study was to use AMMI (additive main effects and multiplicative interaction) and GGE biplot (genotype main effects + genotype x environment interaction) analyses to identify high-yield, stable strawberry genotypes grown at three locations in Espírito Santo for two agricultural years. We evaluated seven strawberry genotypes (Dover, Camino Real, Ventana, Camarosa, Seascape, Diamante, and Aromas) at three locations (Domingos Martins, Iúna, and Muniz Freire) in agricultural years 2006 and 2007, totaling six study environments. Joint analysis of variance was calculated using yield data (t/ha), and AMMI and GGE biplot analysis was conducted following the detection of a significant genotypes x agricultural years x locations (G x A x L) interaction. During the two agricultural years, evaluated locations were allocated to different regions on biplot graphics using both methods, indicating distinctions among them. Based on the results obtained from the two methods used in this study to investigate the G x A x L interaction, we recommend growing the Camarosa genotype for production at the three locations assessed due to the high frequency of favorable alleles, which were expressed in all localities evaluated regardless of the agricultural year.

  20. Drug-resistant Neisseria gonorrhoeae: latest developments.

    PubMed

    Suay-García, B; Pérez-Gracia, M T

    2017-02-16

    Gonorrhea is the second most frequently reported notifiable disease in the United States and is becoming increasingly common in Europe. The purpose of this review was to assess the current state of drug-resistant Neisseria gonorrhoeae in order to evaluate future prospects for its treatment. An exhaustive literature search was conducted to include the latest research regarding drug resistance and treatment guidelines for gonorrhea. Gonococci have acquired all known resistance mechanisms to all antimicrobials used for treatment. Currently, the European Union, the United States, and the United Kingdom have established surveillance programs to assess, on a yearly basis, the development of gonococcal resistance. Current treatment guidelines are being threatened by the increasing number of ceftriaxone-, cefixime-, and azithromycin-resistant N. gonorrhoeae strains being detected worldwide. This has led the scientific community to develop new treatment options with new molecules in order to persevere in the battle against this "superbug".

  1. 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

  2. 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

  3. Genotype-guided drug prescribing: a systematic review and meta-analysis of randomized control trials

    PubMed Central

    Goulding, Rebecca; Dawes, Diana; Price, Morgan; Wilkie, Sabrina; Dawes, Martin

    2015-01-01

    Aim Adverse drug events lead to increased morbidity, mortality and health care costs. Pharmacogenetic testing that guides drug prescribing has the potential to reduced adverse drug events and increase drug effectiveness. Our aim was to quantify the clinical effectiveness of genotype-guided prescribing. Methods Three electronic databases were searched from January 1980 through December 2013. Studies were eligible if they were RCTs comparing genotype-guided prescribing with non-genetic informed prescribing, reported drug specific adverse drug events and clinical effectiveness outcomes. Two reviewers independently screened titles and abstracts, extracted data and assessed study quality. Meta-analyses of specific outcomes were conducted where data allowed. Results Fifteen studies, involving 5688 patients and 19 drugs, met the inclusion and exclusion criteria. Eight studies had statistically significant results for their primary outcome in favour of genotype-guided prescribing. Nine studies evaluated genotype-guided warfarin dosing. Analysis of percentage of time in therapeutic international normalized ratio range (1952 individuals) showed a statistically significant benefit in favour of genotype-guided warfarin dosing (mean difference = 6.67; 95% CI 1.34, 12.0, I2 = 80%). There was a statistically significant reduction in numbers of warfarin-related minor bleeding, major bleeding and thromboembolisms associated with genotype guided warfarin dosing, relative risk 0.57 (95% CI 0.33, 0.99; I2 = 60%). It was not possible to meta-analyze genotype-guided dosing for other drugs. Of the six non-warfarin genotype-guided trials, two demonstrated a statistically significant benefit for their primary outcome, odds ratio 0.03 (95% CI 0.00, 0.62, P < 0.001) for abacavir. Conclusions There is evidence of improved clinical effectiveness associated with genotype-guided warfarin dosing. PMID:25060532

  4. 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.

  5. 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.

  6. 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

  7. Genotyping and phylogenetic analysis of Pneumocystis jirovecii isolates from India.

    PubMed

    Gupta, Rashmi; Mirdha, Bijay Ranjan; Guleria, Randeep; Agarwal, Sanjay Kumar; Samantaray, Jyotish Chandra; Kumar, Lalit; Kabra, Sushil Kumar; Luthra, Kalpana; Sreenivas, Vishnubhatla

    2010-08-01

    Pneumocystis jirovecii is the cause of Pneumocystis pneumonia (PCP) in immuno-compromised individuals. The aim of this study was to describe the genotypes/haplotypes of P. jirovecii in immuno-compromised individuals with positive polymerase chain reaction (PCR) result for PCP. The typing was based on sequence polymorphism at internal transcribed spacer (ITS) regions of rRNA operon. Phylogenetic relationship between Indian and global haplotypes was also studied. Between January 2005 to October 2008, 43 patients were found to be positive for Pneumocystis using PCR targeting mitochondrial large subunit rRNA (mt LSU rRNA) and ITS region. Genotyping of all the positive samples was performed at the ITS locus by direct sequencing. Nine ITS1 alleles (all previously known) and 11 ITS2 alleles (nine previously defined and two new) were observed. A total of 19 ITS haplotypes, including five novel haplotypes (DEL1r, Edel2, Hr, Adel3 and SYD1a), were observed. The most prevalent type was SYD1g (16.3%), followed by types Ea (11.6%), Ec (9.3%), Eg (6.9%), DEL1r (6.9%), Ne (6.9%) and Ai (6.9%). To detect mixed infection, 30% of the positive isolates were cloned and 4-5 clones were sequenced from each specimen. Cloning and sequencing identified two more haplotypes in addition to the 19 types. Mixed infection was identified in 3 of the 13 cloned samples (23.1%). Upon construction of a haplotype network of 21 haplotypes, type Eg was identified as the most probable ancestral type. The present study is the first study that describes the haplotypes of P. jirovecii based on the ITS gene from India. The study suggests a high diversity of P. jirovecii haplotypes in the population.

  8. Genotype analysis of hepatitis E virus from sporadic hepatitis E cases in northern China.

    PubMed

    Geng, Yansheng; Zhao, Chenyan; Fan, Jinping; Harrison, Tim J; Zhang, Hongxin; Lian, Haichen; Geng, Kunjing; Wang, Youchun

    2013-12-01

    Hepatitis E is an important public health problem in many countries. However, there is no definite conclusion about the zoonotic reservoir, transmission patterns and risk factors of hepatitis E in the human population. The aim of this study was to analyze the epidemiological and viral genotype characteristics of hepatitis E cases in northern China. Surveillance was conducted in two hospitals in Liaoning and Hebei province from July 2010 to June 2012. Out of a total of 116 diagnosed patients, 88 (75.9%) were male and 28 (24.1%) were female and most (73%) were in the age group 40-70 years. In both hospitals, cases were diagnosed more frequently in March than in other months. HEV RNA was amplified from 41 patients and characterized by nucleotide sequencing and phylogenetic analysis. Most of the isolates (37 strains, 90.3%) were genotype 4, including subgenotypes 4a, 4b, 4d, 4h, 4i and a new subgenotype. One subgenotype 3a strain was isolated from Baoding, Hebei province. Three genotype 1b strains were found from patients in Jinzhou, Liaoning province. Most of the genotype 4 strains and the genotype 3 strains were phylogenetically related to known swine isolates. In conclusion, the finding that HEV infects mostly middle-aged and elderly men and that the incidence spiked in March may reflect the zoonotic transmission characteristics of HEV infection. Pigs, but not rabbits, were the important reservoirs in this area, because genotype 4 HEV was found to be responsible for the majority hepatitis E cases. However, genotype 1 is still present in northern China. Also, the first isolation of genotype 3 HEV in this area indicates that alternative routes of HEV transmission might exist.

  9. 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

  10. 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

  11. The dam replacing gene product enhances Neisseria gonorrhoeae FA1090 viability and biofilm formation.

    PubMed

    Kwiatek, Agnieszka; Bacal, Pawel; Wasiluk, Adrian; Trybunko, Anastasiya; Adamczyk-Poplawska, Monika

    2014-01-01

    Many Neisseriaceae do not exhibit Dam methyltransferase activity and, instead of the dam gene, possess drg (dam replacing gene) inserted in the leuS/dam locus. The drg locus in Neisseria gonorrhoeae FA1090 has a lower GC-pairs content (40.5%) compared to the whole genome of N. gonorrhoeae FA1090 (52%). The gonococcal drg gene encodes a DNA endonuclease Drg, with GmeATC specificity. Disruption of drg or insertion of the dam gene in gonococcal genome changes the level of expression of genes as shown by transcriptome analysis. For the drg-deficient N. gonorrhoeae mutant, a total of 195 (8.94% of the total gene pool) genes exhibited an altered expression compared to the wt strain by at least 1.5 fold. In dam-expressing N. gonorrhoeae mutant, the expression of 240 genes (11% of total genes) was deregulated. Most of these deregulated genes were involved in translation, DNA repair, membrane biogenesis and energy production as shown by cluster of orthologous group analysis. In vivo, the inactivation of drg gene causes the decrease of the number of live neisserial cells and long lag phase of growth. The insertion of dam gene instead of drg locus restores cell viability. We have also shown that presence of the drg gene product is important for N. gonorrhoeae FA1090 in adhesion, including human epithelial cells, and biofilm formation. Biofilm produced by drg-deficient strain is formed by more dispersed cells, compared to this one formed by parental strain as shown by scanning electron and confocal microscopy. Also adherence assays show a significantly smaller biomass of formed biofilm (OD570 = 0.242 ± 0.038) for drg-deficient strain, compared to wild-type strain (OD570 = 0.378 ± 0.057). Dam-expressing gonococcal cells produce slightly weaker biofilm with cells embedded in an extracellular matrix. This strain has also a five times reduced ability for adhesion to human epithelial cells. In this context, the presence of Drg is more advantageous for N. gonorrhoeae biology than

  12. The dam replacing gene product enhances Neisseria gonorrhoeae FA1090 viability and biofilm formation

    PubMed Central

    Kwiatek, Agnieszka; Bacal, Pawel; Wasiluk, Adrian; Trybunko, Anastasiya; Adamczyk-Poplawska, Monika

    2014-01-01

    Many Neisseriaceae do not exhibit Dam methyltransferase activity and, instead of the dam gene, possess drg (dam replacing gene) inserted in the leuS/dam locus. The drg locus in Neisseria gonorrhoeae FA1090 has a lower GC-pairs content (40.5%) compared to the whole genome of N. gonorrhoeae FA1090 (52%). The gonococcal drg gene encodes a DNA endonuclease Drg, with GmeATC specificity. Disruption of drg or insertion of the dam gene in gonococcal genome changes the level of expression of genes as shown by transcriptome analysis. For the drg-deficient N. gonorrhoeae mutant, a total of 195 (8.94% of the total gene pool) genes exhibited an altered expression compared to the wt strain by at least 1.5 fold. In dam-expressing N. gonorrhoeae mutant, the expression of 240 genes (11% of total genes) was deregulated. Most of these deregulated genes were involved in translation, DNA repair, membrane biogenesis and energy production as shown by cluster of orthologous group analysis. In vivo, the inactivation of drg gene causes the decrease of the number of live neisserial cells and long lag phase of growth. The insertion of dam gene instead of drg locus restores cell viability. We have also shown that presence of the drg gene product is important for N. gonorrhoeae FA1090 in adhesion, including human epithelial cells, and biofilm formation. Biofilm produced by drg-deficient strain is formed by more dispersed cells, compared to this one formed by parental strain as shown by scanning electron and confocal microscopy. Also adherence assays show a significantly smaller biomass of formed biofilm (OD570 = 0.242 ± 0.038) for drg-deficient strain, compared to wild-type strain (OD570 = 0.378 ± 0.057). Dam-expressing gonococcal cells produce slightly weaker biofilm with cells embedded in an extracellular matrix. This strain has also a five times reduced ability for adhesion to human epithelial cells. In this context, the presence of Drg is more advantageous for N. gonorrhoeae biology than

  13. HPV genotype analysis for women in Shaanxi Province of China.

    PubMed

    Li, J; Wang, Y Y; Tian, X F; Nan, X; Yan, T; Wang, P; Fu, Y L; Wang, G Q

    2016-11-03

    The aim of this study was to examine the subtype distribution of human papilloma virus (HPV) in women in the Shaanxi Province of China. A DNA chip, along with polymerase chain reaction amplification and reverse dot blot technology, was adopted to analyze the HPV genotypes of 22,937 cases of cervical cell specimens. The HPV infection rate was 18.70%, wherein high-risk, low-risk, and high- and low-risk multiple infection rates were 15.75, 2.96 and 1.91%, respectively. High-risk infections accounted for 84.20% of total infections. The rate of HPV infection in women with rural residence, high school education or less, a low income, or age over 40 years was significantly higher than that in the control group (negative HPV infection women). Of the 18 detected high-risk HPV subtypes, the most common in single infections were, in the order of prevalence, HPV16, 58, 18, 52, 33, and 56. For multiple high-risk infections, the most common subtypes in the order of prevalence were HPV16, 52, 58, 18, 56, and 33. Age was a factor in the rate of infection, as the 41-50-year age group had a significantly higher risk of infection than the other groups (P < 0.05). In multiple infections, double infections were common, accounting for 77.10% of multiple infections, and triple or more infections were more common in women aged 51-60 years. In Shaanxi Province, high-risk HPV infection in women was mainly attributed to rural residence, age over 40 years, low income, and low education level.

  14. 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

  15. 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.

  16. Genotypic and Spatial Analysis of Mycobacterium tuberculosis Transmission in a High-Incidence Urban Setting

    PubMed Central

    Ribeiro, Fabíola Karla Correa; Pan, William; Bertolde, Adelmo; Vinhas, Solange Alves; Peres, Renata Lyrio; Riley, Lee; Palaci, Moisés; Maciel, Ethel Leonor

    2015-01-01

    Background. Genotyping Mycobacterium tuberculosis isolates allows study of dynamics of tuberculosis transmission, while geoprocessing allows spatial analysis of clinical and epidemiological data. Here, genotyping data and spatial analysis were combined to characterize tuberculosis transmission in Vitória, Brazil, to identify distinct neighborhoods and risk factors associated with recent tuberculosis transmission. Methods. From 2003 to 2007, 503 isolates were genotyped by IS6110 restriction fragment length polymorphism (RFLP) and spoligotyping. The analysis included kernel density estimation, K-function analysis, and a t test distance analysis. Mycobacterium tuberculosis isolates belonging to identical RFLP patterns (clusters) were considered to represent recent tuberculosis infection (cases). Results. Of 503 genotyped isolates, 242 (48%) were categorized into 70 distinct clusters belonging to 12 RFLP families. The proportion of recent transmission was 34.2%. Kernel density maps indicated 3 areas of intense concentration of cases. K-function analysis of the largest RFLP clusters and families showed they co-localized in space. The distance analysis confirmed these results and demonstrated that unique strain patterns (controls) randomly distributed in space. A logit model identified young age, positive smear test, and lower Index of Quality of Urban Municipality as risk factors for recent transmission. The predicted probabilities for each neighborhood were mapped and identified neighborhoods with high risk for recent transmission. Conclusions. Spatial and genotypic clustering of M. tuberculosis isolates revealed ongoing active transmission of tuberculosis caused by a small subset of strains in specific neighborhoods of the city. Such information provides an opportunity to target tuberculosis transmission control, such as through rigorous and more focused contact investigation programs. PMID:25948063

  17. Genotyping and phylogenetic analysis of bovine viral diarrhea virus (BVDV) isolates in Kosovo.

    PubMed

    Goga, Izedin; Berxholi, Kristaq; Hulaj, Beqe; Sylejmani, Driton; Yakobson, Boris; Stram, Yehuda

    2014-01-01

    Three serum samples positive in Antigen ELISA BVDV have been tested to characterise genetic diversity of bovine viral diarrhea virus (BVDV) in Kosovo. Samples were obtained in 2011 from heifers and were amplified by reverse transcription-polymerase chain reaction, sequenced and analysed by computer-assisted phylogenetic analysis. Amplified products and nucleotide sequence showed that all 3 isolates belonged to BVDV 1 genotype and 1b sub genotype. These results enrich the extant knowledge of BVDV and represent the first documented data about Kosovo BVDV isolates.

  18. Comparative transcriptomic analysis of salt adaptation in roots of contrasting Medicago truncatula genotypes.

    PubMed

    Zahaf, Ons; Blanchet, Sandrine; de Zélicourt, Axel; Alunni, Benoît; Plet, Julie; Laffont, Carole; de Lorenzo, Laura; Imbeaud, Sandrine; Ichanté, Jean-Laurent; Diet, Anouck; Badri, Mounawer; Zabalza, Ana; González, Esther M; Delacroix, Hervé; Gruber, Véronique; Frugier, Florian; Crespi, Martin

    2012-09-01

    Evolutionary diversity can be driven by the interaction of plants with different environments. Molecular bases involved in ecological adaptations to abiotic constraints can be explored using genomic tools. Legumes are major crops worldwide and soil salinity is a main stress affecting yield in these plants. We analyzed in the Medicago truncatula legume the root transcriptome of two genotypes having contrasting responses to salt stress: TN1.11, sampled in a salty Tunisian soil, and the reference Jemalong A17 genotype. TN1.11 plants show increased root growth under salt stress as well as a differential accumulation of sodium ions when compared to A17. Transcriptomic analysis revealed specific gene clusters preferentially regulated by salt in root apices of TN1.11, notably those related to the auxin pathway and to changes in histone variant isoforms. Many genes encoding transcription factors (TFs) were also differentially regulated between the two genotypes in response to salt. Among those selected for functional studies, overexpression in roots of the A17 genotype of the bHLH-type TF most differentially regulated between genotypes improved significantly root growth under salt stress. Despite the global complexity of the differential transcriptional responses, we propose that an increase in this bHLH TF expression may be linked to the adaptation of M. truncatula to saline soil environments.

  19. 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.

  20. Genotype analysis of ORF 62 identifies varicella-zoster virus infections caused by a vaccine strain in children.

    PubMed

    Kwak, Byung Ok; Lee, Hoan Jong; Kang, Hyun Mi; Oh, Chi Eun; Choi, Eun Hwa

    2017-02-15

    This study was performed to differentiate vaccine-type strains from wild-type strains and determine the genotype of varicella-zoster virus (VZV) in 51 Korean children. A sequencing analysis of ORF 62 identified two cases of herpes zoster caused by the vaccine-type virus, without a previous history of varicella, 22 months and 5 months after VZV vaccination. The wild-type strain was identified in the remaining children. A genotype analysis of ORF 22 amino acids revealed genotype J in all children except one. Genotype E was identified in an infant with varicella imported from Egypt.

  1. Detection of Neisseria gonorrhoeae Isolates from Tonsils and Posterior Oropharynx

    PubMed Central

    Whiley, D. M.; Lee, D. M.; Snow, A. F.; Fairley, C. K.; Peel, J.; Bradshaw, C. S.; Hocking, J. S.; Lahra, M. M.; Chen, M. Y.

    2015-01-01

    We examined the factors influencing gonorrhea detection at the pharynx. One hundred men infected with Neisseria gonorrhoeae were swabbed from the tonsils and posterior oropharynx. N. gonorrhoeae was reisolated from the tonsils and posterior oropharynx in 62% and 52%, respectively (P = 0.041). Culture positivity was greater with higher gonococcal DNA loads at the tonsils (P = 0.001) and oropharynx (P < 0.001). N. gonorrhoeae can be cultured from the tonsils and posterior oropharynx with greater isolation rates where gonococcal loads are higher. PMID:26292303

  2. 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.

  3. Cigarette smoking, N-acetyltransferase 2 genotypes, and breast cancer risk: pooled analysis and meta-analysis.

    PubMed

    Ambrosone, Christine B; Kropp, Silke; Yang, Jun; Yao, Song; Shields, Peter G; Chang-Claude, Jenny

    2008-01-01

    Approximately 10 years ago, it was noted that smoking increased risk of breast cancer among women with N-acetyltransferase 2 (NAT2) slow acetylation genotypes. This report was followed by a number of studies to address this question. We pooled data from 10 existing studies and also conducted a meta-analysis of 13 studies published from 1996 to October 2006 that were conducted among women, were published in English, and had adequate information on smoking and NAT2 genotyping. Raw data were requested from authors. Unconditional logistic regression was done for pooled analysis, and random effect models was done for meta-analysis. Study heterogeneity was assessed, and sensitivity tests were done when subgroups were excluded from the analysis. In the pooled analysis, there was a significant interaction between smoking, NAT2 genotype, and risk of breast cancer [pack-years (continuous variable, P(interaction) = 0.03)], with higher pack-years significantly associated with an increased risk of breast cancer among women with NAT2 slow genotypes (pooled analysis relative risk, 1.49; 95% confidence interval, 1.08-2.04). These findings were supported by the meta-analysis including all studies; pack-years were significantly associated with risk among slow acetylators in a dose-dependent fashion (meta-analysis relative risk, 1.44; 95% confidence interval, 1.23-1.68 for > or =20 pack-years versus never smokers), but not among rapid acetylators. Similar relationships were noted for smoking status (ever, never) and duration of smoking. Our results show that cigarette smoking is associated with an increase in breast cancer risk among women with NAT2 slow acetylation genotypes. Because slow NAT2 genotypes are present in 50% to 60% of Caucasian populations, smoking is likely to play an important role in breast cancer etiology.

  4. 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

  5. Species status of Neisseria gonorrhoeae: evolutionary and epidemiological inferences from multilocus sequence typing

    PubMed Central

    Bennett, Julia S; Jolley, Keith A; Sparling, P Frederick; Saunders, Nigel J; Hart, C Anthony; Feavers, Ian M; Maiden, Martin CJ

    2007-01-01

    Background Various typing methods have been developed for Neisseria gonorrhoeae, but none provide the combination of discrimination, reproducibility, portability, and genetic inference that allows the analysis of all aspects of the epidemiology of this pathogen from a single data set. Multilocus sequence typing (MLST) has been used successfully to characterize the related organisms Neisseria meningitidis and Neisseria lactamica. Here, the same seven locus Neisseria scheme was used to characterize a diverse collection of N. gonorrhoeae isolates to investigate whether this method would allow differentiation among isolates, and to distinguish these three species. Results A total of 149 gonococcal isolates were typed and submitted to the Neisseria MLST database. Although relatively few (27) polymorphisms were detected among the seven MLST loci, a total of 66 unique allele combinations (sequence types, STs), were observed, a number comparable to that seen among isolate collections of the more diverse meningococcus. Patterns of genetic variation were consistent with high levels of recombination generating this diversity. There was no evidence for geographical structuring among the isolates examined, with isolates collected in Liverpool, UK, showing levels of diversity similar to a global collection of isolates. There was, however, evidence that populations of N. meningitidis, N. gonorrhoeae and N. lactamica were distinct, with little support for frequent genetic recombination among these species, with the sequences from the gdh locus alone grouping the species into distinct clusters. Conclusion The seven loci Neisseria MLST scheme was readily adapted to N. gonorrhoeae isolates, providing a highly discriminatory typing method. In addition, these data permitted phylogenetic and population genetic inferences to be made, including direct comparisons with N. meningitidis and N. lactamica. Examination of these data demonstrated that alleles were rarely shared among the three

  6. Serosorting and recreational drug use are risk factors for diagnosis of genital infection with chlamydia and gonorrhoea among HIV-positive men who have sex with men: results from a clinical cohort in Ontario, Canada

    PubMed Central

    Grewal, Ramandip; Allen, Vanessa G; Gardner, Sandra; Moravan, Veronika; Raboud, Janet; Bayoumi, Ahmed M; Kaul, Rupert; Mazzulli, Tony; McGee, Frank; Rourke, Sean B; Burchell, Ann N

    2017-01-01

    Objectives Rates of chlamydia and gonorrhoea have been rising in urban centres in Canada, particularly among HIV-positive men who have sex with men (MSM). Our objective was to identify behavioural risk factors for diagnosis with chlamydia and gonorrhoea in this population, with a focus on the HIV status of sexual partners. Methods The OHTN Cohort Study follows people in HIV care across Ontario. We restricted the analysis to 1997 MSM who completed questionnaires in 2010–2013 at one of seven clinics that submit all chlamydia and gonorrhoea tests to the provincial public health laboratory; we obtained test results via record linkage. We estimated cumulative incidences using Kaplan–Meier methods and identified risk factors for diagnosis of a composite outcome (chlamydia or gonorrhoea infection) using Cox regression. Results At follow-up, there were 74 new chlamydia/gonorrhoea diagnoses with a 12-month cumulative incidence of 1.7% (95% CI 1.1% to 2.2%). Risk factors for chlamydia/gonorrhoea diagnosis were: 5+ HIV-positive partners (HR=3.3, 95% CI 1.4 to 7.8; reference=none) and recreational drug use (HR=2.2, 95% CI 1.2 to 3.9). Conclusions Heightened risks with recreational drug use and multiple HIV-positive partners suggest that chlamydia/gonorrhoea may have achieved high prevalence in certain sexual networks among HIV-positive MSM. Interventions to promote safer sex and timely testing among MSM are needed. PMID:27154185

  7. Genotyping of Brucella melitensis by rpoB gene analysis and re-evaluation of conventional serotyping method.

    PubMed

    Sayan, Murat; Yumuk, Zeki; Bilenoglu, Onur; Erdenlig, Sevil; Willke, Ayse

    2009-03-01

    In Turkey, where brucellosis is endemic, a comparison of conventional and molecular genotyping methods has not been published to date. In this study, we investigated the efficacy of single nucleotide polymorphism (SNP) analysis of rpoB gene in the genotyping of Brucella melitensis strains by sequencing. In light of the molecular genotyping method available now in Turkey, the adequacy of serological typing alone should be re-evaluated as a tool for epidemiologic studies of B. melitensis.

  8. 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.

  9. Assessment of high resolution melting analysis as a potential SNP genotyping technique in forensic casework.

    PubMed

    Venables, Samantha J; Mehta, Bhavik; Daniel, Runa; Walsh, Simon J; van Oorschot, Roland A H; McNevin, Dennis

    2014-11-01

    High resolution melting (HRM) analysis is a simple, cost effective, closed tube SNP genotyping technique with high throughput potential. The effectiveness of HRM for forensic SNP genotyping was assessed with five commercially available HRM kits evaluated on the ViiA™ 7 Real Time PCR instrument. Four kits performed satisfactorily against forensically relevant criteria. One was further assessed to determine the sensitivity, reproducibility, and accuracy of HRM SNP genotyping. The manufacturer's protocol using 0.5 ng input DNA and 45 PCR cycles produced accurate and reproducible results for 17 of the 19 SNPs examined. Problematic SNPs had GC rich flanking regions which introduced additional melting domains into the melting curve (rs1800407) or included homozygotes that were difficult to distinguish reliably (rs16891982; a G to C SNP). A proof of concept multiplexing experiment revealed that multiplexing a small number of SNPs may be possible after further investigation. HRM enables genotyping of a number of SNPs in a large number of samples without extensive optimization. However, it requires more genomic DNA as template in comparison to SNaPshot®. Furthermore, suitably modifying pre-existing forensic intelligence SNP panels for HRM analysis may pose difficulties due to the properties of some SNPs.

  10. 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.

  11. 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.

  12. 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.

  13. A highly conserved interaction involving the middle residue of the SXN active-site motif is crucial for function of class B penicillin-binding proteins: mutational and computational analysis of PBP 2 from N. gonorrhoeae.

    PubMed

    Tomberg, Joshua; Temple, Brenda; Fedarovich, Alena; Davies, Christopher; Nicholas, Robert A

    2012-04-03

    Insertion of an aspartate residue at position 345a in penicillin-binding protein 2 (PBP 2), which lowers the rate of penicillin acylation by ~6-fold, is commonly observed in penicillin-resistant strains of Neisseria gonorrhoeae. Here, we show that insertions of other amino acids also lower the penicillin acylation rate of PBP 2, but none supported growth of N. gonorrhoeae, indicating loss of essential transpeptidase activity. The Asp345a mutation likely acts by altering the interaction between its adjacent residue, Asp346, in the β2a-β2d hairpin loop and Ser363, the middle residue of the SXN active site motif. Because the adjacent aspartate creates ambiguity in the position of the insertion, we also examined if insertions at position 346a could confer decreased susceptibility to penicillin. However, only aspartate insertions were identified, indicating that only an Asp-Asp couple can confer resistance and retain transpeptidase function. The importance of the Asp346-Ser363 interaction was assessed by mutation of each residue to Ala. Although both mutants lowered the acylation rate of penicillin G by 5-fold, neither could support growth of N. gonorrhoeae, again indicating loss of transpeptidase function. Interaction between a residue in the equivalent of the β2a-β2d hairpin loop and the middle residue of the SXN motif is observed in crystal structures of other Class B PBPs, and its importance is also supported by multisequence alignments. Overall, these results suggest that this conserved interaction can be manipulated (e.g., by insertion) to lower the acylation rate by β-lactam antibiotics and increase resistance, but only if essential transpeptidase activity is preserved.

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

    PubMed

    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.

  15. 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

  16. 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.

  17. Pharyngeal Gonorrhoea: The Willingness of Australian Men Who Have Sex with Men to Change Current Sexual Practices to Reduce Their Risk of Transmission—A Qualitative Study

    PubMed Central

    Bellhouse, Clare; Fairley, Christopher K.; Bilardi, Jade E.; Chow, Eric P. F.

    2016-01-01

    Background The pharynx is a common site of gonorrhoea among men who have sex with men (MSM) and may serve as a reservoir for infection, with saliva implicated in transmission possibly through oral sex, kissing, and rimming. Reducing sexual activities involving saliva may reduce pharyngeal gonorrhoea. This study aimed to explore MSM’s views and knowledge of pharyngeal gonorrhoea and their willingness to change saliva transmitting sexual practices. MSM were also asked their views on using alcohol-containing mouthwash to potentially reduce transmission. Methods Using a qualitative descriptive approach, 30 MSM who were part of a larger study (GONE) conducted at the Melbourne Sexual Health Centre agreed to take part in semi-structured interviews between 14th May and 8th September 2015. The 10 interviews conducted face to face and 20 by telephone, lasted between 20–45 minutes. Data were analysed using qualitative content analysis. Results Most men considered pharyngeal gonorrhoea to be a non-serious sexually transmitted infection and attributed transmission primarily to oral sex. Almost all men reported they would not stop kissing, oral sex, or consider using condoms for oral sex to reduce their risk of pharyngeal gonorrhoea. Kissing and oral sex were commonly practised and considered enjoyable low risk sexual activities. Men were more likely to consider stopping sexual activities they did not enjoy or practice often, in particular insertive rimming. If proven effective, the majority of men reported they would use alcohol-containing mouthwash to reduce or prevent their risk of pharyngeal gonorrhoea. Conclusion Findings from this study suggest MSM are unlikely to stop saliva transmitting sexual practices they enjoy and consider low risk. Men would, however, consider using alcohol-containing mouthwash if found to be effective, highlighting the importance of exploring innovative strategies to reduce pharyngeal gonorrhoea. PMID:27992427

  18. Genotype-phenotype correlations analysis of mutations in the phenylalanine hydroxylase (PAH) gene.

    PubMed

    Bercovich, Dani; Elimelech, Arava; Zlotogora, Joel; Korem, Sigal; Yardeni, Tal; Gal, Nurit; Goldstein, Nurit; Vilensky, Bela; Segev, Roni; Avraham, Smadar; Loewenthal, Ron; Schwartz, Gerard; Anikster, Yair

    2008-01-01

    The aims of our research were to define the genotype-phenotype correlations of mutations in the phenylalanine hydroxylase (PAH) gene that cause phenylketonuria (PKU) among the Israeli population. The mutation spectrum of the PAH gene in PKU patients in Israel is described, along with a discussion on genotype-phenotype correlations. By using polymerase chain reaction/denaturing high-performance liquid chromatography (PCR/dHPLC) and DNA sequencing, we screened all exons of the PAH gene in 180 unrelated patients with four different PKU phenotypes [classic PKU, moderate PKU, mild PKU, and mild hyperphenylalaninemia (MHP)]. In 63.2% of patient genotypes, the metabolic phenotype could be predicted, though evidence is also found for both phenotypic inconsistencies among subjects with more than one type of mutation in the PAH gene. Data analysis revealed that about 25% of patients could participate in the future in (6R)-L: -erythro-5, 6, 7, 8-tetrahydrobiopterin (BH4) treatment trials according to their mutation genotypes. This study enables us to construct a national database in Israel that will serve as a valuable tool for genetic counseling and a prognostic evaluation of future cases of PKU.

  19. [Genetic characterization analysis on the first imported measles virus of genotype D8 in Chinese mainland].

    PubMed

    Sun, Xiao-Dong; Li, Chong-Shan; Tang, Xian; Li, Zhi; Zhang, Yan; Tang, Wei; Wang, Jing; Wang, Hui-Ling; Yang, Yan-Ji; Li, Jia; Yuan, Zheng-An; Xu, Wen-Bo

    2013-11-01

    This study analyzed the genetic characterization on first imported measles virus of genotype D8 in Chinese mainland. Serums were collected from the suspicious MV patients to detect IgM antibody in ELISA. Throat swabs were cultured in Vero/SLAM cell line to get measles virus isolates. Part of the nucleotide sequence of the 3' terminus of nucleoprotein (N) gene of these isolates were amplified by RT-PCR, and the amplicons were directly sequenced. The phylogenetic analysis was based on the nucleotide sequence about 456 base pairs of the 3' terminus of nucleoprotein (N) gene. Results showed that it reported 1 105 suspicious measles cases in shanghai, 2012, including 590 confirmed cases and 2 clinical case. The reported morbidity was 2.52 per one hundred thousand. 247 measles viruses were isolated from 984 throat swabs specimen. Most of them belonged to sub-genotype H1a except Shanghai12-239 was genotype D8. The homology of nucleotide and amino acid sequences were 97.8% and 98.6% respectively between Shanghai12-239 and WHO reference strain (Manchester. UNK30.94(D8)AF280803). Those were 89.6%-94.5% and 88.7%-95.3% between Shanghai12-239 and WHO reference strains of other genotypes.

  20. 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.

  1. Characterization of Brassica napus L. genotypes utilizing sequence-related amplified polymorphism and genotyping by sequencing in association with cluster analysis.

    PubMed

    Lees, Corey J; Li, Genyi; Duncan, Robert W

    2016-01-01

    Identifying parental combinations that exhibit high heterosis is a constant target for commercial Brassica napus L. hybrid development programs. Finding high heterotic parental combinations can require hundreds of test crosses and years of yield evaluation. Heterotic pool development could be used to divide breeding material into specific breeding pools and focus the number of parental combinations created. Here, we report the genotypic characterization of 79 B. napus genotypes by calculating genetic distance based on sequence-related amplified polymorphism (SRAP) and genotyping by sequencing (GBS) in association with a neighbour-joining clustering algorithm. Despite the different genotypic analyses, neighbour-joining cluster analysis based on genetic distance of SRAP and GBS produced similar clusters. Homology between SRAP and GBS clusters was approximately 77 % when manually comparing clusters and 68 % when comparing clusters using Compare2Trees. This research demonstrates that SRAP can have similar efficacy when compared to next-generation sequencing technology for heterotic pool classification. This information may provide an important breeding scaffold for the development of hybrid cultivars based upon genetic distance and cluster analysis.

  2. Genotypic stability and adaptability in tropical maize based on AMMI and GGE biplot analysis.

    PubMed

    Balestre, M; Von Pinho, R G; Souza, J C; Oliveira, R L

    2009-11-03

    We evaluated the phenotypic and genotypic stability and adaptability of hybrids using the additive main effect and multiplicative interaction (AMMI) and genotype x genotype-environment interaction (GGE) biplot models. Starting with 10 single-cross hybrids, a complete diallel was done, resulting in 45 double-cross hybrids that were appraised in 15 locations in Southeast, Center-West and Northeast Brazil. In most cases, when the effects were considered as random (only G effects or G and GE simultaneously) in AMMI and GGE analysis, the distances between predicted values and observed values were smaller than for AMMI and GGE biplot phenotypic means; the best linear unbiased predictors of G and GE generally showed more accurate predictions in AMMI and GGE analysis. We found the GGE biplot method to be superior to the AMMI 1 graph, due to more retention of GE and G + GE in the graph analysis. However, based on cross-validation results, the GGE biplot was less accurate than the AMMI 1 graph, inferring that the quantity of GE or G + GE retained in the graph analysis alone is not a good parameter for choice of stabilities and adaptabilities when comparing AMMI and GGE analyses.

  3. Genetic diversity analysis of Bt cotton genotypes in Pakistan using simple sequence repeat markers.

    PubMed

    Ullah, I; Iram, A; Iqbal, M Z; Nawaz, M; Hasni, S M; Jamil, S

    2012-03-14

    The popularity of genetically modified insect resistant (Bt) cotton has promoted large scale monocultures, which is thought to worsen the problem of crop genetic homogeneity. Information on genetic diversity among Bt cotton varieties is lacking. We evaluated genetic divergence among 19 Bt cotton genotypes using simple sequence repeat (SSR) markers. Thirty-seven of 104 surveyed primers were found informative. Fifty-two primers selected on the basis of reported intra-hirsutum polymorphism in a cotton marker database showed a high degree of polymorphism, 56% compared to 13% for randomly selected primers. A total of 177 loci were amplified, with an average of 1.57 loci per primer, generating 38 markers. The amplicons ranged in size from 98 to 256 bp. The genetic similarities among the 19 genotypes ranged from 0.902 to 0.982, with an average of 0.947, revealing a lack of diversity. Similarities among genotypes from public sector organizations were higher than genotypes developed by private companies. Hybrids were found to be more distant compared to commercial cultivars and advanced breeding lines. Cluster analysis grouped the 19 Bt cotton genotypes into three major clusters and two independent entries. Cultivars IR-3701, Ali Akbar-802 and advanced breeding line VH-259 grouped in subcluster B2, with very narrow genetic distances despite dissimilar parentage. We found a very high level of similarity among Pakistani-bred Bt cotton varieties, which means that genetically diverse recurrent parents should be included to enhance genetic diversity. The intra-hirsutum polymorphic SSRs were found to be highly informative for molecular genetic diversity studies in these cotton varieties.

  4. 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

  5. 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.

  6. Modest rise in chlamydia and gonorrhoea testing did not increase case detection in a clinical HIV cohort in Ontario, Canada

    PubMed Central

    Burchell, Ann N; Grewal, Ramandip; Allen, Vanessa G; Gardner, Sandra L; Moravan, Veronika; Bayoumi, Ahmed M; Kaul, Rupert; McGee, Frank; Millson, Margaret (Peggy) E; Remis, Robert S; Raboud, Janet; Mazzulli, Tony; Rourke, Sean B

    2014-01-01

    Objectives We described patterns of testing for chlamydia and gonorrhoea infection among persons in specialty HIV care in Ontario, Canada, from 2008 to 2011. Methods We analysed data from 3165 participants in the OHTN Cohort Study attending one of seven specialty HIV care clinics. We obtained chlamydia and gonorrhoea test results via record linkage with the provincial public health laboratory. We estimated the proportion of participants who underwent testing annually, the positivity rate among those tested and the proportion diagnosed with chlamydia or gonorrhoea among all under observation. We explored risk factors for testing and diagnosis using multiple logistic regression analysis. Results The proportion tested annually rose from 15.2% (95% CI 13.6% to 16.7%) in 2008 to 27.0% (95% CI 25.3% to 28.6%) in 2011 (p<0.0001). Virtually all were urine-based nucleic acid amplification tests. Testing was more common among men who have sex with men (MSM), younger adults, Toronto residents, persons attending primary care clinics and persons who had tested in the previous year or who had more clinic visits in the current year. We observed a decrease in test positivity rates over time. However, the annual proportion diagnosed remained stable and in 2011 this was 0.97% (95% CI 0.61% to 1.3%) and 0.79% (95% CI 0.46% to 1.1%) for chlamydia and gonorrhoea, respectively. Virtually all cases were among MSM. Conclusions Chlamydia and gonorrhoea testing increased over time while test positivity rates declined and the overall proportion diagnosed remained stable, suggesting that the modest increase in testing did not improve case detection. PMID:25178285

  7. 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

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

    PubMed

    Lithio, Andrew; Nettleton, Dan

    2015-12-01

    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.

