Molecular characterization of Mycobacterium massiliense and Mycobacterium bolletii in isolates collected from outbreaks of infections after laparoscopic surgeries and cosmetic procedures

An outbreak of infections affecting 311 patients who had undergone different invasive procedures occurred in 2004 and 2005 in the city of Belem, in the northern region of Brazil. Sixty-seven isolates were studied; 58 were from patients who had undergone laparoscopic surgeries, 1 was from a patient with a postinjection abscess, and 8 were from patients who had undergone mesotherapy. All isolates were rapidly growing nonpigmented mycobacteria and presented a pattern by PCR-restriction enzyme analysis of the hsp65 gene with BstEII of bands of 235 and 210 bp and with HaeIII of bands of 200, 70, 60, and 50 bp, which is common to Mycobacterium abscessus type 2, Mycobacterium bolletii, and Mycobacterium massiliense. hsp65 and. rpoB gene sequencing of a subset of 20 isolates was used to discriminate between these three species. hsp65 and rpoB sequences chosen at random from 11 of the 58 isolates from surgical patients and the postinjection abscess isolate presented the highest degrees of similarity with the corresponding sequences of M. massiliense. In the same way, the eight mesotherapy isolates were identified as M. bolletii. Molecular typing by pulsed-field gel electrophoresis (PFGE) grouped all 58 surgical isolates, while the mesotherapy isolates presented three different PFGE patterns and the postinjection abscess isolate showed a unique PFGE pattern. In conclusion, molecular techniques for identification and typing were essential for the discrimination of two concomitant outbreaks and one case, the postinjection abscess, not related to either outbreak all of which were originally attributed to a single strain of M. abscessus.

Mycobacterium bovis identification by a molecular method from post-mortem inspected cattle obtained in abattoirs of Mato Grosso do Sul, Brazil

The presence of Mycobacterium bovis in bovine carcasses with lesions suggestive of tuberculosis was evaluated. Seventy-two carcass samples were selected during slaughter inspection procedures in abattoirs in the state of Mato Grosso do Sul, Brazil. Seventeen (23.6%) of samples showed colonies suggestive of mycobacteria that were confirmed to be acid-fast bacilli by Ziehl-Neelsen staining. Polymerase chain reaction (PCR) using primers specific for M. bovis identified M. bovis in 13 (76.5%) isolates. The PCR-restriction enzyme pattern analysis using gene encoding for the 65-kDa protein and two restriction enzymes identified the remaining four isolates that were represented by two M. tuberculosis complex and two nontuberculous mycobacteria. The results are indicative of infection of slaughter cattle by M. bovis and other mycobacteria in the state of Mato Grosso do Sul.

Identification of lubricant contamination by biodiesel using vibration analysis and neural network

Purpose - The purpose of this paper is to provide information on lubricant contamination by biodiesel using vibration and neural network.Design/methodology/approach - The possible contamination of lubricants is verified by analyzing the vibration and neural network of a bench test under determinated conditions.Findings - Results have shown that classical signal analysis methods could not reveal any correlation between the signal and the presence of contamination, or contamination grade. on other hand, the use of probabilistic neural network (PNN) was very successful in the identification and classification of contamination and its grade.Research limitations/implications - This study was done for some specific kinds of biodiesel. Other types of biodiesel could be analyzed.Practical implications Contamination information is presented in the vibration signal, even if it is not evident by classical vibration analysis. In addition, the use of PNN gives a relatively simple and easy-to-use detection tool with good confidence. The training process is fast, and allows implementation of an adaptive training algorithm.Originality/value - This research could be extended to an internal combustion engine in order to verify a possible contamination by biodiesel.

Identification of Mechanical Systems through Kautz Filter

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Proposta metodológica para o imageamento, caracterização, parametrização e geração de modelos virtuais de afloramentos

