Phenotypic and genotypic characteristics of Salmonella Typhi were studied in 30 strains, isolated in different years, from some areas in Brazil. Conventional typing methods were performed by biochemical tests, Vi phage-typing scheme, and antimicrobial susceptibility test. Moleculartyping methods were performed by analysis of plasmid DNA and by random amplified polymorphic DNA (RAPD-PCR). For the latter, an optimization step was performed to ensure the reproducibility of the process in genetic characterization of S. Typhi. The predominance of 76.7% of biotype I (xylose +, arabinose -) was noticed in all studied areas. Three phage types were recognized, with prominence for the phage types A (73.3%) and I+IV (23.3%). All the strains were susceptible to the drugs used. However, 36.7% of the strains contained plasmids, with predominance of the 105 Kb plasmid. RAPD was capable of grouping the strains in 8 genotypic patterns using primer 784, in 6, using primer 787 and in 7, using primer 797. Conventional phenotypic typing methods, as well as the DNA plasmid analysis, presented nonsignificant discriminatory power; however, RAPD- PCR analysis showed discriminatory power, reproducibility, easy interpretation and performance, being considered as a promising alternative typing method for S. Typhi.
Leptospirosis is an emerging infectious disease that has been identified as both a human and animal health problem worldwide. Regular outbreaks associated with specific risk factors have been reported in Argentina. However, there are no available data concerning the genetic population level for this pathogen. Therefore, the aim of this work was to describe the genetic diversity of Leptospira interrogans through the application of two moleculartyping strategies: variable number of tandem repeats (VNTR) and multilocus sequence typing (MLST). For this purpose, seven refer- ence strains and 18 non-epidemiologically related isolates from diverse hosts and Argentinean regions were analysed. Among them, nine genotypes and seven sequence types (STs), including three unreported STs, were described using VNTR and MLST, respectively. eBURST analysis demonstrated that ST37 was the most frequent and founder genotype of a clonal complex (CCs) containing STN1 and STN3, suggesting the importance of studying the serovars belonging to this CC in Argentina. The data from maximum parsimony analysis, which combined both techniques, achieved intra- serovar discrimination, surmounted microscopic agglutination test discrepancies and increased the discriminatory power of each technique applied separately. This study is the first to combine both strategies for L. interrogans typing to generate a more comprehensive molecular genotyping of isolates from Argentina in a global context.
Moleculartyping techniques are widly applied in stud- ies of S. aureus epidemiology, especially MRSA, with the aim of distinguishing isolates that are epidemiologi- cally related from those unrelated. S. aureus outbreaks in hospitals are frequently considered as short duration events of localized epidemiology and, in these situations, most of the moleculartyping methods are able to distin- guish the isolates that belong to the outbreak (Shopsin & Kreiswirth 2001). However, the study of isolates that do not share a temporal or geographic origin, to define an- cestors and lineages, is a major challenge. To date, sev- eral techniques allowed for the investigation and charac-
patients enrolled in this study are particularly interesting since C. albicans were isolated from different sites. In all cases the analysis confirmed that the clinical isolates were identical to each other (Table 3) indicating the migration of yeasts from colonization (urine catheters, tracheal secretion) into the blood, suggesting the source of systemic infection. This result indicates that each isolated pair has genotypic identity, suggesting clonal origin. This fact has been demonstrated by moleculartyping, in several studies 10,24,29,37 and helps confirm that previous colonization is an
Mycobacterium bovis is the main causative agent of animal tuberculosis (TB) and it may cause TB in humans. Moleculartyping of M. bovis isolates provides precise epidemiological data on issues of inter- or intra-herd transmission and wildlife reservoirs. Techniques used for typing M. bovis have evolved over the last 2 decades, and PCR-based methods such as spoligotyping and mycobacterial in- terspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) have been extensively used. These techniques can provide epidemiological information about isolates of M. Bovis that may help control bovine TB by indicating possible links between diseased animals, detecting and sam- pling outbreaks, and even demonstrating cases of laboratory cross-contamination between samples. This review will focus on techniques used for the moleculartyping of M. bovis and discuss their gen- eral aspects and applications.
11. Kersulyte D, Struelens MJ, Deplano A, Berg DE. Comparison of arbitrarily primed PCR and macrorestriction (pulsed-fi eld gel electrophoresis) typing of Pseudomonas aeruginosa strains from cystic fi brosis patients. J Clin Microbiol 1995; 33:2216-9. 12. Liu Y, Davin-Regli A, Bosi C et al. Epidemiological investiga- tion of Pseudomonas aeruginosa nosocomial bacteraemia iso- lates by PCR-based DNA fi ngerprinting analysis. J Med Micro- biol 1996; 45:359-65.
