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A 58-year-old man with Crohn's disease received adalimumab for 13 months after screening results for tuberculosis were found to be negative. He was diagnosed with de novo mediastinal lymph-node tuberculosis, which was proved to be bacteriologically identical to that of an individual with smear positive lung tuberculosis by a variable number of tandem repeat analyses. After initiating anti-tuberculosis therapy, the patient developed immune reconstitution syndrome, which was improved by the re-administration of adalimumab. Even in countries with an intermediate tuberculosis burden, including Japan, we need to be alert for de novo tuberculosis as well as its reactivation during tumor necrosis factor-α inhibitor therapy.

Barbara Wysoczanska,1 Marta Dratwa,1 Katarzyna Gebura,1 Jakub Mizgala,1 Grzegorz Mazur,2 Tomasz Wrobel,3 Katarzyna Bogunia-Kubik11Laboratory of Clinical Immunogenetics and Pharmacogenetics, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw 53-114, Poland; 2Department of Internal Medicine, Occupational Diseases, Hypertension and Clinical Oncology, Wroclaw Medical University, Wroclaw, 50-001, Poland; 3Department of Haematology, Blood Neoplasms and Bone Marrow Transplantation, Wroclaw Medical University, Wroclaw 50-367, PolandBackground: The human telomerase reverse transcriptase (TERT) gene encodes the catalytic subunit of telomerase that is essential for maintenance of telomere length. We aimed to find out whether variability within the TERT gene could be associated with telomere length and development of the disease in non-treated patients with chronic lymphocytic leukemia (CLL).Materials and methods: Telomere length, rs2736100, rs2853690, rs33954691, rs35033501 single-nucleotide polymorphisms, and variable number of tandem repeats (VNTR-MNS16A) were assessed in patients at diagnosis. In addition, blood donors served as controls for the polymorphism studies.Results: The minor rs35033501 A variant was more frequent among CLL patients than in healthy controls (OR=3.488, p=0.039). CLL patients over 60 years of age were characterized with lower disease stage at diagnosis (p=0.001 and p=0.008, for the Rai and Binet criteria, respectively). The MNS16A VNTR-243 short allele was more frequent in patients with a low disease stage (p=0.020 and p=0.028, for the Rai and Binet staging system) and also among older patients having longer telomeres (p=0.046). Patients with Rai 0–I stage were characterized with longer telomeres than those with more advanced disease (p=0.030). This relationship was especially pronounced in patients carrying the rs2736100 C allele, independently of the criteria used, ie, Binet (p=0.048) or Rai (p=0.001).Conclusion: Our results showed that the genetic variation within the TERT gene seems to play a regulatory role in CLL and telomere length.Keywords: telomere length, human telomerase reverse transcriptase, variable number of tandem repeats, single-nucleotide polymorphism, chronic lymphocytic leukemia

The pks island codes for the enzymes necessary for synthesis of the genotoxin colibactin, which contributes to the virulence of Escherichia coli strains and is suspected of promoting colorectal cancer. From a collection of 785 human and bovine E. coli isolates, we identified 109 strains carrying a highly conserved pks island, mostly from phylogroup B2, but also from phylogroups A, B1 and D. Different scenarios of pks acquisition were deduced from whole genome sequence and phylogenetic analysis. In the main scenario, pks was introduced and stabilized into certain sequence types (STs) of the B2 phylogroup, such as ST73 and ST95, at the asnW tRNA locus located in the vicinity of the yersiniabactin-encoding High Pathogenicity Island (HPI). In a few B2 strains, pks inserted at the asnU or asnV tRNA loci close to the HPI and occasionally was located next to the remnant of an integrative and conjugative element. In a last scenario specific to B1/A strains, pks was acquired, independently of the HPI, at a non-tRNA locus. All the pks-positive strains except 18 produced colibactin. Sixteen strains contained mutations in clbB or clbD, or a fusion of clbJ and clbK and were no longer genotoxic but most of them still produced low amounts of potentially active metabolites associated with the pks island. One strain was fully metabolically inactive without pks alteration, but colibactin production was restored by overexpressing the ClbR regulator. In conclusion, the pks island is not restricted to human pathogenic B2 strains and is more widely distributed in the E. coli population, while preserving its functionality.