The aim of this work was to describe the methodological procedures that were mandatory to develop a 3D digital imaging of the external and internal geometry of the analogue outcrops from reservoirs and to build a Virtual Outcrop Model (VOM). The imaging process of the external geometry was acquired by using the Laser Scanner, the Geodesic GPS and the Total Station procedures. On the other hand, the imaging of the internal geometry was evaluated by GPR (Ground Penetrating Radar).The produced VOMs were adapted with much more detailed data with addition of the geological data and the gamma ray and permeability profiles. As a model for the use of the methodological procedures used on this work, the adapted VOM, two outcrops, located at the east part of the Parnaiba Basin, were selected. On the first one, rocks from the aeolian deposit of the Piaui Formation (Neo-carboniferous) and tidal flat deposits from the Pedra de Fogo Formation (Permian), which arises in a large outcrops located between Floriano and Teresina (Piauí), are present. The second area, located at the National Park of Sete Cidades, also at the Piauí, presents rocks from the Cabeças Formation deposited in fluvial-deltaic systems during the Late Devonian. From the data of the adapted VOMs it was possible to identify lines, surfaces and 3D geometry, and therefore, quantify the geometry of interest. Among the found parameterization values, a table containing the thickness and width, obtained in canal and lobes deposits at the outcrop Paredão and Biblioteca were the more relevant ones. In fact, this table can be used as an input for stochastic simulation of reservoirs. An example of the direct use of such table and their predicted radargrams was the identification of the bounding surface at the aeolian sites from the Piauí Formation. In spite of such radargrams supply only bi-dimensional data, the acquired lines followed of a mesh profile were used to add a third dimension to the imaging of the internal geometry. This phenomenon appears to be valid for all studied outcrops. As a conclusion, the tool here presented can became a new methodology in which the advantages of the digital imaging acquired from the Laser Scanner (precision, accuracy and speed of acquisition) were combined with the Total Station procedure (precision) using the classical digital photomosaic technique

Replication of classical infectious bursal disease virus in the chicken embryo related cell line

Infectious bursal disease (IBD) is an acute, highly contagious viral disease. The diagnosis of IBD depends on time-consuming and costly procedures, like virus isolation on chick embryos and histopathological examination, A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), immunoperoxidase and reverse transcription polymerase chain reaction (RT-PCR) were applied in this study to detect classical IBD virus (IBDV) after three blind passages of the Lukert strain on chicken embryo related (CER) cell monolayer after different periods of infection: 6, 12, 24 and 48 h, Cytophatic effects were most evident 12 h post-infection (p.i.) but were observed at 6 h p.i. The maximum discrimination between IBDV-infected and uninfected cell suspensions obtained by the use of DAS-ELISA for virus detection corresponded to 0.597+/-0.02 and 0.010+/-0.01 after 12h p.i., respectively. The RT-PCR was performed using the set of primers A3.1 and A3.2 to amplify the VP2 region of the IBDV genome, This molecular technique demonstrated that from 6 h p.i., it was possible to detect the viral RNA. The results show that the CER cell line can be used for classical IBDV propagation, confirmed by the DAS-ELISA, immunoperoxidase and RT-PCR assay.

Identification of Mycobacteria by Thin Layer Chromatographic Analysis of Mycolic Acids and Conventional Biochemical Method: Four Years of Experience

Mycolic acids analysis by thin-layer chromatography (TLC) has been employed by several laboratories worldwide as a method for fast identification of mycobacteria. This method was introduced in Brazil by our laboratory in 1992 as a routine identification technique. Up to the present, 861 strains isolated were identified by mycolic acids TLC and by standard biochemical tests; 61% out of these strains came as clinical samples, 4% isolated from frogs and 35% as environmental samples. Mycobacterium tuberculosis strains identified by classical methods were confirmed by their mycolic acids contents (I, III and IV). The method allowed earlier differentiation of M. avium complex - MAC (mycolic acids I, IV and VI) from M. simiae (acids I, II and IV), both with similar biochemical properties. The method also permitted to distinguish M. fortuitum (acids I and V) from M. chelonae (acids I and II) , and to detect mixed mycobacterial infections cases as M. tuberculosis with MAC and M. fortuitum with MAC. Concluding, four years experience shows that mycolic acids TLC is an easy, reliable, fast and inexpensive method, an important tool to put together conventional mycobacteria identification methods.

Isolation and identification of mycobacteria from livestock specimens and milk obtained in Brazil

The prevalence of Mycobacterium bovis and other mycobacterial species in livestock specimens and milk was evaluated. An emphasis was placed upon the distribution of these organisms in milk that is readily available to the public that was either untreated, pasteurized, or treated using ultra high temperature. Twenty-two pathologic specimens from livestock (bovine, swine and bubaline) in five Brazilian states and 128 bovine milk samples from retail markets in the State of São Paulo were examined for mycobacteria. Identification was made by classical biochemical tests, thin layer chromatography of mycolic acids and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Mycobacteria were isolated from 15 (68.2%) caseous lesions and from 23 (18%) milk samples. Eleven isolates were identified as M. bovis, and the remaining 27 nontuberculous mycobacterial isolates were represented by five species and six unidentified rapidly growing mycobacterial strains. The data demonstrate that animal products in Brazil are frequent reservoirs of mycobacteria and may pose a risk to the public.