  9. 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.

  10. 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

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

    PubMed

    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.

  12. Genotyping of dairy Bacillus licheniformis isolates by high resolution melt analysis of multiple variable number tandem repeat loci.

    PubMed

    Dhakal, Rajat; Chauhan, Kanika; Seale, R Brent; Deeth, Hilton C; Pillidge, Christopher J; Powell, Ian B; Craven, Heather; Turner, Mark S

    2013-06-01

    In dairy foods, the sporeformer Bacillus licheniformis can be the cause of spoilage or specification compliance issues. Currently used methods for genotyping B. licheniformis have limited discrimination with only 2 or 3 different subgroups being identified. Here, we have developed a multi-locus variable number tandem repeat analysis (MLVA) method and combined it with high resolution melt analysis (MLV-HRMA) for genotyping B. licheniformis. Five repetitive loci were identified and used as markers for genotyping 52 isolates from two milk powder processing plants and retail samples. Nineteen genotypes could be identified using both MLVA and MLV-HRMA leading to Hunter-Gaston discrimination indices (D-value) of 0.93 each. It was found that all 5 MLVA loci were stable following 10 days of sub-culturing of 8 representative isolates. All isolates were also genotyped using previously used methods including randomly amplified polymorphic DNA-PCR (RAPD) and partial rpoB sequencing. Five different RAPD profiles and 5 different partial rpoB sequence types were identified resulting in corresponding D-values of 0.6 and 0.46, respectively. Analysis of the genotypes from dairy samples revealed that dairy B. licheniformis isolates are more heterogeneous than previously thought and that this new method can potentially allow for more discriminatory tracking and monitoring of specific genotypes.

  13. 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.

  14. Epidemiology and Genotype Analysis of Emerging Sapovirus-Associated Infections across Europe▿

    PubMed Central

    Svraka, Sanela; Vennema, Harry; van der Veer, Bas; Hedlund, Kjell-Olof; Thorhagen, Margareta; Siebenga, Joukje; Duizer, Erwin; Koopmans, Marion

    2010-01-01

    Sapoviruses (SaVs) belong to the Caliciviridae family and can cause gastroenteritis in humans and swine. Despite extensive testing, human sapoviruses have been found only in sporadic cases and in one mixed outbreak in children between 1994 and 2007 in the Netherlands. Here we describe a change in sapovirus epidemiology in the Netherlands resulting in sapovirus outbreaks and infections in adults. From November 2007 to January 2009, 478 outbreaks of acute gastroenteritis were reported to the National Institute for Public Health and the Environment in the Netherlands as a part of ongoing surveillance. Sapoviruses were found to be the most likely cause of 19 outbreaks (4%). During the same 2-year period, sapovirus infections were reported in Sweden, Slovenia, and Hungary. In the Netherlands, further characterization of outbreak strains showed that 12 (63%) sapovirus outbreaks were caused by genotype I.2 viruses. Most patients were adults older than 60 years (range, 1 to 100 years). Phylogenetic analysis using all presently available SaV sequences showed high homology between genotype I.2 strains detected in different geographical regions (Sweden, Slovenia, Taiwan, Japan, and Russia) since 2007. These first reported outbreaks of sapovirus infections in adults in the Netherlands were remarkable. Detection of identical genotypes in many samples might suggest that these viruses have the same origin, and since the infection is spreading fast, the prevalence of sapovirus infection may be increasing. The incidence of sapovirus infections in these countries suggests that a substantial part of Europe is affected by this virus. PMID:20392905

  15. Haplotag: Software for Haplotype-Based Genotyping-by-Sequencing Analysis

    PubMed Central

    Tinker, Nicholas A.; Bekele, Wubishet A.; Hattori, Jiro

    2016-01-01

    Genotyping-by-sequencing (GBS), and related methods, are based on high-throughput short-read sequencing of genomic complexity reductions followed by discovery of single nucleotide polymorphisms (SNPs) within sequence tags. This provides a powerful and economical approach to whole-genome genotyping, facilitating applications in genomics, diversity analysis, and molecular breeding. However, due to the complexity of analyzing large data sets, applications of GBS may require substantial time, expertise, and computational resources. Haplotag, the novel GBS software described here, is freely available, and operates with minimal user-investment on widely available computer platforms. Haplotag is unique in fulfilling the following set of criteria: (1) operates without a reference genome; (2) can be used in a polyploid species; (3) provides a discovery mode, and a production mode; (4) discovers polymorphisms based on a model of tag-level haplotypes within sequenced tags; (5) reports SNPs as well as haplotype-based genotypes; and (6) provides an intuitive visual “passport” for each inferred locus. Haplotag is optimized for use in a self-pollinating plant species. PMID:26818073

  16. High-resolution melt analysis without DNA extraction affords rapid genotype resolution and species identification.

    PubMed

    Rugman-Jones, Paul F; Stouthamer, Richard

    2016-09-22

    Extracting and sequencing DNA from specimens can impose major time and monetary costs to studies requiring genotyping, or identification to species, of large numbers of individuals. As such, so-called direct PCR methods have been developed enabling significant savings at the DNA extraction step. Similarly, real-time quantitative PCR techniques (qPCR) offer very cost-effective alternatives to sequencing. High-resolution melt analysis (HRM) is a qPCR method that incorporates an intercalating dye into a double-stranded PCR amplicon. The dye fluoresces brightly, but only when it is bound. Thus, after PCR, raising the temperature of the amplicon while measuring the fluorescence of the reaction results in the generation of a sequence-specific melt curve, allowing discrimination of genotypes. Methods combining HRM (or other qPCR methods) and direct PCR have not previously been reported, most likely due to concerns that any tissue in the reaction tube would interfere with detection of the fluorescent signal. Here, we couple direct PCR with HRM and, by way of three examples, demonstrate a very quick and cost-effective method for genotyping large numbers of specimens, using Rotor-Gene HRM instruments (QIAGEN). In contrast to the heated-block design of most qPCR/HRM instruments, the Rotor-Gene's centrifugal rotor and air-based temperature-regulation system facilitate our method by depositing tissues away from the pathway of the machine's fluorescence detection optics.

  17. 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.

  18. Rapid genotyping of beak and feather disease virus using high-resolution DNA melt curve analysis.

    PubMed

    Sarker, Subir; Ghorashi, Seyed A; Forwood, Jade K; Raidal, Shane R

    2014-11-01

    Beak and feather disease virus (BFDV) is a significant pathogen both for wild and captive psittacine birds globally. Genotypic differentiation of BFDV isolates is crucial to establish effective control strategies for the conservation of endangered species and epidemiological investigations of disease outbreaks. The technique developed in this study is a simple, rapid and inexpensive genotyping method for BFDV using PCR and subsequent high-resolution melt (HRM) curve analysis. This was achieved using PCR amplification of the conserved Rep gene in the presence of a fluorescent DNA intercalating dye (SYTO9). HRM curve analysis of the resultant amplicon could readily differentiate between reference strain (92-SR14) and 18 other BFDV isolates used in this study. Analysis of the nucleotide sequences of the amplicon from each isolate revealed that each melt curve profile was related to a unique DNA sequence. The potential of the PCR-HRM curve analysis to differentiate inter-host genetic variation among critically endangered orange-bellied parrots, lorikeets and cockatoos was also evaluated. Phylogenetic tree topology based on partial Rep gene sequences used in this study showed that BFDV Rep gene sequence patterns were correlated with the results of HRM curve analysis. The results presented in this study indicate that this technique could be used in both clinical research and differentiation of BFDV isolates in a fraction of time without further nucleotide sequencing and provides a novel approach for the genetic screening of BFDV in clinical virology laboratories.

  19. 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.

  20. Viability of in-house datamarting approaches for population genetics analysis of SNP genotypes

    PubMed Central

    Amigo, Jorge; Phillips, Christopher; Salas, Antonio; Carracedo, Ángel

    2009-01-01

    Background Databases containing very large amounts of SNP (Single Nucleotide Polymorphism) data are now freely available for researchers interested in medical and/or population genetics applications. While many of these SNP repositories have implemented data retrieval tools for general-purpose mining, these alone cannot cover the broad spectrum of needs of most medical and population genetics studies. Results To address this limitation, we have built in-house customized data marts from the raw data provided by the largest public databases. In particular, for population genetics analysis based on genotypes we have built a set of data processing scripts that deal with raw data coming from the major SNP variation databases (e.g. HapMap, Perlegen), stripping them into single genotypes and then grouping them into populations, then merged with additional complementary descriptive information extracted from dbSNP. This allows not only in-house standardization and normalization of the genotyping data retrieved from different repositories, but also the calculation of statistical indices from simple allele frequency estimates to more elaborate genetic differentiation tests within populations, together with the ability to combine population samples from different databases. Conclusion The present study demonstrates the viability of implementing scripts for handling extensive datasets of SNP genotypes with low computational costs, dealing with certain complex issues that arise from the divergent nature and configuration of the most popular SNP repositories. The information contained in these databases can also be enriched with additional information obtained from other complementary databases, in order to build a dedicated data mart. Updating the data structure is straightforward, as well as permitting easy implementation of new external data and the computation of supplementary statistical indices of interest. PMID:19344481

  1. 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

  2. Analysis of clinical and environmental Candida parapsilosis isolates by microsatellite genotyping--a tool for hospital infection surveillance.

    PubMed

    Sabino, R; Sampaio, P; Rosado, L; Videira, Z; Grenouillet, F; Pais, C

    2015-10-01

    Candida parapsilosis emerged as an important opportunistic pathogen, causing candidaemia worldwide. Nosocomial outbreaks triggered by this species have been frequently described, particularly in cancer patients. For a better understanding of its epidemiology, several typing methods are used and microsatellite analysis has been reported as highly discriminant. The main objective of this work was to study C. parapsilosis isolates by application of microsatellite genotyping to distinguish epidemiologically related strains, compare clinical and environmental isolates and determine possible routes of dispersion of the isolates in the hospital setting. A total of 129 C. parapsilosis isolates from different origins, including hospital environment and hands of healthcare workers, were genotyped using four microsatellite markers. The isolates were recovered from different health institutions. Analysis of C. parapsilosis isolates from hospital environment showed great genotypic diversity; however, the same or very similar genotypes were also found. The same multilocus genotype was shared by isolates recovered from the hand of a healthcare worker, from the hospital environment and from patients of the same healthcare institution, suggesting that these could be possible routes of transmission and that infections due to C. parapsilosis may be mainly related with exogenous transmission to the patient. Examination of sequential isolates from the same patients showed that colonizing and bloodstream isolates had the same multilocus genotype in the majority of cases. We demonstrate that this typing method is able to distinguish clonal clusters from genetically unrelated genotypes and can be a valuable tool to support epidemiologic investigations in the hospital setting.

  3. Evaluation of an rRNA-derived oligonucleotide probe for culture confirmation of Neisseria gonorrhoeae.

    PubMed Central

    Rossau, R; Duhamel, M; Van Dyck, E; Piot, P; Van Heuverswyn, H

    1990-01-01

    The reliability of an rRNA-derived oligonucleotide probe for Neisseria gonorrhoeae was tested with 187 N. gonorrhoeae isolates, 81 Neisseria meningitidis isolates, and several strains of other bacterial species. The probe proved to be 100% specific and 100% sensitive. N. gonorrhoeae cells could also be reliably identified in contaminated cultures with the oligonucleotide probe. The 2.6-megadalton cryptic plasmid used as a probe for N. gonorrhoeae was shown to be less sensitive, detecting 179 of 181 N. gonorrhoeae isolates. Images PMID:1693630

  4. 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

  5. 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.

  6. AN APPRAISAL OF THE FLUORESCENT ANTIBODY METHOD IN GONORRHOEA.

    PubMed

    OVCINNIKOV, N M

    1963-01-01

    Fluorescent antibody procedures have in a short time become valued techniques for the detection of many pathogenic micro-organisms, and are used in syphilis, for instance. A fluorescent antibody test has also been proposed for use in gonorrhoea, but the author of this paper suggests that there are still many questions to be answered before that test can be recommended for widespread practical application. In particular, large-scale further research is necessary on the antigenic structure of Neisseria gonorrhoeae, and other organisms of the Neisseria group, before reliance can be placed on the strict specificity of this test.The author also describes the fluorescent antibody technique for gonorrhoea used in the USSR, discussing its advantages and disadvantages.

  7. 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.

  8. Differences in Neisseria gonorrhoeae population structure and antimicrobial resistance pattern between men who have sex with men and heterosexuals.

    PubMed

    Serra-Pladevall, J; Barberá, M J; Callarisa, A E; Bartolomé-Comas, R; Andreu, A

    2017-01-01

    This study compared the antimicrobial susceptibility and genotypes of strains of Neisseria gonorrhoeae isolated from men who have sex with men (MSM) and from heterosexuals. One hundred and eleven strains were characterized from 107 patients, comprising 57 strains from 54 heterosexuals and 54 strains from 53 MSM. Antimicrobial resistance rates were higher in strains from heterosexual patients, with resistance to cefixime (P = 0·0159) and ciprofloxacin (P = 0·002) being significantly higher. Typing by N. gonorrhoeae multi-antigen sequence typing (NG-MAST) showed that the most prevalent sequence types (ST) and genogroups (G) respectively were ST2400, ST2992, and ST5793, and G1407, G2992, and G2400. A statistically significant association was observed for MSM and genogroups G2400 (P = 0·0005) and G2992 (P = 0·0488), and G1407 with heterosexuals (P = 0·0002). We conclude that in our region distinct populations of gonococci are circulating among subjects with different sexual practices, with their corresponding transmission patterns. Furthermore, the high prevalence of genotype G2400 in MSM, has not to our knowledge been previously described.

  9. 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.

  10. 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

  11. A Model Based Cost-Effectiveness Analysis of Routine Genotyping for CYP2D6 among Older, Depressed Inpatients Starting Nortriptyline Pharmacotherapy

    PubMed Central

    Luttjeboer, Jos; Wilffert, Bob; Postma, Maarten J.

    2016-01-01

    Objective Genotyping for CYP2D6 has the potential to predict differences in metabolism of nortriptyline. This information could optimize pharmacotherapy. We determined the costs and effects of routine genotyping for old aged Dutch depressed inpatients. Methods With a decision-tree, we modelled the first 12 weeks of nortriptyline therapy. Direct costs of genotyping, hospitalization, therapeutic drug monitoring and drugs were included. Based on genotype, patients could be correctly, sub-, or supratherapeutically dosed. Improvement from sub- or supratherapeutically dosed patients to correctly dosed patients was simulated, assuming that genotyping would prevent under- or overdosing of patients. In the base case, this improvement was assumed to be 35%. A probabilistic sensitivity analysis (PSA) was performed to determine uncertainty around the incremental cost-effectiveness ratio (ICER). Results In the base case analysis, costs for genotyping were assumed €200 per test with a corresponding ICER at €1 333 000 per QALY. To reach a €50 000 per QALY cut-off, genotyping costs should be decreased towards €40 per test. At genotyping test costs < €35 per test, genotyping was dominant. At test costs of €17 per test there was a 95% probability that genotyping was cost-effective at €50 000 per QALY. Conclusions CYP2D6 genotyping was not cost-effective at current genotyping costs at a €50 000 per QALY threshold, however at test costs below €40, genotyping could be costs-effective. PMID:28033366

  12. pilS loci in Neisseria gonorrhoeae are transcriptionally active

    PubMed Central

    Wachter, Jenny; Masters, Thao L.; Wachter, Shaun; Mason, Joanna

    2015-01-01

    Piliation is an important virulence determinant for Neisseria gonorrhoeae. PilE polypeptide is the major protein subunit in the pilus organelle and engages in extensive antigenic variation due to recombination between pilE and a pilS locus. pilS were so-named as they are believed to be transcriptionally silent, in contrast to the pilE locus. In this study, we demonstrate the presence of a small, pil-specific RNA species. Through using a series of pilE deletion mutants, we show by Northern blotting and quantitative reverse transcriptase PCR analysis (qRT-PCR), that these smaller RNA species are not derived from the primary pilE transcript following some processing events, but rather, arose through transcription of the pilS loci. Small transcriptome analysis, in conjunction with analysis of pilS recombinants, identified both sense and anti-sense RNAs originating from most, but not all, of the pilS gene copies. Focusing on the MS11 pilS6 locus, we identified by site-directed mutagenesis a sense promoter located immediately upstream of pilS6 copy 2, as well as an anti-sense promoter immediately downstream of pilS6 copy 1. Whole transcriptome analysis also revealed the presence of pil-specific sRNA in both gonococci and meningococci. Overall, this study reveals an added layer of complexity to the pilE/pilS recombination scheme by demonstrating pil-specific transcription within genes that were previously thought to be transcriptionally silent. PMID:25701734

  13. 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

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

    PubMed

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

    2013-08-01

    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.

  15. 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.

  16. Kinase genotype analysis of gastric gastrointestinal stromal tumor cytology samples using targeted next-generation sequencing.

    PubMed

    Gleeson, Ferga C; Kipp, Benjamin R; Kerr, Sarah E; Voss, Jesse S; Graham, Rondell P; Campion, Michael B; Minot, Douglas M; Tu, Zheng J; Klee, Eric W; Lazaridis, Konstantinos N; Henry, Michael R; Levy, Michael J

    2015-01-01

    Gastric gastrointestinal stromal tumors (GISTs) usually contain the mast/stem cell growth factor receptor Kit gene (KIT) or platelet-derived growth factor receptor A (PDGFRA) mutations that can be targeted by, or mediate resistance to, imatinib. Diagnostic material often is obtained by endoscopic ultrasound-guided fine-needle aspiration, which often is unsuitable for molecular analysis. We investigated whether targeted next-generation sequencing (NGS) can be used in multiplex genotype analysis of cytology samples collected by endoscopic ultrasound-guided fine-needle aspiration. We used the Ion AmpliSeq V2 Cancer Hotspot NGS Panel (Life Technologies, Carlsbad, CA) to identify mutations in more than 2800 exons from 50 cancer-associated genes in GIST samples from 20 patients. We identified KIT mutations in 58% of samples (91% in exon 11 and 9% in exon 17) and PDGFRA mutations in 26% (60% in exon 18 and 40% in exon 12); 16% of samples had no mutations in KIT or PDGFRA. No pathogenic alterations were found in PIK3CA, BRAF, KRAS, NRAS, or FGFR3. We predicted that 32% of patients would have primary resistance to imatinib, based on mutations in exon 17 of KIT, exon 18 of PDGFRA (D842V), or no mutation in either gene. Targeted NGS of cytology samples from GISTs is feasible and provides clinically relevant data about kinase genotypes that can help guide individualized therapy.

  17. 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.

  18. Comparative proteomic analysis of two pathogenic Tritrichomonas foetus genotypes: there is more to the proteome than meets the eye.

    PubMed

    Stroud, Leah J; Šlapeta, Jan; Padula, Matthew P; Druery, Dylan; Tsiotsioras, George; Coorssen, Jens R; Stack, Colin M

    2017-03-01

    Certain clinical isolates of Tritrichomonas foetus infect the urogenital tract of cattle while others infect the gastrointestinal tract of cats. Previous studies have identified subtle genetic differences between these isolates with the term "genotype" adopted to reflect host origin. The aim of this work was to seek evidence of host-specific adaptation and to clarify the relationship between T. foetus genotypes. To do this we characterised the proteomes of both genotypes using two-dimensional gel electrophoresis (2DE) coupled with LC-MS/MS. Our comparative analysis of the data revealed that both genotypes exhibited largely similar proteoform profiles; however differentiation was possible with 24 spots identified as having a four-fold or greater change. Deeper analysis using 2DE zymography and protease-specific fluorogenic substrates revealed marked differences in cysteine protease (CP) expression profiles between the two genotypes. These variances in CP activities could also account for the pathogenic and histopathological differences previously observed between T. foetus genotypes in cross-infection studies. Our findings highlight the importance of CPs as major determinants of parasite virulence and provide a foundation for future host-parasite interaction studies, with direct implications for the development of vaccines or drugs targeting T. foetus.

  19. 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

  20. 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

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

    PubMed

    Rono, Justice Kipkorir; 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.

  2. 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.

  3. Likelihood-based association analysis for nuclear families and unrelated subjects with missing genotype data.

    PubMed

    Dudbridge, Frank

    2008-01-01

    Missing data occur in genetic association studies for several reasons including missing family members and uncertain haplotype phase. Maximum likelihood is a commonly used approach to accommodate missing data, but it can be difficult to apply to family-based association studies, because of possible loss of robustness to confounding by population stratification. Here a novel likelihood for nuclear families is proposed, in which distinct sets of association parameters are used to model the parental genotypes and the offspring genotypes. This approach is robust to population structure when the data are complete, and has only minor loss of robustness when there are missing data. It also allows a novel conditioning step that gives valid analysis for multiple offspring in the presence of linkage. Unrelated subjects are included by regarding them as the children of two missing parents. Simulations and theory indicate similar operating characteristics to TRANSMIT, but with no bias with missing data in the presence of linkage. In comparison with FBAT and PCPH, the proposed model is slightly less robust to population structure but has greater power to detect strong effects. In comparison to APL and MITDT, the model is more robust to stratification and can accommodate sibships of any size. The methods are implemented for binary and continuous traits in software, UNPHASED, available from the author.

  4. SNP genotypes of Mycobacterium leprae isolates in Thailand and their combination with rpoT and TTC genotyping for analysis of leprosy distribution and transmission.

    PubMed

    Phetsuksiri, Benjawan; Srisungngam, Sopa; Rudeeaneksin, Janisara; Bunchoo, Supranee; Lukebua, Atchariya; Wongtrungkapun, Ruch; Paitoon, Soontara; Sakamuri, Rama Murthy; Brennan, Patrick J; Vissa, Varalakshmi

    2012-01-01

    Based on the discovery of three single nucleotide polymorphisms (SNPs) in Mycobacterium leprae, it has been previously reported that there are four major SNP types associated with different geographic regions around the world. Another typing system for global differentiation of M. leprae is the analysis of the variable number of short tandem repeats within the rpoT gene. To expand the analysis of geographic distribution of M. leprae, classified by SNP and rpoT gene polymorphisms, we studied 85 clinical isolates from Thai patients and compared the findings with those reported from Asian isolates. SNP genotyping by PCR amplification and sequencing revealed that all strains like those in Myanmar were SNP type 1 and 3, with the former being predominant, while in Japan, Korea, and Indonesia, the SNP type 3 was found to be more frequent. The pattern of M. leprae distribution in Thailand and Myanmar is quite similar, except that SNP type 2 was not found in Thailand. In addition, the 3-copy hexamer genotype in the rpoT gene is shared among the isolates from these two neighboring countries. On the basis of these two markers, we postulate that M. leprae in leprosy patients from Myanmar and Thailand has a common historical origin. Further differentiation among Thai isolates was possible by assessing copy numbers of the TTC sequence, a more polymorphic microsatellite locus.

  5. Clinical problems in the antibiotic treatment of gonorrhoea

    PubMed Central

    Willcox, R. R.

    1958-01-01

    After briefly reviewing the history of penicillin therapy in gonorrhoea, the author shows that the number of cures effected with the repository penicillins, although originally very high, has diminished considerably in recent years, despite a general tendency to increase the dosage. The reduced efficacy of PAM and benzathine penicillin is demonstrated by an exposition of the current results obtained with these two preparations in the treatment of gonorrhoea patients in London. Some of the difficulties involved in distinguishing between treatment failures and reinfections are discussed. The paper continues with an examination of the possible alternatives to repository penicillin in the treatment of gonorrhoea. Data are given on the comparative efficacy of a number of prepations, including mixed penicillins, phenoxymethyl penicillin and various other antibiotics, such as streptomycin and the tetracycline group. The problem of re-examination of treated gonorrhoea cases is also dealt with, practical reasons being given for restricting the period of follow-up to three weeks. Finally, in a discussion of possible future developments, the author suggests a number of measures designed to prevent a further loss of sensitivity to penicillin in the gonococcus. PMID:13596878

  6. A national quality assurance survey of Neisseria gonorrhoeae testing.

    PubMed

    Trembizki, Ella; Lahra, Monica; Stevens, Kerrie; Freeman, Kevin; Hogan, Tiffany; Hogg, Geoff; Lawrence, Andrew; Limnios, Athena; Pearson, Julie; Smith, Helen; Nissen, Michael; Sloots, Theo; Whiley, David

    2014-01-01

    The aims of this study were to (1) conduct a national survey of Neisseria gonorrhoeae identification by National Neisseria Network (NNN) reference laboratories contributing data to the Australian Gonococcal Surveillance Programme and (2) determine the prevalence in Australia of strains of N. gonorrhoeae lacking gene sequences commonly targeted by in-house PCR assays for confirmation of gonococcal nucleic acid amplification tests. Gonococcal clinical isolates referred to NNN laboratories for the first half of 2012 were screened using in-house real-time PCR assays targeting multicopy opa, porA pseudogene and cppB genes. There were 2455 clinical gonococcal isolates received in the study period; 98.6 % (2420/2455) of isolates harboured all three gene targets, 0.12 % (3/2455) were porA-negative, 0.04 % (1/2455) opa-negative and 1.14 % (28/2455) cppB-negative by PCR. Notably, no isolates were simultaneously negative for two targets. However, three isolates failed to be amplified by all three PCR methods, one isolate of which was shown to be a commensal Neisseria strain by 16S rRNA sequencing. Using PCR as the reference standard the results showed that (1) identification of N. gonorrhoeae isolates by NNN laboratories was highly specific (99.96 %) and (2) strains of N. gonorrhoeae lacking gene sequences commonly targeted by in-house PCR assays are present but not widespread throughout Australia at this point in time.

  7. Clinical problems in the antibiotic treatment of gonorrhoea.

    PubMed

    WILLCOX, R R

    1958-01-01

    After briefly reviewing the history of penicillin therapy in gonorrhoea, the author shows that the number of cures effected with the repository penicillins, although originally very high, has diminished considerably in recent years, despite a general tendency to increase the dosage. The reduced efficacy of PAM and benzathine penicillin is demonstrated by an exposition of the current results obtained with these two preparations in the treatment of gonorrhoea patients in London. Some of the difficulties involved in distinguishing between treatment failures and reinfections are discussed.The paper continues with an examination of the possible alternatives to repository penicillin in the treatment of gonorrhoea. Data are given on the comparative efficacy of a number of prepations, including mixed penicillins, phenoxymethyl penicillin and various other antibiotics, such as streptomycin and the tetracycline group.The problem of re-examination of treated gonorrhoea cases is also dealt with, practical reasons being given for restricting the period of follow-up to three weeks.Finally, in a discussion of possible future developments, the author suggests a number of measures designed to prevent a further loss of sensitivity to penicillin in the gonococcus.

  8. Neisseria gonorrhoeae survives within and modulates apoptosis and inflammatory cytokine production of human macrophages.

    PubMed

    Château, Alice; Seifert, H Steven

    2016-04-01

    The human-adapted organism Neisseria gonorrhoeae is the causative agent of gonorrhoea, a sexually transmitted infection. It readily colonizes the genital, rectal and nasalpharyngeal mucosa during infection. While it is well established that N. gonorrhoeae recruits and modulates the functions of polymorphonuclear leukocytes during infection, how N. gonorrhoeae interacts with macrophages present in infected tissue is not fully defined. We studied the interactions of N. gonorrhoeae with two human monocytic cell lines, THP-1 and U937, and primary monocytes, all differentiated into macrophages. Most engulfed bacteria were killed in the phagolysosome, but a subset of bacteria was able to survive and replicate inside the macrophages suggesting that those cells may be an unexplored cellular reservoir for N. gonorrhoeae during infection. N. gonorrhoeae was able to modulate macrophage apoptosis: N. gonorrhoeae induced apoptosis in THP-1 cells whereas it inhibited induced apoptosis in U937 cells and primary human macrophages. Furthermore, N. gonorrhoeae induced expression of inflammatory cytokines in macrophages, suggesting a role for macrophages in recruiting polymorphonuclear leukocytes to the site of infection. These results indicate macrophages may serve as a significant replicative niche for N. gonorrhoeae and play an important role in gonorrheal pathogenesis.

  9. Integrated Microfluidic Devices for Automated Microarray-Based Gene Expression and Genotyping Analysis

    NASA Astrophysics Data System (ADS)

    Liu, Robin H.; Lodes, Mike; Fuji, H. Sho; Danley, David; McShea, Andrew

    Microarray assays typically involve multistage sample processing and fluidic handling, which are generally labor-intensive and time-consuming. Automation of these processes would improve robustness, reduce run-to-run and operator-to-operator variation, and reduce costs. In this chapter, a fully integrated and self-contained microfluidic biochip device that has been developed to automate the fluidic handling steps for microarray-based gene expression or genotyping analysis is presented. The device consists of a semiconductor-based CustomArray® chip with 12,000 features and a microfluidic cartridge. The CustomArray was manufactured using a semiconductor-based in situ synthesis technology. The micro-fluidic cartridge consists of microfluidic pumps, mixers, valves, fluid channels, and reagent storage chambers. Microarray hybridization and subsequent fluidic handling and reactions (including a number of washing and labeling steps) were performed in this fully automated and miniature device before fluorescent image scanning of the microarray chip. Electrochemical micropumps were integrated in the cartridge to provide pumping of liquid solutions. A micromixing technique based on gas bubbling generated by electrochemical micropumps was developed. Low-cost check valves were implemented in the cartridge to prevent cross-talk of the stored reagents. Gene expression study of the human leukemia cell line (K562) and genotyping detection and sequencing of influenza A subtypes have been demonstrated using this integrated biochip platform. For gene expression assays, the microfluidic CustomArray device detected sample RNAs with a concentration as low as 0.375 pM. Detection was quantitative over more than three orders of magnitude. Experiment also showed that chip-to-chip variability was low indicating that the integrated microfluidic devices eliminate manual fluidic handling steps that can be a significant source of variability in genomic analysis. The genotyping results showed

  10. Meta-analysis of Randomized Controlled Trials of Genotype-Guided vs Standard Dosing of Warfarin

    PubMed Central

    Sharma, Sharan P.; Fung, Erik; Lee, Juyong; Moore, Jason H.; Unterborn, John N.; Williams, Scott M.

    2015-01-01

    BACKGROUND: Warfarin is a widely prescribed anticoagulant, and its effect depends on various patient factors including genotypes. Randomized controlled trials (RCTs) comparing genotype-guided dosing (GD) of warfarin with standard dosing have shown mixed efficacy and safety outcomes. We performed a meta-analysis of all published RCTs comparing GD vs standard dosing in adult patients with various indications of warfarin use. METHODS: We searched MEDLINE, EMBASE, Cochrane Central Register of Controlled Trials (CENTRAL), and relevant references for English language RCTs (inception through March 2014). We performed the meta-analysis using a random effects model. RESULTS: Ten RCTs with a total of 2,505 patients were included in the meta-analysis. GD compared with standard dosing resulted in a similar % time in therapeutic range (TTR) at ≤ 1 month follow-up (39.7% vs 40.2%; mean difference [MD], −0.52 [95% CI, −3.15 to 2.10]; P = .70) and higher % TTR (59.4% vs 53%; MD, 6.35 [95% CI, 1.76-10.95]; P = .007) at > 1 month follow-up, a trend toward lower risk of major bleeding (risk ratio, 0.46 [95% CI, 0.19-0.1.11]; P = .08) at ≤ 1 month follow-up and lower risks of major bleeding (0.34 [95% CI, 0.16-0.74], P = .006) at > 1-month follow-up, and shorter time to maintenance dose (TMD) (24.6 days vs 34.1 days; MD, −9.54 days [95% CI, −18.10 to −0.98]; P = .03) at follow-up but had no effects on international normalized ratio [INR] > 4.0, nonmajor bleeding, thrombotic outcomes, or overall mortality. CONCLUSIONS: In the first month of genotype-guided warfarin therapy, compared with standard dosing, there were no improvements in % TTR, INR > 4.0, major or minor bleeding, thromboembolism, or all-cause mortality. There was a shorter TMD, and, after 1 month, improved % TTR and major bleeding incidence, making this a cost-effective strategy in patients requiring longer anticoagulation therapy. PMID:25811981

  11. 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.

  12. [Ciprofloxacin resistance of Neisseria gonorrhoeae according to sexual habits].

    PubMed

    García, Susana; Casco, Ricardo; Perazzi, Beatriz; De Mier, Carmen; Vay, Carlos; Famiglietti, Angela

    2008-01-01

    The first isolates of Neisseria gonorrhoeae resistant to fluoroquinolones in Argentina were reported in 2000. Since January 2005 to June 2007 Neisseria gonorrhoeae was studied in 595 men who have sex with men (MSM) and 571 heterosexual men. The gonorrhea prevalence in MSM and heterosexual men was 0.091(91/1000) and the Neisseria gonorrhoeae ciprofloxacin resistant (CRNG) was 20% in MSM and 3.8% in heterosexual men (p: 0.0416). Thirteen out of 106 isolates from 11 MSM and 2 heterosexual men were CRNG. Six out of eleven MSM had urethritis, one also carried Neisseria gonorrhoeae in rectum and 5 patients were asymptomatic carriers (rectum 2, pharynx 2, urethra 1). No epidemiological relation was found among the patients. Two heterosexual men had urethritis. The 8 symptomatic men were treated with ciprofloxacin but treatment failed in all of them. These patients and the asymptomatic ones were treated with ceftriaxone, 500 mg IM. The post treatment microbiological controls were negative. The CRNG isolates had ciprofloxacin MIC between 2 and 32 (microg/ml), all were negative to penicillinase, 4 out of 13 were chromosomally resistant to penicillin (MIC: 1 microg/ml). The MICs (microg/ml) ranges for several antimicrobial agents were: penicillin: 0.016-1; tetracycline: 0.125-2; ceftriaxone: 0.004-0.008; erythromycin: 0.032-2; azithromycin: 0.032-0.5; spectinomycin: 8-32. Due to the high level of ciprofloxacin-resistant N. gonorrhoeae isolated from MSM in our hospital, another antimicrobial agent for empirical therapy should be used in these patients.

  13. Clinical manifestation, imaging, and genotype analysis of two pedigrees with spinocerebellar ataxia.

    PubMed

    Peipei, Liu; Yang, Liu; Weihong, Gu; Xiaonan, Song

    2011-12-01

    The objective of this study was to analyze the clinical manifestation, imaging characteristics, genotype, and the relationship between the three aforementioned parameters in two pedigrees suffering from spinocerebellar ataxia. To evaluate the clinical manifestation of the two pedigrees and to compare the characteristics, we performed the MRI analysis of some patients from both pedigrees, while 2 ml of the peripheral blood sample was collected for gene analysis. The gene analysis data showed that pedigree 1 was certified spinocerebellar ataxia type-2 (SCA2); the CAG repeats in the proband, proband's mother, and proband's brother were 44, 36, and 38, respectively. The MRI revealed brainstem cerebellar atrophy and "cross sign" and "ordinate sign" of pons. Pedigree 2 was certified SCA1; the CAG repeats of the proband, proband's aunt, and proband's asymptomatic cousin were 60, 51, and 52, respectively. The MRI revealed cerebellar atrophy in these individuals. We, therefore, concluded that it was difficult to diagnose the SCA subset solely through the clinical manifestation. The imaging characteristics analysis and final diagnosis depended basically on gene analysis data.

  14. 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

  15. Neisseria gonorrhoeae and Chlamydia trachomatis among Women Reporting Extragenital Exposures

    PubMed Central

    Trebach, Joshua D.; Chaulk, C. Patrick; Page, Kathleen R.; Tuddenham, Susan; Ghanem, Khalil G.

    2015-01-01

    Introduction The CDC recommends pharyngeal screening of Neisseria gonorrhoeae (GC) and rectal screening of GC and Chlamydia trachomatis (CT) in HIV-infected and at-risk men who have sex with men (MSM). There are currently no recommendations to routinely screen women at extragenital sites. We define the prevalence of extragenital GC and CT in women attending two urban STD clinics in Baltimore City and compare it to the prevalence of extragenital infections in MSM and men who have sex with women (MSW). Methods All patients who reported extragenital exposures in the preceding 3 months, who presented for care between 6/1/2011 and 5/31/2013, and were tested for GC and CT using nucleic acid amplification tests at all sites of exposure were included in the analyses. We used logistic regression models to identify risk factors for extragenital infections. Results 10,389 patients were included in this analysis (88% African American, mean age 29 years, 42% women, 7% MSM, 2.5% HIV infected). The prevalence estimates of any extragenital GC and CT were: 2.4% GC and 3.7% CT in women; 2.6% GC and 1.6% CT in MSW; 18.9% GC and 11.8% CT in MSM. Among women, 30.3% of GC infections and 13.8% of CT infections would have been missed with urogenital-only testing. Unlike MSM, age ≤ 18 years was the strongest predictor of extragenital infections in women. Conclusions Although the prevalence of extragenital gonorrhea and chlamydia is highest in MSM, a significant number of GC and CT infections in young women would be missed with genital-only testing. Cost-effectiveness analyses are needed to help inform national guidelines on extragenital screening in young women. PMID:25868133

  16. Genotyping and coalescent phylogenetic analysis of Pneumocystis jiroveci from South Africa.

    PubMed

    Robberts, Frans J L; Liebowitz, Lynne D; Chalkley, Lynda J

    2004-04-01

    Sequence analysis of Pneumocystis jiroveci internal transcribed spacer (ITS) regions has become an important epidemiological tool. The objectives of the present study were to investigate sequence variations in the ITS1-5.8S ribosomal DNA (rDNA)-ITS2 regions; determine the P. jiroveci genotypes present in Cape Town, South Africa; and resolve the lineage evolution of the types by use of the coalescent theory. ITS regions were amplified from samples collected from 19 patients. PCR products were cloned, and four to five clones were sequenced from each specimen. Statistical parsimony was applied for coalescence-based network genotype analysis. The most prevalent type was Eg (14 of 19 patients, 33 of 83 clones), followed by Gg (4 of 19 patients, 7 of 83 clones), Eu (3 of 19 patients, 5 of 83 clones), and Gh (2 of 19 patients, 2 of 83 clones). Four new combinations (Eo, Je, Ge, and No), 11 new ITS1 sequences, and 13 new ITS2 sequences were identified. A new ITS2 type was detected in three patients and was designated type u. Coinfection appeared to be common, with 15 of 19 patients harboring more than one type and with up to six types per specimen. The resultant parsimony network identified Eg as the most probable ancestral haplotype and supported the occurrence of recombinational events within the population studied. Although the 5.8S rDNA region revealed only 13 clones containing one to two nucleotide polymorphisms, it may assist in defining types. Coalescent theory proposed that Eg is an ancestral type from which microevolutionary subtypes radiate.

  17. 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).

  18. 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

  19. Multiplexed nanoplasmonic biosensor for one-step simultaneous detection of Chlamydia trachomatis and Neisseria gonorrhoeae in urine.

    PubMed

    Soler, Maria; Belushkin, Alexander; Cavallini, Andrea; Kebbi-Beghdadi, Carole; Greub, Gilbert; Altug, Hatice

    2017-03-21

    Development of rapid and multiplexed diagnostic tools is a top priority to address the current epidemic problem of sexually transmitted diseases. Here we introduce a novel nanoplasmonic biosensor for simultaneous detection of the two most common bacterial infections: Chlamydia trachomatis and Neisseria gonorrhoeae. Our plasmonic microarray is composed of gold nanohole sensor arrays that exhibit the extraordinary optical transmission (EOT), providing highly sensitive analysis in a label-free configuration. The integration in a microfluidic system and the precise immobilization of specific antibodies on the individual sensor arrays allow for selective detection and quantification of the bacteria in real-time. We achieved outstanding sensitivities for direct immunoassay of urine samples, with a limit of detection of 300 colony forming units (CFU)/mL for C. trachomatis and 1500CFU/mL for N. gonorrhoeae. The multiplexing capability of our biosensor was demonstrated by analyzing different urine samples spiked with either C. trachomatis or N. gonorrhoeae, and also containing both bacteria. We could successfully detect, identify and quantify the levels of the two bacteria in a one-step assay, without the need for DNA extraction or amplification techniques. This work opens up new possibilities for the implementation of point-of-care biosensors that enable fast, simple and efficient diagnosis of sexually transmitted infections.