Background Bovine tuberculosis (TB) is caused by Mycobacterium bovis, a well-known cause of zoonotic tuberculosis in cattle and deer, and has been investigated in many physiological and molecular studies. However, detailed genome-level studies of M. bovis have not been performed in Korea. Objectives To survey whole genome-wide single-nucleotide polymorphism (SNP) variants in Korean M. bovis field isolates and to define M. bovis groups in Korea by comparing SNP typing with spoligotyping and variable number tandem repeat typing. Methods A total of 46 M. bovis field isolates, isolated from laryngopharyngeal lymph nodes and lungs of Korean cattle, wild boar, and Korean water deer, were used to identify SNPs by performing whole-genome sequencing. SNP sites were confirmed via polymerase chain reaction using 87 primer pairs. Results We identified 34 SNP sites with different frequencies across M. bovis isolates, and performed SNP typing and epidemiological analysis, which divided the 46 field isolates into 16 subtypes. Conclusions Through SNP analysis, detailed differences in samples with identical spoligotypes could be detected. SNP analysis is, therefore, a useful epidemiological tracing tool that could enable better management of bovine TB, thus preventing further outbreaks and reducing the impact of this disease.

Purpose of Review: The worldwide increase in skin infections due to Mycobacterium abscessus, especially in Latin America, and its difficult diagnosis and treatment, makes a review of literature crucial in reminding and updating physicians about its presentation, diagnosing techniques, and management. Recent Findings: Outbreaks are associated with cosmetic surgeries and medical tourism, but any kind of skin lesion can become infected presenting as negative typical cultures and unsuccessful empirical treatments; therefore, a high clinical suspicion is required. Molecular techniques are promising new diagnosing alternatives and treatment should be guided by susceptibility tests. Summary: Mycobacterium abscessus complex skin infections are concerning due to difficult diagnosis, burdensome treatment, drug resistance, and high cost of management. Usually associated to cosmetic procedures, especially medical tourism, it can also be related to other incisions (C-section, injections, laparoscopy, etc.). Diagnosis requires clinical suspicion and consists of phenotypic and molecular methods to recognize the organisms to a subspecies level. Drug treatment frequently involves surgical debridement and antibiotics with special concern about resistance and adverse events.

Objective: To investigate the association between IL-1Ra variable number of tandem repeat (rs2234663), IL-6 -597GA (rs1800797), IL-6 -572GC (rs1800796) and the risk of Crimean-Congo hemorrhagic fever (CCHF) in the Turkish patients. Methods: This study included 50 patients infected with CCHF and 50 healthy controls. These variants were genotyped using polymerase chain reaction and/or restriction fragment length polymorphism method. Results: The distribution of the IL-6 -572GC genotypes and alleles varied significantly between the patients and the controls. The subjects carrying IL-6 -572GC GG genotype and G allele had increased risk of developing CCHF compared to the control group (P=0.006, P=0.014, respectively). IL-6 -572GC GC genotype was higher in the controls than the patients (P=0.006). For the triple genotype combinations, the 1/2-GC-GG genotype combination was detected more frequently in the control group than CCHF patients (P=0.016). IL-6 (-572/-597) GG-GG genotype was significantly higher in the patient group (P=0.015), while the GC-GG genotype was significantly lower in the patient group (P=0.005). Additionally, the G-G haplotype was significantly higher in the patient group (P=0.042), whereas C-G was found to be significantly lower in the patients than the control group (P=0.037). Conclusions: The results of this study suggest the IL-6 -572GC variant might be genetic markers of sensitivity to CCHF in the Turkish population and may facilitate greater protection against the disease. © 2019 Asian Pacific Journal of Tropical Biomedicine Produced by Wolters Kluwer-Medknow. 2-s2.0-85061101415