Identification and characterization of novel mutations of the aspartoacylase gene in non-Jewish patients with Canavan disease

Canavan disease, an inherited leukodystrophy, is caused by mutations in the aspartoacylase (ASPA) gene. It is most common among children of Ashkenazi Jewish descent but has been diagnosed in many diverse ethnic groups. Two mutations comprise the majority of mutant alleles in Jewish patients, while mutations in the ASPA gene among non-Jewish patients are different and more diverse. In the present study, the ASPA gene was analysed in 22 unrelated non-Jewish patients with Canavan disease, and 24 different mutations were found. of these,14 are novel, including five missense mutations (E24G, D68A, D249V, C152W, H244R), two nonsense mutations (Q184X, E214X), three deletions (923delT, 33del13, 244delA), one insertion mutation (698insC), two sequence variations in one allele ([10T>G; 11insG]), an elimination of the stop codon (941A>G, TAG-->TGG, X314W), and one splice acceptor site mutation (IVS1 - 2A>T). The E24G mutation resulted in substitution of an invariable amino acid residue (Glu) in the first esterase catalytic domain consensus sequence. The IVS1 - 2A>T mutation caused the retention of 40 nucleotides of intron 1 upstream of exon 2. The results of transient expression of the mutant ASPA cDNA containing these mutations in COS-7 cells and assays for ASPA activity of patient fibroblasts indicated that these mutations were responsible for the enzyme deficiency. In addition, patients with the novel D249V mutation manifested clinically at birth and died early. Also, patients with certain other novel mutations, including C152W, E214X, X314W, and frameshift mutations in both alleles, developed clinical manifestations at an earlier age than in classical Canavan disease.

A comparison of mycolic acid analysis for nontuberculous mycobacteria identification by thin-layer chromatography and molecular methods

The development of fast, inexpensive, and reliable tests to identify nontuberculous mycobacteria (NTM) is needed. Studies have indicated that the conventional identification procedures, including biochemical assays, are imprecise. This study evaluated a proposed alternative identification method in which 83 NTM isolates, previously identified by conventional biochemical testing and in-house M. avium IS1245-PCR amplification, were submitted to the following tests: thin-layer chromatography (TLC) of mycolic acids and PCR-restriction enzyme analysis of hsp65 (PRA). High-performance liquid chromatography (HPLC) analysis of mycolic acids and Southern blot analysis for M. avium IS1245 were performed on the strains that evidenced discrepancies on either of the above tests. Sixty-eight out of 83 (82%) isolates were concordantly identified by the presence of IS1245 and PRA and by TLC mycolic acid analysis. Discrepant results were found between the phenotypic and molecular tests in 12/83 (14.4%) isolates. Most of these strains were isolated from non-sterile body sites and were most probably colonizing in the host tissue. While TLC patterns suggested the presence of polymycobacterial infection in 3/83 (3.6%) cultures, this was the case in only one HPLC-tested culture and in none of those tested by PRA. The results of this study indicated that, as a phenotypic identification procedure, TLC mycolic acid determination could be considered a relatively simple and cost-effective method for routine screening of NTM isolates in mycobacteriology laboratory practice with a potential for use in developing countries. Further positive evidence was that this method demonstrated general agreement on MAC and M. simiae identification, including in the mixed cultures that predominated in the isolates of the disseminated infections in the AIDS patients under study. In view of the fact that the same treatment regimen is recommended for infections caused by these two species, TLC mycolic acid analysis may be a useful identification tool wherever molecular methods are unaffordable.

Identification of lattices from genus of compact surface

In this work we propose procedures for the identification of structure of group associate lattices from fundamental region F4g of regular tessellations {4g; 4g} in the Euclidian plane and hyperbolic plane, where g denote genus of compact surface. © 2006 SBrT.

Measurement of melatonin in body fluids: Standards, protocols and procedures

Background and Purpose: The circadian rhythm of melatonin in saliva or plasma, or of the melatonin metabolite 6-sulfatoxymelatonin (a6MTs) in urine, is a defining feature of suprachiasmatic nucleus (SCN) function, the body's endogenous oscillatory pacemaker. The primary objective of this review is to ascertain the clinical benefits and limitations of current methodologies employed for detection and quantification of melatonin in biological fluids and tissues. Data Identification: A search of the English-language literature (Medline) and a systematic review of published articles were carried out. Study Selection: Articles that specified both the methodology for quantifying melatonin and indicated the clinical purpose were chosen for inclusion in the review. Data Extraction: The authors critically evaluated the methodological issues associated with various tools and techniques (e.g. standards, protocols, and procedures). Results of Data Synthesis: Melatonin measurements are useful for evaluating problems related to the onset or offset of sleep and for assessing phase delays or advances of rhythms in entrained individuals. They have also become an important tool for psychiatric diagnosis, their use being recommended for phase typing in patients suffering from sleep and mood disorders. Additionally, there has been a continuous interest in the use of melatonin as a marker for neoplasms of the pineal region. Melatonin decreases such as found with aging are or post pinealectomy can cause alterations in the sleep/wake cycle. The development of sensitive and selective methods for the precise detection of melatonin in tissues and fluids has increasingly been shown to have direct relevance for clinical decision making. Conclusions: Due to melatonin's low concentration, as well as the coexistence of numerous other compounds in the blood, the routine determination of melatonin has been an analytical challenge. The available evidence indicates however that these challenges can be overcome and consequently that evaluation of melatonin's presence and activity can be an accessible and useful tool for clinical diagnosis. © Springer-Verlag 2010.