  20. 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

  1. Previous history of gonococcal infection as a risk factor in patients presenting with gonorrhoea.

    PubMed

    Fowler, T; Caley, M; Johal, R; Brown, R; Ross, J D C

    2010-04-01

    Recidivism is common in patients infected with gonorrhoea. Identifying the factors most closely associated with recurrent gonococcal infection can help to target health promotion and disease prevention interventions. A case-control study design was used to quantify the importance of past infection as a risk marker for gonorrhoea while controlling for other demographic and behavioural factors. Data were available for 134 cases of gonorrhoea and 150 controls. A history of gonorrhoea (odds ratio [OR] 4.36 [95% CI 1.78-10.71]) was the strongest predictor of current infection. The number of partners in the last month (OR 2.19 [95% CI 1.20-4.02]) was also significantly associated with a diagnosis of gonorrhoea. Patients presenting with gonorrhoea are a specific high-risk group who require additional interventions and should be prioritized for evidence-based, enhanced and interactive counselling.

  2. High Resolution Melt (HRM) analysis is an efficient tool to genotype EMS mutants in complex crop genomes

    PubMed Central

    2011-01-01

    Background Targeted Induced Loci Lesions IN Genomes (TILLING) is increasingly being used to generate and identify mutations in target genes of crop genomes. TILLING populations of several thousand lines have been generated in a number of crop species including Brassica rapa. Genetic analysis of mutants identified by TILLING requires an efficient, high-throughput and cost effective genotyping method to track the mutations through numerous generations. High resolution melt (HRM) analysis has been used in a number of systems to identify single nucleotide polymorphisms (SNPs) and insertion/deletions (IN/DELs) enabling the genotyping of different types of samples. HRM is ideally suited to high-throughput genotyping of multiple TILLING mutants in complex crop genomes. To date it has been used to identify mutants and genotype single mutations. The aim of this study was to determine if HRM can facilitate downstream analysis of multiple mutant lines identified by TILLING in order to characterise allelic series of EMS induced mutations in target genes across a number of generations in complex crop genomes. Results We demonstrate that HRM can be used to genotype allelic series of mutations in two genes, BraA.CAX1a and BraA.MET1.a in Brassica rapa. We analysed 12 mutations in BraA.CAX1.a and five in BraA.MET1.a over two generations including a back-cross to the wild-type. Using a commercially available HRM kit and the Lightscanner™ system we were able to detect mutations in heterozygous and homozygous states for both genes. Conclusions Using HRM genotyping on TILLING derived mutants, it is possible to generate an allelic series of mutations within multiple target genes rapidly. Lines suitable for phenotypic analysis can be isolated approximately 8-9 months (3 generations) from receiving M3 seed of Brassica rapa from the RevGenUK TILLING service. PMID:22152063

  3. Therapy for treatment-refractory chronic hepatitis C virus genotype 1b infection: A retrospective analysis

    PubMed Central

    Cindoruk, Mehmet; Karakan, Tarkan; Unal, Selahattin

    2005-01-01

    Background: The most effective current therapy for hepatitis C virus (HCV) infection is the combination of pegylated interferon (peg-IFN) plus ribavirin (RBV). Objective: The aim of this retrospective analysis was to determine the rateof response to this therapy, and the factors affecting outcome, in patients with treatment-refractory chronic HCV genotype l b. Methods: The records of patients with chronic HCV infection and HCV geno-type1b who failed (nonresponse or relapse) previous treatment with standard interferon (IFN) + RSV were retrospectively analyzed for demographic data, virologic load, liver histology, biochemistry, treatment-related adverse effects (AEs), and the effects of dose reduction during treatment with peg-IFN + RBV for 48 weeks. Early virologic response (EVR) was defined as ≥2-log (copies/mL) decrease from baseline in serum HCV RNA concentration or the absence of detectable serum HCV RNA at treatment week 12. End-of-treatment response (ETR) was defined as the absence of detectable serum HCV RNA at treatment week 48. Sustained virologic response (SVR) was defined as the absence of detectable serum HCV RNA 24 weeks after treatment was discontinued. Factors affecting treatment outcome were determined using correlation analyses. Results: Data from the files of 17 patients (12 men, 5 women; mean [SD] age, 48 [2] years) were analyzed. EVR was achieved in 7 patients; however, viral breakthrough occurred in 2 of these patients during the treatment period, and 5 of these patients discontinued treatment because of severe treatment-related AEs (depression [1 patient] and neutropenia [4]). Seven patients achieved ETR, but HCV infection relapsed during the follow-up period. Three (18%) patients achieved SVR. Data concerning previous patterns of response to IFN + RBV therapy were available in 10 patients. Of these, 3 of 6 patients who had experienced relapse with the previous treatment achieved SVR with peg-IFN + RBV; neither of the 2 patients with

  4. Conservation of peptide structure of outer membrane protein-macromolecular complex from Neisseria gonorrhoeae.

    PubMed Central

    Hansen, M V; Wilde, C E

    1984-01-01

    The structural conservation of an outer membrane protein of Neisseria gonorrhoeae called OMP-MC (outer membrane protein-macromolecular complex) was investigated by determining the isoelectric point and amino-terminal amino acid sequence of the protein and by using high-performance liquid chromatography for comparative tryptic peptide mapping. The 76,000-dalton subunits generated by reduction and alkylation of the native 800,000-dalton complex from six test strains focused in ultrathin gels as bands of restricted heterogeneity at an approximate pI of 7.6. Dansyl chloride labeling indicated that all strains shared glycine as the amino-terminal amino acid. Sequence analysis of OMP-MC from two strains revealed no amino acid differences within the first 11 residues. Dual-label peptide maps revealed an extremely high degree of conservation of peptide structure. The results indicate that (i) OMP-MCs isolated from various strains of N. gonorrhoeae share structural homology and (ii) the 800,000-dalton complex is a homopolymer composed of 10 to 12 apparently identical 76,000-dalton subunits. Images PMID:6421738

  5. 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.

  6. Phase variable DNA repeats in Neisseria gonorrhoeae influence transcription, translation, and protein sequence variation

    PubMed Central

    Zelewska, Marta A.; Pulijala, Madhuri; Spencer-Smith, Russell; Mahmood, Hiba-Tun-Noor A.; Norman, Billie; Churchward, Colin P.; Calder, Alan

    2016-01-01

    There are many types of repeated DNA sequences in the genomes of the species of the genus Neisseria, from homopolymeric tracts to tandem repeats of hundreds of bases. Some of these have roles in the phase-variable expression of genes. When a repeat mediates phase variation, reversible switching between tract lengths occurs, which in the species of the genus Neisseria most often causes the gene to switch between on and off states through frame shifting of the open reading frame. Changes in repeat tract lengths may also influence the strength of transcription from a promoter. For phenotypes that can be readily observed, such as expression of the surface-expressed Opa proteins or pili, verification that repeats are mediating phase variation is relatively straightforward. For other genes, particularly those where the function has not been identified, gathering evidence of repeat tract changes can be more difficult. Here we present analysis of the repetitive sequences that could mediate phase variation in the Neisseria gonorrhoeae strain NCCP11945 genome sequence and compare these results with other gonococcal genome sequences. Evidence is presented for an updated phase-variable gene repertoire in this species, including a class of phase variation that causes amino acid changes at the C-terminus of the protein, not previously described in N. gonorrhoeae. PMID:28348872

  7. Difference in DNA-binding abilities of Fur-homolog DNA binding protein from Neisseria gonorrhoeae.

    PubMed

    Bagchi, Angshuman

    2016-10-01

    Gonorrhea is a severe disease infecting both men and women worldwide. The causative agent of the disease is Neisseria gonorrhoeae. The organism mostly affects human beings in iron restricted environments. In such an environment the organism produces a set of proteins which are mostly absent in iron rich environments. The expressions of the genes for the proteins are regulated by the transcription factor (TF) belonging to the Fur family. Interestingly, the same TF acts as the activator and repressor of genes. In this present work, an attempt has been made to analyze the molecular details of the differential DNA-binding activities of the TF from Neisseria gonorrhoeae to come up with a plausible molecular reason behind the difference DNA binding activities of the same TF. Computational modelling technique was used to build the three dimensional structure of the TF. Molecular docking and molecular dynamics simulations were employed to determine the binding interactions between the TF and the promoter DNA. With the help of the computational techniques, the biochemical reason behind the different modes of DNA binding by the TF was analyzed. Results from this analysis may be useful to future drug development endeavours to curtail the spread of Gonorrhea.

  8. A novel high-resolution melting analysis-based method for Yersinia pseudotuberculosis genotyping.

    PubMed

    Souza, Roberto A; Falcão, Juliana P

    2012-12-01

    Yersinia pseudotuberculosis is an enteric pathogen that is environmentally widespread and is known to cause human and animal infections. The development of a fast and inexpensive typing system is necessary to facilitate epidemiological studies of Y. pseudotuberculosis infections. In this study, we aimed to develop a method of Y. pseudotuberculosis genotyping based on determining differences in single-nucleotide polymorphisms (SNPs) using a high-resolution melting analysis (HRMA). Using a set of nine primer pairs, ten SNPs were screened from sequences in the 16S rRNA, glnA, gyrB and recA sequences of 12 Y. pseudotuberculosis strains that were deposited in the GenBank database. The genetic diversity of a collection of 40 clinical Y. pseudotuberculosis strains was determined using the HRMA method and the multilocus sequence typing (MLST) technique was used for comparison. Different melting profiles were found in five out of a total of nine analyzed fragments. A phylogenetic tree was constructed from the nucleotides that were identified in the nine analyzed fragments, and the tree demonstrated that Y. pseudotuberculosis strains were separated into two groups. The first cluster was composed of strains from the 1/O:1a serogroup and the second of strains from the 2/O:3 serogroup. The separation into two clusters based on distinct bio-serogroups of Y. pseudotuberculosis was consistent with the results in the MLST database. The simple and highly reproducible HRMA assay developed by us may be used as a rapid and cost-effective method to genotype Y. pseudotuberculosis strains of O:1 and O:3 serogroups and it can complement sequence-based methods facilitating epidemiological studies of this Yersinia species.

  9. Genotype-Phenotype Analysis in Congenital Adrenal Hyperplasia due to P450 Oxidoreductase Deficiency

    PubMed Central

    Krone, Nils; Reisch, Nicole; Idkowiak, Jan; Dhir, Vivek; Ivison, Hannah E.; Hughes, Beverly A.; Rose, Ian T.; O'Neil, Donna M.; Vijzelaar, Raymon; Smith, Matthew J.; MacDonald, Fiona; Cole, Trevor R.; Adolphs, Nicolai; Barton, John S.; Blair, Edward M.; Braddock, Stephen R.; Collins, Felicity; Cragun, Deborah L.; Dattani, Mehul T.; Day, Ruth; Dougan, Shelley; Feist, Miriam; Gottschalk, Michael E.; Gregory, John W.; Haim, Michaela; Harrison, Rachel; Haskins Olney, Ann; Hauffa, Berthold P.; Hindmarsh, Peter C.; Hopkin, Robert J.; Jira, Petr E.; Kempers, Marlies; Kerstens, Michiel N.; Khalifa, Mohamed M.; Köhler, Birgit; Maiter, Dominique; Nielsen, Shelly; O'Riordan, Stephen M.; Roth, Christian L.; Shane, Kate P.; Silink, Martin; Stikkelbroeck, Nike M. M. L.; Sweeney, Elizabeth; Szarras-Czapnik, Maria; Waterson, John R.; Williamson, Lori; Hartmann, Michaela F.; Taylor, Norman F.; Wudy, Stefan A.; Malunowicz, Ewa M.; Shackleton, Cedric H. L.

    2012-01-01

    Context: P450 oxidoreductase deficiency (PORD) is a unique congenital adrenal hyperplasia variant that manifests with glucocorticoid deficiency, disordered sex development (DSD), and skeletal malformations. No comprehensive data on genotype-phenotype correlations in Caucasian patients are available. Objective: The objective of the study was to establish genotype-phenotype correlations in a large PORD cohort. Design: The design of the study was the clinical, biochemical, and genetic assessment including multiplex ligation-dependent probe amplification (MLPA) in 30 PORD patients from 11 countries. Results: We identified 23 P450 oxidoreductase (POR) mutations (14 novel) including an exonic deletion and a partial duplication detected by MLPA. Only 22% of unrelated patients carried homozygous POR mutations. p.A287P was the most common mutation (43% of unrelated alleles); no other hot spot was identified. Urinary steroid profiling showed characteristic PORD metabolomes with variable impairment of 17α-hydroxylase and 21-hydroxylase. Short cosyntropin testing revealed adrenal insufficiency in 89%. DSD was present in 15 of 18 46,XX and seven of 12 46,XY individuals. Homozygosity for p.A287P was invariably associated with 46,XX DSD but normal genitalia in 46,XY individuals. The majority of patients with mild to moderate skeletal malformations, assessed by a novel scoring system, were compound heterozygous for missense mutations, whereas nearly all patients with severe malformations carried a major loss-of-function defect on one of the affected alleles. Conclusions: We report clinical, biochemical, and genetic findings in a large PORD cohort and show that MLPA is a useful addition to POR mutation analysis. Homozygosity for the most frequent mutation in Caucasians, p.A287P, allows for prediction of genital phenotype and moderate malformations. Adrenal insufficiency is frequent, easily overlooked, but readily detected by cosyntropin testing. PMID:22162478

  10. 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.

  11. 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

  12. Assessment of fluorescent amplified fragment length polymorphism analysis for epidemiological genotyping of Legionella pneumophila serogroup 1.

    PubMed

    Fry, N K; Afshar, B; Visca, P; Jonas, D; Duncan, J; Nebuloso, E; Underwood, A; Harrison, T G

    2005-09-01

    This study assessed the reproducibility and epidemiological concordance of double-enzyme fluorescent amplified fragment length polymorphism (fAFLP) analysis for genotyping of Legionella pneumophila serogroup (sg) 1. fAFLP fragment analysis was performed on three different sequencing platforms (one gel- and two capillary-based) in different laboratories with a well-characterised set of 50 strains of L. pneumophila sg 1. fAFLP data were analysed with the Pearson correlation similarity coefficient, using a range of parameters, and dendrogram outputs were converted to arbitrary types after selection of a specified percentage similarity threshold. The results obtained were compared with those obtained by the standard non-fluorescent AFLP method and were found to be broadly concordant. Using optimised settings for each fAFLP method to analyse the panel of 50 strains, epidemiological concordance (E) and reproducibility (R) values of 1.00 were obtained, and the number of types ranged from nine to 15, compared with E=1.00 and R=1.00, with 16 types, for the non-fluorescent AFLP protocol. The study demonstrated the potential of fAFLP for typing strains of L. pneumophila sg 1 on all three platforms; however, inter-platform comparison of fAFLP data was not achieved. fAFLP analysis may have a role in the fingerprinting of multiple isolates during Legionella outbreak investigations, but further work is required before type designations and identification libraries can be developed.

  13. A flexible model for association analysis in sibships with missing genotype data.

    PubMed

    Dudbridge, Frank; Holmans, Peter A; Wilson, Scott G

    2011-05-01

    A common design in family-based association studies consists of siblings without parents. Several methods have been proposed for analysis of sibship data, but they mostly do not allow for missing data, such as haplotype phase or untyped markers. On the other hand, general methods for nuclear families with missing data are computationally intensive when applied to sibships, since every family has missing parents that could have many possible genotypes. We propose a computationally efficient model for sibships by conditioning on the sets of alleles transmitted into the sibship by each parent. This means that the likelihood can be written only in terms of transmitted alleles and we do not have to sum over all possible untransmitted alleles when they cannot be deduced from the siblings. The model naturally accommodates missing data and admits standard theory of estimation, testing, and inclusion of covariates. Our model is quite robust to population stratification and can test for association in the presence of linkage. We show that our model has similar power to FBAT for single marker analysis and improved power for haplotype analysis. Compared to summing over all possible untransmitted alleles, we achieve similar power with considerable reductions in computation time.

  14. 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

  15. 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.

  16. 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...

  17. [Selection of resistant peanut genotypes to Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae) supported by multivariate analysis].

    PubMed

    Pitta, Rafael M; Boiça, Arlindo L; Jesus, Flávio G de; Tagliari, Sônia R A

    2010-01-01

    The velvetbean caterpillar Anticarsia gemmatalis Hübner attacks peanut leaves, and the use of resistant varieties has directly contributed to ecological and economic aspects of pest control. The aim of this work was to select resistant peanut genotypes to A. gemmatalis using cluster analyses (dendogram obtained by Ward's methods and K-means) and Principal Components analysis for data interpretation. The evaluated genotypes were: IAC 5, IAC 8112, IAC 22 and IAC Tatu ST with upright growth habit, and IAC 147, IAC 125, IAC Caiapó and IAC Runner 886 with runner growth habit, and soybean genotype BR 16 as a susceptible control. The biological parameters: leaf consumption, larval (4 masculine instar) and pupal (24h old) weight, larval and pupal development time and adult longevity were evaluated at laboratory conditions. The genotypes IAC 147 and IAC Runner 886 were resistant to A. gemmatalis in both cluster tests, grouping apart from most of the other genotypes. Both dendrogram and K-means methods provided satisfactory biological explanation, and they can be complementary used together with Principal Component and vice-versa. These results suggest that cluster analyses may be an important statistical tool in the selection of host plant resistance.

  18. Multiplex agarose gel electrophoresis system for variable number of tandem repeats genotyping: analysis example using Mycobacterium tuberculosis.

    PubMed

    Wada, Takayuki; Maeda, Shinji

    2013-04-01

    As one genotyping method for Mycobacterium tuberculosis, variable number of tandem repeats (VNTR) is a promising tool to trace the undefined transmission of tuberculosis, but it often requires large equipment such as a genetic analyzer for DNA fragment analysis or CE system to conduct systematic analyses. For convenient genotyping at low cost in laboratories, we designed a multiplex PCR system that is applicable to agarose gel electrophoresis using fluorescent PCR primers. For tuberculosis genotyping by VNTR, the copy quantities of minisatellite DNA must be determined in more than 12 loci. The system can halve laborious electrophoresis processes by presenting an image of two VNTR amplicons on a single lane. No expensive equipment is necessary for this method. Therefore, it is useful even in developing countries.

  19. APOE genotype and neuroimaging markers of Alzheimer’s disease: systematic review and meta-analysis

    PubMed Central

    Liu, Ying; Yu, Jin-Tai; Wang, Hui-Fu; Han, Pei-Ran; Tan, Chen-Chen; Wang, Chong; Meng, Xiang-Fei; Risacher, Shannon L; Saykin, Andrew J

    2015-01-01

    Objective We aimed to examine the association of apolipoprotein E (APOE) ε4 genotype with neuroimaging markers of Alzheimer’s disease: hippocampal volume, brain amyloid deposition and cerebral metabolism. Methods We performed a systematic review and meta-analysis of 14 cross-sectional studies identified in Pubmed from 1996 to 2014 (n=1628). The pooled standard mean difference (SMD) was used to estimate the association between APOE and hippocampal volume and amyloid deposition. Meta-analysis was performed using effect size signed differential mapping using coordinates extracted from clusters with statistically significant difference in cerebral metabolic rate for glucose between APOE ε4+ and ε4− groups. Results APOE ε4 carrier status was associated with atrophic hippocampal volume (pooled SMD: −0.47; 95% CI −0.82 to −0.13; p=0.007) and increased cerebral amyloid positron emission tomography tracer (pooled SMD: 0.62, 95% CI 0.27 to 0.98, p=0.0006). APOE ε4 was also associated with decreased cerebral metabolism, especially in right middle frontal gyrus. Conclusions APOE ε4 was associated with atrophic hippocampal volume in MRI markers, increased cerebral amyloid deposition and cerebral hypometabolism. Theses associations may indicate the potential role of the APOE gene in the pathophysiology of Alzheimer’s disease. PMID:24838911

  20. Optimization of the genotyping-by-sequencing strategy for population genomic analysis in conifers.

    PubMed

    Pan, Jin; Wang, Baosheng; Pei, Zhi-Yong; Zhao, Wei; Gao, Jie; Mao, Jian-Feng; Wang, Xiao-Ru

    2015-07-01

    Flexibility and low cost make genotyping-by-sequencing (GBS) an ideal tool for population genomic studies of nonmodel species. However, to utilize the potential of the method fully, many parameters affecting library quality and single nucleotide polymorphism (SNP) discovery require optimization, especially for conifer genomes with a high repetitive DNA content. In this study, we explored strategies for effective GBS analysis in pine species. We constructed GBS libraries using HpaII, PstI and EcoRI-MseI digestions with different multiplexing levels and examined the effect of restriction enzymes on library complexity and the impact of sequencing depth and size selection of restriction fragments on sequence coverage bias. We tested and compared UNEAK, Stacks and GATK pipelines for the GBS data, and then developed a reference-free SNP calling strategy for haploid pine genomes. Our GBS procedure proved to be effective in SNP discovery, producing 7000-11 000 and 14 751 SNPs within and among three pine species, respectively, from a PstI library. This investigation provides guidance for the design and analysis of GBS experiments, particularly for organisms for which genomic information is lacking.

  1. Comparative RNA-seq analysis of the Tritrichomonas foetus PIG30/1 isolate from pigs reveals close association with Tritrichomonas foetus BP-4 isolate 'bovine genotype'.

    PubMed

    Morin-Adeline, Victoria; Mueller, Kai; Conesa, Ana; Šlapeta, Jan

    2015-09-15

    Tritrichomonas foetus was described as a commensal of the stomach, caecum and nasal cavity of pigs before it was recognised as the cause of reproductive tract disease of cattle. T. foetus also causes chronic large bowel diarrhoea in domestic cats. Multi-locus genotyping and comparative transcriptome analysis has previously revealed that T. foetus isolated from cat and cattle hosts are genetically distinct, referred to as the 'feline genotype' and 'bovine genotype', respectively. Conversely, multi-locus genotyping has grouped porcine T. foetus with the 'bovine genotype'. To compare the extent of the similarity between porcine T. foetus and cattle 'bovine genotype' isolates, RNA-sequencing (RNA-seq) was used to produce the first cell-wide transcriptome library of porcine T. foetus PIG30/1. Comparative transcriptome analysis of the PIG30/1 with the published bovine (BP-4) and feline (G10/1) transcriptomes revealed that the porcine T. foetus shares a 4.7 fold greater number of orthologous genes with the bovine T. foetus than with the feline T. foetus. Comparing transcription of the virulence factors, cysteine proteases (CP) between the three isolates, the porcine T. foetus was found to preferentially transcribe CP8 like the 'bovine genotype' T. foetus, compared to thehigh transcription of CP7 seen for 'feline genotype' T. foetus. At the cell-wide transcriptome level, the porcine T. foetus isolate (PIG30/1) groups closer with the 'bovine genotype' T. foetus rather than the 'feline genotype' T. foetus.

  2. Comparative assessment of CDS, CLSI disc diffusion and Etest techniques for antimicrobial susceptibility testing of Neisseria gonorrhoeae: a 6-year study

    PubMed Central

    Singh, Vikram; Kakran, Monika; Ramesh, V

    2012-01-01

    Background A variety of techniques are available for antimicrobial susceptibility testing of Neisseria gonorrhoeae. Objective The aim of this study was to find a cost-effective, reliable and easily applicable microbiological method to detect antimicrobial susceptibilities of N. gonorrhoeae in resource-poor countries. Design Prospective study. Setting Male and female STD clinic of Regional STD Teaching, Training and Research Centre, New Delhi, India. Participants N. gonorrhoeae isolates from all male and female patients presenting with acute gonococcal urethritis and cervical discharge. Material and methods A total of 295 consecutive N. gonorrhoeae isolates during 2005–2010 was used to compare the Clinical and Laboratory Standards Institute (CLSI) and CDS disc diffusion technique with Etest by performing antimicrobial susceptibility testing in parallel for penicillin, tetracycline, ceftriaxone, ciprofloxacin and spectinomycin. WHO reference strains were used as controls. Results CDS disc diffusion zones of inhibition showed that complete percentage agreement for penicillin, ciprofloxacin and tetracycline was high with their analogous Etest minimal inhibitory concentrations in comparison to CLSI disc diffusion technique, that is, 91.5%, 92.9% and 99.3% versus 87.5%, 88.5% and 74.9%, respectively. CDS results had less number of major and minor category discrepancies in comparison to CLSI and CDS method showed excellent correlation coefficient (r=1) with Etest for all five antimicrobial agents tested in comparison to CLSI (r=0.92). It was very poor (r=0.61) by CLSI method for tetracycline. The correlation coefficients between the two methods and the Etest were identical if tetracycline was removed from the CLSI analysis. Conclusions The CDS technique is an attractive alternative for N. gonorrhoeae susceptibility testing and is recommended for monitoring the antimicrobial susceptibility in less developed and resource-poor settings to facilitate enhanced antimicrobial

  3. 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

  4. 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.

  5. Antioxidative enzymes and isozymes analysis of taro genotypes and their implications in Phytophthora blight disease resistance.

    PubMed

    Sahoo, Manas Ranjan; DasGupta, Madhumita; Kole, Paresh C; Bhat, Jayant S; Mukherjee, Archana

    2007-04-01

    Assessment of the differential expression of antioxidative enzymes and their isozymes, was done in 30 day-old ex vitro raised plants of three highly resistant (DP-25, Jhankri and Duradim) and one highly susceptible (N-118) genotypes of taro [Colocasia esculenta (L.) Schott]. Antioxidative enzymes were assayed in the ex vitro plants, 7 days after inoculation with the spores (15,000 spores ml(-1) water) of Phytophthora colocasiae Raciborski to induce taro leaf blight disease. Uninoculated ex vitro plants in each genotype were used as control. The activity of superoxide dismutase (SOD) and guaiacol peroxidase (GPX) increased under induced blight condition when compared with control. Increase in antioxidative enzymes was more (67-92%) in the resistant genotypes than that (21-29%) of the susceptible genotype. The zymograms of SOD and GPX in the resistant genotypes, with pathogenic infection, showed increased activity for anodal isoform of SOD and increased expression and/or induction of either POX 1 or POX 2 isoforms of GPX. In susceptible genotype, expression of the above isoforms was faint for SOD and nearly absent for GPX under both blight free and induced blight conditions. Induction and/or increased activity of particular isoform of SOD and GPX against infection of Phytophthora colocasiae in the resistant genotypes studied led to the apparent conclusion of linkage of isozyme expression with blight resistance in taro. This might be an important criterion in breeding of taro for Phytophthora leaf blight resistance.

  6. Analysis of phenotype and genotype information for the diagnosis of Marfan syndrome.

    PubMed

    Sheikhzadeh, S; Kade, C; Keyser, B; Stuhrmann, M; Arslan-Kirchner, M; Rybczynski, M; Bernhardt, A M; Habermann, C R; Hillebrand, M; Mir, T; Robinson, P N; Berger, J; Detter, C; Blankenberg, S; Schmidtke, J; von Kodolitsch, Y

    2012-09-01

    Marfan syndrome is considered a clinical diagnosis. Three diagnostic classifications comprising first, Marfan genotype with a causative FBN1 gene mutation; second, Marfan phenotype with clinical criteria of the original Ghent nosology (Ghent-1); and third, phenotype with clinical criteria of its current revision (Ghent-2) in 300 consecutive persons referred for confirmation or exclusion of Marfan syndrome (150 men, 150 women aged 35 ± 13 years) were used. Sequencing of TGBR1/2 genes was performed in 128 persons without FBN1 mutation. Marfan genotype was present in 140, Ghent-1 phenotype in 139, and Ghent-2 phenotype in 124 of 300 study patients. Marfan syndrome was confirmed in 94 and excluded in 129 persons consistently by all classifications, but classifications were discordant in 77 persons. With combined genotype and phenotype information confirmation of Marfan syndrome was finally achieved in 126 persons by Ghent-1 and in 125 persons by Ghent-2 among 140 persons with Marfan genotype, and exclusion was accomplished in 139 persons by Ghent-1 and in 141 persons by Ghent-2 among 160 persons without Marfan genotype. In total, genotype information changed final diagnoses in 22 persons with Ghent-1, and in 32 persons with Ghent-2. It is concluded that genotype information is essential for diagnosis or exclusion of Marfan syndrome.

  7. [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.

  8. Inhibition of Neisseria gonorrhoeae by a Bacteriocin from Pseudomonas aeruginosa

    PubMed Central

    Morse, Stephen A.; Vaughan, Patrick; Johnson, Deanne; Iglewski, Barbara H.

    1976-01-01

    Supernatants from broth-grown cultures of Pseudomonas aeruginosa PA 103 exhibited bactericidal activity against Neisseria gonorrhoeae. The concentration of the bactericidal substance increased significantly after induction by mitomycin C. Purification was effected by salt fractionation, chromatography on diethylaminoethyl-cellulose, and sedimentation by centrifugation at 100,000 × g for 90 min. Electron microscopy of this purified preparation revealed structures resembling R-type pyocins in both the contracted and uncontracted state. Pyocins in the contracted state were observed in association with the gonococcal cell surface. No loss of bactericidal activity was observed after treatment with proteolytic enzymes. Standard pyocin typing procedures identified the pyocin pattern as 611 131. The bactericidal activity of this pyocin was examined on various species of Neisseria. Out of 56 strains of N. gonorrhoeae from disseminated and nondisseminated infections, all were susceptible to pyocin 611 131. However, only 3 of 20 strains of N. meningitidis and 5 of 16 strains of N. lactamica were susceptible. The bactericidal activity that pyocin 611 131 has for N. gonorrhoeae and other species of Neisseria is significant because it departs from the expected specificity that heretofore has distinguished bacteriocins from most “classical” antibiotics. Images PMID:825024

  9. First report of Tasmanian sheep strain (G2) genotype isolated from Iranian goat using the high resolution melting (HRM) analysis

    PubMed Central

    Hosseini-Safa, Ahmad; Mohag, hegh, Mohammad Ali; Pestechian, Nader; Ganji, Maryam; Mohammadi, Rasoul; Mahmoudi Lamouki, Reza; Rostami-Nejad, Mohammad

    2016-01-01

    Aim: The present study was aimed to evaluate E. granulosus genotypes isolated from goats using HRM analysis in Isfahan province. Background: Cystic echincoccosis, so-called hydatidosis, is widespread infection caused by the larval stage of Echinococcus granulosus. This is an important zoonotic disease worldwide, especially in the developing countries such as Iran. To date, molecular studies mainly based on the mitochondrial DNA sequences have identified distinct genotypes termed G1-G10 which can differ in some characteristics such as the growth and infectivity to different intermediate hosts or the survival rate in the definitive hosts that are important for the development of control strategies. Methods: From August to December 2014, 1341 goats were investigated and hydatid cysts were collected from the liver and lungs of 43 infected goats in Isfahan province abattoirs, Isfahan, Iran. Total genomic DNA was extracted from each sample, amplified for the presence of polymorphism of mitochondrial gene coding for cytochrome c oxidase subunit 1 (CO1), using high resolution melting curve (HRM) method. Results: the results of HRM analysis using the sequence of CO1 gene for 43 Echinococcus granulosus isolates from goats showed 31, 2 and 10 isolates were identified as G1, G2, and G3 genotypes, respectively. Conclusion: G1 is the predominant genotype in the isolated goat samples in Isfahan province, and the presence of G2 strain was reported for the first time in goat in Iran. PMID:28224031

  10. Interferon λ 3 and 4 Genotyping Using High-Resolution Melt Curve Analysis Suitable for Multiple Clinical Sample Types.

    PubMed

    Lamoury, François M J; Bartlett, Sofia; Jacka, Brendan; Hajarizadeh, Behzad; Grebely, Jason; Matthews, Gail V; Dore, Gregory J; Applegate, Tanya L

    2015-09-01

    Many people living with hepatitis C virus (HCV) infection will continue to rely on interferon-based regimens until effective strategies to minimize the cost of directly acting antivirals (DAAs) and to improve treatment access are implemented. Host single-nucleotide polymorphisms related to IFNL3 and IFNL4 are associated with spontaneous clearance of HCV, and pegylated interferon- and DAA-based treatment outcomes. We describe a simple and rapid genotyping method for IFNL rs12979860, rs8099917, and rs368234815 using high-resolution melting analysis for DNA extracted from whole blood, buffy coat, plasma, serum, and dried blood spots. This assay successfully detected all three polymorphisms on DNA extracted by the automated platform easyMAG from all samples when compared to sequenced amplicons. Analysis of 126 participants with recent HCV infection from the Australian Trial in Acute Hepatitis C study demonstrated the prevalence of favorable single-nucleotide polymorphisms were 62%, 51%, and 45% for rs8099917 TT, rs12979860 CC, and rs368234815 TT/TT, respectively. The genotyping assay described here provides a rapid and affordable IFNL3 and IFNL4 genotyping method for a range of clinical sample types. Until global access to DAAs is achieved, IFNL3 and IFNL4 genotyping could identify those likely to clear naturally and in whom treatment could be delayed, or help prioritize DAA treatment to those less likely to respond to interferon-containing regimens.

  11. Self-incompatibility genotypes in almond re-evaluated by PCR, stylar ribonucleases, sequencing analysis and controlled pollinations.

    PubMed

    López, Mercè; Mnejja, Mourad; Rovira, Mercè; Collins, Graham; Vargas, Francisco J; Arús, Pere; Batlle, Ignasi

    2004-09-01

    As part of the almond breeding programme at IRTA, we investigated the S genotypes of several cultivars using a combination of RNase zymograms, testcrosses, pollen-tube growth analysis and molecular identification by PCR analysis. For some of the cultivars examined, discrepancies appeared between their S alleles as reported in the literature and those found in this investigation, leading to a re-evaluation of their S genotypes. Analysis of the stylar ribonucleases (RNases), which are known to correlate with S alleles, of cvs. Achaak, Ardechoise, Desmayo Largueta, Ferrastar, Gabaix, Garbi, Glorieta, Languedoc, Primorskiy and Texas revealed inconsistencies with respect to the S5 and S10 alleles. However, PCR with the conserved primer pair AS1II/AmyC5R failed to detect any of these inconsistencies. When the S alleles from Desmayo Largueta, Gabaix, Primorskiy and Texas were sequenced, Texas and Primorskiy were found to carry the reported S5 allele, while Desmayo Largueta and Gabaix carried a new allele, which has been tentatively denoted as S25 This new S allele, previously reported to be S10, was also identified in Achaak, Ardechoise and Ferrastar. The proposed new S genotypes are Achaak (S2S25), Ardechoise (S1S25), Desmayo Largueta (S1S25), Ferrastar (S2S25) and Gabaix (S10S25). The S alleles of Garbi, Glorieta, Languedoc, Texas and Primorskiy remain as reported in the literature. Testcrosses in the field and laboratory confirmed the new S genotypes. One cultivar (Gabaix) could be assigned to the existing cross-incompatibility group O of unique genotypes, and two new groups were established (XVI and XVII) consisting of two cultivars each. The clarification of these S alleles will be useful in almond breeding programmes and for planning new commercial orchards in the future.

  12. CYP2D6 genotype and adjuvant tamoxifen: meta-analysis of heterogeneous study populations.

    PubMed

    Province, M A; Goetz, M P; Brauch, H; Flockhart, D A; Hebert, J M; Whaley, R; Suman, V J; Schroth, W; Winter, S; Zembutsu, H; Mushiroda, T; Newman, W G; Lee, M-T M; Ambrosone, C B; Beckmann, M W; Choi, J-Y; Dieudonné, A-S; Fasching, P A; Ferraldeschi, R; Gong, L; Haschke-Becher, E; Howell, A; Jordan, L B; Hamann, U; Kiyotani, K; Krippl, P; Lambrechts, D; Latif, A; Langsenlehner, U; Lorizio, W; Neven, P; Nguyen, A T; Park, B-W; Purdie, C A; Quinlan, P; Renner, W; Schmidt, M; Schwab, M; Shin, J-G; Stingl, J C; Wegman, P; Wingren, S; Wu, A H B; Ziv, E; Zirpoli, G; Thompson, A M; Jordan, V C; Nakamura, Y; Altman, R B; Ames, M M; Weinshilboum, R M; Eichelbaum, M; Ingle, J N; Klein, T E

    2014-02-01

    The International Tamoxifen Pharmacogenomics Consortium was established to address the controversy regarding cytochrome P450 2D6 (CYP2D6) status and clinical outcomes in tamoxifen therapy. We performed a meta-analysis on data from 4,973 tamoxifen-treated patients (12 globally distributed sites). Using strict eligibility requirements (postmenopausal women with estrogen receptor-positive breast cancer, receiving 20 mg/day tamoxifen for 5 years, criterion 1); CYP2D6 poor metabolizer status was associated with poorer invasive disease-free survival (IDFS: hazard ratio = 1.25; 95% confidence interval = 1.06, 1.47; P = 0.009). However, CYP2D6 status was not statistically significant when tamoxifen duration, menopausal status, and annual follow-up were not specified (criterion 2, n = 2,443; P = 0.25) or when no exclusions were applied (criterion 3, n = 4,935; P = 0.38). Although CYP2D6 is a strong predictor of IDFS using strict inclusion criteria, because the results are not robust to inclusion criteria (these were not defined a priori), prospective studies are necessary to fully establish the value of CYP2D6 genotyping in tamoxifen therapy.

  13. 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.

  14. 5-HTTLPR Genotype and Anxiety-Related Personality Traits: A meta-analysis and new data

    PubMed Central

    Munafò, Marcus R.; Freimer, Nelson B.; Ng, Whitney; Ophoff, Roel; Veijola, Juha; Miettunen, Jouko; Järvelin, Marjo-Riitta; Taanila, Anja; Flint, Jonathan

    2008-01-01

    We investigated the strength of evidence for association of the 5-HTTLPR polymorphism and the personality trait of Harm Avoidance. We used new primary data from a large sample of adults drawn from the Finnish population. We also applied meta-analytic techniques to synthesize existing published data. The large number studies of the 5-HTTLPR polymorphism allowed us to apply a formal test of publication bias, as well as formally investigate the impact of potential moderating factors such as measurement instrument. Univariate ANOVA of primary data (n = 3,872), with 5-HTTLPR genotype as a between-groups factor, indicated no evidence of association with Harm Avoidance (p = 0.99). Meta-analysis indicated no evidence of significant association of 5-HTTLPR with Harm Avoidance (d = 0.02, p = 0.37), or EPQ Neuroticism (d = 0.01, p = 0.71), although there was evidence of association with NEO Neuroticism (d = 0.18, p < 0.001). Our analyses indicate that the 5-HTTLPR variant is not associated with Harm Avoidance. Together with our previous analyses of a large sample of participants with extreme Neuroticism scores (defined by the EPQ), we have data that excludes a meaningful genetic effect of the 5-HTTLPR on two measures of anxiety-related personality traits. There remains the possibility that the variant influences the NEO personality questionnaire measure of Neuroticism. However, a large, well-powered primary study is required to test this hypothesis directly and adequately. PMID:18546120

  15. Phylogenetic analysis of 626 hepatitis E virus (HEV) isolates from humans and animals in China (1986-2011) showing genotype diversity and zoonotic transmission.

    PubMed

    Liu, Peng; Li, Lingjun; Wang, Ling; Bu, Qiuning; Fu, Hongwei; Han, Jian; Zhu, Yonghong; Lu, Fengmin; Zhuang, Hui

    2012-03-01

    Hepatitis E is considered as a public health problem in China. To determine the overall molecular epidemiology of hepatitis E virus (HEV) and analyze the situation of cross-species transmission between humans and swine in China over the last 25 years (1986-2011), 626 HEV complete and partial sequences (89 isolates identified by our group) isolated from humans and animals in China were retrieved from GenBank and subjected to phylogenetic analysis. There were three genotypes and 11 sub-genotypes of HEV prevailing in China. Furthermore, rabbit HEVs, of which the genotype is controversial, are also widespread in China. Genotype 1 was the most isolated genotype prior to 2000 and mainly detected in Xinjiang, Beijing and East China. However, genotype 4, which was identified in most regions of China during the last 10 years, has overtaken genotype 1 in frequency of isolation nationwide. Genotype 3 HEV strains have been found only in eastern China and were thought to be imported from Japan. Both genotypes 3 and 4 were found in humans and swine and cross-species transmission from pigs to humans of the two genotypes may have occurred in Northeast, Northwest, North, East and South China. These results indicate that HEV strains with considerable genetic diversity are widespread and the zoonotic transmission between swine and humans appears ubiquitous in China.