The first part of this study consisted in whole genome sequencing of three pathogenic and three carriage strains of S. lugdunensis and comparison with the 15 genomes available in the NCBI. No genetic determinant was associated to the pathogenic or carriage context. However, we have highlighted the presence of mobile genetic elements and MultiLocus Sequence Typing clonal complex dependent variations within loci potentially associated with virulence. Variations wereobserved in the ess locus homologous to that of Staphylococcus aureus encoding the type VII secretion system (T7SS). We showed eight genetic organizations in this species with a clonal population structure. The second part of our work consisted in a phenotypic and molecular characterization of T7SS in S. lugdunensis by construction of a deletion essC gene mutant. This gene encodes a protein requiredfor protein secretion. Our results suggest that T7SS could be involved in translocation of proteins predicted as implicated in virulence in silico. Nevertheless, no virulence attenuation was observed in cells cytotoxicity assay and Caenorhabditis elegans virulence assays between wild-type and mutant strains which yet has lost the ability to lyse erythrocytes. We also developed and evaluated discriminating power of three new typing methods, which are very promising tools for the molecular epidemiology of S. lugdunensis infections.; La première partie de nos travaux a consisté au séquençage de génomes complets de trois souches pathogènes et de trois souches de portage de Staphylococcus lugdunensis pour les comparer aux 15 génomes complets disponibles sur NCBI. Aucun déterminant génétique associé au contexte de virulence ou de portage de S. lugdunensis n’a été identifié. Cependant, nous avons mis en évidence la présence d’éléments génétiques mobiles et des variations dépendantes des complexes clonaux,définis par MultiLocus Sequence Typing, au sein de loci potentiellement associés à la virulence. Des variations ont été observées dans un locus homologue à celui de Staphylococcus aureus codant le système de sécrétion Ess/type VII (SST7). Nous avons mis en évidence huit organisations génétiques chez cette espèce présentant pourtant une structure de population clonale. La seconde partie de nos travaux a consisté à la caractérisation phénotypique et moléculaire du SST7 chez S. lugdunensis par la formation d’un mutant de délétion du gène essC codant une protéine essentielle à la sécrétion. Nos résultats suggèrent que le SST7 serait impliqué dans la translocation de protéines prédites in silico comme impliquées dans la virulence. Néanmoins, dans des modèles de cytotoxicité cellulaire et d’infection du nématode Caenorhabditis elegans, aucune atténuation de la virulence n’a été observée chez la souche mutante malgré une perte de sa capacité à lyser les erythrocytes, comparativement à la souche sauvage. Nos travaux ont également permis de développer et d’évaluer le pouvoir discriminant de trois nouvelles méthodes de typage constituant des outils très prometteurs pour l’épidémiologie moléculaire des infections à S. lugdunensis.

Introduction: Genetic variance of attention deficit hyperactivity disorder (ADHD) is a strong determinant of this disorder. The 40 bp variable number tandem repeat (VNTR) located in the 3′ untranslated region (UTR) of DAT1 gene increases the expression of the dopamine transporter. Therefore, DAT1 has been associated with susceptibility to ADHD. Objective: To determine the association between the VNTR of DAT1 and the phenotype of ADHD or its endophenotypes in a sample of children aged between 6 and 15 years from Bogotá. Subjects and methods: We selected 73 patients with ADHD and 54 controls. WISC test was applied in all subjects and executive functions were assessed. The VNTR of DAT1 was amplified by polymerase chain reaction. Data regarding population genetics and statistical analysis were obtained. Correlation and association tests between genotype and neuropsychological testing were performed. Results: The DAT1 polymorphism was not associated with ADHD (P = .85). Nevertheless, the 10/10 genotype was found to be correlated with the processing speed index (P

Mycobacterium avium subsp. hominissuis (MAH) is an important zoonotic pathogen with raising global health concerns. In humans, MAH is one of the most widespread non-tuberculous mycobacterial species responsible for lung disease. In animals, MAH is frequently isolated from pigs; however, it is also an opportunistic pathogen for other mammals including cattle. To elucidate the genetic diversity of MAH in cattle, a molecular characterization of isolates (n = 26) derived from lymph nodes was performed. Fourteen isolates originated from slaughtered cattle with visible altered lymph nodes at meat inspection, whereas 12 isolates were from lymph nodes without any gross pathological changes of healthy slaughtered cattle. Variable number of tandem repeat (VNTR) analysis was performed at 20 loci to examine genetic differences of isolates and to compare to previously reported VNTR data of human isolates from different countries. Genetic elements IS901, IS1245, IS1311, LSPA17, ITS1 sequevar, and hsp65 code were determined. Interestingly, two bovine MAH isolates harbored ISMav6 and hsp65 code 15, which so far has only been observed in human isolates. We supposed that VNTR data of Swiss samples would show clustering with European samples. Minimum spanning tree and unweighted pair group method using arithmetic averages analyses based on the VNTR data indicated a specific cluster of MAH isolates obtained from lymph nodes without any gross pathological changes of healthy slaughtered cattle. Comparing Swiss isolates with isolates from different other countries, no geographical clustering was observed; however, four Swiss isolates had an identical VNTR profile as human isolates from the Netherlands, the United States, and Japan. These findings indicate a possible public health issue.