Several alternative PCR-based methods have been developed in order to overcome these problems. Spoligotyping is often used as a secondary method for typing low-copy-number IS6110 isolates, but it does little to improve strain differentiation in population-based studies (Allix-Béguec et al., 2008; Cronin et al., 2001). Spoligotyping is a technique related to DNA polymorphisms within direct repeat (DR) locus of M. tuberculosis, pointing out the presence or absence of a set of target spacer sequences between the DRs. The octonal pat- tern of spacers between the conserved DRs in the region was used to differentiate M. tuberculosis strains (Mendes et al., 2011; Sola et al., 2001).
[2,4,5,7,8]. Phenotypic typing methodologies have limited discriminative capacities. Genotypic methods are usually more discriminative and have been useful in demonstrating that many outbreaks of infections with RGM involved mul- tiple species and ⁄ or multiple strains of the same species [4,5,9,10]. Plasmid analysis has been used to characterise M. fortuitum isolates responsible for infections following augmentation mamma- plasty and cardiac surgery in the USA [4,5]. Newer and more discriminative approaches in- clude pulsed-ﬁeld gel electrophoresis (PFGE), which has been used to type a range of different bacterial species, including M. fortuitum . Several PCR-based techniques are also being used with increasing frequency to investigate out- breaks involving non-tuberculous mycobacteria. Randomly ampliﬁed polymorphic DNA (RAPD) PCR analysis has been used to type isolates of Mycobacterium avium, M. abscessus and Myco- bacterium szulgai [8,11,12]. Enterobacterial repet- itive intergenic consensus (ERIC) PCR analysis has been reported to be a useful tool for typing Mycobacterium tuberculosis isolates , and has been shown to differentiate Mycobacteri- um paratuberculosis from other species of myco- bacteria, including M. fortuitum ; however, to date, there are no reports on the use of this technique to evaluate clonal relationships among RGM.
Currently, according to the second taxonomic consensus for T. cruzi approved during the XXV Protozoology Meet- ing held in Buzios, RJ, Brazil, the species is subdivided into six discrete typing units (DTU) named TcI, TcII, TcIII, TcIV, TcV and TcVI , related to several previous classifications based on different molecular markers. Regarding the geo- graphical distribution of the T. cruzi genotypes, it has been demonstrated that TcI has the largest distribution in all America. In Colombia, Mexico, Guatemala, Venezuela, Panama and Bolivia there is evidence of a predominance of
Ion AmpliSeq Colon and Lung Cancer Panel sensitivity resulted higher than that of routine molecular determinations, enabling the detection of sequence variants down to 1% allele frequency, which corresponds to 2% cancer cells in a sample. This is important in the assessment of clonal heterogeneity, i.e., the existence and quantification of multiple clones in the tumor mass . This is imperative in the therapeutic management and is not achievable by conventional sequencing, which requires a sample with at least 10% tumor cells for accurate detection of mutations. In some cases, the presence of a major driver mutation coexisting with other variants displaying lower allele frequencies, as in case #23 showing EGFR mutation in 73% of alleles and at lower frequency in TP53 (16%) and MAP2K1 (4%) sustains the hypothesis of tumor molecular heterogeneity and further underlines the demand of a NGS approach to characterize the samples. In addition, case #26 may represent a good example of a patient that may be candidate to a target therapy with a BRAF inhibitor instead of conventional therapy as the neoplasm shows a dominant BRAF mutation in 83% of alleles.