  16. Analysis of complete genomes of the rubella virus genotypes 1E and 2B which circulated in China, 2000–2013

    PubMed Central

    Zhu, Zhen; Chen, Min-hsin; Abernathy, Emily; Icenogle, Joseph; Zhou, Shujie; Wang, Changyin; Zhao, Chunfang; Wang, Yan; Chen, Haiyun; Si, Yuan; Xu, Wenbo

    2016-01-01

    Rubella viruses of genotypes 1E and 2B are currently the most frequently detected wild-type viruses in the world. Genotype 1E viruses from China have been genetically distinct from genotype 1E viruses found elsewhere, while genotype 2B viruses found in China are not distinguishable from genotype 2B viruses from other areas. Genetic clusters of viruses of both genotypes were defined previously using sequences of the 739-nt genotyping window. Here we report phylogenic analysis using whole genomic sequences from seven genotype 1E and three genotype 2B viruses which were isolated in China between 2000 and 2013 and confirm the subgrouping of current circulating genotypes 1E and 2B viruses. In addition, the whole genomic characterization of Chinese rubella viruses was clarified. The results indicated that the Chinese rubella viruses were highly conserved at the genomic level, and no predicted amino acid variations were found at positions where functional domains of the proteins were identified. Therefore, it gives us the idea that the rubella control and elimination goal should be achieved if vaccine immunization coverage continues maintaining at the high level. PMID:27959338

  17. 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

  18. Analysis of genetic diversity among selected grasspea (Lathyrus sativus L.) genotypes using RAPD markers.

    PubMed

    Barik, Durga P; Acharya, Laxmikanta; Mukherjee, Arup K; Chand, Pradeep K

    2007-01-01

    Randomly amplified polymorphic DNA (RAPD) technique was applied to assess the genetic variability among five selected genotypes of grasspea. Out of 30 random decamer primers tested for the present investigation 20 showed reproducible DNA amplification. A total of 257 loci were amplified of which 159 were polymorphic including 57 genotype-specific unique bands. Amplicons had molecular weights ranging from 3.0 kb to 0.1 kb. Majority amplicons were shared by most of the genotypes which indicated a very narrow genetic gap between them. The dendrogram constructed on the basis of RAPD data showed two clusters. The local genotype collected from Nayagarh was grouped along with IC-120451 and IC-120453, sharing a common node at an 82% similarity level. The other genotypes, IC-120478 and IC-120487, were located in the second clade having a common node at 84% similarity level. The investigation showed that though all the genotypes of grasspea were of apparently similar morphology there exists polymorphism at the molecular level, which can be exploited in breeding programmes aimed at crop improvement.

  19. 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

  20. 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

  1. 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.

  2. Phenotypic and Genotypic Analysis of Newly Obtained Interspecific Hybrids in the Campanula Genus

    PubMed Central

    Röper, Anna-Catharina; Orabi, Jihad; Lütken, Henrik; Christensen, Brian; Thonning Skou, Anne-Marie; Müller, Renate

    2015-01-01

    Interspecific hybridisation creates new phenotypes within several ornamental plant species including the Campanula genus. We have employed phenotypic and genotypic methods to analyse and evaluate interspecific hybridisation among cultivars of four Campanula species, i.e. C. cochleariifolia, C. isophylla, C. medium and C. formanekiana. Hybrids were analysed using amplified fragment length polymorphism (AFLP), flow cytometry and biometrical measurements. Results of correlation matrices demonstrated heterogeneous phenotypes for the parental species, which confirmed our basic premise for new phenotypes of interspecific hybrids. AFLP assays confirmed the hybridity and identified self-pollinated plants. Limitation of flow cytometry analysis detection was observed while detecting the hybridity status of two closely related parents, e.g. C. cochleariiafolia × C. isophylla. Phenotypic characteristics such as shoot habitus and flower colour were strongly influenced by one of the parental species in most crosses. Rooting analysis revealed that inferior rooting quality occurred more often in interspecific hybrids than in the parental species. Only interspecific hybrid lines of C. formanekiana ‘White’ × C. medium ‘Pink’ showed a high rooting level. Phenotype analyses demonstrated a separation from the interspecific hybrid lines of C. formanekiana ‘White’ × C. medium ‘Pink’ to the other clustered hybrids of C. formanekiana and C. medium. In our study we demonstrated that the use of correlation matrices is a suitable tool for identifying suitable cross material. This study presents a comprehensive overview for analysing newly obtained interspecific hybrids. The chosen methods can be used as guidance for analyses for further interspecific hybrids in Campanula, as well as in other ornamental species. PMID:26352688

  3. 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.

  4. 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.

  5. 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

  6. 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

  7. 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.

  8. Estimating gonorrhoea prevalence in young heterosexual men and women attending community-based sexual health services to inform decisions on gonorrhoea testing.

    PubMed

    Town, K; Furegato, M; Field, N; Hughes, G

    2017-03-03

    In England, dual tests detecting chlamydia and gonorrhoea are used in specialist and community-based sexual health services (SHSs). Test performance is poor when prevalence is low, therefore UK national guidelines recommend against opportunistic gonorrhoea screening unless there is a clear local public health need. While surveillance data on gonorrhoea prevalence is comprehensive in specialist SHSs, it is sparse in community SHSs. We aimed to estimate gonorrhoea prevalence in heterosexual men and women aged 15-24 attending community SHSs to inform testing care pathways. We used linear and quadratic regression to model the relationship between prevalence in community and specialist SHSs in local authorities (LAs) with available surveillance data. We applied best-fitting models to predict prevalence in community SHSs in remaining LAs. Data from community SHSs were available for 102/326 LAs. There was a weak positive association between gonorrhoea prevalence in community and specialist SHSs in corresponding LAs within (R 2 = 0·13, P = 0·058) and outside (R 2 = 0·07, P = 0·02) London. Applying best-fitting models, we estimated a median gonorrhoea prevalence of 0·5% (mean 0·6%; range 0·2%-2·7%) in heterosexuals attending community SHSs. Despite some unexplained variation, our analyses suggest gonorrhoea prevalence in young heterosexuals attending community SHSs is below 1% in most English LAs. Our findings re-inforce the current national guidelines that recommend care pathways for gonorrhoea testing in community SHSs include confirmatory testing to reduce the risk of misdiagnosis and inappropriate management.

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

    PubMed

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

    2013-05-01

    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.

  10. 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.

  11. Emergence of Hepatitis C Virus Genotype 4: Phylogenetic Analysis Reveals Three Distinct Epidemiological Profiles ▿

    PubMed Central

    de Bruijne, Joep; Schinkel, Janke; Prins, Maria; Koekkoek, Sylvie M.; Aronson, Sem J.; van Ballegooijen, Marijn W.; Reesink, Hendrik W.; Molenkamp, Richard; van de Laar, Thijs J. W.

    2009-01-01

    Hepatitis C virus (HCV) genotype 4 (HCV-4) infection is considered to be difficult to treat and has become increasingly prevalent in European countries, including The Netherlands. Using a molecular epidemiological approach, the present study investigates the genetic diversity and evolutionary origin of HCV-4 in Amsterdam, The Netherlands. Phylogenetic analysis of the NS5B sequences (668 bp) obtained from 133 patients newly diagnosed with HCV-4 infection over the period from 1999 to 2008 revealed eight distinct HCV-4 subtypes; the majority of HCV-4 isolates were of subtypes 4d (57%) and 4a (37%). Three distinct monophyletic clusters were identified, with each one having a specific epidemiological profile: (i) Egyptian immigrants infected with HCV-4a (n = 46), (ii) Dutch patients with a history of injecting drug use infected with HCV-4d (n = 44), and (iii) Dutch human immunodeficiency virus (HIV)-positive men who have sex with men (MSM) infected with HCV-4d (n = 26). Subsequent molecular clock analyses confirmed that the emergence of HCV-4 within these three risk groups coincided with (i) the parenteral antischistosomal therapy campaigns in Egypt (1920 to 1960), (ii) the popularity of injecting drug use in The Netherlands (1960 to 1990), and (iii) the rise in high-risk sexual behavior among MSM after the introduction of highly active antiretroviral therapy (1996 onwards). Our data show that in addition to the influx of HCV-4 strains from countries where HCV-4 is endemic, the local spread of HCV-4d affecting injecting drug users and, in recent years, especially HIV-positive MSM will further increase the relative proportion of HCV-4-infected patients in The Netherlands. HCV-4-specific agents are drastically needed to improve treatment response rates and decrease the future burden of HCV-4-related disease. PMID:19794040

  12. Hypercontrols in Genotype-Phenotype Analysis Reveal Ancestral Haplotypes Associated With Essential Hypertension

    PubMed Central

    Balam-Ortiz, Eros; Esquivel-Villarreal, Adolfo; Huerta-Hernandez, David; Fernandez-Lopez, Juan Carlos; Alfaro-Ruiz, Luis; Muñoz-Monroy, Omar; Gutierrez, Ruth; Figueroa-Genis, Enrique; Carrillo, Karol; Elizalde, Adela; Hidalgo, Alfredo; Rodriguez, Mauricio; Urushihara, Maki; Kobori, Hiroyuki; Jimenez-Sanchez, Gerardo

    2012-01-01

    The angiotensinogen gene locus has been associated with essential hypertension in most populations analyzed to date. Increased plasma angiotensinogen levels have been proposed as an underlying cause of essential hypertension in whites; however, differences in the genetic regulation of plasma angiotensinogen levels have also been reported for other populations. The aim of this study was to analyze the relationship between angiotensinogen gene polymorphisms and haplotypes with plasma angiotensinogen levels and the risk of essential hypertension in the Mexican population. We genotyped 9 angiotensinogen gene polymorphisms in 706 individuals. Four polymorphisms, A-6, C4072, C6309, and G12775, were associated with increased risk, and the strongest association was found for the C6309 allele (χ2 = 23.9; P = 0.0000009), which resulted in an odds ratio of 3.0 (95% CI: 1.8–4.9; P = 0.000006) in the recessive model. Two polymorphisms, A-20C (P = 0.003) and C3389T (P = 0.0001), were associated with increased plasma angiotensinogen levels but did not show association with essential hypertension. The haplotypes H1 (χ2 = 8.1; P = 0.004) and H5 (χ2 = 5.1; P = 0.02) were associated with essential hypertension. Using phylogenetic analysis, we found that haplotypes 1 and 5 are the human ancestral haplotypes. Our results suggest that the positive association between angiotensinogen gene polymorphisms and haplotypes with essential hypertension is not simply explained by an increase in plasma angiotensinogen concentration. Complex interactions between risk alleles suggest that these haplotypes act as “superalleles.” PMID:22371359

  13. Hypercontrols in genotype-phenotype analysis reveal ancestral haplotypes associated with essential hypertension.

    PubMed

    Balam-Ortiz, Eros; Esquivel-Villarreal, Adolfo; Huerta-Hernandez, David; Fernandez-Lopez, Juan Carlos; Alfaro-Ruiz, Luis; Muñoz-Monroy, Omar; Gutierrez, Ruth; Figueroa-Genis, Enrique; Carrillo, Karol; Elizalde, Adela; Hidalgo, Alfredo; Rodriguez, Mauricio; Urushihara, Maki; Kobori, Hiroyuki; Jimenez-Sanchez, Gerardo

    2012-04-01

    The angiotensinogen gene locus has been associated with essential hypertension in most populations analyzed to date. Increased plasma angiotensinogen levels have been proposed as an underlying cause of essential hypertension in whites; however, differences in the genetic regulation of plasma angiotensinogen levels have also been reported for other populations. The aim of this study was to analyze the relationship between angiotensinogen gene polymorphisms and haplotypes with plasma angiotensinogen levels and the risk of essential hypertension in the Mexican population. We genotyped 9 angiotensinogen gene polymorphisms in 706 individuals. Four polymorphisms, A-6, C4072, C6309, and G12775, were associated with increased risk, and the strongest association was found for the C6309 allele (χ(2)=23.9; P=0.0000009), which resulted in an odds ratio of 3.0 (95% CI: 1.8-4.9; P=0.000006) in the recessive model. Two polymorphisms, A-20C (P=0.003) and C3389T (P=0.0001), were associated with increased plasma angiotensinogen levels but did not show association with essential hypertension. The haplotypes H1 (χ(2)=8.1; P=0.004) and H5 (χ(2)=5.1; P=0.02) were associated with essential hypertension. Using phylogenetic analysis, we found that haplotypes 1 and 5 are the human ancestral haplotypes. Our results suggest that the positive association between angiotensinogen gene polymorphisms and haplotypes with essential hypertension is not simply explained by an increase in plasma angiotensinogen concentration. Complex interactions between risk alleles suggest that these haplotypes act as "superalleles."

  14. 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.

  15. Prevalence and predictors of chlamydia co-infection among patients infected with gonorrhoea at a sexual health clinic in Sydney.

    PubMed

    Templeton, David J; Manokaran, Niveditha; O'Connor, Catherine C

    2012-09-01

    Anogenital gonorrhoea (Neisseria gonorrhoeae) is commonly diagnosed at sexual health clinics by on-site microscopy. Whether to add anti-chlamydial therapy in such situations is unclear. The medical records of all patients diagnosed with gonorrhoea between May 2005 and April 2010 at RPA Sexual Health were reviewed. Of 165 patients diagnosed with anogenital gonorrhoea, 27 (16.4%, 95% confidence interval (CI) 11.1-22.9%) were co-infected with chlamydia (Chlamydia trachomatis). Compared with those only infected with anogenital gonorrhoea, there was no correlation of anogenital gonorrhoea-chlamydia co-infection with any demographic, behavioural or clinical variables examined. Anti-chlamydial therapy should be considered for all patients with gram stain diagnosed anogenital gonorrhoea at the initial clinic visit.

  16. 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

  17. Organization and transcription of the division cell wall (dcw) cluster in Neisseria gonorrhoeae.

    PubMed

    Francis, F; Ramirez-Arcos, S; Salimnia, H; Victor, C; Dillon, J R

    2000-06-27

    A cluster of genes involved in cell division and cell wall (dcw) biosynthesis was identified in Neisseria gonorrhoeae using genomic analysis and through verification of gene order by polymerase chain reaction (PCR) analysis. The gonococcal dcw cluster consists of 17 genes, in the order 5'-mraZ-mraW-ftsI-murE-hyp1-murF- mraY-hyp2-murD-ftsW-murG-murC-ddl -ft sQ-ftsA-ftsZ-hyp3-3'. The gene organization of the dcw cluster of N. gonorrhoeae is more similar to that observed in Gram-negative rods such as Escherichia coli and Haemophilus influenzae than in Gram-positive bacteria. The cluster is characterized by several intergenic spaces. Compared with E. coli, two genes, ftsL and envA, are absent in the gonococcal dcw cluster and three hypothetical genes are novel to the cluster. The cluster is flanked by two transcriptional terminators consisting of paired neisserial uptake sequences and also includes four internal terminators, three of which are paired neisserial uptake sequences. We also found that a repeated sequence on the gonococcal genome, commonly called a Correia element, acts as the fourth transcriptional terminator. All termination sequences were shown to be fully functional by using reverse transcription PCR experiments. Transcriptional start sites upstream of ftsQ, ftsA and ftsZ were determined by primer extension and six promoters were identified; three promoters were located upstream of ftsZ in the intergenic space, two were upstream of ftsA within ftsQ and one was upstream of ftsQ within ddl. Some of these promoters were preferentially used under anaerobic conditions. The location of these promoters differed from those described in E. coli indicating dissimilar transcriptional regulation.

  18. 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

  19. The use of the MegaBACE for sequencing and genotype analysis.

    PubMed

    Burger, Pamela A

    2013-01-01

    Despite the advent of next generation sequencing techniques, which provide access to an enormous amount of genomic information in a relatively short time, the conventional Sanger sequencing and microsatellite genotyping analyses present a straightforward method to answer clearly defined questions in population genetics, phylogeography, or forensics. The MegaBACE is a platform that provides both applications with equally reliable performance. In this overview, protocols for the classical techniques of Sanger sequencing and microsatellite genotyping are described. This chapter aims to supply the user of the MegaBACE with methodological tools and some "insider" knowledge of this highly sensitive apparatus.

  20. 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.

  1. [Antimicrobal resistance of Neisseria gonorrhoeae strains in Hungary].

    PubMed

    Nemes-Nikodém, Éva; Brunner, Alexandra; Tóth, Béla; Tóth, Veronika; Bánvölgyi, András; Ostorházi, Eszter

    2015-02-08

    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.

  2. Location Contributions Determined via GGE Biplot Analysis of Multievironment Sugarcane Genotype-Performance Trials

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Selection for productive sugarcane (Saccharum spp.) cultivars in Florida has been more successful for organic than sand soils. The objectives of this study were to assess the contributions of the location with a sand soil to the final stage of multi-environment testing of sugarcane genotypes in Flor...

  3. Non-destructive Analysis Chlorophyll Content of Different Genotypes of Poplars Based on Hyperspectral Reflectance Data

    NASA Astrophysics Data System (ADS)

    Jin, S.; Dian, Y.; Wang, R.; Peng, L.; Liu, X.; Zhou, Z.; Zhong, S.; Wang, Y.

    2016-11-01

    Leaf Chlorophyll content (Ct) indicates plant physiological status and can be detected by hyperspectral measurements. However, it is difficult to conclude that different genotypes of same species have the same relationship with the hyperspectral data. The aim of this paper was to test that whether the different genotypes of same species have the similar relationship with hyperspectral reflectance. First of all, spectral reflectance of populus simonii (Populus simonii Carr) and I-72 poplar (Populus euramericana cv. ‘San Martino I-72/58’) were collected by spectrometric meter, and then extract chlorophyll index (CI) and other 11 types of vegetation indices from the hyperspectral reflectance data. At last, relationships between different vegetation indices and Ct of the two genotypes of poplar were compared. Results show that (1) the relationships between SPAD value and Ct are different in the low and high Ct level, we can choose proper vegetation index, REPIG, mSR705 and SDr/SDb et al to predict the Ct value. (2) Meanwhile, we can use PSSRb and PRI to distinguish fine difference between different genotypes.

  4. 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…

  5. 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...

  6. 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.

  7. Analysis of the entire nucleotide sequence of hepatitis B virus genotype B in the Philippines reveals a new subgenotype of genotype B.

    PubMed

    Nagasaki, Futoshi; Niitsuma, Hirofumi; Cervantes, Julieta G; Chiba, Masanori; Hong, Shan; Ojima, Toshiaki; Ueno, Yoshiyuki; Bondoc, Edgardo; Kobayashi, Koju; Ishii, Motoyasu; Shimosegawa, Tooru

    2006-05-01

    The entire nucleotide sequences were determined for hepatitis B virus (HBV) genotype B (HBV/B) genomes extracted from five patients in the Philippines and designated GenBank AB219426, AB219427, AB219428, AB219429 and AB219430. The serotype of the first four isolates was ayw and that of GenBank AB219430 was adw. Divergences of entire sequences were 1.0-2.0 % between the first four isolates and 3.8-4.2 % between these four and GenBank AB219430. Phylogenetic-tree analysis revealed that, worldwide, HBV/B comprises five subgenotypes: B1, B2, B3, B4 and the new Philippines group, designated B5. Divergences of the entire genome sequences between four isolates in subgenotype B5 and isolates from other countries (subgenotypes) were 4.4-4.8 % with Vietnam (B4), 2.9-3.5 % with Indonesia (B3), 4.7-5.1 % with China (B2) and 5.4-6.0 % with Japan (B1). Similarly, GenBank AB219430 showed the lowest divergences: 3.4 % with the isolate from Indonesia (B3), 5.0 % with Vietnam (B4), 5.4 % with China (B2) and 6.1 % with Japan (B1). This is the first report of entire nucleotide sequences of HBV/B from the Philippines and the results show that these sequences belong to a new subgenotype, B5. The present study identified that HBV/B isolates throughout the world are divided genetically into five subgenotypes, the relationships between geographical distances and the genetic distances of HBV/B being well-correlated.

  8. Protocol for the molecular detection of antibiotic resistance mechanisms in Neisseria gonorrhoeae.

    PubMed

    Goire, Namraj; Sloots, Theo P; Nissen, Michael D; Whiley, David M

    2012-01-01

    Gonorrhoea is no longer an easily treatable ailment but rather is now a challenging disease in terms of antimicrobial resistance (AMR) with treatment options rapidly diminishing. The causative agent of gonorrhoea, Neisseria gonorrhoeae, has managed to develop resistance to almost every single drug used against it with the sole exception of extended spectrum cephalosporins. The situation is further exacerbated by the fact that not only are the rates of gonococcal infections on a steady rise globally, but tracking AMR is being undermined by the growing popularity of molecular methods at the expense of traditional bacterial culture in diagnostic laboratories. Recently, concerns have been raised over the emergence of a multi-resistant gonococci and the potential for untreatable gonorrhoea. Maintaining optimal epidemiological surveillance of gonococcal AMR remains an important aspect of gonorrhoea control. The development of molecular tools for tracking AMR in N. gonorrhoeae has the potential to further enhance such surveillance. In this chapter, we discuss nucleic acid amplification-based detection of AMR in gonorrhoea with a particular emphasis on chromosomal-mediated resistance to beta-lactam antibiotics.

  9. Proteolysis of bacterial membrane proteins by Neisseria gonorrhoeae type 2 immunoglobulin A1 protease.

    PubMed Central

    Shoberg, R J; Mulks, M H

    1991-01-01

    The immunoglobulin A1 (IgA1) proteases of Neisseria gonorrhoeae have been defined as having human IgA1 as their single permissive substrate. However, in recent years there have been reports of other proteins which are susceptible to the proteolytic activity of these enzymes. To examine the possibility that gonococcal membrane proteins are potential substrates for these enzymes, isolated outer and cytoplasmic membranes of N. gonorrhoeae were treated in vitro with exogenous pure IgA1 protease. Analysis of silver-stained sodium dodecyl sulfate-polyacrylamide gels of outer membranes indicated that there were two outer membrane proteins of 78 and 68 kDa which were cleaved by IgA1 protease in vitro in GCM 740 (a wild-type strain) and in two isogenic IgA1 protease-negative variants. Similar results were observed with a second gonococcal strain, F62, and its isogenic IgA1 protease-negative derivative. When GCM 740 cytoplasmic membranes were treated with protease, three minor proteins of 24.5, 23.5, and 21.5 kDa were cleaved. In addition, when outer membranes of Escherichia coli DH1 were treated with IgA1 protease, several proteins were hydrolyzed. While the identities of all of these proteolyzed proteins are unknown, the data presented indicate that there are several proteins found in the isolated membranes of gram-negative bacteria which are permissive in vitro substrates for gonococcal IgA1 protease. Images PMID:1713195

  10. 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

  11. 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.

  12. Prediction of HIV-1 coreceptor usage (tropism) by sequence analysis using a genotypic approach.

    PubMed

    Sierra, Saleta; Kaiser, Rolf; Lübke, Nadine; Thielen, Alexander; Schuelter, Eugen; Heger, Eva; Däumer, Martin; Reuter, Stefan; Esser, Stefan; Fätkenheuer, Gerd; Pfister, Herbert; Oette, Mark; Lengauer, Thomas

    2011-12-01

    Maraviroc (MVC) is the first licensed antiretroviral drug from the class of coreceptor antagonists. It binds to the host coreceptor CCR5, which is used by the majority of HIV strains in order to infect the human immune cells (Fig. 1). Other HIV isolates use a different coreceptor, the CXCR4. Which receptor is used, is determined in the virus by the Env protein (Fig. 2). Depending on the coreceptor used, the viruses are classified as R5 or X4, respectively. MVC binds to the CCR5 receptor inhibiting the entry of R5 viruses into the target cell. During the course of disease, X4 viruses may emerge and outgrow the R5 viruses. Determination of coreceptor usage (also called tropism) is therefore mandatory prior to administration of MVC, as demanded by EMA and FDA. The studies for MVC efficiency MOTIVATE, MERIT and 1029 have been performed with the Trofile assay from Monogram, San Francisco, U.S.A. This is a high quality assay based on sophisticated recombinant tests. The acceptance for this test for daily routine is rather low outside of the U.S.A., since the European physicians rather tend to work with decentralized expert laboratories, which also provide concomitant resistance testing. These laboratories have undergone several quality assurance evaluations, the last one being presented in 2011. For several years now, we have performed tropism determinations based on sequence analysis from the HIV env-V3 gene region (V3). This region carries enough information to perform a reliable prediction. The genotypic determination of coreceptor usage presents advantages such as: shorter turnover time (equivalent to resistance testing), lower costs, possibility to adapt the results to the patients' needs and possibility of analysing clinical samples with very low or even undetectable viral load (VL), particularly since the number of samples analysed with VL < 1000 copies/μl roughly increased in the last years (Fig. 3). The main steps for tropism testing (Fig. 4) demonstrated in

  13. Recall of LCx Neisseria gonorrhoeae assay and implications for laboratory testing for N. gonorrhoeae and Chlamydia trachomatis.

    PubMed

    2002-08-16

    On July 18, 2002, Abbott Laboratories (Abbott Park, IL) initiated a voluntary recall of its LCx Neisseria gonorrhoeae Assay (List Numbers 8A48-81 and 8A48-82) because, during routine quality assurance testing, several reagent lots failed to meet the analytical sensitivity described in the product insert. The cause of the failure is under investigation by the company. Abbott Laboratories has sent a letter to its customers informing them of this recall and the specific reagent lot numbers not meeting the analytical sensitivity.

  14. Genotyping of present-day and historical Geobacillus species isolates from milk powders by high-resolution melt analysis of multiple variable-number tandem-repeat loci.

    PubMed

    Seale, R Brent; Dhakal, Rajat; Chauhan, Kanika; Craven, Heather M; Deeth, Hilton C; Pillidge, Christopher J; Powell, Ian B; Turner, Mark S

    2012-10-01

    Spores of thermophilic Geobacillus species are a common contaminant of milk powder worldwide due to their ability to form biofilms within processing plants. Genotyping methods can provide information regarding the source and monitoring of contamination. A new genotyping method was developed based on multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) in conjunction with high-resolution melt analysis (MLV-HRMA) and compared to the currently used method, randomized amplified polymorphic DNA PCR (RAPD-PCR). Four VNTR loci were identified and used to genotype 46 Geobacillus isolates obtained from retailed powder and samples from 2 different milk powder processing plants. These 46 isolates were differentiated into 16 different groups using MLV-HRMA (D = 0.89). In contrast, only 13 RAPD-PCR genotypes were identified among the 46 isolates (D = 0.79). This new method was then used to analyze 35 isolates obtained from powders with high spore counts (>10(4) spores · g(-1)) from a single processing plant together with 27 historical isolates obtained from powder samples processed in the same region of Australia 17 years ago. Results showed that three genotypes can coexist in a single processing run, while the same genotypes observed 17 years ago are present today. While certain genotypes could be responsible for powders with high spore counts, there was no correlation to specific genotypes being present in powder plants and retailed samples. In conclusion, the MLV-HRMA method is useful for genotyping Geobacillus spp. to provide insight into the prevalence and persistence of certain genotypes within milk powder processing plants.

  15. UK national guideline for the management of gonorrhoea in adults, 2011.

    PubMed

    Bignell, C; Fitzgerald, M

    2011-10-01

    The British Association for Sexual Health and HIV (BASHH) UK gonorrhoea guideline has been updated in 2011. It offers advice on diagnosis, treatment and health promotion for anogenital and pharyngeal gonorrhoea. Nucleic acid amplification tests (NAATs) are now being used more for diagnosis and are increasing detection rates in the pharynx and rectum. First line treatment using ceftriaxone with azithromycin is now advised, along with routine test of cure (TOC). The aim is to slow the spread of resistant gonorrhoea now that fewer antibiotics remain effective. A patient information leaflet has been developed.

  16. Comparative transcriptional analysis of human macrophages exposed to animal and human isolates of Mycobacterium avium subspecies paratuberculosis with diverse genotypes.

    PubMed

    Motiwala, Alifiya S; Janagama, Harish K; Paustian, Michael L; Zhu, Xiaochun; Bannantine, John P; Kapur, Vivek; Sreevatsan, Srinand

    2006-11-01

    Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease in animals and has been hypothesized to be associated with Crohn's disease in humans. Recently, M. avium subsp. paratuberculosis isolates recovered from Crohn's disease patients were shown to have limited diversity, implying the existence of human disease-associated genotypes and strain sharing with animals (A. H. Ghadiali et al., J. Clin. Microbiol. 42:5345-5348, 2004). To explore whether these genotypic differences or similarities among human and animal isolates translated to functionally significant attributes such as variance in host preference and/or difference in magnitude of infections, we performed a global scale analysis of M. avium subsp. paratuberculosis isolates that were representative of different genotypes and host species using DNA microarrays. Genome-wide characterization of the transcriptional changes was carried out using a human monocytic cell line (THP-1 cells) in response to different genotypes of M. avium subsp. paratuberculosis isolates recovered from various hosts. We identified several differentially expressed genes during early intracellular infection, including those involved in common canonical pathways such as NF-kappaB, interleukin-6 (IL-6), mitogen-activated protein kinase/extracellular signal-regulated kinase, and Jun N-terminal protein kinase signaling, as well as genes involved in T helper type 1 (Th1) responses (such as CCL5 ligand) and those that encode several proinflammatory cytokines and chemokine receptors. The cattle and human isolates of M. avium subsp. paratuberculosis, regardless of their short sequence repeat (SSR) genotype, induced similar global gene expression patterns in THP-1 cells. They differentially regulated genes necessary for cell survival without causing major alterations in proinflammatory genes. In contrast, the sheep isolates representing diverse SSR genotypes closely resembled the global gene expression pattern of an M

  17. 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.

  18. Analysis of HCV genotypes from blood donors shows three new HCV type 6 subgroups exist in Myanmar.

    PubMed

    Shinji, Toshiyuki; Kyaw, Yi Yi; Gokan, Katsunori; Tanaka, Yasuhito; Ochi, Koji; Kusano, Nobuchika; Mizushima, Takaaki; Fujioka, Shin-ichi; Shiraha, Hidenori; Lwin, Aye Aye; Shiratori, Yasushi; Mizokami, Masashi; Khin, Myo; Miyahara, Masayuki; Okada, Shigeru; Koide, Norio

    2004-06-01

    The prevalence of hepatitis C virus (HCV) genotypes in Myanmar in comparison with the rest of Southeast Asia is not well known. Serum samples were obtained from 201 HCV antibody-positive volunteer blood donors in and around the Myanmar city of Yangon. Of these, the antibody titers of 101 samples were checked by serial dilution using HCV antibody PA test II and Terasaki microplate as a low-cost method. To compare antibody titers by this method and RNA identification, we also checked HCV-RNA using the Amplicor 2.0 test. Most high-titer groups were positive for HCV-RNA. Of the 201 samples, 110 were successfully polymerase chain reaction (PCR) amplified. Among them, 35 (31.8%) were of genotype 1, 52 (47.3%) were of genotype 3, and 23 (20.9%) were of type 6 variants, and phylogenetic analysis of these type 6 variants revealed that 3 new type 6 subgroups exist in Myanmar. We named the subgroups M6-1, M6-2, and M6-3. M6-1 and M6-2 were relatively close to types 8 and 9, respectively. M6-3, though only found in one sample, was a brand-new subgroup. These subtypes were not seen in Vietnam, where type 6 group variants are widely spread. These findings may be useful for analyzing how and when these subgroups were formed.

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

    PubMed Central

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

    2014-01-01

    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 15N 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. PMID:24186940

  20. Antigenic and physical diversity of Neisseria gonorrhoeae lipooligosaccharides.

    PubMed Central

    Mandrell, R; Schneider, H; Apicella, M; Zollinger, W; Rice, P A; Griffiss, J M

    1986-01-01

    We used mouse monoclonal antibodies (MAbs) to characterize Neisseria gonorrhoeae lipooligosaccharide (LOS). LOSs that bound two or more MAbs in a solid-phase radioimmunoassay usually bound them to different LOS components, as separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); strains with multiple LOS components on SDS-PAGE usually bound more than one MAb. However, the LOS of some strains bound the same MAb to two LOS components with different relative molecular weights, and some individual LOS components bound more than one MAb. LOSs from different strains bound different amounts of the same MAb at saturation, reflecting differences in the quantitative expression of individual LOS components. Not all components recognized by MAbs were stained by silver after periodate oxidation. Treatment with NaOH variously affected epitopes defined by different MAbs. MAb 3F11 completely inhibited and MAb 2-1-L8 partially inhibited the binding of 125I-labeled 06B4 MAb to WR220 LOS and WR220 outer membranes in competitive binding studies. Other MAbs did not compete with the binding of 125I-labeled 06B4 to either antigen. We conclude that a strain of N. gonorrhoeae elaborates multiple LOSs that can be separated by SDS-PAGE and that are antigenically distinct. Epitope expression within these glycolipids is complex. Images PMID:2428752

  1. Microscopy detection of rectal gonorrhoea in asymptomatic men.

    PubMed

    Forni, J; Miles, K; Hamill, M

    2009-11-01

    This audit aimed to determine the usefulness of microscopy to detect presumptive rectal gonorrhoea (GC) infection in asymptomatic men. We retrospectively audited more than 400 male patients attending a London genitourinary medicine clinic from January 2005 to March 2007 who tested rectal culture positive for Neisseria gonorrhoeae and compared this with the microscopy detection rate. In total, 123/423 (29%) of culture positive samples were microscopy positive. Of those that tested microscopy negative (300/423), 64 (21%) were symptomatic and 236 (79%) asymptomatic. In addition, a time and motion study examined 81 rectal slides over a two-week period to identify microscopy reading time required to make a presumptive diagnosis of GC. Three slides were positive, resulting in six hours and 45 minutes to detect one positive sample. Given the low sensitivity for rectal microscopy coupled with the length of time required to obtain a presumptive positive rectal GC result, we believe rectal microscopy is no longer a cost-effective tool screening for asymptomatic men, and this report supports the BASHH guideline that it is not recommended in the management of asymptomatic rectal infection.

  2. 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.

  3. Molecular epidemiology in relation to azithromycin resistance in Neisseria gonorrhoeae isolates from Amsterdam, the Netherlands, between 2008 and 2015-a case-control study.

    PubMed

    Wind, Carolien M; Bruisten, Sylvia M; Schim van der Loeff, Maarten F; Dierdorp, Mirjam; de Vries, Henry J C; van Dam, Alje P

    2017-04-03

    Neisseria gonorrhoeae resistance to ceftriaxone and azithromycin increases, which threatens recommended dual therapy. We used molecular epidemiology to identify N. gonorrhoeae clusters, and associations with azithromycin resistance in Amsterdam, the Netherlands. N. gonorrhoeae isolates (n=143) were selected from patients visiting the Sexually Transmitted Infections Clinic, from January 2008 through September 2015. We included all 69 azithromycin resistant isolates (minimum inhibitory concentration [MIC] ≥2.0 mg/L), and 74 frequency matched susceptible controls (MIC ≤0.25 mg/L). Methods used were 23S rRNA and mtrR sequencing, N. gonorrhoeae multiantigen sequence typing (NG-MAST), multilocus variable-number of tandem repeat analysis (NG-MLVA) and a specific PCR to detect mosaic penA genes. A hierarchical cluster analysis of NG-MLVA related to resistance and epidemiological characteristics was performed. Azithromycin resistant isolates had significantly more often C2611T mutations of 23S rRNA (n=62, 89.9%, P<0.001), an NG-MAST genogroup G2992 (P<0.001), G5108 (P<0.001), or G359 (P=0.02), and were more often part of NG-MLVA clusters (P<0.001). Two resistant isolates (2.9%) had A2059G mutations, and five (7.3%) were wild-type 23S rRNA. No association was found between mtrR mutations and azithromycin resistance. Twenty-four isolates showed reduced susceptibility to extended-spectrum cephalosporins, including 10 azithromycin-resistant isolates. Of these, five contained a penA mosaic gene. Four of the five NG-MLVA clusters contained resistant and susceptible isolates. Two clusters consisting mainly of resistant isolates, included strains from MSM, heterosexual males and females. Co-occurrence of resistant and susceptible strains in NG-MLVA clusters and frequent occurrence of resistant strains outside of clusters suggests that azithromycin resistance develops independently from the 'background genome'.

  4. 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

  5. A novel high-resolution melting analysis-based method for Yersinia enterocolitica genotyping.

    PubMed

    Souza, Roberto A; Falcão, Juliana P

    2014-11-01

    Pathogenic Yersinia enterocolitica strains are associated with biotypes 1B, 2-5, while environmental strains with biotype 1A. In this work a method for Y. enterocolitica genotyping based on HRMA to determine SNPs was developed and the genetic diversity of 50 strains was determined. The strains were clustered into three groups consistent with the pathogenic profile of each biotype. The results provided a better understanding of the Y. enterocolitica genetic variability.

  6. Genomic characterization of Alzheimer's disease and genotype-related phenotypic analysis of biological markers in dementia.

    PubMed

    Cacabelos, Ramón

    2004-12-01

    More than 180 genes distributed across the human genome are potentially involved in the pathogenesis of Alzheimer's disease (AD). The AD population shows a higher genetic variation rate than the control population. Significant differences in allelic distribution and frequency exist when AD-related polygenic clusters are compared with other forms of dementia, indicating that the genetic component in neurodegenerative dementia differs from that of other CNS disorders. The characterization of AD genotype-related phenotypic profiles reveals substantial differences in biological markers among AD clusters associated with different genes and/or allelic combinations. AD and dementia with vascular component (DVC) are the most prevalent forms of dementia. Both clinical entities share many similarities, but they differ in their major phenotypic and genotypic profiles, as revealed by structural and functional genomics studies. Comparative phenotypic studies have identified significant differences in 25% of more than 100 parametric variables, including anthropometric values, cardiovascular function, blood pressure, lipid metabolism, uric acid metabolism, peripheral calcium homeostasis, liver function, alkaline phosphatase, lactate dehydrogenase, red and white blood cells, regional brain atrophy, and brain blood flow velocity. Functional genomic studies incorporating apolipoprotein E (APOE)-related changes in biological markers extended the difference between AD and DVC by up to 57%. Structural genomic studies with AD-related genes, including APP, MAPT, APOE, PS1, PS2, A2M, ACE, AGT, cFOS, and PRNP, demonstrate different genetic profiles in AD and DVC, with an absolute genetic variation rate in the range of 30-80%, depending upon genes and genetic clusters. The relative polymorphic variation in genetic clusters integrated by two, three or four genes associated with AD ranges from 1 to 3%. The main phenotypic differences in AD are genotype dependent, indicating a powerful

  7. Ice-cap. A high-throughput method for capturing plant tissue samples for genotype analysis.

    PubMed

    Krysan, Patrick

    2004-07-01

    High-throughput genotype screening is rapidly becoming a standard research tool in the post-genomic era. A major bottleneck currently exists, however, that limits the utility of this approach in the plant sciences. The rate-limiting step in current high-throughput pipelines is that tissue samples from living plants must be collected manually, one plant at a time. In this article I describe a novel method for harvesting tissue samples from living seedlings that eliminates this bottleneck. The method has been named Ice-Cap to reflect the fact that ice is used to capture the tissue samples. The planting of seeds, growth of seedlings, and harvesting of tissue are all performed in a 96-well format. I demonstrate the utility of this system by using tissue harvested by Ice-Cap to genotype a population of Arabidopsis seedlings that is segregating a previously characterized mutation. Because the harvesting of tissue is performed in a nondestructive manner, plants with the desired genotype can be transferred to soil and grown to maturity. I also show that Ice-Cap can be used to analyze genomic DNA from rice (Oryza sativa) seedlings. It is expected that this method will be applicable to high-throughput screening with many different plant species, making it a useful technology for performing marker assisted selection.