Background Enterococcus faecium is ranked worldwide as one of the top ten pathogens identified in healthcare-associated infections (HAI) and is classified as one of the high priority pathogens for research and development of new antibiotics worldwide. Due to molecular biology techniques’ higher costs, the approach for identifying and controlling infectious diseases in developing countries has been based on clinical and epidemiological perspectives. Nevertheless, after an abrupt vancomycin-resistant Enterococcus faecium dissemination in the Méderi teaching hospital, ending up in an outbreak, further measures needed to be taken into consideration. The present study describes the vancomycin-resistant Enterococcus faecium pattern within Colombian’s largest installed-bed capacity hospital in 2016. Methods Thirty-three vancomycin-resistant Enterococcus faecium isolates were recovered during a 5-month period in 2016. Multilocus variable-number tandem-repeat analysis was used for molecular typing to determine clonality amongst strains. A modified time-place-sequence algorithm was used to trace VREfm spread patterns during the outbreak period and estimate transmission routes. Results Four clonal profiles were identified. Chronological clonal profile follow-up suggested a transitional spread from profile “A” to profile “B”, returning to a higher prevalence of “A” by the end of the study. Antibiotic susceptibility indicated high-level vancomycin-resistance in most isolates frequently matching vanA gene identification. Discussion Transmission analysis suggested cross-contamination via healthcare workers. Despite epidemiological control of the outbreak, post-outbreak isolates were still being identified as having outbreak-related clonal profile (A), indicating reduction but not eradication of this clonality. This study supports the use of combined molecular and epidemiological strategies in an approach to controlling infectious diseases. It contributes towards a more accurate evaluation of the effectiveness of the epidemiological measures taken regarding outbreak control and estimates the main cause related to the spread of this microorganism. Electronic supplementary material The online version of this article (10.1186/s12879-019-3877-7) contains supplementary material, which is available to authorized users.

Aim: SNPs in the gene for TPMT exemplify one of the most successful translations of pharmacogenomics into clinical practice. This study explains the correlation between common SNPs and variable number of tandem repeats (VNTR) in promoter of the gene. Materials & methods: We determined VNTR polymorphisms, as well as TPMT*2 and TPMT*3 SNPs and TPMT activity in Slovenian and Italian individuals and lymphoblastoid cell lines. Results: We observed a previously unreported VNTR allele, AB7C, in a TPMT*3A heterozygous individual. VNTRs with two (AB2C) and three or more (ABnC, n ≥ 3) B motifs were statistically significant in complete linkage disequilibrium (D′ = 1, r2 = 1, p

Mycobacterium avium subsp. hominissuis (MAH) is an important zoonotic pathogen with raising global health concerns. In humans, MAH is one of the most widespread non-tuberculous mycobacterial species responsible for lung disease. In animals, MAH is frequently isolated from pigs; however, it is also an opportunistic pathogen for other mammals including cattle. To elucidate the genetic diversity of MAH in cattle, a molecular characterization of isolates (n = 26) derived from lymph nodes was performed. Fourteen isolates originated from slaughtered cattle with visible altered lymph nodes at meat inspection, whereas 12 isolates were from lymph nodes without any gross pathological changes of healthy slaughtered cattle. Variable number of tandem repeat (VNTR) analysis was performed at 20 loci to examine genetic differences of isolates and to compare to previously reported VNTR data of human isolates from different countries. Genetic elements IS901, IS1245, IS1311, LSPA17, ITS1 sequevar, and hsp65 code were determined. Interestingly, two bovine MAH isolates harbored ISMav6 and hsp65 code 15, which so far has only been observed in human isolates. We supposed that VNTR data of Swiss samples would show clustering with European samples. Minimum spanning tree and unweighted pair group method using arithmetic averages analyses based on the VNTR data indicated a specific cluster of MAH isolates obtained from lymph nodes without any gross pathological changes of healthy slaughtered cattle. Comparing Swiss isolates with isolates from different other countries, no geographical clustering was observed; however, four Swiss isolates had an identical VNTR profile as human isolates from the Netherlands, the United States, and Japan. These findings indicate a possible public health issue.