Damage detection in nonlinear structures using discrete-time volterra series

Structural damage identification is basically a nonlinear phenomenon; however, nonlinear procedures are not used currently in practical applications due to the complexity and difficulty for implementation of such techniques. Therefore, the development of techniques that consider the nonlinear behavior of structures for damage detection is a research of major importance since nonlinear dynamical effects can be erroneously treated as damage in the structure by classical metrics. This paper proposes the discrete-time Volterra series for modeling the nonlinear convolution between the input and output signals in a benchmark nonlinear system. The prediction error of the model in an unknown structural condition is compared with the values of the reference structure in healthy condition for evaluating the method of damage detection. Since the Volterra series separate the response of the system in linear and nonlinear contributions, these indexes are used to show the importance of considering the nonlinear behavior of the structure. The paper concludes pointing out the main advantages and drawbacks of this damage detection methodology. © (2013) Trans Tech Publications.

Isolation, X-ray crystal structure and theoretical calculations of the new compound 8-Eepicordatin and identification of others terpenes and steroids from the bark and leaves of Croton palanostigma Klotzsch

Estudos fitoquímicos com as cascas do caule e com as folhas de Croton palanostigma Klotzsch (Euphorbiaceae) levaram ao isolamento do novo diterpeno clerodânico 8-epicordatina (2), além de éster metílico do ácido 12-oxohardwickiico (3), aparisthmano, cordatina (1), ácido ent-trachiloban-18-óico, óxido de ent-13-epimanoila, óxido de ent-3-oxo-13-epimanoila, óxido de ent-3β-hidroxi-13-epimanoila, sitosterol, estigmasterol, estigmastan-3-ona, 6β-hidroxiestigmast-4-en-3-ona, 6β-hidroxiestigmasta-4,22-dien-3-ona, estigmast-4-en-3-ona, estigmasta-4,22-dien-3-ona, ácido 3-O-acetilaleuritolico, 11α-hidroxiurs-12-en-3-ona, α-amirenona, 24-metilenocicloartenona e lupenona. Estas substâncias foram isoladas através de procedimentos fitoquímicos usuais e suas estruturas foram deduzidas por estudos espectroscópicos, incluindo experimentos em 2D. Adicionalmente, a estrutura cristalina de 8-epicordatina (2) foi determinada por difração de raios-X. Cálculos teóricos de RMN ao nível B3PW91/DGDZVP foram usados para confirmação dos assinalamentos dos deslocamentos químicos dos hidrogênios H-7α e H-7β de 8-epicordatina.

Environmental isolation, biochemical identification, and antifungal drug susceptibility of Cryptococcus species

Introduction The incidence of opportunistic fungal infections has increased in recent years and is considered an important public health problem. Among systemic and opportunistic mycoses, cryptococcosis is distinguished by its clinical importance due to the increased risk of infection in individuals infected by human immunodeficiency virus. Methods To determine the occurrence of pathogenic Cryptococcus in pigeon excrement in the City of Araraquara, samples were collected from nine environments, including state and municipal schools, abandoned buildings, parks, and a hospital. The isolates were identified using classical tests, and susceptibility testing for the antifungal drugs (fluconazole, itraconazole, voriconazole, and amphotericin B) independently was also performed. After collection, the excrement samples were plated on Niger agar and incubated at room temperature. Results A total of 87 bird dropping samples were collected, and 66.6% were positive for the genus Cryptococcus. The following species were identified: Cryptococcus neoformans (17.2%), Cryptococcus gattii (5.2%), Cryptococcus ater (3.5%), Cryptococcus laurentti (1.7%), and Cryptococcus luteolus (1.7%). A total of 70.7% of the isolates were not identified to the species level and are referred to as Cryptococcus spp. throughout the manuscript. Conclusions Although none of the isolates demonstrated resistance to antifungal drugs, the identification of infested areas, the proper control of birds, and the disinfection of these environments are essential for the epidemiological control of cryptococcosis.