  8. Drought stress response in wheat: physiological and molecular analysis of resistant and sensitive genotypes.

    PubMed

    Rampino, Patrizia; Pataleo, Stefano; Gerardi, Carmela; Mita, Giovanni; Perrotta, Carla

    2006-12-01

    Water deficit is a severe environmental stress and the major constraint on plant productivity with an evident effect on plant growth. The aim of this work was to study Triticum and Aegilops seedlings differing in their response to drought stress at the physiological and molecular levels. The identification of resistant and sensitive genotypes was firstly based on the relative water content (RWC) measurement. Further characterization of genotypes contrasting in their response to water stress was performed at the physiological level by determination of RWC, water loss rate (WLR) and free proline content after different hours of dehydration. Modification in the expression level of five dehydrin (DHN) genes was also analysed by reverse transcription-polymerase chain reaction (RT-PCR). Five cDNAs coding for different DHNs were identified and characterized. These genes are not expressed in the well-watered plants, but only in the stressed plants. Four of these cDNAs are related to novel DHN sequences. The results obtained clearly indicate a relation between the expression of these genes and tissue water content. In particular, in the resistant genotypes the expression of DHN genes is initiated even though tissue hydration levels are still high, indicating also in wheat the involvement of these proteins in water retention.

  9. Analysis of HLA DQ alpha allele and genotype frequencies in populations from Florida.

    PubMed

    Crouse, C A; Feuer, W J; Nippes, D C; Hutto, S C; Barnes, K S; Coffman, D; Livingston, S H; Ginsberg, L; Glidewell, D E

    1994-05-01

    HLA DQ alpha allele and genotype frequencies for Caucasian, African American, Haitian, and Hispanic populations in Florida have been estimated. The Florida laboratories involved in these studies collected donor samples from a variety of sites including clinical laboratories, victim and suspect standards, blood banks, county jail detainees, and laboratory personnel. We have determined that the Caucasian and African American DQ alpha genotype frequencies do not deviate significantly from Hardy-Weinberg expectations and as a result of this heterogeneity analyses, data from the four Florida Caucasian populations may be combined and data from the four Florida African American populations may be combined to form two large HLA DQ alpha genotype frequency databanks. Further, data from the Florida Haitian population may be combined with the Florida African American population. Comparison of the combined Florida Caucasian populations, combined Florida African American populations, the Palm Beach Sheriff's Office (PBSO) Hispanic, and PBSO Haitian population with other databases does not support combination because allele frequency distributions are heterogeneous.

  10. 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.

  11. Genotyping of the protozoan pathogen Toxoplasma gondii using high-resolution melting analysis of the repeated B1 gene.

    PubMed

    Costa, Jean-Marc; Cabaret, Odile; Moukoury, Sandrine; Bretagne, Stéphane

    2011-09-01

    Genetic studies of the protozoan parasite Toxoplasma gondii have identified three main distinct types according to virulence in some hosts. Several methods have been developed to differentiate genotypes currently dominated by microsatellite markers targeting single-copy loci. We analyzed the possibility of using the 35-fold repetitive B1 gene via high-resolution melting (HRM) curve analysis. Sequencing of the B1 gene of 14 reference strains (four Type I, six Type II, and four Type III strains) identified 18 single nucleotide polymorphisms (SNP). Primers were designed to amplify eight of them for HRM analysis and for relative quantification of each nucleotide variation using SNaPshot mini-sequencing. Genotyping with five microsatellite markers was performed for comparison. Two to four HRM profiles were obtained depending on the SNP tested. The differences observed relied on the different ratios of nucleotides at the SNP locus as evidenced via SNaPshot mini-sequencing. The three main lineages could be distinguished by using several HRM profiles. Some HRM profiles proved more informative than the analysis based on five microsatellite markers, showing additional differences in Type I and Type II strains. Using HRM analysis, we obtained at least an equally good discrimination of the main lineages than that based on five microsatellite markers.

  12. GSTM1 null genotype and gastric cancer risk in the Chinese population: an updated meta-analysis and review.

    PubMed

    Zhang, Xi-Liang; Cui, Yong-Hui

    2015-01-01

    Although a number of studies have been conducted on the association between the GSTM1 null genotype and gastric cancer in People's Republic of China, this association remains elusive and controversial. To clarify the effects of the GSTM1 null genotype on the risk of gastric cancer, an updated meta-analysis was performed in the Chinese population. Related studies were identified from PubMed, Springer Link, Ovid, Chinese Wanfang Data Knowledge Service Platform, Chinese National Knowledge Infrastructure (CNKI), and Chinese Biology Medicine (CBM) up to November 5, 2014. A total of 25 studies including 3,491 cases and 5,921 controls were included in this meta-analysis. Overall, a significant association (odds ratio [OR] =1.47, 95% CI: 1.28-1.69) was found between the null GSTM1 and gastric cancer risk when all studies in Chinese population were pooled into the meta-analysis. In subgroup analyses stratified by quality score, geographic area, and source of controls, the same results were observed. Additionally, a significant association was found both in smokers and non-smokers. This meta-analysis showed that the null GSTM1 may be a potential biomarker for gastric cancer risk in Chinese, and further studies with gene-gene and gene-environment interactions are required for definite conclusions.

  13. GSTM1 null genotype and gastric cancer risk in the Chinese population: an updated meta-analysis and review

    PubMed Central

    Zhang, Xi-Liang; Cui, Yong-Hui

    2015-01-01

    Although a number of studies have been conducted on the association between the GSTM1 null genotype and gastric cancer in People’s Republic of China, this association remains elusive and controversial. To clarify the effects of the GSTM1 null genotype on the risk of gastric cancer, an updated meta-analysis was performed in the Chinese population. Related studies were identified from PubMed, Springer Link, Ovid, Chinese Wanfang Data Knowledge Service Platform, Chinese National Knowledge Infrastructure (CNKI), and Chinese Biology Medicine (CBM) up to November 5, 2014. A total of 25 studies including 3,491 cases and 5,921 controls were included in this meta-analysis. Overall, a significant association (odds ratio [OR] =1.47, 95% CI: 1.28–1.69) was found between the null GSTM1 and gastric cancer risk when all studies in Chinese population were pooled into the meta-analysis. In subgroup analyses stratified by quality score, geographic area, and source of controls, the same results were observed. Additionally, a significant association was found both in smokers and non-smokers. This meta-analysis showed that the null GSTM1 may be a potential biomarker for gastric cancer risk in Chinese, and further studies with gene–gene and gene–environment interactions are required for definite conclusions. PMID:25995643

  14. 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.

  15. 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

  16. 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

  17. Transfer of plasmid-mediated ampicillin resistance from Haemophilus to Neisseria gonorrhoeae requires an intervening organism.

    PubMed

    McNicol, P J; Albritton, W L; Ronald, A R

    1986-01-01

    Haemophilus species have been implicated as the source of plasmid-mediated ampicillin resistance in Neisseria gonorrhoeae. Previous attempts to transfer conjugally the resistance plasmids from Haemophilus species to N. gonorrhoeae have met with limited success. Using both biparental and triparental mating systems, it was found that transfer will occur if the commensal Neisseria species, Neisseria cinerea, is used as a transfer intermediate. This organism stably maintains resistance plasmids of Haemophilus and facilitates transfer of these plasmids to N. gonorrhoeae, in a triparental mating system, at a transfer frequency of 10(-8). Both Haemophilus ducreyi and N. gonorrhoeae carry mobilizing plasmids capable of mediating conjugal transfer of the same resistance plasmids. However, restriction endonuclease mapping and DNA hybridization studies indicate that the mobilizing plasmids are distinctly different molecules. Limited homology is present within the transfer region of these plasmids.

  18. 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

  19. 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

    2013-01-02

    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.

  20. High-resolution melting analysis for detection of a single-nucleotide polymorphism and the genotype of the myostatin gene in warmblood horses.

    PubMed

    Serpa, Priscila B S; Garbade, Petra; Natalini, Cláudio C; Pires, Ananda R; Tisotti, Tainor M

    2017-01-01

    OBJECTIVE To develop a high-resolution melting (HRM) assay to detect the g.66493737C>T polymorphism in the myostatin gene (MSTN) and determine the frequency of 3 previously defined g.66493737 genotypes (T/T, T/C, and C/C) in warmblood horses. SAMPLES Blood samples from 23 horses. PROCEDURES From each blood sample, DNA was extracted and analyzed by standard PCR methods and an HRM assay to determine the MSTN genotype. Three protocols (standard protocol, protocol in which a high-salt solution was added to the reaction mixture before the first melting cycle, and protocol in which an unlabeled probe was added to the reaction mixture before analysis) for the HRM assay were designed and compared. Genotype results determined by the HRM protocol that generated the most consistent melting curves were compared with those determined by sequencing. RESULTS The HRM protocol in which an unlabeled probe was added to the reaction mixture generated the most consistent melting curves. The genotypes of the g.66493737C>T polymorphism were determined for 22 horses (16 by HRM analysis and 20 by sequencing); 14, 7, and 1 had the T/T, T/C, and C/C genotypes, respectively. The genotype determined by HRM analysis agreed with that determined by sequencing for 14 of 16 horses. The frequency of alleles T and C was 79.5% and 20.5%, respectively. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that HRM analysis may be a faster and more economical alternative than PCR methods for genotyping. Genotyping results might be useful as predictors of athletic performance for horses.

  1. Examination of Neisseria Gonorrhoeae Opacity Protein Expression During Experimental Murine Genital Tract Infection

    DTIC Science & Technology

    2005-01-01

    of wild-type Chinese hamster ovary ( CHO ) cells and isogenic mutants with deficiencies in HSPG biosynthesis was used to identify the HSPG-binding...34Vitronectin mediates internalization of Neisseria gonorrhoeae by Chinese hamster ovary cells ." Infect Immun 65(3): 964-70. 57. Duensing, T. D. and J. P...Seifert, Northwestern University) was implemented to insert the opaB::phoA fusion into a non- essential locus of the genome of N. gonorrhoeae strain

  2. Efficacy of a Novel Tricyclic Topoisomerase Inhibitor in a Murine Model of Neisseria gonorrhoeae Infection

    PubMed Central

    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.

    2016-01-01

    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

  3. 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.

  4. Molecular Tracing of Hepatitis C Virus Genotype 1 Isolates in Iran: A NS5B Phylogenetic Analysis with Systematic Review

    PubMed Central

    Hesamizadeh, Khashayar; Alavian, Seyed Moayed; Najafi Tireh Shabankareh, Azar; Sharafi, Heidar

    2016-01-01

    Context Hepatitis C virus (HCV) is characterized by a high degree of genetic heterogeneity and classified into 7 genotypes and different subtypes. It heterogeneously distributed through various risk groups and geographical regions. A well-established phylogenetic relationship can simplify the tracing of HCV hierarchical strata into geographical regions. The current study aimed to find genetic phylogeny of subtypes 1a and 1b of HCV isolates based on NS5B nucleotide sequences in Iran and other members of Eastern Mediterranean regional office of world health organization, as well as other Middle Eastern countries, with a systematic review of available published and unpublished studies. Evidence Acquisition The phylogenetic analyses were performed based on the nucleotide sequences of NS5B gene of HCV genotype 1 (HCV-1), which were registered in the GenBank database. The literature review was performed in two steps: 1) searching studies evaluating the NS5B sequences of HCV-1, on PubMed, Scopus, and Web of Science, and 2) Searching sequences of unpublished studies registered in the GenBank database. Results In this study, 442 sequences from HCV-1a and 232 from HCV-1b underwent phylogenetic analysis. Phylogenetic analysis of all sequences revealed different clusters in the phylogenetic trees. The results showed that the proportion of HCV-1a and -1b isolates from Iranian patients probably originated from domestic sources. Moreover, the HCV-1b isolates from Iranian patients may have similarities with the European ones. Conclusions In this study, phylogenetic reconstruction of HCV-1 sequences clearly indicated for molecular tracing and ancestral relationships of the HCV genotypes in Iran, and showed the likelihood of domestic origin for HCV-1a and various origin for HCV-1b. PMID:28123445

  5. Oropharyngeal gonorrhoea: rate of co-infection with sexually transmitted infection, antibiotic susceptibility and treatment outcome.

    PubMed

    Manavi, K; Zafar, F; Shahid, H

    2010-02-01

    The aim of the present study is to investigate the rate of co-infections with other sexually transmitted infections (STIs), antibiotic susceptibility and management of oropharyngeal gonorrhoea diagnosed in a busy genitourinary medicine clinic. The method involved a retrospective study on consecutive patients diagnosed with oropharyngeal gonorrhoea. A total of 131 patients were diagnosed with oropharyngeal gonorrhoea over the study period. The median age of the infected patients was 28 (interquartile range: 22 to 35) years. Forty-one (31%) of patients were younger than 24 years. High rates of co-infection with urethral gonorrhoea (37%), rectal gonorrhoea (37%) or chlamydial infection (16%) were identified. Thirty patients (23%) had only oropharyngeal infection. Twenty-two (17%) patients' isolates showed resistance to at least one antibiotic. Antibiotic resistance among oropharyngeal gonococcal isolates was above 5% between 2000 and 2009. Test-of-cure (TOC) was carried out for only 63 (48%) of patients; none had positive culture. Among 46 isolates treated with cefixime 400 mg/stat, 27 (59%) had TOC; all were negative. Repeat TOC was not carried out for any of the patients. In conclusion, successful management of oropharyngeal gonorrhoea should comprise of counselling, partner notification and TOC after treatment with appropriate antibiotic regimen.

  6. Insights into the Enhanced in vivo Fitness of Neisseria gonorrhoeae Driven by a Fluoroquinolone Resistance-Conferring Mutant DNA Gyrase

    DTIC Science & Technology

    2015-02-05

    Insights into the Enhanced in vivo Fitness of Neisseria gonorrhoeae Driven by a Fluoroquinolone Resistance-Conferring Mutant DNA Gyrase...gonorrhoeae Driven by a Fluoroquinolone Resistance-Conferring Mutant DNA Gyrase" Name of Candidate: MAJ Jonathan D’ Ambrozio Doctor of Philosophy Degree...gonorrhoeae Driven by a Fluoroquinolone Resistance-Conferring Mutant DNA Gyrase" is appropriately acknowledged and, bey9nd brief excerpts, is with

  7. [Molecular epidemiology of Neisseria gonorrhoeae: effect of sex and sexual orientation on the distribution of gonococcal types].

    PubMed

    Kohl, P K; Henze, I; Kamionek, I; Krahl, D; Petzoldt, D

    1994-05-01

    Auxotype/serovar (A/S) classification enables precise characterisation of Neisseria gonorrhoeae. In the present study we evaluated whether sex and sexual preference of the patient influence the auxotype/serovar class of the infecting gonococcal strain. In male patients prototrophic/IB-3 was the most frequently isolated A/S class. By contrast, in female patients the A/S class (P)AH(U)/IA-1/2 was significantly (p < 0.005) more frequently isolated than in male patients. Analysis of our data according to sexual preference of the patients showed that in heterosexual patients the two mentioned A/S classes were leading, whereas in homo- and bisexual patients A/S classes prototrophic/IB-2 (p < 0.0001) and Pro/IB-2/16 (p < 0.0001) were isolated significantly more often. Our data are a strong indication that the host environment is also responsible for the selection of N. gonorrhoeae strains with certain typing characteristics.

  8. The risk of transmission of genital Chlamydia trachomatis infection is less than that of genital Neisseria gonorrhoeae infection.

    PubMed

    Lycke, E; Löwhagen, G B; Hallhagen, G; Johannisson, G; Ramstedt, K

    1980-01-01

    A total of 211 men with 237 female sexual partners and a total of 155 women with 156 male consorts were examined for genital infection with Chlamydia trachomatis and Neisseria gonorrhoeae. The index patients had either single chlamydial or gonococcal infections or dual infections with both microorganisms. Analysis of recovery rates for groups of sexual consorts indicated that gonorrhea was contracted more frequently than chlamydial infection. Thus, when index patients had dual infections, 45% and 28% of their female and male consorts, respectively, had chlamydial infection, but 64% and 77%, respectively, had gonorrhea. When index patients had single infections with C. trachomatis or N. gonorrhoeae, chlamydial infections were observed in consorts of 45% (women) and 28% (men), but gonococcal infections were observed in 80% (women) and 81% (men). Moreover, a significantly larger proportion of consorts of patients with chlamydial infection eluded infection than did partners of patients with gonorrhea. Women who used an intrauterine contraceptive device had chlamydial and gonococcal infections more often than those who used other forms of contraception, or no contraceptive.

  9. Genomic Epidemiology and Molecular Resistance Mechanisms of Azithromycin-Resistant Neisseria gonorrhoeae in Canada from 1997 to 2014

    PubMed Central

    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

    2016-01-01

    The emergence of Neisseria gonorrhoeae strains with decreased susceptibility to cephalosporins and azithromycin (AZM) resistance (AZMr) 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 AZMr strains. The genomes of 213 AZMr 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 AZMr (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 AZMr 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 AZMr (MICs = 2 to 4 and 8 to 32 μg/ml, respectively). Low-level AZMr 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 AZMr strains arise independently and can then rapidly expand clonally in a region through local sexual networks. PMID:26935729

  10. NK cell immunophenotypic and genotypic analysis of infants with severe respiratory syncytial virus infection.

    PubMed

    Noyola, Daniel E; Juárez-Vega, Guillermo; Monjarás-Ávila, César; Escalante-Padrón, Francisco; Rangel-Ramírez, Verónica; Cadena-Mota, Sandra; Monsiváis-Urenda, Adriana; García-Sepúlveda, Christian A; González-Amaro, Roberto

    2015-07-01

    Respiratory syncytial virus (RSV) is the leading cause of severe lower respiratory tract infection in infants. Reduced numbers of NK cells have been reported in infants with severe RSV infection; however, the precise role of NK cells during acute RSV infection is unclear. In this study the NK and T cell phenotypes, LILRB1 gene polymorphisms and KIR genotypes of infants hospitalized with RSV infection were analyzed. Compared to controls, infants with acute RSV infection showed a higher proportion of LILRB1+ T cells; in addition, a subgroup of infants with RSV infection showed an increase in LILRB1+ NK cells. No differences in NKG2C, NKG2A, or CD161 expression between RSV infected infants and controls were observed. LILRB1 genotype distribution of the rs3760860 A>G, and rs3760861 A>G single nucleotide polymorphisms differed between infants with RSV infection and healthy donors, whereas no differences in any of the KIR genes were observed. Our results suggest that LILRB1 participates in the pathogenesis of RSV infection. Further studies are needed to define the role of LILRB1+ NK in response to RSV and to confirm an association between LILRB1 polymorphisms and the risk of severe RSV infection.

  11. Combined high-resolution genotyping and geospatial analysis reveals modes of endemic urban typhoid fever transmission

    PubMed Central

    Baker, Stephen; Holt, Kathryn E.; Clements, Archie C. A.; Karkey, Abhilasha; Arjyal, Amit; Boni, Maciej F.; Dongol, Sabina; Hammond, Naomi; Koirala, Samir; Duy, Pham Thanh; Nga, Tran Vu Thieu; Campbell, James I.; Dolecek, Christiane; Basnyat, Buddha; Dougan, Gordon; Farrar, Jeremy J.

    2011-01-01

    Typhoid is a systemic infection caused by Salmonella Typhi and Salmonella Paratyphi A, human-restricted bacteria that are transmitted faeco-orally. Salmonella Typhi and S. Paratyphi A are clonal, and their limited genetic diversity has precluded the identification of long-term transmission networks in areas with a high disease burden. To improve our understanding of typhoid transmission we have taken a novel approach, performing a longitudinal spatial case–control study for typhoid in Nepal, combining single-nucleotide polymorphism genotyping and case localization via global positioning. We show extensive clustering of typhoid occurring independent of population size and density. For the first time, we demonstrate an extensive range of genotypes existing within typhoid clusters, and even within individual households, including some resulting from clonal expansion. Furthermore, although the data provide evidence for direct human-to-human transmission, we demonstrate an overwhelming contribution of indirect transmission, potentially via contaminated water. Consistent with this, we detected S. Typhi and S. Paratyphi A in water supplies and found that typhoid was spatially associated with public water sources and low elevation. These findings have implications for typhoid-control strategies, and our innovative approach may be applied to other diseases caused by other monophyletic or emerging pathogens. PMID:22645647

  12. 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

  13. Hepatitis E Virus Genotype 3 Diversity: Phylogenetic Analysis and Presence of Subtype 3b in Wild Boar in Europe

    PubMed Central

    Vina-Rodriguez, Ariel; Schlosser, Josephine; Becher, Dietmar; Kaden, Volker; Groschup, Martin H.; Eiden, Martin

    2015-01-01

    An increasing number of indigenous cases of hepatitis E caused by genotype 3 viruses (HEV-3) have been diagnosed all around the word, particularly in industrialized countries. Hepatitis E is a zoonotic disease and accumulating evidence indicates that domestic pigs and wild boars are the main reservoirs of HEV-3. A detailed analysis of HEV-3 subtypes could help to determine the interplay of human activity, the role of animals as reservoirs and cross species transmission. Although complete genome sequences are most appropriate for HEV subtype determination, in most cases only partial genomic sequences are available. We therefore carried out a subtype classification analysis, which uses regions from all three open reading frames of the genome. Using this approach, more than 1000 published HEV-3 isolates were subtyped. Newly recovered HEV partial sequences from hunted German wild boars were also included in this study. These sequences were assigned to genotype 3 and clustered within subtype 3a, 3i and, unexpectedly, one of them within the subtype 3b, a first non-human report of this subtype in Europe. PMID:26008708

  14. Hepatitis E virus genotype 3 diversity: phylogenetic analysis and presence of subtype 3b in wild boar in Europe.

    PubMed

    Vina-Rodriguez, Ariel; Schlosser, Josephine; Becher, Dietmar; Kaden, Volker; Groschup, Martin H; Eiden, Martin

    2015-05-22

    An increasing number of indigenous cases of hepatitis E caused by genotype 3 viruses (HEV-3) have been diagnosed all around the word, particularly in industrialized countries. Hepatitis E is a zoonotic disease and accumulating evidence indicates that domestic pigs and wild boars are the main reservoirs of HEV-3. A detailed analysis of HEV-3 subtypes could help to determine the interplay of human activity, the role of animals as reservoirs and cross species transmission. Although complete genome sequences are most appropriate for HEV subtype determination, in most cases only partial genomic sequences are available. We therefore carried out a subtype classification analysis, which uses regions from all three open reading frames of the genome. Using this approach, more than 1000 published HEV-3 isolates were subtyped. Newly recovered HEV partial sequences from hunted German wild boars were also included in this study. These sequences were assigned to genotype 3 and clustered within subtype 3a, 3i and, unexpectedly, one of them within the subtype 3b, a first non-human report of this subtype in Europe.

  15. A new method for ABO genotyping using fluorescence melting curve analysis based on peptide nucleic acid probes.

    PubMed

    Lee, Kyungmyung; Park, Hyun-Chul; An, Sanghyun; Ahn, Eu-Ree; Lee, Yang-Han; Kim, Mi-Jung; Lee, Eun-Jung; Park, Jae Sin; Jung, Jin Wook; Lim, Sikeun

    2015-09-01

    ABO genotyping has been routinely used to identify suspects or unknown remains in crime investigations. Probe-based fluorescence melting curve analysis (FMCA) is a powerful tool for mutation detection and is based on melting temperature shifts due to thermal denaturation. In the present study, we developed a new method for ABO genotyping using peptide nucleic acid (PNA) probe-based FMCA. This method allowed for the simultaneous detection of three single nucleotide polymorphism (SNP) sites in the ABO gene (nucleotide positions 261, 526, and 803) and the determination of 14 ABO genotypes (A/A, A/O01 or A/O02, A/O03, B/B, B/O01 or B/O02, B/O03, O01/O01 or O01/O02 or O02/O02, O01/O03 or O02/O03, O03/O03, A/B, cis-AB01/A, cis-AB01/B, cis-AB01/O01 or cis-AB01/O02, and cis-AB01/cis-AB01). Using this method, we analyzed 80 samples and successfully identified ABO genotypes (A/A [n=5], A/O01 or A/O02 [n=23], B/B [n=3], B/O01 or B/O02 [n=18], A/B [n=9], O01/O01 or O01/O02 or O02/O02 [n=20], cis-AB01/A [n=1], and cis-AB01/O01 or cis-AB01/O02 [n=1]). In addition, all steps in the method, including polymerase chain reaction, PNA probe hybridization, and FMCA, could be performed in one single closed tube in less than 3h. Since no processing or separation steps were required during analysis, this method was more convenient and rapid than traditional methods and reduced the risk of contamination. Thus, this method may be an effective and helpful tool in forensic investigations.

  16. Detecting re-infection in patients after an initial diagnosis of gonorrhoea: is routine recall for re-screening useful?

    PubMed

    Fernando, K A; Fowler, T; Harding, J; Flew, S; Caley, M; Phattey, J; Ross, Jdc

    2015-08-01

    To assess the outcome of routine sexually transmitted infection re-screening after a three-month interval in unselected patients diagnosed with gonorrhoea, we sought to assess whether this active approach would result in an increase in the number of people attending clinic and subsequently diagnosed with gonorrhoea re-infection, compared with normal re-presentation rates. A recall group of patients were invited for re-screening three months after their initial diagnosis of gonorrhoea. Permission was sought to send a reminder two weeks prior to their scheduled recall appointment. Comparisons were made with a historical control group of patients with gonorrhoea in the absence of any formal recall. Of the 242 patients in the intervention arm, 96 (40%) re-attended within six months, and 15 (6%) tested gonorrhoea positive. Two hundred and two patients were assessed in the control group, of whom 45 (22%) re-attended within six months, and 13 (6%) tested gonorrhoea positive. Women were more likely than men to re-attend following active recall, but they were not at higher risk of re-attending while re-infected with gonorrhoea. Active recall following a gonorrhoea diagnosis significantly increases re-attendance rates for repeat screening but did not result in an increased number of subsequent gonorrhoea diagnoses.

  17. High-level tetracycline resistant Neisseria gonorrhoeae isolated in Portugal.

    PubMed

    Ferreira, E; Louro, D; Gomes, J P; Catry, M A; Pato, M V

    1997-05-01

    The first high-level tetracycline resistance (MIC > or = 16 mg/l) isolates of Neisseria gonorrhoeae (TRNG) were reported in 1990 from patients attending a Sexual Transmitted Disease (STD) Center in Lisbon. The TRNG prevalence was 4% in 1991, 5.3% in 1992 and 10,8% in 1994, exploding to 52.2% in 1995. The tet M determinant was evaluated by PCR. The digests of PCRP using HpaII produced the restriction pattern 2 for all the strains, except one (pattern 3). 78.3% of the TRNG strains were beta-lactamase producers and the 4.5 MDa penicillinase plasmid was the dominant (83%), 90% and 93.3% of the TRNG strains belonged to the auxotype NR and to the serogroup IA, respectively. The IA-8/NR class represented 58.3% of the TRNG isolates, suggesting a clonal spreading.

  18. 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.

  19. The Distribution of Hepatitis C Virus Genotypes in Middle Eastern Countries: A Systematic Review and Meta-Analysis

    PubMed Central

    Ghaderi-Zefrehi, Hossein; Gholami-Fesharaki, Mohammad; Sharafi, Heidar; Sadeghi, Farzin; Alavian, Seyed Moayed

    2016-01-01

    Context The hepatitis C virus (HCV) is classified into seven genotypes and more than 100 subtypes. The treatment regimen, duration and efficacy of HCV therapy may vary according to the HCV genotype. Therefore, the HCV genotype should be determined prior to antiviral therapy. The objective of the current study was to review systematically all studies reporting the distribution of HCV genotypes in the countries that make up the Middle East. Evidence Acquisition Articles were identified by searching electronic databases, including Scopus, PubMed and Google scholar, with timeline limits (articles published between 1995 and 2016). We carried out a systematic search regarding the distribution of HCV genotypes in Middle Eastern countries. Results A total of 579 studies were identified by the electronic search. Of these, a total of 187 were identified as eligible papers including 60,319 patients who were meta-analyzed for pooled distribution of HCV genotypes. In Turkey, Israel, Cyprus, and Iran, genotype 1 was the most prevalent HCV genotype with rates of 82% (95% CI, 82%-83%), 68% (95% CI, 67%-69%), 68% (95% CI, 59%-77%), and 55% (95% CI, 54%-55%), respectively. In Egypt, Iraq, Saudi Arabia, and Syria, HCV genotype 4 was the most common genotype with rates of 86% (95% CI, 85%-88%), 60% (95% CI, 56%-64%), 56% (95% CI, 54%-55%), and 57% (95% CI, 54%-61%), respectively. On the basis of adjusted data, HCV genotype 4 was the most prevalent genotype in the Middle East region, with a rate of 74.7% (95% CI, 73.4%-76%), followed by genotype 1 at 15.1% (95% CI, 14.1%-16%). Conclusions Our results showed that HCV genotype 4 is the most prevalent genotype in the Middle East region. However, HCV genotype 1 is the most prevalent among non-Arab countries in the region including Turkey, Iran, Cyprus, and Israel. PMID:27826320

  20. Understanding the differential nitrogen sensing mechanism in rice genotypes through expression analysis of high and low affinity ammonium transporter genes.

    PubMed

    Gaur, Vikram Singh; Singh, U S; Gupta, Atul K; Kumar, Anil

    2012-03-01

    Two rice genotypes, Kalanamak 3119 (KN3119) and Pusa Basmati 1(PB1) differing in their optimum nitrogen requirements (30 and 120 kg/ha, respectively) were undertaken to study the expression of both high and low affinity ammonium transporter genes responsible for ammonium uptake. Exposing the roots of the seedlings of both the genotypes to increasing (NH(4))(2)SO(4) concentrations revealed that all the three families of rice AMT genes are expressed, some of which get altered in a genotype and concentration specific manner. This indicates that individual ammonium transporter genes have defined contributions for ammonium uptake and plant growth. Interestingly, in response to increasing nitrogen concentrations, a root specific high affinity gene, AMT1;3, was repressed in the roots of KN3119 but not in PB1 indicating the existence of a differential ammonium sensing mechanism. This also indicates that not only AMT1;3 is involved not only in ammonium uptake but may also in ammonium sensing. Further, if it can differentiate and could be used as a biomarker for nitrogen responsiveness. Expression analysis of low affinity AMT genes showed that, both AMT2;1 and AMT2;2 have high levels of expression in both roots and shoots and in KN3119 are induced at low ammonium concentrations. Expressions of AMT3 family genes were higher shoots than in the roots indicating that these genes are probably involved in the translocation and distribution of ammonium ions in leaves. The expression of the only high affinity AMT gene, AMT1;1, along with six low affinity AMT genes in the shoots suggests that low affinity AMTs in the shoots leaves are involved in supporting AMT1;1 to carry out its activities/function efficiently.

  1. 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.

  2. 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

  3. Simultaneous Detection and Genotype Determination of HSV 1 and 2 by Real-time PCR Using Melting Curve Analysis and a Unique Pair of Primers.

    PubMed

    Paryan, Mahdi; Mohammadi-Yeganeh, Samira; Rezvan, Houri; Kia, Vahid; Mansouri, Ardalan; Mirab Samiee, Siamak

    2017-02-01

    Herpes simplex virus (HSV) is a human pathogen that causes different pathologic manifestations. Rapid and feasible detection and discrimination methods for HSV genotyping is a challenge in clinical laboratories, especially in children suffering from herpetic encephalitis. A quantitative real-time polymerase chain reaction (PCR)-based genotyping assay using SYBR Green I was established. We designed only 1 pair of primer for HSV 1 and 2, targeting thymidine kinase gene conserved region. HSV genotypes were determined by PCR using melting curve analysis with LightCycler. Different HSV genotypes were successfully detected in all clinical samples. The melting temperature for HSV 1 and 2 was 85.5±0.78°C and 89±0.53°C, respectively. These 2 genotypes were completely distinguished by means of the accurate melting assay. Importantly, detection was reliably performed within only 1 hour. The assay had no cross-reactivity across species, an excellent dynamic range from 10 to 10 copies per reaction, a good intra-assay and interassay reproducibility, and a detection limit of a single copy per reaction. Our homebrew designed and validated quantitative real-time PCR followed by a melting curve analysis provided a rapid and convenient screening test for differential identification of HSV genotypes 1 and 2. We recommend the large-scale application of this method for HSV 1 and 2 detection.

  4. Comparative analysis of the complete genome sequences of Kenyan African swine fever virus isolates within p72 genotypes IX and X.

    PubMed

    Bishop, Richard P; Fleischauer, Clare; de Villiers, Etienne P; Okoth, Edward A; Arias, Marisa; Gallardo, Carmina; Upton, Chris

    2015-04-01

    Twelve complete African swine fever virus (ASFV) genome sequences are currently publicly available and these include only one sequence from East Africa. We describe genome sequencing and annotation of a recent pig-derived p72 genotype IX, and a tick-derived genotype X isolate from Kenya using the Illumina platform and comparison with the Kenya 1950 isolate. The three genomes constitute a cluster that was phylogenetically distinct from other ASFV genomes, but 98-99 % conserved within the group. Vector-based compositional analysis of the complete genomes produced a similar topology. Of the 125 previously identified 'core' ASFV genes, two ORFs of unassigned function were absent from the genotype IX sequence which was 184 kb in size as compared to 191 kb for the genotype X. There were multiple differences among East African genomes in the 360 and 110 multicopy gene families. The gene corresponding to 360-19R has transposed to the 5' variable region in both genotype X isolates. Additionally, there is a 110 ORF in the tick-derived genotype X isolate formed by fusion of 13L and 14L that is unique among ASFV genomes. In future, functional analysis based on the variations in the multicopy families may reveal whether they contribute to the observed differences in virulence between genotpye IX and X viruses.

  5. Prevalence of EBV genotypes in Polish, Taiwanese and Arabic healthy students and association between genotypes and 30-bp deletion in the LMP-1 gene phylogenetic analysis.

    PubMed

    Polz, Dorota; Podsiadło, Łukasz; Stec, Agnieszka; Polz-Dacewicz, Małgorzata

    2014-01-01

    The aim of this study was to compare the prevalence of EBV genotype and del-LMP-1 in saliva from Polish, Taiwanese and Arabic healthy students. The study group consisted of 56 healthy students; 24 of them Polish, 25 Taiwanese, and 7 Arabic. Typing was carried out using PCR with EBNA-2 primers. A detection of LMP-1 variants was also performed using PCR. EBV DNA was detected in 22 investigated samples (39.3%). Type 1 of the virus was dominant in both Polish and Taiwanese group. Among 62.5% Taiwanese with EBV 1 and 55.6% Polish detected EBV with 30-bp deletion in LMP-1 gene.

  6. Comparison of multilocus sequence analysis and virulence genotyping of Escherichia coli from live birds, retail poultry meat, and human extraintestinal infection.

    PubMed

    Danzeisen, Jessica L; Wannemuehler, Yvonne; Nolan, Lisa K; Johnson, Timothy J

    2013-03-01

    To examine the correlations between virulence genotyping and multilocus sequence analysis of Escherichia coli from poultry and humans, 88 isolates were examined. The isolates were selected from a population of over 1000 based on their assignment to nine different virulence genotyping clusters. Clustering based on multilocus sequence analysis mostly correlated with virulence genotyping, although multilocus sequence analysis demonstrated higher discriminatory ability and greater reliability related to inferred phylogenetic relationships. No distinct patterns in host source were observed using inferred phylogeny through multilocus sequence analysis, indicating that human, avian, and retail meat isolates are diverse, and some belong to multiple shared clonal complexes. Clonal complexes with host source overlap included ST95 and ST23 and additional novel groups, underscoring the diversity of avian pathogenic E. coli and the potential importance of these novel groups as avian and zoonotic pathogens.

  7. Comparative genomic hybridization and transcriptome analysis with a pan-genome microarray reveal distinctions between JP2 and non-JP2 genotypes of Aggregatibacter actinomycetemcomitans.

    PubMed

    Huang, Y; Kittichotirat, W; Mayer, M P A; Hall, R; Bumgarner, R; Chen, C

    2013-02-01

    It was postulated that the highly virulent JP2 genotype of Aggregatibacter actinomycetemcomitans may possess a constellation of distinct virulence determinants not found in non-JP2 genotypes. This study compared the genome content and the transcriptome of the serotype b JP2 genotype and the closely related serotype b non-JP2 genotype of A. actinomycetemcomitans. A custom-designed pan-genomic microarray of A. actinomycetemcomitans was constructed and validated against a panel of 11 sequenced reference strains. The microarray was subsequently used for comparative genomic hybridization of serotype b strains of JP2 (six strains) and non-JP2 (six strains) genotypes, and for transcriptome analysis of strains of JP2 (three strains) and non-JP2 (two strains). Two JP2-specific and two non-JP2-specific genomic islands were identified. In one instance, distinct genomic islands were found to be inserted into the same locus among strains of different genotypes. Transcriptome analysis identified five operons, including the leukotoxin operon, to have at least two genes with an expression ratio of 2 or greater between genotypes. Two of the differentially expressed operons were members of the membrane-bound nitrate reductase system (nap operon) and the Tol-Pal system of gram-negative bacterial species. This study is the first to demonstrate the differences in the full genome content and gene expression between A. actinomycetemcomitans strains of JP2 and non-JP2 genotypes. The information is essential for designing hypothesis-driven experiments to examine the pathogenic mechanisms of A. actinomycetemcomitans.

  8. Genotypic analysis of the earliest known prehistoric case of tuberculosis in Britain.

    PubMed

    Taylor, G Michael; Young, Douglas B; Mays, Simon A

    2005-05-01

    The earliest known case of human tuberculosis in Britain dates to the middle period of the Iron Age, approximately 2,200 years before present. Bone lesions on the spine of a male skeleton excavated at Tarrant Hinton in Dorset, United Kingdom, show evidence of Pott's disease and are supported by molecular evidence of Mycobacterium tuberculosis complex DNA amplified by IS6110 PCR (19). In the present study, we used a further series of sensitive PCR methods to confirm the diagnosis of tuberculosis and to determine the genotype of the infecting strain. These tests demonstrated that this individual was infected with a strain of M. tuberculosis rather than Mycobacterium bovis. The strain had undergone the tuberculosis D1 deletion affecting the mmpS6 and mmpL6 genes and can therefore be identified as a member of the family of "modern" M. tuberculosis isolates. All evidence obtained was consistent with surviving mycobacterial DNA being highly fragmented in this case.

  9. Genotype Correlation Analysis Reveals Pathway-Based Functional Disequilibrium and Potential Epistasis in the Human Interactome

    PubMed Central

    Bush, William S.; Haines, Jonathan L.

    2016-01-01

    Epistasis is thought to be a pervasive part of complex phenotypes due to the dynamics and complexity of biological systems, and a further understanding of epistasis in the context of biological pathways may provide insight into the etiology of complex disease. In this study, we use genotype data from the International HapMap Project to characterize the functional dependencies between alleles in the human interactome as defined by KEGG pathways. We performed chi-square tests to identify non-independence between functionally-related SNP pairs within parental Caucasian and Yoruba samples. We further refine this list by testing for skewed transmission of pseudo-haplotypes to offspring using a haplotype-based TDT test. From these analyses, we identify pathways enriched for functional disequilibrium, and a set of 863 SNP pairs (representing 453 gene pairs) showing consistent non-independence and transmission distortion. These results represent gene pairs with strong evidence of epistasis within the context of a biological function.

  10. Genetic diversity analysis of Gossypium arboreum germplasm accessions using genotyping-by-sequencing.

    PubMed

    Li, Ruijuan; Erpelding, John E

    2016-10-01

    The diploid cotton species Gossypium arboreum possesses many favorable agronomic traits such as drought tolerance and disease resistance, which can be utilized in the development of improved upland cotton cultivars. The USDA National Plant Germplasm System maintains more than 1600 G. arboreum accessions. Little information is available on the genetic diversity of the collection thereby limiting the utilization of this cotton species. The genetic diversity and population structure of the G. arboreum germplasm collection were assessed by genotyping-by-sequencing of 375 accessions. Using genome-wide single nucleotide polymorphism sequence data, two major clusters were inferred with 302 accessions in Cluster 1, 64 accessions in Cluster 2, and nine accessions unassigned due to their nearly equal membership to each cluster. These two clusters were further evaluated independently resulting in the identification of two sub-clusters for the 302 Cluster 1 accessions and three sub-clusters for the 64 Cluster 2 accessions. Low to moderate genetic diversity between clusters and sub-clusters were observed indicating a narrow genetic base. Cluster 2 accessions were more genetically diverse and the majority of the accessions in this cluster were landraces. In contrast, Cluster 1 is composed of varieties or breeding lines more recently added to the collection. The majority of the accessions had kinship values ranging from 0.6 to 0.8. Eight pairs of accessions were identified as potential redundancies due to their high kinship relatedness. The genetic diversity and genotype data from this study are essential to enhance germplasm utilization to identify genetically diverse accessions for the detection of quantitative trait loci associated with important traits that would benefit upland cotton improvement.