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:28:45Z No. of bitstreams: 0 Made available in DSpace on 2014-05-27T11:28:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-04-01 This study describes the comparison of three methods for genotyping of Mycobacterium tuberculosis, namely MIRU-VNTR (mycobacterial interspersed repetitive units-variable number of tandem repeats), spoligotyping and, for the first time, MLST (Multilocus Sequence Typing). In order to evaluate the discriminatory power of these methods, a total of 44 M. tuberculosis isolates obtained from sputum specimens of patients from Brazil were genotyped. Among the three methods, MLST showed the lowest discriminatory power compared to the other two techniques. MIRU-VNTR showed better discriminatory power when compared to spoligotyping, however, the combination of both methods provides the greatest level of discrimination and therefore this combination is the most useful genotyping tool to be applied to M. tuberculosis isolates. © 2013 Elsevier B.V. Departamento de Análises Clínicas, Toxicológicas e Bromatológicas Faculdade de Ciências Farmacêuticas de Ribeirão Preto Universidade de São Paulo Departamento de Ciências Biológicas Faculdade de Ciências Farmacêuticas Universidade Estadual Paulista Júlio de Mesquita Filho Department of Zoology University of Oxford, Oxford Departamento de Ciências Biológicas Faculdade de Ciências Farmacêuticas Universidade Estadual Paulista Júlio de Mesquita Filho

The fungus Mycosphaerella fijiensis is the causative agent of black sigatoka, which is one of the most destructive diseases of banana plants. Infection with this pathogen results in underdeveloped fruit, with no commercial value. We analyzed the distribution of the M. fijiensis mating-type system and its genetic variability using M13 phage DNA markers. We found a 1:1 distribution of mating-type alleles, indicating MAT1-1 and MAT1-2 idiomorphs. A polymorphism analysis using three different primers for M13 markers showed that only the M13 minisatellite primers generated polymorphic products. We then utilized this polymorphism to characterize 40 isolates from various Brazilian states. The largest genetic distances were found between isolates from the same location and between isolates from different parts of the country. Therefore, there was no correlation between the genetic similarity and the geographic origin of the isolates. The M13 marker was used to generate genetic fingerprints for five isolates; these fingerprints were compared with the band profiles obtained from inter-simple sequence repeat (UBC861) and inter-retrotransposon amplified polymorphism analyses. We found that the M13 marker was more effective than the other two markers for differentiating these isolates. The fungus Mycosphaerella fijiensis is the causative agent of black sigatoka, which is one of the most destructive diseases of banana plants. Infection with this pathogen results in underdeveloped fruit, with no commercial value. We analyzed the distribution of the M. fijiensis mating-type system and its genetic variability using M13 phage DNA markers. We found a 1:1 distribution of mating-type alleles, indicating MAT1-1 and MAT1-2 idiomorphs. A polymorphism analysis using three different primers for M13 markers showed that only the M13 minisatellite primers generated polymorphic products. We then utilized this polymorphism to characterize 40 isolates from various Brazilian states. The largest genetic distances were found between isolates from the same location and between isolates from different parts of the country. Therefore, there was no correlation between the genetic similarity and the geographic origin of the isolates. The M13 marker was used to generate genetic fingerprints for five isolates; these fingerprints were compared with the band profiles obtained from inter-simple sequence repeat (UBC861) and inter-retrotransposon amplified polymorphism analyses. We found that the M13 marker was more effective than the other two markers for differentiating these isolates.