The plasmid profile analysis showed that all strains carrying intl1 had 2e8 bands of high (>15 kb) and low (<15 kb) molecular weights. Southern blot hybridization assays indicated that a class 1 integron was located on plas- mids in five of the seven strains (Fig. 2A,B). To see if hybridization signal was detectable in chromosomal DNA, genomic DNA incorporated in agarose plugs was digested with I-CeuI and DNA fragments were separated by PFGE (Shu-Lin et al., 1993). Southern blot hybridization indicated that intl1 was not present in chromosomal DNA (data not shown). To further confirm intl1-carrying plasmid, conjugation experiments were performed using STEC strain Ec120/00 as donor and E. coli HS as recipient. STEC strain Ec120/00 showed the presence of intl1 and resistance to streptomycin and tetracycline (Table 1). Likewise, the transconjugant strain
With the public attention being focused mainly on HIV and tuberculosis, there are only few data available on other issues such as, e.g., antibiotic resistance in various pathogens such as Staphylococcus aureus. Since its first description roughly 50 years ago, methicillin-resistant S. aureus (MRSA) has globally become a public health threat. While there is an abundance of epidemio- logical and typing data from Western Europe, the United States and Australia, relatively few data are available for other parts of the world. Unfortunately, this is also true for Eastern Europe including Romania. It is known that the MRSA rates are extremely high, ranging from approximately 30% up to 70% in recent Romanian studies [5,6,7,8,9,10,11,12], thus reaching the highest prevalence levels reported anywhere in the world. According to the ECDC antimicrobial resistance surveillance (formerly EARSS), the MRSA rate among invasive infections was ‘‘equal to or above 25%’’ in 2008 and slightly above 50% in 2011/2012 ; [http://www.ecdc.europa.eu/en/publications/ publications/antimicrobial-resistance-surveillance-europe-2011. pdf, http://www.ecdc.europa.eu/en/publications/publications/ antimicrobial-resistance-surveillance-europe-2012.pdf]. PVL-posi-
A tipagem de cepas é uma ferramenta fundamental para a análise epidemiológica molecular e pode fornecer informações importantes sobre a dispersão dos vírus dengue. Neste trabalho, foi realizada a caracterização molecular de amostras de vírus DEN-2 isoladas no Brasil entre 1990- 2000, de áreas geograficamente e temporalmente distintas, com o objetivo de investigar a distribuição genética deste sorotipo circulante no país. A reação em cadeia pela polimerase baseada em sítios de restrição específicos (RSS-PCR) apresentou o mesmo padrão para as 52 amostras Brasileiras, mostrando a circulação de apenas uma variante de vírus DEN- 2. A análise filogenética utilizando alinhamento progressivo de sequências de 240 nucleotídeos da junção E/NS1 de 15 cepas mostrou que estas pertencem ao genotipo III (genotipo Jamaica).
This study describes a new multilocus variable number tandem-repeat (VNTR) analysis (MLVA) typing system for the discrimination of Chlamydia trachomatis genovar D to K isolates or specimens. We focused our MLVA scheme on genovar E which predominates in most populations worldwide. This system does not require culture and therefore can be performed directly on DNA extracted from positive clinical specimens. Our method was based on GeneScan analysis of five VNTR loci labelled with fluorescent dyes by multiplex PCR and capillary electrophoresis. This MLVA, called MLVA-5, was applied to a collection of 220 genovar E and 94 non-E genovar C. trachomatis isolates and specimens obtained from 251 patients and resulted in 38 MLVA-5 types. The genetic stability of the MLVA-5 scheme was assessed for results obtained both in vitro by serial passage culturing and in vivo using concomitant and sequential isolates and specimens. All anorectal genovar E isolates from men who have sex with men exhibited the same MLVA-5 type, suggesting clonal spread. In the same way, we confirmed the clonal origin of the Swedish new variant of C. trachomatis. The MLVA-5 assay was compared to three other moleculartyping methods, ompA gene sequencing, multilocus sequence typing (MLST) and a previous MLVA method called MLVA-3, on 43 genovar E isolates. The discriminatory index was 0.913 for MLVA-5, 0.860 for MLST and 0.622 for MLVA-3. Among all of these genotyping methods, MLVA-5 displayed the highest discriminatory power and does not require a time- consuming sequencing step. The results indicate that MLVA-5 enables high-resolution molecular epidemiological characterisation of C. trachomatis genovars D to K infections directly from specimens.
9. Goker H, Haznedaroglu IC, Chao NJ. Acute graft-vs-host disease: pathobiology and management. Exp Hematol. 2001;29(3):259-77. 10. Nademanee A, Schmidt GM, Parker P, Dagis AC, Stein A, Snyder DS et al. The outcome of matched unrelated donor bone marrow transplantation in patients with hematologic malignancies using moleculartyping for donor selection and graft-versus-host disease prophylaxis regimen of cyclosporine, methotrexate, and prednisone. Blood. 1995;86(3):1228-34.
The epidemiologic typing of bacterial pathogens can be applied to answer a number of different questions: in case of outbreak, what is the extent and mode of transmission of epidemic clone(s )? In case of long-term surveillance, what is the prevalence over time and the geographic spread of epidemic and endemic clones in the population? A number of moleculartyping methods can be used to classify bacteria based on genomic diversity into groups of closely-related isolates (presumed to arise from a common ancestor in the same chain of transmission) and divergent, epidemiologically-unrelated iso- lates (arising from independent sources of infection). Ribotyping, IS-RFLP fingerprinting, macrorestriction analysis of chromosomal DNA and PCR-fingerprinting using arbitrary sequence or repeat element primers are useful methods for outbreak investigations and regional surveillance. Library typing systems based on multilocus sequence-based analysis and strain-specific probe hybridization schemes are in develop- ment for the international surveillance of major pathogens like Mycobacterium tuberculosis. Accurate epidemiological interpretation of data obtained with moleculartyping systems still requires additional research on the evolution rate of polymorphic loci in bacterial pathogens.