  11. 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.

  12. Phenotypic and genotypic analysis of pathogenic Escherichia coli virulence genes recovered from Riyadh, Saudi Arabia.

    PubMed

    Al-Arfaj, Abdullah A; Ali, Mohamed S; Hessain, Ashgan M; Zakri, Adel M; Dawoud, Turki M; Al-Maary, Khalid S; Moussa, Ihab M

    2016-11-01

    The current study was carried out to evaluate the phenotypic and genotypic characterization of avian pathogenic Escherichia coli recovered from Riyadh, Saudi Arabia. During the period of 10th February-30th May 2015, 70 E. coli strains were isolated from chicken farms located in Riyadh, Saudi Arabia. All strains were tested phenotypically by standard microbiological techniques, serotyped and the virulence genes of such strains were detected by polymerase chain reaction (PCR). Most of the recovered strains from chickens belonged to serotype O111:K58 25 strains (35.7%), followed by serotype O157:H7 13 strains (18.57%), followed by serotype O114:K90 10 strains (14.29%), then serotype O126:K71 9 strains (12.9%), serotype O78:K80 8 strains (11.43%) and in lower percentage serotype O114:K90 and O119:K69 5 strains (7.14%). The virulence genotyping of E. coli isolates recovered from broilers revealed the presence of the uidA gene in all the field isolates (6 serovars) examined in an incidence of 100%, as well as the cvaC gene was also present in all field isolates (6 serovars), while the iutA gene and the iss gene were detected in 5 out of 6 field serovars in an incidence of 81.43% and 64.29%, respectively. Phenotypical examination of the other virulence factors revealed that 65 isolates were hemolytic (92.9%), as well as 15 isolates (21.42%) were positive for enterotoxin production. Meanwhile, 21 isolates (30%) were positive for verotoxin production, 58 isolates (82.86%) for the invasiveness and 31 isolates (44.29%) for Congo red binding activities of the examined serotypes.

  13. Analysis of the 5'UTR of HCV genotype 3 grown in vitro in human B cells, T cells, and macrophages

    PubMed Central

    2010-01-01

    Background Previously, we have reported the isolation and molecular characterization of human Hepatitis C virus genotype 1 (HCV-1) from infected patients. We are now reporting an analysis of HCV obtained from patients infected with HCV genotype 3 (HCV-3) as diagnosed by clinical laboratories. Results HCV was cultured in vitro using our system. HCV RNA was isolated from patients' blood and from HCV cultured in various cell types for up to three months. The 5'UTR of these isolates were used for comparisons. Results revealed a number of sequence changes as compared to the serum RNA. The HCV RNA produced efficiently by infected macrophages, B-cells, and T-cells had sequences similar to HCV-1, which suggests that selection of the variants was performed at the level of macrophages. Virus with sequences similar to HCV-1 replicated better in macrophages than HCV having a 5'UTR similar to HCV-3. Conclusions Although HCV-3 replicates in cell types such as B-cells, T-cells, and macrophages, it may require a different primary cell type for the same purpose. Therefore, in our opinion, HCV-3 does not replicate efficiently in macrophages, and patients infected with HCV-3 may contain a population of HCV-1 in their blood. PMID:20626910

  14. 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.

  15. Genotype-Based Bayesian Analysis of Gene-Environment Interactions with Multiple Genetic Markers and Misclassification in Environmental Factors

    PubMed Central

    Lobach, Iryna; Fan, Ruzong

    2015-01-01

    A key component to understanding etiology of complex diseases, such as cancer, diabetes, alcohol dependence, is to investigate gene-environment interactions. This work is motivated by the following two concerns in the analysis of gene-environment interactions. First, multiple genetic markers in moderate linkage disequilibrium may be involved in susceptibility to a complex disease. Second, environmental factors may be subject to misclassification. We develop a genotype based Bayesian pseudolikelihood approach that accommodates linkage disequilibrium in genetic markers and misclassification in environmental factors. Since our approach is genotype based, it allows the observed genetic information to enter the model directly thus eliminating the need to infer haplotype phase and simplifying computations. Bayesian approach allows shrinking parameter estimates towards prior distribution to improve estimation and inference when environmental factors are subject to misclassification. Simulation experiments demonstrated that our method produced parameter estimates that are nearly unbiased even for small sample sizes. An application of our method is illustrated using a case-control study of interaction between early onset of drinking and genes involved in dopamine pathway. PMID:26180529

  16. Genotype-Based Bayesian Analysis of Gene-Environment Interactions with Multiple Genetic Markers and Misclassification in Environmental Factors.

    PubMed

    Lobach, Iryna; Fan, Ruzong

    A key component to understanding etiology of complex diseases, such as cancer, diabetes, alcohol dependence, is to investigate gene-environment interactions. This work is motivated by the following two concerns in the analysis of gene-environment interactions. First, multiple genetic markers in moderate linkage disequilibrium may be involved in susceptibility to a complex disease. Second, environmental factors may be subject to misclassification. We develop a genotype based Bayesian pseudolikelihood approach that accommodates linkage disequilibrium in genetic markers and misclassification in environmental factors. Since our approach is genotype based, it allows the observed genetic information to enter the model directly thus eliminating the need to infer haplotype phase and simplifying computations. Bayesian approach allows shrinking parameter estimates towards prior distribution to improve estimation and inference when environmental factors are subject to misclassification. Simulation experiments demonstrated that our method produced parameter estimates that are nearly unbiased even for small sample sizes. An application of our method is illustrated using a case-control study of interaction between early onset of drinking and genes involved in dopamine pathway.

  17. Grazoprevir and Elbasvir in Patients with Genotype 1 Hepatitis C Virus Infection: A Comprehensive Efficacy and Safety Analysis

    PubMed Central

    Yao, Yinan; Yue, Ming; Wang, Jie; Chen, Hongbo; Liu, Mei; Zang, Feng; Zhang, Yun

    2017-01-01

    Background. It is urgent for patients with hepatitis C virus (HCV) infection to find a safe, effective, and interferon-free regimen to optimize therapy. A comprehensive analysis was performed to evaluate the efficacy and safety of the grazoprevir combined with elbasvir, with or without ribavirin (RBV), in 777 treatment-naive and treatment-experienced patients with HCV genotype 1 infection from 3 randomized controlled trials (RCTs). Method. We collected data from the following trials: C-WORTHY (NCT01717326), C-SALVAGE (NCT02105454), and C-EDGE (NCT02105467). All patients received grazoprevir plus elbasvir with or without RBV for 12 or 18 weeks. The sustained virological response (SVR) 12 weeks after end of treatment was calculated for overall and subgroups. Results. 568 (73%) patients were treatment-naive. Overall, 95% (95% CI: 93–96) patients achieved SVR12, 95% (95% CI: 92–96) for treatment-naive and 96% (95% CI: 92–98) for previously treated patients, respectively. Treatment duration and treatment regimen did not have great difference in SVR12 rates. The most common AEs were fatigue (18%–29%), headache (20%), nausea (8%–14%), and asthenia (4%–12%). One patient (<1%) receiving grazoprevir plus elbasvir alone and one (<1%) receiving grazoprevir plus elbasvir plus RBV had treatment-related serious AEs. Conclusions. The result shows that 12-week grazoprevir plus elbasvir therapy is safe and effective for treatment-naive patients with HCV genotype 1. PMID:28164081

  18. Fast Technology Analysis (FTA) Enables Identification of Species and Genotypes of Latent Microsporidia Infections in Healthy Native Cameroonians

    PubMed Central

    Ndzi, Edward S.; Asonganyi, Tazoacha; Nkinin, Mary Bello; Xiao, Lihua; Didier, Elizabeth S.; Bowers, Lisa C.; Nkinin, Stephenson W.; Kaneshiro, Edna S.

    2015-01-01

    Several enteric microsporidia species have been detected in humans and other vertebrates and their identifications at the genotype level are currently being elucidated. As advanced methods, reagents, and disposal kits for detecting and identifying pathogens become commercially available, it is important to test them in settings other than in laboratories with “state-of-the-art” equipment and well-trained staff members. In the present study, we sought to detect microsporidia DNA preserved and extracted from FTA (fast technology analysis) cards spotted with human fecal suspensions obtained from Cameroonian volunteers living in the capital city of Yaoundé to preclude the need for employing spore-concentrating protocols. Further, we tested whether amplicon nucleotide sequencing approaches could be used on small aliquots taken from the cards to elucidate the diversity of microsporidia species and strains infecting native residents. Of 196 samples analyzed, 12 (6.1%) were positive for microsporidia DNA; Enterocytozoon bieneusi (Type IV and KIN-1), Encephalitozoon cuniculi, and Encephalitozoon intestinalis were identified. These data demonstrate the utility of the FTA cards in identifying genotypes of microsporidia DNA in human fecal samples that may be applied to field testing for prevalence studies. PMID:26303263

  19. Evaluation of the new AmpliSens multiplex real-time PCR assay for simultaneous detection of Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, and Trichomonas vaginalis.

    PubMed

    Rumyantseva, Tatiana; Golparian, Daniel; Nilsson, Christian S; Johansson, Emma; Falk, My; Fredlund, Hans; Van Dam, Alje; Guschin, Alexander; Unemo, Magnus

    2015-10-01

    In this study, we performed an evaluation of the new CE-marked multiplex real-time AmpliSens N.gonorrhoeae/C.trachomatis/M.genitalium/T.vaginalis-MULTIPRIME-FRT PCR assay compared to APTIMA tests, i.e., APTIMA COMBO 2 assay, APTIMA Trichomonas vaginalis assay (FDA-approved), and two different APTIMA Mycoplasma genitalium assays (research use only; one of them only used for discrepancy analysis). Vaginal swabs (n = 209) and first-void urine (FVU) specimens from females (n = 498) and males (n = 554), consecutive attendees (n = 1261) at a dermatovenerological clinic in Sweden, were examined. The sensitivity of the AmpliSens PCR assay for detection of C. trachomatis (6.3% prevalence), M. genitalium (5.7% prevalence), N. gonorrhoeae (0.3% prevalence), and T. vaginalis (0.08% prevalence) was 97.5% (95% confidence interval (CI): 91.2-99.6%), 81.9% (95% CI: 70.7-89.7%), 100% (95% CI: 40.2-100%) and 100% (95% CI: 16.5-100%), respectively. The specificity of the AmpliSens PCR assay was 100% (95% CI: 99.6-100%) for all agents. The analytical sensitivity and specificity for N. gonorrhoeae detection was excellent, i.e., 55 international gonococcal strains detected and 135 isolates of 13 non-gonococcal Neisseria species were negative. In conclusion, the multiplex real-time AmpliSens N.gonorrhoeae/C.trachomatis/M.genitalium/T.vaginalis-MULTIPRIME-FRT PCR assay demonstrated high sensitivity and excellent specificity for the detection of C. trachomatis, N. gonorrhoeae, and T. vaginalis, and excellent specificity but suboptimal sensitivity for M. genitalium detection.

  20. Hexa-acylated lipid A is required for host inflammatory response to Neisseria gonorrhoeae in experimental gonorrhea.

    PubMed

    Zhou, Xiyou; Gao, Xi; Broglie, Peter M; Kebaier, Chahnaz; Anderson, James E; Thom, Natalie; Apicella, Michael A; Sempowski, Gregory D; Duncan, Joseph A

    2014-01-01

    Neisseria gonorrhoeae causes gonorrhea, a sexually transmitted infection characterized by inflammation of the cervix or urethra. However, a significant subset of patients with N. gonorrhoeae remain asymptomatic, without evidence of localized inflammation. Inflammatory responses to N. gonorrhoeae are generated by host innate immune recognition of N. gonorrhoeae by several innate immune signaling pathways, including lipooligosaccharide (LOS) and other pathogen-derived molecules through activation of innate immune signaling systems, including toll-like receptor 4 (TLR4) and the interleukin-1β (IL-1β) processing complex known as the inflammasome. The lipooligosaccharide of N. gonorrhoeae has a hexa-acylated lipid A. N. gonorrhoeae strains that carry an inactivated msbB (also known as lpxL1) gene produce a penta-acylated lipid A and exhibit reduced biofilm formation, survival in epithelial cells, and induction of epithelial cell inflammatory signaling. We now show that msbB-deficient N. gonorrhoeae induces less inflammatory signaling in human monocytic cell lines and murine macrophages than the parent organism. The penta-acylated LOS exhibits reduced toll-like receptor 4 signaling but does not affect N. gonorrhoeae-mediated activation of the inflammasome. We demonstrate that N. gonorrhoeae msbB is dispensable for initiating and maintaining infection in a murine model of gonorrhea. Interestingly, infection with msbB-deficient N. gonorrhoeae is associated with less localized inflammation. Combined, these data suggest that TLR4-mediated recognition of N. gonorrhoeae LOS plays an important role in the pathogenesis of symptomatic gonorrhea infection and that alterations in lipid A biosynthesis may play a role in determining symptomatic and asymptomatic infections.

  1. 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

  2. Whole genome analysis of porcine astroviruses detected in Japanese pigs reveals genetic diversity and possible intra-genotypic recombination.

    PubMed

    Ito, Mika; Kuroda, Moegi; Masuda, Tsuneyuki; Akagami, Masataka; Haga, Kei; Tsuchiaka, Shinobu; Kishimoto, Mai; Naoi, Yuki; Sano, Kaori; Omatsu, Tsutomu; Katayama, Yukie; Oba, Mami; Aoki, Hiroshi; Ichimaru, Toru; Mukono, Itsuro; Ouchi, Yoshinao; Yamasato, Hiroshi; Shirai, Junsuke; Katayama, Kazuhiko; Mizutani, Tetsuya; Nagai, Makoto

    2017-02-09

    Porcine astroviruses (PoAstVs) are ubiquitous enteric virus of pigs that are distributed in several countries throughout the world. Since PoAstVs are detected in apparent healthy pigs, the clinical significance of infection is unknown. However, AstVs have recently been associated with a severe neurological disorder in animals, including humans, and zoonotic potential has been suggested. To date, little is known about the epidemiology of PoAstVs among the pig population in Japan. In this report, we present an analysis of nearly complete genomes of 36 PoAstVs detected by a metagenomics approach in the feces of Japanese pigs. Based on a phylogenetic analysis and pairwise sequence comparison, 10, 5, 15, and 6 sequences were classified as PoAstV2, PoAstV3, PoAstV4, and PoAstV5, respectively. Co-infection with two or three strains was found in individual fecal samples from eight pigs. The phylogenetic trees of ORF1a, ORF1b, and ORF2 of PoAstV2 and PoAstV4 showed differences in their topologies. The PoAstV3 and PoAstV5 strains shared high sequence identities within each genotype in all ORFs; however, one PoAstV3 strain and one PoAstV5 strain showed considerable sequence divergence from the other PoAstV3 and PoAstV5 strains, respectively, in ORF2. Recombination analysis using whole genomes revealed evidence of multiple possible intra-genotype recombination events in PoAstV2 and PoAstV4, suggesting that recombination might have contributed to the genetic diversity and played an important role in the evolution of Japanese PoAstVs.

  3. Monitoring of Transmission of Tuberculosis between Wild Boars and Cattle: Genotypical Analysis of Strains by Molecular Epidemiology Techniques

    PubMed Central

    Serraino, Andrea; Marchetti, Giulia; Sanguinetti, Valeria; Rossi, Maria Cristina; Zanoni, Renato Giulio; Catozzi, Lidia; Bandera, Alessandra; Dini, Walter; Mignone, Walter; Franzetti, Fabio; Gori, Andrea

    1999-01-01

    An epidemiological survey for the monitoring of bovine tuberculosis transmission was carried out in western Liguria, a region in northern Italy. Fifteen Mycobacterium bovis strains were isolated from 63 wild boar samples (62 from mandibular lymph nodes and 1 from a liver specimen). Sixteen mediastinal lymph nodes of 16 head of cattle were collected, and 15 Mycobacterium bovis strains were subsequently cultured. All M. bovis strains isolated from cattle and wild boars were genotyped by spoligotyping and by restriction fragment length polymorphism (RFLP) analysis with the IS6110 and IS1081 probes. All M. bovis strains showed the typical spoligotype characterized by the absence of the 39 to 43 spacers in comparison with the number in M. tuberculosis. A total of nine different clusters were identified by spoligotyping. The largest cluster included 9 strains isolated from wild boars and 11 strains isolated from cattle, thus confirming the possibility of transmission between the two animal species. Fingerprinting by RFLP analysis with the IS6110 probe showed an identical single-band pattern for 29 of 30 strains analyzed, and only 1 strain presented a five-band pattern. The use of IS1081 as a second probe was useful for differentiation of M. bovis from M. bovis BCG but not for differentiation among M. bovis strains, which presented the same undifferentiated genomic profile. In relation to the epidemiological investigation, we hypothesized that the feeding in pastures contaminated by cattle discharges could represent the most probable route of transmission of M. bovis between the two animal species. In conclusion, our results confirmed the higher discriminatory power of spoligotyping in relation to that of RFLP analysis for the differentiation of M. bovis genomic profiles. Our data showed the presence of a common M. bovis genotype in both cattle and wild boars, confirming the possible interspecies transmission of M. bovis. PMID:10449449

  4. [Analysis of newborn screening for galactosemia and genotype-phenotype of confirmed galatosemia cases].

    PubMed

    Yang, R L; Tong, F; Hong, F; Qian, G L; Wu, D W; Zhao, Z Y

    2017-02-02

    Objective: To investigate the prevalence of galactosemia(GAL), and the characteristics of genotype and phenotype of newborns who were confirmed with GAL in newborn screening in Zhejiang province. Method: The number of all live births, newborn screened infants and all clinical data of confirmed newborns with GAL from October 2013 to March 2015 were retrospectively analyzed by reviewing the data of Zhejiang Province screening center database. And the characteristics of genes and the clinical data of GAL cases who were confirmed by correlative gene test and enzyme activity measurement were analyzed. Result: The prevalence of GAL in Zhejiang province was 1/189 857. Among them, there was 1 case confirmed with GAL typeⅠ (prevalence, 1/759 428), with mutations of c. 904+ 1G>T and c. 687G>A, the enzyme activity of galactose-1-phosphate uridyltransferase (GALT) was 56.4% of controls. And there was 1 case of GAL typeⅡ(prevalence, 1/759 428), with mutations of c. 85G>T and c. 502G>A. There were 2 cases confirmed with GAL type Ⅲ(prevalence, 1/379 714), with mutations of c. 505C>T, c. 452G>A, c. 280G>A and c. 925G>A, the enzyme activity of UDP-galactose-4'-epimerase (GALE) were 42% and 38% of controls, respectively. All cases had different abnormal biochemical marks of liver function, and 1 case had combined hyperlactacidemia or hyperammonemia or increase of multiple kinds of amino acids, respectively. The newborn of GAL type Ⅱ had phacoscotasmus before treatment. All the cases were fed with lactose free milk powder, and all the abnormal parameters were improved during following up. Conclusion: The disease of GAL is rare in Zhejiang province, and its genotype distribution is scattered with comparatively mind clinical manifestations, and the cases with early treatment with lactose free milk powder have good prognosis. All cases needed to be treated and followed up for a life-long time. It is recommended that the high risk cases with GAL should be screened as soon as

  5. 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.

  6. Analysis of EEG patterns and genotypes in patients with Angelman syndrome.

    PubMed

    Vendrame, Martina; Loddenkemper, Tobias; Zarowski, Marcin; Gregas, Matt; Shuhaiber, Hans; Sarco, Dean P; Morales, Augusto; Nespeca, Mark; Sharpe, Cia; Haas, Kevin; Barnes, Gregory; Glaze, Daniel; Kothare, Sanjeev V

    2012-03-01

    We prospectively analyzed EEGs from participants in the ongoing NIH Rare Diseases Clinical Research Network Angelman Syndrome Natural History Study. Of the one-hundred-sixty enrolled patients (2006-2010), 115 had complete data (58 boys, median age 3.6 years). Distinct EEG findings were intermittent rhythmic delta waves (83.5%), interictal epileptiform discharges (74.2%), intermittent rhythmic theta waves (43.5%), and posterior rhythm slowing (43.5%). Centro-occipital and centro-temporal delta waves decreased with age (p=0.01, p=0.03). There were no specific correlations between EEG patterns and genotypes. A classification tree allowed the prediction of deletions class-1 (5.9 Mb) in patients with intermittent theta waves in <50% of EEG and interictal epileptiform abnormalities; UPD, UBE3A mutation or imprinting defects in patients with intermittent theta in <50% of EEG without interictal epileptiform abnormalities; deletions class-2 (5.0 Mb) in patients with >50% theta and normal posterior rhythm; atypical deletions in patients with >50% theta but abnormal posterior rhythm. EEG patterns are important biomarkers in Angelman syndrome and may suggest the underlying genetic etiology.

  7. 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.

  8. 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

  9. Quantitative polymerase chain reaction analysis of DNA from noninvasive samples for accurate microsatellite genotyping of wild chimpanzees (Pan troglodytes verus).

    PubMed

    Morin, P A; Chambers, K E; Boesch, C; Vigilant, L

    2001-07-01

    Noninvasive samples are useful for molecular genetic analyses of wild animal populations. However, the low DNA content of such samples makes DNA amplification difficult, and there is the potential for erroneous results when one of two alleles at heterozygous microsatellite loci fails to be amplified. In this study we describe an assay designed to measure the amount of amplifiable nuclear DNA in low DNA concentration extracts from noninvasive samples. We describe the range of DNA amounts obtained from chimpanzee faeces and shed hair samples and formulate a new efficient approach for accurate microsatellite genotyping. Prescreening of extracts for DNA quantity is recommended for sorting of samples for likely success and reliability. Repetition of results remains extensive for analysis of microsatellite amplifications beginning from low starting amounts of DNA, but is reduced for those with higher DNA content.

  10. 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.

  11. Integrative Variation Analysis Reveals that a Complex Genotype May Specify Phenotype in Siblings with Syndromic Autism Spectrum Disorder

    PubMed Central

    Kitajima, João Paulo; Tahira, Ana Carolina; Feio-dos-Santos, Ana Cecília; Fock, Rodrigo Ambrósio; Lisboa, Bianca Cristina Garcia; Simões, Sérgio Nery; Krepischi, Ana C. V.; Rosenberg, Carla; Lourenço, Naila Cristina; Passos-Bueno, Maria Rita; Brentani, Helena

    2017-01-01

    It has been proposed that copy number variations (CNVs) are associated with increased risk of autism spectrum disorder (ASD) and, in conjunction with other genetic changes, contribute to the heterogeneity of ASD phenotypes. Array comparative genomic hybridization (aCGH) and exome sequencing, together with systems genetics and network analyses, are being used as tools for the study of complex disorders of unknown etiology, especially those characterized by significant genetic and phenotypic heterogeneity. Therefore, to characterize the complex genotype-phenotype relationship, we performed aCGH and sequenced the exomes of two affected siblings with ASD symptoms, dysmorphic features, and intellectual disability, searching for de novo CNVs, as well as for de novo and rare inherited point variations—single nucleotide variants (SNVs) or small insertions and deletions (indels)—with probable functional impacts. With aCGH, we identified, in both siblings, a duplication in the 4p16.3 region and a deletion at 8p23.3, inherited by a paternal balanced translocation, t(4, 8) (p16; p23). Exome variant analysis found a total of 316 variants, of which 102 were shared by both siblings, 128 were in the male sibling exome data, and 86 were in the female exome data. Our integrative network analysis showed that the siblings’ shared translocation could explain their similar syndromic phenotype, including overgrowth, macrocephaly, and intellectual disability. However, exome data aggregate genes to those already connected from their translocation, which are important to the robustness of the network and contribute to the understanding of the broader spectrum of psychiatric symptoms. This study shows the importance of using an integrative approach to explore genotype-phenotype variability. PMID:28118382

  12. [The estimation of possibilities for the application of the laser capture microdissection technology for the molecular-genetic expert analysis (genotyping) of human chromosomal DNA].

    PubMed

    Ivanov, P L; Leonov, S N; Zemskova, E Iu

    2012-01-01

    The present study was designed to estimate the possibilities of application of the laser capture microdissection (LCM) technology for the molecular-genetic expert analysis (genotyping) of human chromosomal DNA. The experimental method employed for the purpose was the multiplex multilocus analysis of autosomal DNA polymorphism in the preparations of buccal epitheliocytes obtained by LCM. The key principles of the study were the application of physical methods for contrast enhancement of the micropreparations (such as phase-contrast microscopy and dark-field microscopy) and PCR-compatible cell lysis. Genotyping was carried out with the use of AmpFISTR Minifiler TM PCR Amplification Kits ("Applied Biosynthesis", USA). It was shown that the technique employed in the present study ensures reliable genotyping of human chromosomal DNA in the pooled preparations containing 10-20 dissected diploid cells each. This result fairly well agrees with the calculated sensitivity of the method. A few practical recommendations are offered.

  13. 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.

  14. Genetic signatures of Mycobacterium tuberculosis Nonthaburi genotype revealed by whole genome analysis of isolates from tuberculous meningitis patients in Thailand.

    PubMed

    Coker, Olabisi Oluwabukola; Chaiprasert, Angkana; 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

  15. The threat of untreatable gonorrhoea: implications and consequences for reproductive and sexual morbidity.

    PubMed

    Ndowa, Francis; Lusti-Narasimhan, Manjula

    2012-12-01

    Gonorrhoea (caused by the organism Neisseria gonorrhoeae) is one of the most commonly reported sexually transmitted infections (STIs), with 106 million new cases per year globally, according to 2008 estimates by the World Health Organization (WHO). There is growing global concern about antimicrobial resistance in N. gonorrhoeae. Only third-generation cephalosporins, the last available class of antibiotics to treat this condition, currently remain as the recommended first line treatment. If gonococcal infections become untreatable, they will cause a wide range of reproductive morbidities, including pelvic inflammatory disease, infertility and neonatal blindness. Furthermore, infection with N. gonorrhoeae facilitates the transmission of HIV. Thus, there is an urgent need to contain the threat of untreatable gonorrhoea within the framework of WHO's policy package to combat antimicrobial resistance, launched in April 2011. Interventions should take cognisance of sexual networks, international travel and reproductive commodity supplies, e.g. male and female condoms. There is also an urgent need for the identification of alternative effective treatment regimens for gonococcal infections; concerted efforts to prescribe antibiotics appropriately and ensure treatment compliance; strengthened programmes for primary prevention of STIs, including the importance of protected oral sex (fellatio); enhanced screening; development of affordable and accurate screening tests; and better surveillance and monitoring of resistance.

  16. Neisseria gonorrhoeae downregulates expression of the human antimicrobial peptide LL-37.

    PubMed

    Bergman, Peter; Johansson, Linda; Asp, Vendela; Plant, Laura; Gudmundsson, Gudmundur H; Jonsson, Ann-Beth; Agerberth, Birgitta

    2005-07-01

    Neisseria gonorrhoeae is a human pathogen causing the sexually transmitted disease gonorrhoeae. The bacteria preferentially attach to and invade epithelial cells of the genital tract. As these cells previously have been shown to express the human cathelicidin LL-37, we wanted to investigate the role of LL-37 during N. gonorrhoeae infection. The cervical epithelial cell line ME180 was utilized and the expression of LL-37 was confirmed on both peptide and transcriptional levels. Moreover, LL-37 exhibited potent in vitro activity against N. gonorrhoeae. Interestingly, the transcript and peptide levels of LL-37 were downregulated during infection, according to quantitative real-time polymerase chain reaction (PCR) and immunocyto-chemistry. The downregulation was most prominent with pathogenic strains of Neisseria, while non-pathogenic strains such as Neisseria lactamica and Escherichia coli only exhibited moderate effects. Heat-killed N. gonorrhoeae had no impact on the downregulation, emphasizing the importance of live bacteria. The results in this study suggest that pathogenic Neisseria may gain a survival advantage in the female genital tract by downregulating LL-37 expression.

  17. 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.

  18. Nested PCR for detection and genotyping of Ehrlichia ruminantium: use in genetic diversity analysis.

    PubMed

    Martinez, Dominique; Vachiéry, Nathalie; Stachurski, Frederic; Kandassamy, Yane; Raliniaina, Modestine; Aprelon, Rosalie; Gueye, Arona

    2004-10-01

    Ehrlichia ruminantium, the agent of cowdriosis transmitted by Amblyomma ticks, presents an extensive genetic and antigenic diversity of key importance for vaccine formulation. Two means of nested polymerase chain reaction (PCR) targeting were developed to conduct molecular epidemiology studies in the Caribbean and Africa. The first used a conserved DNA fragment for detection of the pathogen in animals and vectors, and the second relied on the polymorphic map1 gene for genotyping. As compared to a PCR, the nested PCR showed a 2-Log10 improvement of sensitivity and allowed amplification from ticks, blood, brain, and lungs from infected animals, providing a more accurate picture of the tick infection rate. In Guadeloupe, this rate reached 36% (N = 212) instead of 1.7% (N = 224), as previously estimated. Genetic typing was done by restriction fragment length polymorphism or sequencing of map1 amplification products. Molecular epidemiology studies conducted in field sites selected for vaccination trials with inactivated vaccine, revealed the circulation of genetically divergent strains in limited geographical areas. It is known, then, that genetic clustering based on map1 has no predictive value regarding the protective value of a given strain against a new strain. However, tracing the strains by this technique revealed the extent of E. ruminantium diversity that one can expect in a given region, and the method allows differentiation between an inadequate immune response and the challenge by a breakthrough strain on animals dying despite vaccination. Up to now, genetic typing does not avoid cross-protection studies, which were conducted in parallel, although on a more limited scale. The importance of pathogen diversity studies for optimization of vaccine design is discussed as well as the research for new polymorphic genes. These genes may allow better predictions on cross-protection, given the recent completion of the sequence of the full genome of two E. ruminantium

  19. Sequence analysis of cox1 and nad1 genes in Echinococcus granulosus G3 genotype in camels (Camelus dromedarius) from central Iran.

    PubMed

    Sharbatkhori, Mitra; Fasihi Harandi, Majid; Mirhendi, Hossein; Hajialilo, Elham; Kia, Eshrat Beigom

    2011-03-01

    Nineteen hydatid cyst isolates collected from camels in central Iran were subjected to sequences analysis of mitochondrial cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1) genes. A consensus sequence obtained containing 366 nucleotides for cox1 and 471 nucleotides for nad1 genes. Overall, the camel isolates indicated five different sequences in cox1 and nine in nad1 genes. The sequences analysis indicated that 26.3%, 42.1%, and 31.6% of isolates belonging to G1, G3, and G6 genotypes of Echinococcus granulosus, respectively. The isolates with G3 genotype indicated one cox1 sequence having 100% homology with reference G3 sequence (AN: M84663) and two different nad1 sequences, one having 100% homology with reference G3 sequence (AN: AJ237634) and the other with a silent mutation (G to A) in position 279. The presence of G3 genotype (buffalo strain) of E. granulosus as dominant genotype in camels is emphasized. As G3 genotype has formerly been reported in human, the epidemiological role of camels is warranted in future surveys.

  20. Neisseria gonorrhoeae isolates from four centres in Papua New Guinea remain susceptible to amoxycillin-clavulanate therapy.

    PubMed

    Toliman, Pamela J; Lupiwa, Tony; Law, Gregory J; Reeder, John C; Siba, Peter M

    2010-01-01

    Antibiotic-resistant strains of Neisseria gonorrhoeae have the potential to undermine treatment and control of gonorrhoea, which remains a highly prevalent sexually transmitted infection (STI) in Papua New Guinea (PNG). The standard treatment regimen for gonorrhoea in PNG based on amoxycillin and clavulanic acid (amoxycillin-clavulanate) was introduced about 15 years ago and there is some concern that over time circulating strains may have developed resistance to this therapy. To investigate this, N. gonorrhoeae isolates (n = 52) were collected from STI clinics in geographically representative centres in PNG and tested for their in vitro susceptibility to a range of antibiotics. All 52 isolates tested were found susceptible to amoxycillin-clavulanate, despite 40% (n = 21) being penicillinase producers and thus resistant to penicillin. These findings indicate that amoxycillin-clavulanate therapy remains an effective treatment for gonococcal infections in PNG, and support the maintenance of the present standard treatment for gonorrhoea in PNG.

  1. 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

  2. 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.

  3. Genomic fingerprinting of penicillinase-producing strains of Neisseria gonorrhoeae in Valencia, Spain.

    PubMed Central

    Dasi, M A; Nogueira, J M; Camarena, J J; Gil, C; García-Verdú, R; Barberá, J L; Barberá, J

    1992-01-01

    OBJECTIVE--To compare the value of different markers and their combinations with the restriction enzyme technique in the differentiations of penicillinase-producing N. gonorrhoeae (PPNG) strains. MATERIALS AND METHODS--17 PPNG strains isolated from symptomatic, untreated male patients with urethritis were characterised by antibiotic sensitivity testing, auxotyping, serotyping, plasmid profile, and restriction endonuclease fingerprinting (Hind III digestion). Cluster analysis with the method of unweighted pair-group average (UPGMA) linkage was used to calculate similarity or dissimilarity for PPNG strains. MAIN RESULTS--Either auxotyping or plasmid profile alone differentiated three groups of PPNG strains, whereas the combination auxotyping/serotyping identified 10. Although the combination auxotyping/serotyping/plasmid profile and the restriction enzyme technique showed a similar discrimination ability (differentiation of 11 PPNG strains), genomic fingerprinting gave highly specific restriction patterns on individual gonococcal isolates. CONCLUSIONS--The combination of different markers gave more epidemiological information than the use of only one. The sequence of discriminating ability for PPNG strains was: auxotyping/serotyping less than auxotyping/serotyping/plasmid profile less than restriction patterns of genomic DNA. Images PMID:1607193

  4. Manganese regulation of virulence factors and oxidative stress resistance in Neisseria gonorrhoeae

    PubMed Central

    Wu, Hsing-Ju; Seib, Kate L.; Srikhanta, Yogitha N.; Edwards, Jennifer; Kidd, Stephen P.; Maguire, Tina L .; Hamilton, Amanda; Pan, Kuan-Tin; Hsiao, He-Hsuan; Yao, Chen-Wen; Grimmond, Sean M.; Apicella, Michael A.; McEwan, Alastair G.; Wang, Andrew H-J.; Jennings, Michael P.

    2014-01-01

    Neisseria gonorrhoeae has evolved a complex and novel network of oxidative stress responses, including defense mechanisms that are dependent on manganese (Mn). We performed systematic analyses at the transcriptomic and proteomic (1D SDS-PAGE and Isotope-Coded Affinity Tag [ICAT]) levels to investigate the global expression changes that take place in a high Mn environment, which results in a Mn-dependent oxidative stress resistance phenotype. These studies revealed that 97 proteins are regulated at the post-transcriptional level under conditions of increased Mn concentration, including proteins involved in virulence (eg. Pilin, a key adhesin), oxidative stress defence (eg. superoxide dismutase), cellular metabolism, protein synthesis, RNA processing and cell division. Mn regulation of inorganic pyrophosphatase (Ppa) indicated the potential involvement of phosphate metabolism in the Mn-dependent oxidative stress defense. A detailed analysis of the role of Ppa and polyphosphate kinase (Ppk) in the gonococcal oxidative stress response revealed that ppk and ppa mutant strains showed increased resistance to oxidative stress. Investigation of these mutants grown with high Mn suggests that phosphate and pyrophosphate are involved in Mn-dependent oxidative stress resistance. PMID:20004262

  5. 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.

  6. Genotypic and phenotypic analysis of the polymorphic thiopurine S-methyltransferase gene (TPMT) in a European population

    PubMed Central

    Spire-Vayron de la Moureyre, Catherine; Debuysere, Hervé; Mastain, Bruno; Vinner, Elizabeth; Marez, Delphine; Lo Guidice, Jean-Marc; Chevalier, Dany; Brique, Serge; Motte, Kokou; Colombel, Jean-Frédéric; Turck, Dominique; Noel, Christian; Flipo, René-Marc; Pol, Annie; Lhermitte, Michel; Lafitte, Jean-Jacques; Libersa, Christian; Broly, Franck

    1998-01-01

    Characterization of allelic variants of the TPMT gene (TPMT) responsible for changes in TPMT activity, and elucidation of the mechanism by which these alleles act, are required because of the clinical importance of this polymorphism for patients receiving thiopurine drugs.We defined the mutational and allelic spectrum of TPMT in a group of 191 Europeans. Using PCR–SSCP, we screened for mutation the entire coding sequence, the exon-intron boundaries, the promoter region and the 3′-flanking region of the gene. Six mutations were detected throughout the ten exons and seven TPMT alleles were characterized. Four of them, TPMT*2, *3A, *3C and *7, harbouring the known mutations, G238C, G460A, A719G or T681G, were nonfunctional and accounted for 0.5, 5.7, 0.8 and 0.3% of the allele totality, respectively.Within the promoter region, six alleles corresponding to a variable number of tandem repeats (VNTR), were identified. VNTR*V4 and *V5a which harbour four or five repeats of a 17–18 bp unit, were the most frequent (55% and 34%, respectively). The other VNTR alleles, having from five to eight repeats, were rarer.The TPMT phenotype was correctly predicted by genotyping for 87% of individuals. A clear negative correlation between the total number of repeats from both alleles and the TPMT activity level was observed, indicating that VNTRs contribute to interindividual variations of TPMT activity. Therefore, additional analysis of the promoter region of TPMT can improve the phenotype prediction rate by genotyping. PMID:9831928

  7. Development of a Rapid High-Throughput Method for High-Resolution Melting Analysis for Routine Detection and Genotyping of Noroviruses▿

    PubMed Central

    Tajiri-Utagawa, Etsuko; Hara, Masayuki; Takahashi, Kuniaki; Watanabe, Mayumi; Wakita, Takaji

    2009-01-01

    We developed a simple, rapid, high-throughput detection and genotyping method for noroviruses using real-time reverse transcription-PCR (RT-PCR) and high-resolution melting (HRM) analysis to create a difference plot. The capsid gene was amplified by real-time RT-PCR in the presence of ResoLight HRM dye, a saturating DNA dye. Following optimization of the HRM assay conditions, the major norovirus genotypes were selected. Because we had only small quantities of the patient stool samples used in this study, we constructed plasmids for each genotype and used these to optimize the HRM assay. We selected six stool samples, each positive for one of the six dominant subtypes of noroviruses that have been circulating in Japan, namely, genotypes 4, 8, and 9 from genogroup 1 and genotypes 3, 4, and 10 from genogroup 2. The specific high-resolution derivate plot of the HRM assay for each plasmid was constructed by subtracting the melting-curve shape of the plasmid from the reference or base curve. The RNAs extracted from 14 clinical samples positive for small round structured viruses were then directly analyzed using the HRM assay. The HRM data from the clinical RNA samples corresponded with the genotype results obtained by RT-PCR and sequencing of the clinical samples. In addition, the HRM data from the clinical RNA samples corresponded with the HRM data from the six reference plasmid DNAs, indicating that this assay is useful for the direct detection and genotyping of noroviruses in clinical samples. This assay requires no multiplexing or hybridization probes and provides a new approach to the genetic screening of noroviruses in clinical virology laboratories. PMID:19073870

  8. 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

  9. Proposed interpretive criteria and quality control parameters for ofloxacin susceptibility testing of Neisseria gonorrhoeae.