Four mitochondrial minisatellites were used to study cytoplasmic diversity in leaf and garden beet germplasm resources. Eleven multi-locus haplotypes were identified, of which one (named mitochondrial minisatellite haplotype 4, hereafter min04) was associated with male-sterile Owen cytoplasm and two others (min09 and min18), with a normal fertile cytoplasm. European leaf beet germplasm exhibited the greatest haplotype diversity, with min09 and min18 predominating. In North African leaf beet accessions, only these two haplotypes were observed, making it likely that North African accessions were descended from European genotypes. The prevalence of min18 was also noted in leaf beet from the Middle East and western Asia. Such a pattern contrasts with that found in east Asian leaf beet where the two haplotypes were extremely rare. The geographical structure of the mitochondrial haplotypes allowed us to infer possible dissemination pathways of leaf beet. Additionally, we showed that mitochondrial genome diversity was low in garden beet germplasm, with min18 being highly predominant. An explanation of this limited diversity may lie in the geographically restricted origin of as well as relatively short cultivation histories of garden beet.

The variable numbers of tandem repeats (VNTR) locus D1S80, located on chromosome 1 (1p35-36), has a repeat unit 16 bp in length, and different numbers of these repeat units have been observed for populations of different origins and ethnicity. We used a molecular identification method based on capillary electrophoresis separation to analyze D1S80 locus polymorphism among 74 subjects from Denizli province, Turkey, finding an amplified fragment length size of 379-635 bp. Allele repeat numbers were deduced from these sizes and sequence comparison. The most common alleles were repeat units 24 (34.3%) and 18 (22.4%), with frequencies of 0.414 and 0.207, respectively. Other alleles were 25 (7.86%), 28 (5.71%), 22 (4.25%), and 29 (2.86%). The allele with 23 repeat units was not observed. Results were in Hardy-Weinberg linkage disequilibrium. Observed heterozygosity was 0.614, and expected heterozygosity was 0.787. Theta(k) value was 4.86 (95% confidence interval limits). Capillary electrophoresis is a powerful approach for accurate identification of VNTR loci, especially for low base pair units like D1S80, for prenatal diagnosis, linkage analysis, forensic identification, paternity testing, anthropological research, and phylogenetic studies. © 2009 Springer Science+Business Media, LLC.

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:22:42Z No. of bitstreams: 0Bitstream added on 2014-05-27T14:40:36Z : No. of bitstreams: 1 2-s2.0-50049126707.pdf: 129217 bytes, checksum: 0cb09d217283856c58d51a557c26c06c (MD5) Made available in DSpace on 2014-05-27T11:22:42Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-12-01 A existência de sistemas que possam diferenciar cepas de Mycobacterium tuberculosis epidemiologicamente relacionadas daquelas não relacionadas, são ferramentas importantes. A tuberculose é uma doença infecciosa, na qual técnicas de biologia molecular permitem a obtenção de informações muito difíceis ou impossíveis de serem alcançadas pela epidemiologia clássica. Um método de tipagem discriminatório, baseado no DNA genômico bacteriano, denominado RFLP (polimorfismo de comprimento de fragmentos de restrição), é empregado no estudo epidemiológico da tuberculose. Entretanto esta técnica é trabalhosa e sua substituição é uma tendência. Assim, outras seqüências têm sido usadas como marcadores epidemiológicos, como na Spoligotyping, a qual é baseada na PCR, com hibridização diferencial subseqüente dos produtos amplificados. O polimorfismo observado nas diferentes amostras é provavelmente produto de recombinação homóloga. A técnica de MIRU (mycobacterial interspersed repetitive unit) é um sistema rápido e reprodutível, onde ocorre a geração de genótipos baseados no estudo de 12 loci contendo VNTRs (número variável de repetições em seqüência) do complexo M. tuberculosis. Ela compara as cepas de áreas geográficas diferentes e permite o rastreamento do movimento de linhagens individuais, como no RFLP. Este tipo de abordagem permite a análise de maior número de cepas e a identificação de um número maior de focos de contaminação dentro da população, propiciando melhores maneiras de frear a transmissão da doença. Palavras-chave: Mycobacterium tuberculosis; tuberculose; epidemiologia molecular; genotipagem. Systems that can distinguish epidemiologically-related Mycobacterium tuberculosis strains from unrelated ones are extremely valuable. Molecular biology techniques have allowed a great deal of information to be acquired about the infectious disease tuberculosis (TB) that was very hard or impossible to obtain by conventional epidemiology. A typing method based on bacterial DNA genome differences, known as RFLP (restriction fragment length polymorphism), is widely used to discriminate strains in the epidemiologic study of TB. However, RFLP is laborious and there is a tendency to replace it by other methods. Thus, other DNA sequences have been employed as epidemiological markers, as in Spoligotyping, a fast technique based on PCR followed by differential hybridization of amplified products. The polymorphism observed among different isolates is probably the product of strain-dependent recombination. MIRU (mycobacterial interspersed repetitive unit) typing is a reproducible and fast assay, involving the generation of genotypes based on the study of 12 loci containing VNTRs (variable-number tandem repeats) in strains of the M. tuberculosis complex. It compares strains from different geographic areas and allows the movement of individual lineages to be tracked, as in RFLP. This approach enables a greater number of isolates to be analyzed, leading to the identification of a larger number of foci of transmission within the population and thus to improved ways of slowing the progress of the disease. Departamento de Ciências Biológicas Faculdade de Ciências Farmacêuticas Universidade Estadual Paulista, Araraquara, SP Faculdade de Ciências Agrárias Universidade Camilo Castelo Branco UNICASTELO, Descalvado, SP Laboratório de Biologia Molecular Aplicada a Tuberculose Fundação Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, RJ Departamento de Ciências Biológicas Faculdade de Ciências Farmacêuticas Universidade Estadual Paulista, Rodovia Araraquara-Jaú, km 01, CEP: 14801-902 - Araraquara - SP Departamento de Ciências Biológicas Faculdade de Ciências Farmacêuticas Universidade Estadual Paulista, Araraquara, SP Departamento de Ciências Biológicas Faculdade de Ciências Farmacêuticas Universidade Estadual Paulista, Rodovia Araraquara-Jaú, km 01, CEP: 14801-902 - Araraquara - SP

Objective - Genetic polymorphisms in the gene for endothelial nitric oxide synthase have been considered as potential risk factors for the development of coronary artery disease in some populations. Methods - We studied two polymorphisms of the NOS3 gene, the VNTR in intron 4 (4VNTR) and the Glu298Asp polymorphism in exon 7, in relation to the existence of premature coronary artery disease and the occurrence of myocardial infarction. A total number of 370 individuals of the Greek population was examined by PCR-RFLP method. The patient group consisted of 209 subjects, aged less than 58 years presenting symptomatic coronary artery disease, documented by coronary angiography. Results - The frequencies for bb, ab and aa genotypes of 4VNTR polymorphism were 0.67, 0.29, 0.04, respectively, for the patient group and 0.73, 0.24, 0.03 for the control group. The frequencies for GG (Glu/ Glu), GT (Glu/Asp), TT (Asp/Asp) of the Glu298Asp polymorphism were 0.52, 0.41, 0.07, respectively, in patients compared to 0.47, 0.46, 0.07, in control subjects. Statistical analysis indicated that there are no significant differences in the frequencies of the genotypes between patients and control subjects for both polymorphisms. The combined analysis of the two polymorphisms indicated no synergistic effect of the a and T alleles on coronary artery disease. Conclusions - We have found no evidence for association between the a allele of the 4VNTR polymorphism, or the T allele of Glu298Asp polymorphism and the risk for premature coronary artery disease or occurrence of myocardial infarction. Furthermore, no synergistic contribution of these polymorphisms to the development of premature coronary artery disease has been observed. 63 609 614 Cited By :17

Background: Brain natriuretic peptide (BNP) acts primarily as a cardiac hormone; it is produced by the ventricle and has both vasodilatory and natriuretic actions. Therefore, the BNP gene is thought to be a candidate gene for essential hypertension (EH). The present study identified variants in the 5'-flanking region of natriuretic peptide precursor B (NPPB) gene and assessed the relationship between gene variants and EH. Methods: The polymerase chain reaction-single strand conformation polymorphism method and nucleotide sequencing were used to identify variants. Results: A novel variable number of tandem repeat (VNTR) polymorphism in the 5'-flanking region (-1241 nucleotides from the major transcriptional initiation site) was discovered. This VNTR polymorphism is a tandem repeat of the 4-nucleotide sequence TTTC. There were 8 alleles, ranging from 9-repeat to 19-repeat. An association study was done involving 317 EH patients and 262 age-matched normotensive (NT) subjects. The 11-repeat allele was the most frequent (88.2%); the 16-repeat allele was the second most frequent (10.5%) in the NT group. The observed and expected genotypes were in agreement with the predicted Hardy-Weinberg equilibrium values (P=0.972). Among females, the overall distribution of genotypes was significantly different between the EH and NT groups (p=0.039). The frequency of the 16-repeat allele was significantly lower in the female EH group (6.5%) than in the female NT group (12.2%, p=0.046). Conclusions: The 16-repeat allele of the VNTR in the 5'-flanking region of NPPB appears to be a useful genetic marker of EH in females.