    PubMed Central

    Fuchs, P C; Barry, A L; Baker, C; Murray, P R; Washington, J A

    1992-01-01

    A multilaboratory study designed to determine the in vitro susceptibility criteria and quality control parameters for ofloxacin against Neisseria gonorrhoeae was conducted according to the guidelines of the National Committee for Clinical Laboratory Standards. Proposed susceptibility breakpoints are MICs of less than or equal to 0.25 microgram/ml for the agar dilution test and greater than or equal to 31 mm for the disk diffusion test. A category for resistance could not be defined. Proposed acceptable quality control MICs for N. gonorrhoeae ATCC 49226 and Staphylococcus aureus ATCC 29213 range from 0.004 to 0.03 microgram/ml and 0.25 to 1.0 microgram/ml, respectively. With 5-micrograms ofloxacin disks, acceptable inhibitory zone diameters for S. aureus ATCC 25923 and the N. gonorrhoeae control strains range from 22 to 27 mm and 43 to 51 mm, respectively. PMID:1572960

  10. Meeting the public health challenge of multidrug- and extensively drug-resistant Neisseria gonorrhoeae.

    PubMed

    Tapsall, John W; Ndowa, Francis; Lewis, David A; Unemo, Magnus

    2009-09-01

    Globally, antimicrobial resistance (AMR) in Neisseria gonorrhoeae is increasing in prevalence, both within and across antibiotic classes, including extended-spectrum cephalosporins, raising concerns that gonorrhea may become untreatable in certain circumstances. The AMR surveillance that is essential to optimize standard treatments is often lacking or of poor quality in countries with high disease rates. Recent initiatives by the WHO to enhance global AMR surveillance that focus on multidrug- and extensively drug-resistant N. gonorrhoeae through revision of surveillance standards and use of a new panel of N. gonorrhoeae control strains are described. Keys to meeting these new challenges posed by gonococcal AMR remain the reduction in global burden of gonorrhea combined with implementation of wider strategies for general AMR control, and better understanding of mechanisms of emergence and spread of AMR.

  11. Effect of environment on sensitivity of Neisseria gonorrhoeae to Pseudomonas aeruginosa bacteriocins.

    PubMed Central

    Stein, D C; Hebeler, B H; Young, F E

    1980-01-01

    The effect of environmental variation on the susceptibility of Neisseria gonorrhoeae to pyocin produced by Pseudomonas aeruginosa was examined. Susceptibility to at least one pyocin was demonstrated in strains of N. gonorrhoeae (99%), N. meningitidis (35%), and N. lactamica (47%). The degree of sensitivity to pyocin displayed by N. gonorrhoeae was affected by varying the pH of the growth environment. Gonococcal strains were more sensitive to growth inhibition by pyocins at an alkaline pH and less sensitive to growth inhibition at an acid pH. Inhibitory titers fluctuated during nonselective subculture of fresh clinical isolates. There was no apparent correlation between auxotype and sensitivity to pyocin. Also, no relationship between colony morphology and pyocin sensitivity was seen. PMID:6783533

  12. 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.

  13. Neisseria gonorrhoeae : Detection and Typing by Probe Hybridization, LCR, and PCR.

    PubMed

    Gaydos, C A; Quinn, T C

    1999-01-01

    Neisseria gonorrhoeae, first described by Neisser in 1879, is a Gram-negative, nonmotile, nonspore-forming diplococcus, belonging to the family Neisseriaceae. It is the etiologic agent of gonorrhea. The other pathogenic species is Neisseria meningitidis, to which N. gonorrhoeae is genetically closely related. Although N. meningitidis is not usually considered to be a sexually transmitted disease, it may infect the mucous membranes of the anogenital area of homosexual men (1). The other members of the genus, which include Neisseria lactamic a, Neisseriapolysaccharea, Neisseria cinerea, and Neisseria flavescens, which are related to Neisseria gonorrhoeae, and saccharolytic strains, such as Neisseria subflava, Neisseria sicca, and Neisseria mucosa, which are less genetically related to the aforementioned, are considered to be nonpathogenic, being normal flora of the nasopharyngeal mucous membranes (2).

  14. Opa Expression Correlates with Elevated Transformation Rates in Neisseria gonorrhoeae

    PubMed Central

    Hill, Stuart A.

    2000-01-01

    Neisseria gonorrhoeae is naturally competent for DNA transformation. Under most conditions encountered in vivo, gonococci express one or more opacity (Opa) proteins on their surfaces. Recently, it was shown that DNA preferentially binds to the surfaces of Opa-expressing organisms compared to those of isogenic Opa-negative strains, presumably due to the numerous cationic residues in the predicted surface-exposed loops of the Opa protein. This study examined whether Opa-DNA interactions actually influence DNA transformation of the gonococcus. The data show that Opa-expressing gonococci are more efficient recipients of DNA for transformation and are more susceptible to exogenous DNase I treatment at early stages during the DNA transformation process than non-Opa expressors. Furthermore, inhibition of the transformation process was demonstrable for Opa+ populations when either nonspecific DNA or the polyanion heparin was used. Overall, the data suggest that Opa expression, with its presumptive positive surface charge contribution, promotes DNA transformation by causing a more prolonged sequestration of donor DNA at the cell surface, which translates into more efficient transformation over time. PMID:10613877

  15. A putatively phase variable gene (dca) required for natural competence in Neisseria gonorrhoeae but not Neisseria meningitidis is located within the division cell wall (dcw) gene cluster.

    PubMed

    Snyder, L A; Saunders, N J; Shafer, W M

    2001-02-01

    A cluster of 18 open reading frames (ORFs), 15 of which are homologous to genes involved in division and cell wall synthesis, has been identified in Neisseria gonorrhoeae and Neisseria meningitidis. The three additional ORFs, internal to the dcw cluster, are not homologous to dcw-related genes present in other bacterial species. Analysis of the N. meningitidis strain MC58 genome for foreign DNA suggests that these additional ORFs have not been acquired by recent horizontal exchange, indicating that they are a long-standing, integral part of the neisserial dcw gene cluster. Reverse transcription-PCR analysis of RNA extracted from N. gonorrhoeae strain FA19 confirmed that all three ORFs are transcribed in gonococci. One of these ORFs (dca, for division cluster competence associated), located between murE and murF, was studied in detail and found to be essential for competence in the gonococcal but not in the meningococcal strains tested. Computer analysis predicts that dca encodes an inner membrane protein similar to hypothetical proteins produced by other gram-negative bacteria. In some meningococcal strains dca is prematurely terminated following a homopolymeric tract of G's, the length of which differs between isolates of N. meningitidis, suggesting that dca is phase variable in this species. A deletion and insertional mutation was made in the dca gene of N. gonorrhoeae strain FA19 and N. meningitidis strain NMB. This mutation abrogated the ability of the gonococci to be transformed with chromosomal DNA. Thus, we conclude that the dca-encoded gene product is an essential competence factor for gonococci.

  16. 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

  17. 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.

  18. 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

  19. Whole-Genome Phylogenomic Heterogeneity of Neisseria gonorrhoeae Isolates with Decreased Cephalosporin Susceptibility Collected in Canada between 1989 and 2013

    PubMed Central

    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.

    2014-01-01

    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. PMID:25378573

  20. Prevalence of cervical Chlamydia trachomatis and Neisseria gonorrhoeae in female adolescents.

    PubMed

    Fraser, J J; Rettig, P J; Kaplan, D W

    1983-03-01

    The prevalence of cervical infection with Chlamydia trachomatis and Neisseria gonorrhoeae was examined in 125 girls receiving primary gynecologic care in a general adolescent clinic. C trachomatis was isolated in 8% of the patients using a microtiter tissue-culture method, and N gonorrhoeae was found in 12%. A significant association was found between the use of oral contraceptives and positive chlamydial cultures. Patients with Chlamydia-positive cultures were frequently asymptomatic and exhibited no positive findings on physical examination. Three of ten women with cervical chlamydial infection developed pelvic inflammatory disease. These results support the use of cervical screening for both of these pathogens in sexually active adolescents.

  1. Multicenter Investigation of Gepotidacin (GSK2140944) Agar Dilution Quality Control Determinations for Neisseria gonorrhoeae ATCC 49226

    PubMed Central

    Fedler, Kelley A.; Scangarella-Oman, Nicole E.; Ross, James E.; Flamm, Robert K.

    2016-01-01

    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

  2. Analysis of grain elements and identification of best genotypes for Fe and P in Afghan wheat landraces

    PubMed Central

    Kondou, Youichi; Manickavelu, Alagu; Komatsu, Kenji; Arifi, Mujiburahman; Kawashima, Mika; Ishii, Takayoshi; Hattori, Tomohiro; Iwata, Hiroyoshi; Tsujimoto, Hisashi; Ban, Tomohiro; Matsui, Minami

    2016-01-01

    This study was carried out with the aim of developing the methodology to determine elemental composition in wheat and identify the best germplasm for further research. Orphan and genetically diverse Afghan wheat landraces were chosen and EDXRF was used to measure the content of some of the elements to establish elemental composition in grains of 266 landraces using 10 reference lines. Four elements, K, Mg, P, and Fe, were measured by standardizing sample preparation. The results of hierarchical cluster analysis using elemental composition data sets indicated that the Fe content has an opposite pattern to the other elements, especially that of K. By systematic analysis the best wheat germplasms for P content and Fe content were identified. In order to compare the sensitivity of EDXRF, the ICP method was also used and the similar results obtained confirmed the EDXRF methodology. The sampling method for measurement using EDXRF was optimized resulting in high-throughput profiling of elemental composition in wheat grains at low cost. Using this method, we have characterized the Afghan wheat landraces and isolated the best genotypes that have high-elemental content and have the potential to be used in crop improvement. PMID:28163583

  3. 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.

  4. Development of a High-Resolution Melting Analysis Method for CYP2C19*17 Genotyping in Healthy Volunteers

    PubMed Central

    Ghasemi, Zahra; Hashemi, Mehrdad; Ejabati, Mahsa; Ebrahimi, Seyyed Meisam; Kheiri Manjili, Hamidreza; Sharafi, Ali; Ramazani, Ali

    2016-01-01

    Background: Genetic polymorphisms of drug metabolisms by cytochrome P450 (P450s) could affect drug response, attracting particular interest in the pharmacogenetics. Due to the importance of CYP2C19* 17 allele and its capability of super- fast metabolism and also lack of information about distribution of the alleles in Iranian population, this research aimed to use High Resolution Melting (HRM) method compared to PCR-RFLP for genotyping healthy Iranian population. Methods: Blood samples were collected from 100 healthy Iranian volunteers. DNA was extracted by salting out method. Real-time PCR was used for amplification of the CYP2C19 gene and the alleles were identified by HRM. Sequencing was used to confirm the amplified DNA fragments and data were analyzed using SPSS software ver.18. Results: The frequency of alleles CYP2C19*1/*1, CYP2C19*1/*17 and CYP2C19*17/*17 were estimated as 58.33, 29.1 and 11.1%, respectively. Specificity and sensitivity of HRM method were 90% and 100%, with respect to PCR-RFLP. Also, HRM analysis has been evaluated as a faster and more effective approach. Conclusion: Comparison of our results based on HRM analysis with PCR-RFLP showed that our developed method is rapid, accurate, fast and economic to study the CYP2C19*17 allele and it is appropriate for other similar population genetic studies. PMID:27920888

  5. Multiple-locus variable-number tandem repeat analysis (MLVA) of Leptospira interrogans serovar Pomona from Argentina reveals four new genotypes.

    PubMed

    Pavan, María Elisa; Cairó, Fabián; Brihuega, Bibiana; Samartino, Luis

    2008-01-01

    Outbreaks of leptospirosis occur regularly in Argentina, but little is known about their epidemiological relationships. We have analyzed the genetic diversity of a collection of 16 strains of Leptospira interrogans serovar Pomona isolated from animals and humans in Argentina during the past 45 years. Genotyping was performed by multiple-locus variable-number tandem repeat analysis (MLVA) using the loci VNTR4, VNTR7, VNTR9, VNTR10, VNTR19, VNTR23 and VNTR31, as described by Majed et al. [Identification of variable-number tandem-repeat loci in Leptospira interrogans sensu stricto. J Clin Microbiol 2005;43:539-45]. Clustering analysis revealed four new distinct MLVA genotypes, with a dominant one. Strains with this genotype were consistently isolated since 1960 to the present, mainly from cows and pigs, but also from humans, representing 75% of the total strains studied. These strains coexisted temporally and geographically with isolates presenting the other new genotypes. VNTR4 locus, with four different alleles, presented the highest diversity between the VNTR loci analyzed. MLVA patterns obtained will be useful for future diagnostic and epidemiological tracing analysis.

  6. Temperature-Dependent Growth Modeling of Environmental and Clinical Legionella pneumophila Multilocus Variable-Number Tandem-Repeat Analysis (MLVA) Genotypes.

    PubMed

    Sharaby, Yehonatan; Rodríguez-Martínez, Sarah; Oks, Olga; Pecellin, Marina; Mizrahi, Hila; Peretz, Avi; Brettar, Ingrid; Höfle, Manfred G; Halpern, Malka

    2017-04-15

    Legionella pneumophila causes waterborne infections resulting in severe pneumonia. High-resolution genotyping of L. pneumophila isolates can be achieved by multiple-locus variable-number tandem-repeat analysis (MLVA). Recently, we found that different MLVA genotypes of L. pneumophila dominated different sites in a small drinking-water network, with a genotype-related temperature and abundance regime. The present study focuses on understanding the temperature-dependent growth kinetics of the genotypes that dominated the water network. Our aim was to model mathematically the influence of temperature on the growth kinetics of different environmental and clinical L. pneumophila genotypes and to compare it with the influence of their ecological niches. Environmental strains showed a distinct temperature preference, with significant differences among the growth kinetics of the three studied genotypes (Gt4, Gt6, and Gt15). Gt4 strains exhibited superior growth at lower temperatures (25 and 30°C), while Gt15 strains appeared to be best adapted to relatively higher temperatures (42 and 45°C). The temperature-dependent growth traits of the environmental genotypes were consistent with their distribution and temperature preferences in the water network. Clinical isolates exhibited significantly higher growth rates and reached higher maximal cell densities at 37°C and 42°C than the environmental strains. Further research on the growth preferences of L. pneumophila clinical and environmental genotypes will result in a better understanding of their ecological niches in drinking-water systems as well as in the human body.IMPORTANCELegionella pneumophila is a waterborne pathogen that threatens humans in developed countries. The bacteria inhabit natural and man-made freshwater environments. Here we demonstrate that different environmental L. pneumophila genotypes have different temperature-dependent growth kinetics. Moreover, Legionella strains that belong to the same species

  7. 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,…

  8. 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…

  9. Transcriptome Analysis of Early Fruit Development in Three Seedy Citrus Genotypes and Their Seedless Mutants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Seedlessness is desirable for most citrus fruit, and identification of spontaneous or irradiated seedless mutants is important in developing citrus cultivars. We conducted a transcriptome analysis in early fruit development of three seedy citrus types (‘Fallglo’, a largely C. reticulata hybrid; ‘Pi...

  10. Path analysis and canonical correlations for indirect selection of Jatropha genotypes with higher oil yield.

    PubMed

    Silva, L A; Peixoto, L A; Teodoro, P E; Rodrigues, E V; Laviola, B G; Bhering, L L

    2017-03-22

    Jatropha is a species with great potential for biodiesel production, and the knowledge on how the main agronomic traits are correlated will contribute to its improvement. Therefore, the objectives of this study were to estimate the genetic parameters of the traits: plant height at 12 and 40 months, canopy projection on the row at 12 and 40 months, canopy projection between the row at 12 and 40 months, number of branches at 40 months, grain yield, and oil yield; to verify the existence of phenotypic correlation between these traits; to verify the influence of the morphological traits on oil yield by means of path analysis; and to evaluate the relationship between the productive traits in Jatropha and the morphological traits measured at different ages. Sixty-seven half-sib families were evaluated using a completely randomized block design with two replications and five plants per plot. Analysis of variance was used to estimate the genetic value. Phenotypic correlations were given by the Pearson correlation between traits. For the canonical correlation analysis, two groups of traits were established: group I, consisting of traits of economic importance for the culture, and group II, consisting of morphological traits. Path analysis was carried out considering oil yield as the main dependent variable. Genetic variability was observed among Jatropha families. Productive traits can be indirectly selected via morphological traits due to the correlation between these two groups of traits. Therefore, canonical correlations and path analysis are two strategies that may be useful in Jatropha-breeding program when the objective is to select productive traits via morphological traits.

  11. 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 Central

    Drali, Rezak; Benkouiten, Samir; Badiaga, Sékéné; Bitam, Idir

    2012-01-01

    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

  12. Genotyping Clostridium botulinum toxinotype A isolates from patients using amplified rDNA restriction analysis.

    PubMed

    Pourshafie, M; Vahdani, P; Popoff, M

    2005-10-01

    In this study, the application of amplified rDNA restriction analysis (ARDRA) for characterizing Clostridium botulinum toxinotype A strains isolated from individuals with botulism was evaluated. Ten restriction enzymes were tested for their suitability in ARDRA as a typing method and HhaI was selected for the best outcome. Analysis of HhaI restriction profiles of the amplified products divided C. botulinum isolates into three clusters. Non-toxigenic Clostridium sporogenes strains showed an ARDRA restriction pattern that was distinct from those observed for C. botulinum. The successful use of ARDRA for subdivision of C. botulinum in this study confirmed that this technique is a powerful method for typing of C. botulinum toxinotype A clonal diversity. In addition, it is rapid, sensitive and simple.

  13. In vitro growth of multidrug-resistant Neisseria gonorrhoeae isolates is inhibited by ETX0914, a novel spiropyrimidinetrione.

    PubMed

    Papp, John R; Lawrence, Kenneth; Sharpe, Samera; Mueller, John; Kirkcaldy, Robert D

    2016-09-01

    Antimicrobial resistance in Neisseria gonorrhoeae has severely limited the number of treatment options, and the emergence of extended-spectrum cephalosporin resistance threatens the effectiveness of the last remaining recommended treatment regimen. This study assessed the in vitro susceptibility of N. gonorrhoeae to ETX0914, a novel spiropyrimidinetrione that inhibits DNA biosynthesis. In vitro activity was determined by agar dilution against 100 N. gonorrhoeae isolates collected from men presenting with urethritis in the USA during 2012-2013 through the Gonococcal Isolate Surveillance Project. The minimum inhibitory concentration (MIC) that inhibited growth in 50% (MIC50) and 90% (MIC90) of isolates was calculated for each antimicrobial agent. ETX0914 demonstrated a high level of antimicrobial activity against N. gonorrhoeae, including isolates with decreased susceptibility or resistance to currently available agents. The ability of ETX0914 to inhibit the growth of N. gonorrhoeae was similar to ceftriaxone, which is currently recommended in combination with azithromycin to treat gonorrhoea. The data presented in this study strongly suggest that ETX0914 should be evaluated in a clinical trial for the treatment of N. gonorrhoeae.

  14. Gonorrhoea Diagnostic and Treatment Uncertainties: Risk Factors for Culture Negative Confirmation after Positive Nucleic Acid Amplification Tests.

    PubMed

    Vyth, Rebecka; Leval, Amy; Eriksson, Björn; Ericson, Eva-Lena; Marions, Lena; Hergens, Maria-Pia

    2016-01-01

    Gonorrhoea incidence has increased substantially in Stockholm during the past years. These increases have coincided with changes in testing practice from solely culture-based to nucleic acid amplification tests (NAAT). Gonorrhoea NAAT is integrated with Chlamydia trachomatis testing and due to opportunistic screening for chlamydia, testing prevalence for gonorrhoea has increased substantially in the Stockholm population. The aim of this study was to examine epidemiological risk-factors for discordant case which are NAAT positive but culture negative. These discordant cases are especially problematic as they give rise to diagnostic and treatment uncertainties with risk for subsequent sequelae. All gonorrhoea cases from Stockholm county during 2011-2012 with at least one positive N. gonorrhoea NAAT test and follow-up cultures were included (N = 874). Data were analysed using multivariate and stratified logistic regression models. Results showed that women were 4-times more likely (OR 4.9; 95% CI 2.4-6.7) than men to have discordant cultures. Individuals tested for gonorrhoea without symptoms were 2.3 times more likely (95% CI 1.5-3.5) than those with symptoms to be discordant. NAAT method and having one week or more between NAAT and culture testing were also indicative of an increased likelihood for discordance. Using NAAT should be based on proper clinical or epidemiological indications and, when positive, followed-up with a culture-based test within one week if possible. Routine gonorrhoea testing is not recommended in low prevalence populations.

  15. Neisseria gonorrhoeae Modulates Cell Death in Human Endocervical Epithelial Cells through Export of Exosome-Associated cIAP2

    PubMed Central

    Massari, Paola

    2015-01-01

    Several bacterial pathogens persist and survive in the host by modulating host cell death pathways. We previously demonstrated that Neisseria gonorrhoeae, a Gram-negative pathogen responsible for the sexually transmitted infection gonorrhea, protects against exogenous induction of apoptosis in human cervical epithelial cells. However, induction of cell death by N. gonorrhoeae has also been reported in other cell types. The mechanisms by which N. gonorrhoeae modulates cell death are not clear, although a role for the inhibitor of apoptosis-2 (cIAP2) has been proposed. In this study, we confirmed that N. gonorrhoeae induces production of cIAP2 in human cervical epithelial cells. High levels of intracellular cIAP2 were detected early after N. gonorrhoeae stimulation, which was followed by a marked decrease at 24 h. At this time point, we observed increased levels of extracellular cIAP2 associated with exosomes and an overall increase in production of exosomes. Inhibition of cIAP2 in N. gonorrhoeae-stimulated epithelial cells resulted in increased cell death and interleukin-1β (IL-1β) production. Collectively these results indicate that N. gonorrhoeae stimulation of human endocervical epithelial cells induces the release of cIAP2, an essential regulator of cell death and immune signaling. PMID:26077759

  16. Neisseria gonorrhoeae Modulates Cell Death in Human Endocervical Epithelial Cells through Export of Exosome-Associated cIAP2.

    PubMed

    Nudel, Kathleen; Massari, Paola; Genco, Caroline A

    2015-09-01

    Several bacterial pathogens persist and survive in the host by modulating host cell death pathways. We previously demonstrated that Neisseria gonorrhoeae, a Gram-negative pathogen responsible for the sexually transmitted infection gonorrhea, protects against exogenous induction of apoptosis in human cervical epithelial cells. However, induction of cell death by N. gonorrhoeae has also been reported in other cell types. The mechanisms by which N. gonorrhoeae modulates cell death are not clear, although a role for the inhibitor of apoptosis-2 (cIAP2) has been proposed. In this study, we confirmed that N. gonorrhoeae induces production of cIAP2 in human cervical epithelial cells. High levels of intracellular cIAP2 were detected early after N. gonorrhoeae stimulation, which was followed by a marked decrease at 24 h. At this time point, we observed increased levels of extracellular cIAP2 associated with exosomes and an overall increase in production of exosomes. Inhibition of cIAP2 in N. gonorrhoeae-stimulated epithelial cells resulted in increased cell death and interleukin-1β (IL-1β) production. Collectively these results indicate that N. gonorrhoeae stimulation of human endocervical epithelial cells induces the release of cIAP2, an essential regulator of cell death and immune signaling.

  17. Estimating the positive predictive value of opportunistic population testing for gonorrhoea as part of the English Chlamydia Screening Programme.

    PubMed

    Fowler, T; Edeghere, O; Inglis, N; Bradshaw, S

    2013-03-01

    Advances in technology have raised the possibility of including gonorrhoea testing as part of chlamydia screening. In England this is recommended only where the positive predictive value (PPV) of the test is ≥90%. This study assessed the PPV for gonorrhoea testing using routine testing data. Routine data (including gonorrhoea testing) from the Greater Manchester Chlamydia Screening Programme (GMCSP) in 2009/2010, were used to estimate the PPV for gonorrhoea testing. Of those screened, 0.3% (59/18044) of men and 0.4% (174/41873) of women tested positive for gonorrhoea. The PPV was 82.3% in women and 73.6% in men, in those who also tested positive for chlamydia. For women and men testing negative for chlamydia the PPV for a positive gonorrhoea test was incalculable. The low PPV observed in most groups suggests that where population testing for gonorrhoea occurs there is a need for further confirmatory testing of positive results before treatment decisions are made. Clinicians should be aware of screening test result limitations in this context.

  18. Multiple Origins and Replication Proteins Influence Biological Properties of β-Lactamase-Producing Plasmids from Neisseria gonorrhoeae

    PubMed Central

    Pagotto, Franco; Dillon, Jo-Anne R.

    2001-01-01

    The β-lactamase-producing Asia-type plasmid pJD4 of Neisseria gonorrhoeae is a 7.4-kb, broad-host-range plasmid. It is part of a family of plasmids which are structurally related yet vary in size, found in both N. gonorrhoeae and Haemophilus ducreyi. Branch-point analysis by electron microscopy indicates that pJD4 carries three clustered but distinguishable origins of replication, which we named ori1, ori2, and ori3. Although pJD4 belongs to incompatibility (Inc) group W, it also carries a silent IncFII determinant which is expressed when ori2 and ori3 are absent. The Africa-type plasmid pJD5, a naturally occurring deletion derivative of pJD4, carries only ori1, belongs to the IncFII group, and, in contrast to pJD4, requires DNA polymerase I (Pol I) for replication. Plasmids constructed from pJD4 which lack ori1 but carry ori2 and ori3 do not require Pol I and are incompatible with IncW plasmids, suggesting that the ori2 or ori3 region contains the IncW determinant. We have cloned a replication initiation protein (RepB) that is necessary for ori2 and ori3 to function. This Rep protein is distinct from RepA, which is necessary for ori1. Thus, pJD4 is unique because it is the smallest plasmid characterized containing three origins of replication and two unique Rep proteins. PMID:11544207

  19. 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.

  20. Genotyping of celiac disease-related-risk haplotypes using a closed-tube polymerase chain reaction analysis of dried blood and saliva disk samples.

    PubMed

    Ollikka, Pia; Raussi, Hanna-Mari; Laitala, Ville; Jaakkola, Lassi; Hovinen, Jari; Hemmilä, Ilkka; Ylikoski, Alice

    2009-03-01

    Expansion of molecular diagnostics more widely into clinical routines requires simplified methods allowing automation. We developed a homogeneous, multilabel polymerase chain reaction (PCR) method based on time-resolved fluorometry, and studied the use of dried disk samples in PCR. Celiac disease-related HLA-DQA1*05, HLA-DQB1*02, and HLA-DQB1*0302 genotyping was used to verify the method with blood and saliva samples dried on S&S 903 and IsoCode sample collection papers. Three sample preparation procedures, including manufacturer's manual elution, an automated elution, and direct use of disk samples, were compared using dried disk samples. The three procedures gave successful amplification and correct genotyping results. Owing to the simplicity of the direct use of disk samples in PCR, this method was chosen for the subsequent homogeneous analysis of blood (n=194) and saliva (n=30) disk samples on S&S 903 paper. The results revealed that, in addition to DNA samples (n=29), both blood and saliva disk samples were successfully amplified and genotyped using the homogeneous PCR assays for HLA-DQA1 and HLA-DQB1. The homogeneous PCR assays developed provide a useful tool to genotype celiac disease-related HLA-DQA1*05, HLA-DQB1*02, and HLA-DQB1*0302 alleles. Furthermore, the method provides a direct way to perform a closed-tube PCR analysis of dried blood and saliva disk samples enabling simple automation.

  1. Systematic review and meta-analysis of serotonin transporter genotype and discontinuation from antidepressant treatment.

    PubMed

    Crawford, Andrew A; Lewis, Glyn; Lewis, Sarah J; Munafò, Marcus R

    2013-10-01

    There is evidence that 5-HTTLPR is associated with response following treatment from selective serotonin reuptake inhibitors (SSRIs). The short (S) allele has reduced serotonin transporter expression, compared to the long (L) allele, and has been reported to be associated with poorer response in Europeans, with the effect in other populations unclear. However the published literature is inconsistent. A systematic review and meta-analysis was performed to investigate the effect of 5-HTTLPR on discontinuation from antidepressant treatment. Data were obtained from 17 studies including 4309 participants. The principal outcome measure was the allelic odds ratio (OR) for the 5-HTTLPR S allele and discontinuation status. A random effects meta-analysis provided no evidence that the S allele was associated with increased odds of discontinuation from SSRIs in Europeans (OR 1.09, 95% CI 0.83-1.42, p=0.53; 10 studies, n=2504) but in East Asians there was evidence of a reduced odds of discontinuation (OR 0.28, 95% CI 0.12-0.64, p=0.002; 2 studies, n=136). There was a suggestion of small study bias (p=0.05). This meta-analysis provides no evidence of an association between 5-HTTLPR and discontinuation from antidepressant treatment in Europeans. The low number of studies in East Asian samples using SSRIs reduces confidence in our evidence that the S allele decreases the odds of discontinuation in this population. At present, there is no evidence of an association between 5-HTTLPR and discontinuation from SSRI treatment in a European population with further studies required to investigate its effects in different populations.

  2. Human and tick spotted fever group Rickettsia isolates from Israel: a genotypic analysis.

    PubMed Central

    Manor, E; Ighbarieh, J; Sarov, B; Kassis, I; Regnery, R

    1992-01-01

    The genomes of spotted fever group rickettsiae isolated in different geographical areas of Israel (two from ticks and four from humans, obtained over a span of 20 years) were studied by polymerase chain reaction (PCR) and restriction endonuclease fragment length polymorphism (RFLP) analysis. The human isolates were obtained from patients suffering from rickettsial disease of different degrees of severity. The PCR products obtained with five pairs of oligonucleotide primers (two primer sets derived from the 190-kDa polypeptide gene and three from the 120-kDa polypeptide gene) and cleaved with restriction endonucleases were used to study the Israeli isolates and reference Rickettsia conorii isolates. Subtle differences between the PCR-RFLP patterns of Israeli isolates and the two R. conorii reference strains (Moroccan and no. 7) were seen when the PCR products derived from the 190-kDa gene-derived primer sets were digested. All of the Israeli isolates were identical by RFLP analysis using all of the primer sets. This study showed that the Israeli spotted fever group isolates (from both ticks and humans) were genetically homogeneous by the criteria used in this study, despite the time and location differences in their original isolation, and different as a group from R. conorii. Images PMID:1356998

  3. 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

  4. Sequence analysis and comparison of avian hepatitis E viruses from Australia and Europe indicate the existence of different genotypes.

    PubMed

    Bilic, Ivana; Jaskulska, Barbara; Basic, Ana; Morrow, Chris J; Hess, Michael

    2009-04-01

    Avian hepevirus infections were detected in chickens suffering from big liver and spleen disease or hepatitis-splenomegaly syndrome in Australia, the USA and Europe. Available data indicate their genetic relationship to mammalian hepatitis E virus (HEV). In the present study, the near-complete genomic sequences of an Australian and a European isolate of avian hepatitis E virus (avian HEV) are reported for the first time. Furthermore, the phylogenetic relationship to other avian HEVs is determined. Sequence analyses of these isolates identified major genetic differences among avian HEVs. Most of them are located within the open reading frame (ORF)1 region, although only a few lie within conserved motifs of predicted domains. Non-silent mutations in the ORF2 region suggest the presence of potentially different epitopes among avian HEV isolates. Finally, phylogenetic analysis confirmed the distant relationship to mammalian HEV and additionally suggested that the avian HEVs can be separated into three different genotypes: 1 (Australia), 2 (USA) and 3 (Europe), indicating a geographical distribution pattern.

  5. Genotypic analysis of HCV 1a by sequencing of the NS3 proteasic region in simeprevir therapy candidates.

    PubMed

    Liberti, Alfonso; Raddi, Adriana; Cuomo, Nunzia

    2016-12-01

    Each phase of the HCV replication cycle can represent a therapy target. In fact, SIMEPREVIR (SMV) acts as NS3/4A protease inhibitor (PI); its efficacy is, however, reduced in HCV1a patients characterized by NS3Q80K polymorphism. The aim of this work was to design a genotypic analysis of NS3 protease in order to characterize viral quasispecies in HCV 1a patients before starting the SMV therapy. In all, 38 peripheral blood-EDTA samples were collected from patients infected with HCV 1a (RNA > 10,000 cp/ml). The samples were sequenced in a region of 543 nucleotides, codifying for 181 amino acids of the NS3 protease with ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems). Of the 38 samples, two showed the Q80K mutation associated with resistance to SMV. In 16 samples mutations associated with a possible resistance to protease inhibitor, TELAPREVIR, were observed. Only one sample showed the T54S mutation, which is responsible for resistance to BOCEPREVIR, a protease inhibitor too. The data reported in this paper show a 5% prevalence of the Q80K mutation in HCV 1a patients. So far, some differences in the percentage of the Q80K mutations were observed within the European population, when compared with its US counterpart. The prevalence study described herein, albeit observed on a low number of samples, could challenge the recommendations reported in the technical data sheet of SMV.

  6. High-resolution melt PCR analysis for genotyping of Ureaplasma parvum isolates directly from clinical samples.

    PubMed

    Payne, Matthew S; Tabone, Tania; Kemp, Matthew W; Keelan, Jeffrey A; Spiller, O Brad; Newnham, John P

    2014-02-01

    Ureaplasma sp. infection in neonates and adults underlies a variety of disease pathologies. Of the two human Ureaplasma spp., Ureaplasma parvum is clinically the most common. We have developed a high-resolution melt (HRM) PCR assay for the differentiation of the four serovars of U. parvum in a single step. Currently U. parvum strains are separated into four serovars by sequencing the promoter and coding region of the multiple-banded antigen (MBA) gene. We designed primers to conserved sequences within this region for PCR amplification and HRM analysis to generate reproducible and distinct melt profiles that distinguish clonal representatives of serovars 1, 3, 6, and 14. Furthermore, our HRM PCR assay could classify DNA extracted from 74 known (MBA-sequenced) test strains with 100% accuracy. Importantly, HRM PCR was also able to identify U. parvum serovars directly from 16 clinical swabs. HRM PCR performed with DNA consisting of mixtures of combined known serovars yielded profiles that were easily distinguished from those for single-serovar controls. These profiles mirrored clinical samples that contained mixed serovars. Unfortunately, melt curve analysis software is not yet robust enough to identify the composition of mixed serovar samples, only that more than one serovar is present. HRM PCR provides a single-step, rapid, cost-effective means to differentiate the four serovars of U. parvum that did not amplify any of the known 10 serovars of Ureaplasma urealyticum tested in parallel. Choice of reaction reagents was found to be crucial to allow sufficient sensitivity to differentiate U. parvum serovars directly from clinical swabs rather than requiring cell enrichment using microbial culture techniques.

  7. Comparative evaluation of New York City and modified Thayer-Martin media for isolation of Neisseria gonorrhoeae.

    PubMed

    Greenwood, J R; Voss, J; Smith, R F; Wallace, H; Peter, C; Nachtigall, M; Maier, T; Wilber, J; Butsumyo, A

    1986-12-01

    Commercially manufactured New York City (NYC) medium and modified Thayer-Martin (MTM) medium were compared for their ability to isolate Neisseria gonorrhoeae from clinical specimens. Twenty-seven public health laboratories throughout California evaluated 4,802 specimens collected from patients attending either sexually transmitted disease or family planning clinics. Total of 726 and 737 N. gonorrhoeae isolates were recovered from NYC and MTM medium, respectively. Although less contamination was noted on NYC medium, MTM medium was equivalent to commercially prepared NYC medium for the isolation of N. gonorrhoeae from clinical specimens.

  8. The development and validation of a rapid genetic method for species identification and genotyping of medically important fungal pathogens using high-resolution melting curve analysis.

    PubMed

    Alnuaimi, A D; Wiesenfeld, D; O'Brien-Simpson, N M; Reynolds, E C; Peng, B; McCullough, M J

    2014-06-01

    Accurate, rapid and economical fungal species identification has been a major aim in mycology. In this study, our goal was to examine the feasibility of a high-resolution melting curve analysis (HRMA) of internal transcribed regions ITS1 and ITS2 in ribosomal DNA (rDNA) for a rapid, simple and inexpensive differentiation of eight clinically relevant Candida species (Candida albicans, Candida glabrata, Candida parapsilosis, Candida krusei, Candida tropicalis, Candida guilliermondii, Candida dubliniensis and Candida lusitaniae). In addition, for the first time, we tested the applicability of HRMA to classify C. albicans strains into four previously described genotypes (A, B, C and D) using a primer set that spans the transposable intron region of 25S of rDNA. Type and unknown clinical oral isolates were used in this study and the melting curve analysis was compared with both amplicons' sequencing and agarose gel electrophoresis analysis. Real-time PCR and subsequent HRMA of the two described rDNA regions generated distinct melting curve profiles that were in accord with sequencing and gel electrophoresis analysis, highly reproducible, and characteristic of each of the eight Candida species and C. albicans genotypes. Moreover, results were obtained in 4 h and without the need for any post-amplification handling, so reducing time and cost. Owing to its simplicity and speed, this technique is a good fit for genotypic analysis of hundreds of clinical strains in large epidemiological settings.

  9. Association of human papillomavirus, Neisseria gonorrhoeae and Chlamydia trachomatis co-infections on the risk of high-grade squamous intraepithelial cervical lesion

    PubMed Central

    de Abreu, André LP; Malaguti, Natália; Souza, Raquel P; Uchimura, Nelson S; Ferreira, Érika C; Pereira, Monalisa W; Carvalho, Maria DB; Pelloso, Sandra M; Bonini, Marcelo G; Gimenes, Fabrícia; Consolaro, Marcia EL

    2016-01-01

    The link between high-risk human Papillomavirus (HR-HPV) and other sexually transmitted diseases (STDs) in the risk of developing cervical cancer still unclear. Thus, in this report we investigated the rates of co-infections between HPV and other important non-HPV STDs in different cervical findings using a multiplex polymerase chain reaction (M-PCR) to simultaneously detect Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Trichomonas vaginalis, HSV-1 and -2, and Treponema pallidum. A total of 838 women aged 18 to 68 years were screened using Papanicolaou smears for cervical abnormalities, HPV and non-HPV STDs using PCR and M-PCR methods. A total of 614 (73.3%) of the women had normal cytology (NILM) and 224 (26.7%) women exhibited abnormal cytology (≥ ASC-US). HPV-DNA prevalence was 33.9%, and HPV-16 was the most prevalent genotype in women with NILM and ≥ ASC-US cytology. Non-HPV STDs were detected in 30.4% women and T. vaginalis was the most prevalent one (11.6%). A higher increased risk of ≥ ASC-US and HSIL occurred in co-infections of HR-HPV with C. trachomatis and N. gonorrhoeae. Co-infections of HPV-DNA and HR-HPV with HSV-2 exhibited a similar increased risk but only with ≥ ASC-US. Co-infections of HPV-DNA and HR-HPV with T. vaginalis demonstrated a similar increased risk of ≥ ASC-US and HSIL. We found that C. trachomatis and N. gonorrhoeae were the primary pathogens associated with HR-HPV for the increased risk for all grades of cervical abnormalities but mainly for HSIL, suggesting a possible synergistic action in cervical lesions progression. Our results reinforce the hypothesis that some non-HPV STDs might play a role as co-factors in HPV-mediated cervical carcinogenesis. These data improve our understanding of the etiology of SCC and may also be useful for disease prevention. PMID:27429850

  10. Association of human papillomavirus, Neisseria gonorrhoeae and Chlamydia trachomatis co-infections on the risk of high-grade squamous intraepithelial cervical lesion.