Sputum samples from new tuberculosis (TB) cases were collected over 2 years as part of a prospective study in the northeastern part of Lima, Peru. To measure the contribution of recent transmission to the high rates of multidrug resistance (MDR) in this area, Mycobacterium tuberculosis complex (MTBc) isolates were tested for drug susceptibility to first-line drugs and were genotyped by spoligotyping and 15-locus mycobacterial interspersed repetitive-unit (MIRU-15)-variable-number tandem repeat (VNTR) analysis. MDR was found in 6.8% of 844 isolates, of which 593 (70.3%) were identified as belonging to a known MTBc lineage, whereas 198 isolates (23.5%) could not be assigned to these lineages and 12 (1.4%) represented mixed infections. Lineage 4 accounted for 54.9% ( n = 463) of the isolates, most of which belonged to the Haarlem family ( n = 279). MIRU-15 analysis grouped 551/791 isolates (69.7%) in 102 clusters, with sizes ranging from 2 to 46 strains. The overall high clustering rate suggests a high level of recent transmission in this population, especially among younger patients (odds ratio [OR], 1.6; P = 0.01). Haarlem strains were more prone to cluster, compared to the other families taken together (OR, 2.0; P < 0.0001), while Beijing (OR, 0.6; P = 0.006) and LAM (OR, 0.7; P = 0.07) strains clustered less. Whereas streptomycin-resistant strains were more commonly found in clusters (OR, 1.8; P = 0.03), clustering rates did not differ between MDR and non-MDR strains (OR, 1.8; P = 0.1). Furthermore, only 16/51 MDR strains clustered with other MDR strains, suggesting that patients with primary MDR infections acquired the infections mostly from index cases outside the study population, such as retreated cases.

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:21:43Z No. of bitstreams: 0Bitstream added on 2014-05-27T14:29:32Z : No. of bitstreams: 1 2-s2.0-30644471195.pdf: 61570 bytes, checksum: 5a070967306409a24270de736e01aec5 (MD5) Made available in DSpace on 2014-05-27T11:21:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2005-12-01 The Pacific white shrimp, Litopenaeus vannamei (Penaeidae), represents about 95% of all Brazilian shrimp production. The Brazilian L. vannamei foundation broodstock was made up of specimens collected from different American Pacific sites, but little information was collected on the genetic structure of the broodstock. We used the fluorescence amplified fragment length polymorphism (fAFLP) method to study the genetic diversity of L. vannamei broodstock lines 03CMF1 and 03CBF1 originally produced by breeder-shrimps imported mainly from Panama and Ecuador, although wild individuals from other localities may also have been used in producing these two lines. Our results showed a total of 93 polymorphic bands ranging from 50 to 500 bp, the mean Nei's genetic diversity calculated for the total sample was 13.4% and identity and genetic distance analyses indicated high genetic homogeneity within and between both the broodstock lineages studied which suggests that they had similar genetic structure. These results may represent an important tool for the appropriate management of L. vannamei broodstocks. Copyright by the Brazilian Society of Genetics. Universidade Federal de São Carlos Departamento de Genética e Evolução, Caixa Postal 676, 13575-905 São Carlos, SP Universidade Estadual Paulista 'Júlio de Mesquita Filho' Departamento de Biologia Aplicada à Agropecuária, Jaboticabal, SP Universidade Federal do Ceará Departamento de Engenharia de Pesca, Fortaleza, CE Universidade Estadual Paulista 'Júlio de Mesquita Filho' Departamento de Biologia Aplicada à Agropecuária, Jaboticabal, SP