    PubMed

    de Abreu, André Lp; Malaguti, Natália; Souza, Raquel P; Uchimura, Nelson S; Ferreira, Érika C; Pereira, Monalisa W; Carvalho, Maria Db; Pelloso, Sandra M; Bonini, Marcelo G; Gimenes, Fabrícia; Consolaro, Marcia El

    2016-01-01

    The link between high-risk human Papillomavirus (HR-HPV) and other sexually transmitted diseases (STDs) in the risk of developing cervical cancer still unclear. Thus, in this report we investigated the rates of co-infections between HPV and other important non-HPV STDs in different cervical findings using a multiplex polymerase chain reaction (M-PCR) to simultaneously detect Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Trichomonas vaginalis, HSV-1 and -2, and Treponema pallidum. A total of 838 women aged 18 to 68 years were screened using Papanicolaou smears for cervical abnormalities, HPV and non-HPV STDs using PCR and M-PCR methods. A total of 614 (73.3%) of the women had normal cytology (NILM) and 224 (26.7%) women exhibited abnormal cytology (≥ ASC-US). HPV-DNA prevalence was 33.9%, and HPV-16 was the most prevalent genotype in women with NILM and ≥ ASC-US cytology. Non-HPV STDs were detected in 30.4% women and T. vaginalis was the most prevalent one (11.6%). A higher increased risk of ≥ ASC-US and HSIL occurred in co-infections of HR-HPV with C. trachomatis and N. gonorrhoeae. Co-infections of HPV-DNA and HR-HPV with HSV-2 exhibited a similar increased risk but only with ≥ ASC-US. Co-infections of HPV-DNA and HR-HPV with T. vaginalis demonstrated a similar increased risk of ≥ ASC-US and HSIL. We found that C. trachomatis and N. gonorrhoeae were the primary pathogens associated with HR-HPV for the increased risk for all grades of cervical abnormalities but mainly for HSIL, suggesting a possible synergistic action in cervical lesions progression. Our results reinforce the hypothesis that some non-HPV STDs might play a role as co-factors in HPV-mediated cervical carcinogenesis. These data improve our understanding of the etiology of SCC and may also be useful for disease prevention.

  11. Metronidazole and spiramycin therapy of mixed Bacteroides spp. and Neisseria gonorrhoeae infection in mice.

    PubMed

    Brook, I

    1989-01-01

    The in vitro and in vivo activity of metronidazole and spiramycin, used singly or in combination, was tested in the eradication of infection caused by Bacteroides spp. and Neisseria gonorrhoeae alone or in combination. The in vitro tests consisted of determinations of the minimal inhibitory concentrations (MIC), carried out with or without the addition of a constant amount of the other antimicrobials. The MIC of both Bacteroides bivius and Bacteroides fragilis for metronidazole were significantly reduced by the addition of spiramycin (from 0.5 to 0.125 micrograms/ml). The in vivo tests were carried out in mice and consisted of measurements of the effects of the antimicrobial agents on the bacterial contents of abscesses induced by subcutaneous injection of bacterial suspension. Synergism between metronidazole and spiramycin was noted against Bacteroides spp. in abscesses caused by either Bacteroides spp. alone, or in combination with N. gonorrhoeae. Furthermore, an additional reduction in the number of N gonorrhoeae was noted in mixed infection with Bacteroides that was treated with metronidazole alone. This study demonstrates the in vitro and in vivo efficacy of the combination of metronidazole and spiramycin in the treatment of infections caused by either Bacteroides spp. alone or in combination with N. gonorrhoeae.

  12. Investigation of the Basis for Persistent Porin Serotypes of Neisseria Gonorrhoeae in Community Infections

    DTIC Science & Technology

    2006-01-01

    community outbreaks. N. gonorrhoeae has a number of highly phase variable surface structures, most notably, opacity (Opa) proteins, lipo- oligosaccharide ...Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms . Proc Natl Acad Sci U S

  13. Biological properties of two distinct pilus types produced by isogenic variants of Neisseria gonorrhoeae P9.

    PubMed Central

    Lambden, P R; Robertson, J N; Watt, P J

    1980-01-01

    Isogenic variants from a single strain of Neisseria gonorrhoeae were shown to produce two distinct types of pili. These pili, designated alpha and beta, differed in both subunit molecular weight and in ability to attach to buccal epithelial cells. Images PMID:6101593

  14. Mutation of a single lytic transglycosylase causes aberrant septation and inhibits cell separation of Neisseria gonorrhoeae.

    PubMed

    Cloud, Karen A; Dillard, Joseph P

    2004-11-01

    The function of lytic peptidoglycan transglycosylases is poorly understood. Single lytic transglycosylase mutants of Escherichia coli have no growth phenotype. By contrast, mutation of Neisseria gonorrhoeae ltgC inhibited cell separation without affecting peptidoglycan monomer production. Thus, LtgC has a dedicated function in gonococcal cell division.

  15. 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

  16. Phenotypic and Genotypic Analysis of Multidrug-Resistant Mycobacterium tuberculosis Isolates from Sudanese Patients

    PubMed Central

    Salih, Mohamed Ahmed; Ali, Manasik; EL-Zaki, Salah-Eldin; Abuzeid, Nadir; Elgadi, Zeinab Abubaker Mohammed; Altayb, Hisham N.; Elegail, Asrar M. A.; Ibrahim, Nuha Y.; Elamin, Bahaeldin K.

    2017-01-01

    Background. Currently, mutations in rpoB, KatG, and rrs genes and inhA promoter were considered to be involved in conferring resistance to rifampicin, isoniazid, and streptomycin in Mycobacterium tuberculosis (MTB). Objective. The aims of this study were to detect the prevalence of first-line tuberculosis (TB) drug resistance among a group of previously treated and newly detected TB patients, to determine the association between prevalence of multidrug resistance (MDR) and demographic information (age and sex), to explain genes correlated with MDR Mycobacterium tuberculosis, and to characterize MTB via 16S ribosomal RNA (16S rRNA) analysis. Methods. A hundred MTB isolates from Sudanese pulmonary TB patients were included in the study. The proportional method of drug susceptibility test was carried out on Löwenstein-Jensen media. Multiplex PCR of rpoB and KatG genes and inhA promoter was conducted; then rrs genes were amplified by conventional PCR and were sequenced. The sequences of the PCR product were compared with known rrs gene sequences in the GenBank database by multiple sequence alignment tools. Result. The prevalence of MDR was 14.7% among old cases and 5.3% among newly diagnosed cases. Conclusion. Mutations in rrs could be considered as a diagnostic marker. PMID:28197340

  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 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

  18. Analysis of the tricho-dento-osseous syndrome genotype and phenotype.

    PubMed

    Wright, J T; Kula, K; Hall, K; Simmons, J H; Hart, T C

    1997-10-17

    The tricho-dento-osseous (TDO) syndrome demonstrates kinky curly hair, thin-pitted enamel, taurodontism, and thickening of cortical bone. The purpose of this investigation was to characterize the phenotypic variation of TDO in 3, previously unreported, kindreds and to examine possible candidates for the genomic TDO locus. Thirty-three affected and 20 unaffected individuals were recruited for prospective analysis. Participants were evaluated clinically and photographed by one examiner. Blood was drawn for genetic linkage analyses and radiographs were taken to assess dental and skeletal characteristics. All TDO individuals with teeth had generalized thin and/or pitted enamel hypoplasia. Taurodontism was present in all affected individuals, but was variably expressed. Unique kinky/curly hair at birth was reported in 85% of affected individuals. The curly hair phenotype was retained in 46% of affected individuals after infancy. Thick cranial bones, lack of visible pneumatization of the mastoid process, and/or obliteration of the calvarial diploë was seen in 97% of affected persons compared with 30% of the unaffected individuals. The findings suggest that curly hair at birth, enamel hypoplasia, and taurodontism are highly penetrant yet clinically variable components of TDO. The ABO, Kell, and Gc loci previously suggested to be linked to TDO were excluded as candidates in this TDO population. This investigation characterizes the marked variability in the expression of skeletal, hair, and dental manifestations. The broad range of TDO phenotypes seen in these families, including a variety of skeletal changes, does not support subdividing TDO into multiple subtypes based on subtle phenotypic differences.

  19. Molecular characterization and genotypic antimicrobial resistance analysis of Campylobacter jejuni and Campylobacter coli isolated from broiler flocks in northern Italy.

    PubMed

    Giacomelli, M; Andrighetto, C; Rossi, F; Lombardi, A; Rizzotti, L; Martini, M; Piccirillo, A

    2012-12-01

    Genetic variability and genotypic antimicrobial resistance (AMR) of Campylobacter jejuni and Campylobacter coli from commercial broiler farms were investigated in this study. Campylobacter isolates were genetically characterized by random amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR) and flaA-SVR and flaB-SVR sequence-based typing. Eight RAPD types were identified in C. jejuni and three in C. coli, while 16 fla profiles were detected among all isolates. Further, 13 flaA-SVR and 13 flaB-SVR alleles were identified. Both typing methods detected a high level of genetic diversity, but fla-SVR typing showed a higher discriminatory power. Indeed, Simpson's index of fla typing (D=0.920) was higher than that of RAPD typing (D=0.814). Moreover, the association of flaA-SVR and flaB-SVR sequence analysis showed a higher discriminatory power compared with the sequence analysis of single loci. Isolates were also analysed by the mismatch amplification mutation assay PCR test and the detection of cmeB gene to determine the occurrence of genetic determinants of AMR to macrolides and fluoroquinolones and multidrug resistance. The A2074C and A2075G mutations in the 23S rRNA gene, the C257T mutation in the gyrA gene, and the cmeB gene were higher in C. coli (19.0%, 67.0%, 100.0% and 100.0%, respectively) than in C. jejuni (0.0%, 3.1%, 48.3% and 48.3%, respectively). This study showed a high degree of genetic diversity and a high prevalence of genetic determinants of macrolide resistance, fluoroquinolone resistance and multidrug resistance among C. jejuni and C. coli isolates from Italian commercial broiler farms.

  20. 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

  1. 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

  2. Sequence and phylogenetic analysis of virulent Newcastle disease virus isolates from Pakistan during 2009-2013 reveals circulation of new sub genotype.

    PubMed

    Siddique, Naila; Naeem, Khalid; Abbas, Muhammad Athar; Ali Malik, Akbar; Rashid, Farooq; Rafique, Saba; Ghafar, Abdul; Rehman, Abdul

    2013-09-01

    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 (112)GRQGRL(117) and velogenic (112)RRQKRF(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.

  3. Proteomic analysis for low and high nitrogen-responsive proteins in the leaves of rice genotypes grown at three nitrogen levels.

    PubMed

    Hakeem, Khalid Rehman; Chandna, Ruby; Ahmad, Altaf; Qureshi, Mohd Irfan; Iqbal, Muhammad

    2012-10-01

    Nitrogen (N) is an essential nutrient for plants. Increase in crop production is associated with increase in N fertilizers. Excessive use of N fertilizers and the low nitrogen utilization efficiency by crop plants is a major cause for environmental damage. Therefore, to reduce the N-fertilizer pollution, there is an urgent need to improve nitrogen use efficiency. Identification and/or development of genotypes which can grow and yield well at low nitrogen levels may provide a solution. Understanding the molecular mechanism of differential nitrogen use efficiency of the genotypes may provide some clues. Keeping the above facts in mind, in this study we have identified the high N-responsive and low N-responsive contrasting rice genotypes, out of 20 genotypes that were grown at low (1 mM), moderate (10 mM), and high (25 mM) levels of N (KNO(3)). Proteome analysis of leaves revealed that the proteins involved in the energy production/regulation and metabolism in plant leaf tissues are differentially expressed under N treatments. Moreover, some disease-resistant and stress-induced proteins were found to be overexpressed at high levels of N. The present study could be useful in identifying proteins responding to different levels of nitrogen fertilization, which may open new avenues for a better understanding of N use efficiency, and for developing new strategies to enhance N efficiency in cereal crops.

  4. Multi-locus variable number tandem repeat analysis for Clostridium botulinum type B isolates in Japan: comparison with other isolates and genotyping methods.

    PubMed

    Umeda, Kaoru; Wada, Takayuki; Kohda, Tomoko; Kozaki, Shunji

    2013-06-01

    Clostridium botulinum produces botulinum neurotoxin (BoNT) and causes botulism in humans and animals. Recently, 15-loci multi-locus variable number tandem repeat analysis (MLVA) for C. botulinum was developed for high-resolution and inter-lab comparative genotyping. This study examines the relation between MLVA and other genotyping methods such as pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST), BoNT/B subtyping and bont/b gene location to evaluate MLVA as a method applicable to the genetic markers for C. botulinum type B. Japanese isolates were genotyped using MLVA and were compared with strains from other sources reported previously. Results show that the discriminatory power of MLVA was comparable to that of PFGE and higher than that of MLST. The topology of the minimum spanning tree (MST) constructed using MLVA data was very consistent with the phylogenetic classifications of PFGE and MLST. The MST topology also represented genetic diversity between the strains possessing bont/b gene on chromosomes and plasmids. Some Japanese isolates including those associated with infant botulism were inferred to be related to isolates of Europe origin from MLVA genotyping results. The MLVA scheme used for this study is apparently useful not only for high-resolution molecular typing, but also for phylogenetic characterization of C. botulinum type B.

  5. 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

  6. 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

  7. Spatial Analysis of Phytophthora infestans Genotypes and Late Blight Severity on Tomato and Potato in the Del Fuerte Valley Using Geostatistics and Geographic Information Systems.

    PubMed

    Jaime-Garcia, R; Orum, T V; Felix-Gastelum, R; Trinidad-Correa, R; Vanetten, H D; Nelson, M R

    2001-12-01

    ABSTRACT Genetic structure of Phytophthora infestans, the causal agent of potato and tomato late blight, was analyzed spatially in a mixed potato and tomato production area in the Del Fuerte Valley, Sinaloa, Mexico. Isolates of P. infestans were characterized by mating type, allozyme analysis at the glucose-6-phosphate isomerase and peptidase loci, restriction fragment length polymorphism with probe RG57, metalaxyl sensitivity, and aggressiveness to tomato and potato. Spatial patterns of P. infestans genotypes were analyzed by geographical information systems and geo-statistics during the seasons of 1994-95, 1995-96, and 1996-97. Spatial analysis of the genetic structure of P. infestans indicates that geographic substructuring of this pathogen occurs in this area. Maps displaying the probabilities of occurrence of mating types and genotypes of P. infestans, and of disease severity at a regional scale, were presented. Some genotypes that exhibited differences in epidemiologically important features such as metalaxyl sensitivity and aggressiveness to tomato and potato had a restricted spread and were localized in isolated areas. Analysis of late blight severity showed recurring patterns, such as the earliest onset of the disease in the area where both potato and tomato were growing, strengthening the hypothesis that infected potato tubers are the main source of primary inoculum. The information that geostatistical analysis provides might help improve management programs for late blight in the Del Fuerte Valley.

  8. Emergence of decreased susceptibility and resistance to extended-spectrum cephalosporins in Neisseria gonorrhoeae in Korea

    PubMed Central

    Lee, Hyukmin; Unemo, Magnus; Kim, Hyo Jin; Seo, Younghee; Lee, Kyungwon; Chong, Yunsop

    2015-01-01

    Objectives Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a major concern globally; however, no comprehensive AMR data for gonococcal isolates cultured after 2006 in Korea have been published internationally. We determined the susceptibility of N. gonorrhoeae isolates cultured in 2011–13, the mechanism of extended-spectrum cephalosporin (ESC) resistance and the molecular epidemiology of gonococcal strains in Korea. Methods In 2011–13, 210 gonococcal isolates were collected in Korea and their AMR profiles were examined by the agar dilution method. The penA, mtrR, penB, ponA and pilQ genes were sequenced in 25 isolates that were resistant to ESCs and 70 randomly selected isolates stratified by year. For molecular epidemiology, N. gonorrhoeae multiantigen sequence typing and MLST were performed. Results None of the N. gonorrhoeae isolates was susceptible to penicillin G and most were resistant to tetracycline (50%) and ciprofloxacin (97%). The rates of resistance to ceftriaxone, azithromycin, cefpodoxime and cefixime were 3%, 5%, 8% and 9%, respectively. However, all isolates were susceptible to spectinomycin. Twenty-one (84%) of the 25 ESC-resistant isolates contained the non-mosaic PBP2 XIII allele; however, the remaining 4 (16%) possessed the mosaic PBP2 X allele, which has been previously associated with ESC resistance including treatment failures. Conclusions In Korea, susceptibility to spectinomycin remains high. However, the recent emergence of ESC-resistant N. gonorrhoeae strains, including strains possessing the PBP2 mosaic X and non-mosaic XIII alleles, is a major concern and enhanced AMR surveillance is necessary to prevent transmission of these strains. PMID:26084303

  9. Neisseria gonorrhoeae Modulates Immunity by Polarizing Human Macrophages to a M2 Profile

    PubMed Central

    Ortiz, María Carolina; Lefimil, Claudia; Rodas, Paula I.; Vernal, Rolando; Lopez, Mercedes; Acuña-Castillo, Claudio; Imarai, Mónica; Escobar, Alejandro

    2015-01-01

    Current data suggest that Neisseria gonorrhoeae is able to suppress the protective immune response at different levels, such as B and T lymphocytes and antigen-presenting cells. The present report is focused on gonococcus evasion mechanism on macrophages (MФ) and its impact in the subsequent immune response. In response to various signals MФ may undergo classical-M1 (M1-MФ) or alternative-M2 (M2-MФ) activation. Until now there are no reports of the gonococcus effects on human MФ polarization. We assessed the phagocytic ability of monocyte-derived MФ (MDM) upon gonococcal infection by immunofluorescence and gentamicin protection experiments. Then, we evaluated cytokine profile and M1/M2 specific-surface markers on MФ challenged with N. gonorrhoeae and their proliferative effect on T cells. Our findings lead us to suggest N. gonorrhoeae stimulates a M2-MФ phenotype in which some of the M2b and none of the M1-MФ-associated markers are induced. Interestingly, N. gonorrhoeae exposure leads to upregulation of a Programmed Death Ligand 1 (PD-L1), widely known as an immunosuppressive molecule. Moreover, functional results showed that N. gonorrhoeae-treated MФ are unable to induce proliferation of human T-cells, suggesting a more likely regulatory phenotype. Taken together, our data show that N. gonorroheae interferes with MФ polarization. This study has important implications for understanding the mechanisms of clearance versus long-term persistence of N. gonorroheae infection and might be applicable for the development of new therapeutic strategies. PMID:26125939

  10. [Laboratory practices: diagnostics and antibiotics resistance testing of Neisseria gonorrhoeae in Germany].

    PubMed

    Loenenbach, Anna; Dudareva-Vizule, S; Buder, S; Sailer, A; Kohl, P K; Bremer, V

    2015-08-01

    Recent years have seen a world-wide increase in antimicrobial resistance (AMR) in cases of infection with Neisseria gonorrhoeae (NG). NG infection is not notifiable in Germany and there is a lack of information available about the spread and AMR of NG infections. The objective of the study was to provide information on diagnostic methods and AMR testing in cases of NG infections in German laboratories. A cross-sectional survey was undertaken in Germany between June and August 2013 using an online questionnaire. Laboratories performing NG diagnostics were identified and described with regard to the diagnostic methods used, the number of tests performed, the antibiotics tested and the AMR observed, in addition to general laboratory information. In total, 188 of the 521 participating laboratories performed NG diagnostics; these were included in the further statistical analysis. 92.6 % of the 188 laboratories performed culture. A median of 60 (IQR 15-270) samples per quarter (SPQ) were tested, with an overall positivity rate of 4.1 and 6.9 % among men. Most (82.1 %) of the 151 laboratories performing NG culture tested for AMR as well. The most frequently tested antibiotics were ciprofloxacin (94.8 %), penicillin (93.1 %), doxycycline (70.0 %) and ceftriaxone (67.2 %). The most frequently observed AMR ever were those against ciprofloxacin (87.1 %), penicillin (78.3 %), doxycycline (56.6 %) and azithromycin (35.1 %; all percentages refer to laboratories). The laboratories used different standards regarding susceptibility criteria. The emergence and spread of AMR shows that it is crucial to assess and monitor the scope and trends of multidrug-resistant gonorrhea. The data collected on diagnostic methods and AMR testing in cases of NG infections in German laboratories constitute an important basis for future monitoring.

  11. Risk factors associated with chlamydia and gonorrhoea infection among female sex workers in two Mexico-USA border cities.

    PubMed

    Loza, O; Strathdee, S A; Martinez, G A; Lozada, R; Ojeda, V D; Staines-Orozco, H; Patterson, T L

    2010-07-01

    Female sex workers (FSWs) aged ≥18 years without known HIV infection living in Tijuana and Ciudad Juarez, Mexico who had recent unprotected sex with clients underwent interviews and testing for chlamydia and gonorrhoea using nucleic acid amplification. Correlates of each infection were identified with logistic regression. Among 798 FSWs, prevalence of chlamydia and gonorrhoea was 13.0% and 6.4%, respectively. Factors independently associated with chlamydia were younger age, working in Tijuana versus Ciudad Juarez and recent methamphetamine injection. Factors independently associated with gonorrhoea were working in Tijuana versus Ciudad Juarez, using illegal drugs before or during sex, and having a recent male partner who injects drugs. Chlamydia and gonorrhoea infection were more closely associated with FSWs' drug use behaviours and that of their sexual partners than with sexual behaviours. Prevention should focus on subgroups of FSWs and their partners who use methamphetamine and who inject drugs.

  12. 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.

  13. People of the British Isles: preliminary analysis of genotypes and surnames in a UK-control population.

    PubMed

    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

    2012-02-01

    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.

  14. 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

  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. Meta-analysis of randomized controlled trials reveals an improved clinical outcome of using genotype plus clinical algorithm for warfarin dosing.

    PubMed

    Liao, Zhenqi; Feng, Shaoguang; Ling, Peng; Zhang, Guoqing

    2015-02-01

    Previous studies have raised interest in using the genotyping of CYP2C9 and VKORC1 to guide warfarin dosing. However, there is lack of solid evidence to prove that genotype plus clinical algorithm provides improved clinical outcomes than the single clinical algorithm. The results of recent reported clinical trials are paradoxical and needs to be systematically evaluated. In this study, we aim to assess whether genotype plus clinical algorithm of warfarin is superior to the single clinical algorithm through a meta-analysis of randomized controlled trials (RCTs). All relevant studies from PubMed and reference lists from Jan 1, 1995 to Jan 13, 2014 were extracted and screened. Eligible studies included randomized trials that compared clinical plus pharmacogenetic algorithms group to single clinical algorithm group using adult (≥ 18 years) patients with disease conditions that require warfarin use. We further used fix-effect models to calculate the mean difference or the risk ratios (RRs) and 95% CIs to analyze the extracted data. The statistical heterogeneity was calculated using I(2). The percentage of time within the therapeutic INR range was considered to be the primary clinical outcome. The initial search strategy identified 50 citations and 7 trials were eligible. These seven trials included 1,910 participants, including 960 patients who received genotype plus clinical algorithm of warfarin dosing and 950 patients who received clinical algorithm only. We discovered that the percentage of time within the therapeutic INR range of the genotype-guided group was improved compared with the standard group in the RCTs when the initial standard dose was fixed (95% CI 0.09-0.40; I(2) = 47.8%). However, for the studies using non-fixed initial doses, the genotype-guided group failed to exhibit statistically significant outcome compared to the standard group. No significant difference was observed in the incidences of adverse events (RR 0.94, 95% CI 0.84-1.04; I(2) = 0%, p

  17. Null genotypes of glutathione S-transferase μ1 and glutathione S-transferase θ1 are associated with osteosarcoma risk: A meta-analysis

    PubMed Central

    HAN, JICHENG; DENG, WEI; WANG, LAIYING; QI, WANLI

    2015-01-01

    Glutathione S-transferase (GST) genetic polymorphisms has been reported to be associated with osteosarcoma; however, the results of previous studies are conflicting. Thus, in the present study, a meta-analysis was conducted to investigate the effects of GSTM1 and GSTT1 polymorphisms on osteosarcoma risk. A literature search was performed in the PubMed, Cochrane Library and China National Knowledge Infrastructure databases to identify case-control studies published prior to March 2014. Data were extracted and pooled odds ratios (OR) with 95% confidence intervals (CI) were calculated. In addition, Begg’s test was used to measure publication bias. Sensitivity analysis were performed to ensure the accuracy of the results. The meta-analysis results demonstrated no significant association between the null genotype of GSTM1 and osteosarcoma risk (OR=0.83; 95% CI, 0.37–1.85). By contrast, the results revealed a significant association for the comparison of null vs. non-null genotypes of GSTT1 (OR=1.54; 95% CI, 1.09–2.19). In conclusion, the GSTT1 null genotype may be associated with an increased risk of developing osteosarcoma. Further studies with larger sample sizes and well-designed methodologies are required to verify these conclusions. PMID:25789067

  18. Spatial distribution pattern analysis of Dof1 transcription factor in different tissues of three Eleusine coracana genotypes differing in their grain colour, yield and photosynthetic efficiency.

    PubMed

    Gupta, Nidhi; Gupta, Atul Kumar; Kumar, Anil

    2012-03-01

    In the present study Dof1 gene of finger millet was cloned and sequenced. In silico analysis reveals 61% identity with the Sorghum bicolor and 57% identity with the Oryza sativa Dof1 sequence. A comparative analysis of gene sequences from different crops and three finger millet genotypes {Brown (PRM-1), Golden (PRM-701) and White (PRM-801)} differing in grain colour, yield and photosynthetic efficiency showed a high degree of sequence identity of Dof1 sequence gene ranging from 22 to 70% as evident from distance matrix of the built phylogenetic tree showing two major clusters. A total of five conserved motifs were observed in Dof1 sequences of different cereals. Motif 1 with multilevel consensus sequence CKNCRRYWTKGGAMRNVPVG contains zinc finger Dof domain. Motif 3 and motif 5 contains protein kinase phosphorylation site. Motif 2 contains Dof domain and zinc finger N-glycosylation site while motif 4 is involved in Zinc finger type profiling. Further, we studied the spatial distribution of Dof1 gene in three vegetative tissues (root, stem and flag leaf) as well as four stages of developing spikes (S1, S2, S3 and S4) of the three finger millet genotypes using qualitative and quantitative PCR based approaches. Physiological parameters (plant height, leaf area, chlorophyll content, SPAD value and photosynthetic efficiency) at the time of flowering was found to be highest in white (PRM-801) genotype followed by golden (PRM-701) and brown (PRM-1) genotype. Semi-quantitative RT-PCR and quantitative real-time PCR analysis revealed that the expression of Dof1 is highest in leaves and lowest in roots, which suggests its role in regulation of photosynthesis-related genes and carbon skeleton synthesis. Also at grain maturity stage, expression of Dof1 was higher in white (PRM-801) genotype followed by golden (PRM-701) and brown (PRM-1) genotype. The result is suggestive of Dof1 role in the accumulation of grain protein and yield attribute through regulation of key enzymes

  19. Genetic analysis of attachment glycoprotein (G) gene in new genotype ON1 of human respiratory syncytial virus detected in Japan.

    PubMed

    Tsukagoshi, Hiroyuki; Yokoi, Hajime; Kobayashi, Miho; Kushibuchi, Izumi; Okamoto-Nakagawa, Reiko; Yoshida, Ayako; Morita, Yukio; Noda, Masahiro; Yamamoto, Norio; Sugai, Kazuko; Oishi, Kazunori; Kozawa, Kunihisa; Kuroda, Makoto; Shirabe, Komei; Kimura, Hirokazu

    2013-09-01

    We studied the evolution of the G gene in the new genotype ON1 of RSV detected from patients with acute respiratory infection in Japan. Phylogenetic analyses and the evolutionary timescale were obtained by the Bayesian MCMC method. We also analyzed p-distance and positive selection sites. A new genotype ON1 emerged around 2001. The evolution rate was rapid (3.57 × 10(-3) substitutions/site per year). The p-distance was short and no positive selection site was found in the present strains. These results suggested that a new genotype ON1 of RSV-A emerged approximately10 years ago and spread to some countries with a high evolution rate.

  20. Recent advances and considerations for the future in forensic analysis of degraded DNA by autosomic miniSTR multiplex genotyping.

    PubMed

    Odriozola, A; Aznar, J M; Celorrio, D; De Pancorbo, M M

    2011-08-01

    STR genotyping from degraded DNA samples requires genetic profiles to be obtained from DNA fragments no bigger than 200-300 bp. It requires the use of miniSTRs, which are smaller than the STRs used in standard typing. This paper reviews recent advances in miniSTR genotyping, beginning with a brief introduction to the processes involved in DNA fragmentation and how it hinders standard STR genotyping before proceeding further to the loci included in the main DNA databases and finishing with the International Workgroups' recommended design strategies for developing miniSTR reactions. The results of the efforts of many laboratories achieving different STR multiplexes and patents are also described and compared. Finally, a consideration of the perspectives for the future in this area is presented.

  1. Evaluation of AMPLICOR Neisseria gonorrhoeae PCR using cppB nested PCR and 16S rRNA PCR.

    PubMed

    Farrell, D J

    1999-02-01

    Certain strains of Neisseria subflava and Neisseria cinerea are known to produce false-positive results with the AMPLICOR Neisseria gonorrhoeae PCR (Roche Diagnostic Systems, Branchburg, N.J.). The analytical sensitivity and analytical specificity of three PCR tests were assessed with 3 geographically diverse N. gonorrhoeae strains and 30 non-N. gonorrhoeae Neisseria spp. The sensitivities of the in-house nested cppB gene and the 16S rRNA PCR methods were greater than that of the AMPLICOR N. gonorrhoeae PCR with purified DNA from all 3 N. gonorrhoeae strains. Six of 14 clinical strains of N. subflava (1 from a vaginal swab, 5 from respiratory sites) produced false-positive AMPLICOR N. gonorrhoeae PCR results and were negative by the two other PCR methods. When applied to 207 clinical specimens selected from a population with a high prevalence ( approximately 9%) of infection, the results for 15 of 96 (15.6%) AMPLICOR-positive specimens and 14 of 17 (82.3%) AMPLICOR-equivocal specimens were not confirmed by the more sensitive nested cppB PCR method. Only 2 of 94 (2.1%) of AMPLICOR N. gonorrhoeae PCR-negative specimens from the same population tested positive by the nested cppB method. These results suggest that for this population the AMPLICOR N. gonorrhoeae PCR test is suitable as a screening test only and all positive results should be confirmed by a PCR method that is more specific and at least as sensitive. This study also illustrates that caution should be used when introducing commercially available nucleic acid amplification-based diagnostic tests into the regimens of tests used for populations not previously tested with these products.

  2. Automated SNP genotype clustering algorithm to improve data completeness in high-throughput SNP genotyping datasets from custom arrays.

    PubMed

    Smith, Edward M; Littrell, Jack; Olivier, Michael

    2007-12-01

    High-throughput SNP genotyping platforms use automated genotype calling algorithms to assign genotypes. While these algorithms work efficiently for individual platforms, they are not compatible with other platforms, and have individual biases that result in missed genotype calls. Here we present data on the use of a second complementary SNP genotype clustering algorithm. The algorithm was originally designed for individual fluorescent SNP genotyping assays, and has been optimized to permit the clustering of large datasets generated from custom-designed Affymetrix SNP panels. In an analysis of data from a 3K array genotyped on 1,560 samples, the additional analysis increased the overall number of genotypes by over 45,000, significantly improving the completeness of the experimental data. This analysis suggests that the use of multiple genotype calling algorithms may be advisable in high-throughput SNP genotyping experiments. The software is written in Perl and is available from the corresponding author.

  3. 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.

  4. 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.

  5. Selection of 29 highly informative InDel markers for human identification and paternity analysis in Chinese Han population by the SNPlex genotyping system.

    PubMed

    Li, Chengtao; Zhang, Suhua; Li, Li; Chen, Jingzhong; Liu, Yan; Zhao, Shumin

    2012-03-01

    The interest of forensic researchers in single nucleotide polymorphism (SNP) has been attracted because of its potential advantages, such as low mutation rates, amenable to high-throughput automated platform and the improved application in the analysis of degraded samples. In this paper, 29 highly informative insertion/deletion (InDel, a special kind of SNP) markers were selected from the dbSNP ( http://www.ncbi.nlm.nih.gov/SNP/ ) according to the given criteria. 109 unrelated Chinese Han subjects were genotyped for the 29 InDels with SNPlex genotyping system. The allele frequency data revealed that the combined power of discrimination for the 29 InDel markers was 0.999999999990867 and the combined probability of paternity exclusion (PE) was 0.9930. Sensitivity studies were performed to evaluate the flexibility of the SNPlex genotyping system on the set of 29 InDels. Highly reproducible results could be obtained with 40-100 ng genomic DNA and the proportion of total allele drop-in was significantly increased when the amount of DNA added to PCR was lower than 35 ng. These results suggested that the set of 29 InDels was useful in paternity analysis or human identification in the future.

  6. Genotypic analysis and latent membrane protein 1 expression of Epstein-Barr virus in extranodal NK/T-cell lymphoma from Northern Chinese patients.

    PubMed

    Wang, Haijuan; Li, Hui; Xing, Xiaoming; Zhao, Chengquan; Luo, Bing

    2015-08-01

    As the most common NK/T-cell lymphoma in Asian countries, extranodal NK/T-cell lymphoma, nasal type (ENKTL), has unique clinical features and a strong association with Epstein-Barr virus (EBV). In order to gain a preliminary understanding of the relationship between ENKTL and EBV, we performed genotypic analysis of EBV and investigated LMP1 expression in extranodal NK/T-cell lymphoma. Our study shows that ENKTL is an EBV-associated malignancy and that A, C and F are the predominant EBV genotypes in northern China. LMP1 expression is stronger in extranasal sites than nasal sites, and the expression level is strongly correlated to ENKTL and may play an important role in the development of ENKTL.

  7. 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 ...

  8. Hepatitis C virus genotype 4d in Southern Italy: reconstruction of its origin and spread by a phylodynamic analysis.

    PubMed

    Ciccozzi, Massimo; Equestre, Michele; Costantino, Angela; Marascio, Nadia; Quirino, Angela; Lo Presti, Alessandra; Cella, Eleonora; Bruni, Roberto; Liberto, Maria Carla; Focà, Alfredo; Pisani, Giulio; Zehender, Gianguglielmo; Ciccaglione, Anna Rita

    2012-10-01

    Hepatitis C Virus (HCV) genotype 4 predominates in Middle East and Central Africa countries. Recently, it has become also prevalent in Southern European countries where it is thought to have been introduced through immigration and the movement of intravenous drug users. In Italy, the prevalence of genotype 4 is particularly high (4.5%) in Southern regions, such as Calabria, and reaches values of 8.4% in specific areas where there appears to be endemic circulation of this genotype. In the present study, the phylogeny of HCV subtype 4d isolated from 19 Italian patients in Calabria was investigated by analysing a fragment of the NS5B viral genomic region. A Bayesian coalescent-based framework was used to estimate origin and spread of the HCV 4d in this area. The mean evolutionary rate HCV 4d NS5B sequences was estimated using a dataset of sequences sampled at known times and a relaxed clock constant model that best fitted the data. By using a Bayesian coalescent method, the Italian 4d isolates collected in Calabria were found to share a common ancestor with reference 4d isolates whose origin was traced back to 1940s. The genotype 4d epidemic in Southern Italy was maintained in a steady non-expanding phase until the late 1970s after that it grew exponentially up to 1990s probably sustained by the vast increase of unsafe blood transfusions and the spread of illicit intravenous drug users.

  9. 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.

  10. Recommendations for the Laboratory-Based Detection of Chlamydia trachomatis and Neisseria gonorrhoeae — 2014

    PubMed Central

    Papp, John R.; Schachter, Julius; Gaydos, Charlotte A.; Van Der Pol, Barbara

    2014-01-01

    Summary This report updates CDC's 2002 recommendations regarding screening tests to detect Chlamydia trachomatis and Neisseria gonorrhoeae infections (CDC. Screening tests to detect Chlamydia trachomatis and Neisseria gonorrhoeae infections—2002. MMWR 2002;51[No. RR-15]) and provides new recommendations regarding optimal specimen types, the use of tests to detect rectal and oropharyngeal C. trachomatis and N. gonorrhoeae infections, and circumstances when supplemental testing is indicated. The recommendations in this report are intended for use by clinical laboratory directors, laboratory staff, clinicians, and disease control personnel who must choose among the multiple available tests, establish standard operating procedures for collecting and processing specimens, interpret test results for laboratory reporting, and counsel and treat patients. The performance of nucleic acid amplification tests (NAATs) with respect to overall sensitivity, specificity, and ease of specimen transport is better than that of any of the other tests available for the diagnosis of chlamydial and gonococcal infections. Laboratories should use NAATs to detect chlamydia and gonorrhea except in cases of child sexual assault involving boys and rectal and oropharyngeal infections in prepubescent girls and when evaluating a potential gonorrhea treatment failure, in which case culture and susceptibility testing might be required. NAATs that have been cleared by the Food and Drug Administration (FDA) for the detection of C. trachomatis and N. gonorrhoeae infections are recommended as screening or diagnostic tests because they have been evaluated in patients with and without symptoms. Maintaining the capability to culture for both N. gonorrhoeae and C. trachomatis in laboratories throughout the country is important because data are insufficient to recommend nonculture tests in cases of sexual assault in prepubescent boys and extragenital anatomic site exposure in prepubescent girls. N

  11. Demographic and anthropometrical analysis and genotype distribution of chronic hepatitis C patients treated in public and private reference centers in Brazil.

    PubMed

    Focaccia, R; Baraldo, D C M; Ferraz, M L G; Martinelli, A L C; Carrilho, F J; Gonçales, F L; Pedroso, M L A; Coelho, H S M; Lacerda, M A; Brandão, C E; Mattos, A A; Lira, L G C; Zamin, I; Pinheiro, J O P; Tovo, C V; Both, C T; Soares, J A S; Dittrich, S

    2004-10-01

    Hepatitis C virus (HCV) infection is a serious public health problem, since 80% to 85% of HCV carriers develop a persistent infection that can progress into liver cirrhosis and hepatocarcinoma. Considering that the response of hepatitis C patients to combination therapy with interferon and ribavirin depends on HCV characteristics as well as on host features, we made a retrospective analysis of demographic and anthropometrical data and HCV genotype distribution of chronic hepatitis C patients treated in public and private reference centers in Brazil. The medical records of 4,996 patients were reviewed, 81% from public and 19% from private institutions. Patients' median age was 46 years, and there was a higher prevalence of male (62%) and white patients (80%). The analysis of HCV-infecting strains showed a predominance of genotype 1 (64%) over genotypes 2 and 3. The patients' mean weight was 70.6 kg, and 65% of the patients weighed less than 77 kg. Overweight and obesity were observed in 37.8% and 13.6% of the patients, respectively. Since a body weight of 75 kg or less has been considered an independent factor that significantly increases the odds of achieving a sustained virological response, the Brazilian population seems to have a more favorable body weight profile to achieve a sustained response than the American and European populations. The finding that 65% of chronic hepatitis C patients have a body weight of 77 kg or less may have a positive pharmacoeconomic impact on the treatment of genotype 1 HCV patients with weight-based doses of peginterferon.

  12. 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.

  13. 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.

  14. 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

  15. Proteomic Analysis of Different Mutant Genotypes of Arabidopsis Led to the Identification of 11 Proteins Correlating with Adventitious Root Development1[W

    PubMed Central

    Sorin, Céline; Negroni, Luc; Balliau, Thierry; Corti, Hélène; Jacquemot, Marie-Pierre; Davanture, Marlène; Sandberg, Göran; Zivy, Michel; Bellini, Catherine

    2006-01-01

    A lack of competence to form adventitious roots by cuttings or explants in vitro occurs routinely and is an obstacle for the clonal propagation and rapid fixation of elite genotypes. Adventitious rooting is known to be a quantitative genetic trait. We performed a proteomic analysis of Arabidopsis (Arabidopsis thaliana) mutants affected in their ability to develop adventitious roots in order to identify associated molecular markers that could be used to select genotypes for their rooting ability and/or to get further insight into the molecular mechanisms controlling adventitious rooting. Comparison of two-dimensional gel electrophoresis protein profiles resulted in the identification of 11 proteins whose abundance could be either positively or negatively correlated with endogenous auxin content, the number of adventitious root primordia, and/or the number of mature adventitious roots. One protein was negatively correlated only to the number of root primordia and two were negatively correlated to the number of mature adventitious roots. Two putative chaperone proteins were positively correlated only to the number of primordia, and, interestingly, three auxin-inducible GH3-like proteins were positively correlated with the number of mature adventitious roots. The others were correlated with more than one parameter. The 11 proteins are predicted to be involved in different biological processes, including the regulation of auxin homeostasis and light-associated metabolic pathways. The results identify regulatory pathways associated with adventitious root formation and represent valuable markers that might be used for the future identification of genotypes with better rooting abilities. PMID:16377752

  16. 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