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Mycobacterium tuberculosis complex differentiation using gyrB-restriction fragment length polymorphism analysis
Chimara, Erica;Ferrazoli, Lucilaine;Le?o, Sylvia Cardoso;
Memórias do Instituto Oswaldo Cruz , 2004, DOI: 10.1590/S0074-02762004000700014
Abstract: mycobacterium tuberculosis complex (mtbc) members are causative agents of human and animal tuberculosis. differentiation of mtbc members is required for appropriate treatment of individual patients and for epidemiological purposes. strains from six mtbc species - m. tuberculosis, m. bovis subsp. bovis, m. bovis bcg, m. africanum, m. pinnipedii, and "m. canetti" - were studied using gyrb-restriction fragment length polymorphism (gyrb-rflp) analysis. a table was elaborated, based on observed restriction patterns and published gyrb sequences. to evaluate applicability of gyrb-rflp at instituto adolfo lutz, s?o paulo, mycobacterial reference laboratory, 311 mtbc clinical isolates, previously identified using traditional methods as m. tuberculosis (306), m. bovis (3), and m. bovis bcg (2), were analyzed by gyrb-rflp. all isolates were correctly identified by the molecular method, but no distinction between m. bovis and m. bovis bcg was obtained. differentiation of m. tuberculosis and m. bovis is of utmost importance, because they require different treatment schedules. in conclusion, gyrb-rflp is accurate and easy-to-perform, with potential to reduce time needed for conventional differentiation methods. however, application for epidemiological studies remains limited, because it cannot differentiate m. tuberculosis from m. africanum subtype ii, and "m. canetti", m. africanum subtype i from m. pinnipedii, and. m. bovis from m. bovis bcg.
Reliable identification of mycobacterial species by PCR-restriction enzyme analysis (PRA)-hsp65 in a reference laboratory and elaboration of a sequence-based extended algorithm of PRA-hsp65 patterns
Erica Chimara, Lucilaine Ferrazoli, Suely Ueky, Maria Martins, Alan Durham, Robert D Arbeit, Sylvia Le?o
BMC Microbiology , 2008, DOI: 10.1186/1471-2180-8-48
Abstract: A total of 434 NTM isolates were obtained from 5019 cultures submitted to the Institute Adolpho Lutz, Sao Paulo Brazil, between January 2000 and January 2001. Species identification was performed for all isolates using conventional phenotypic methods and PRA-hsp65. For isolates for which these methods gave discordant results, definitive species identification was obtained by sequencing a 441 bp fragment of hsp65. Phenotypic evaluation and PRA-hsp65 were concordant for 321 (74%) isolates. These assignments were presumed to be correct. For the remaining 113 discordant isolates, definitive identification was based on sequencing a 441 bp fragment of hsp65. PRA-hsp65 identified 30 isolates with hsp65 alleles representing 13 previously unreported PRA-hsp65 patterns. Overall, species identification by PRA-hsp65 was significantly more accurate than by phenotype methods (392 (90.3%) vs. 338 (77.9%), respectively; p < .0001, Fisher's test). Among the 333 isolates representing the most common pathogenic species, PRA-hsp65 provided an incorrect result for only 1.2%.PRA-hsp65 is a rapid and highly reliable method and deserves consideration by any clinical microbiology laboratory charged with performing species identification of NTM.The genus Mycobacterium comprises organisms that are heterogeneous in terms of metabolism, growth, environmental niche, epidemiology, pathogenicity, geographic distribution and disease association [1]. While there are notable pathogens such as Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium leprae, most are environmental organisms typically acting as opportunistic pathogens. These species, often collectively called nontuberculous mycobacteria (NTM), have been associated with a variety of problems including pulmonary, lymph node, skin, soft tissue, skeletal, and disseminated infections as well as nosocomial outbreaks related to inadequate disinfection/sterilization of medical devices [2]. In recent years, infections due to the subset
MALDI-TOF MS Assessment to Identify Environmental Mycobacteria  [PDF]
Camilla Pereira de Paula Uzam, Urze Adomaitis Brianesi, Camila Romagnoli, Karen Machado Gomes, Rafael Silva Duarte, Erica Chimara, Julio Cezar Franco de Oliveira, Marcelo Affonso Vallim, Renata Castiglioni Pascon, Cristina Viana-Niero
Advances in Microbiology (AiM) , 2015, DOI: 10.4236/aim.2015.59065
Abstract: Over the past few decades, there has been a significant increase in the number of mycobacterial species described. Currently, the genusMycobacteriumconsists of 170 species. Most species are called nontuberculous mycobacteria (NTM) and are potentially or rarely pathogenic and ubiquitous. One of the main challenges in mycobacteriology is the rapid and precise identification of these microorganisms. In this work, we compared two protein extraction protocols for the identification of 38 reference strains and clinical isolates, representing 27 species, by mass spectrometry (MALDI-TOF MS) to subsequently use the best method for identifying environmental mycobacteria. The results obtained with reference strains and clinical isolates showed that protocol A was effective in identifying 92.1% of mycobacterial specimens at the species level and protocol B, 50%. Therefore, protocol A was evaluated for the rapid identification of 27 environmental mycobacterial isolates. These isolates were subjected to PCR-restriction enzyme analysis (PRA-hsp65). Two isolates were misidentified by PRA-hsp65, whereas MALDI-TOF MS was able to identify them correctly. The results were confirmed byhsp65 and 16S rRNA gene sequencing. Mass spectrometry has the advantage of being a simpler and faster technique than PRA-hsp65, and our results showed that MALDI-TOF MS is a valuable tool for the identification of environmental mycobacterial isolates.
Micobactérias n?o-tuberculosas: diversidade das espécies no estado de S?o Paulo
Ueki, Suely Yoko Mizuka;Martins, Maria Concei??o;Telles, Maria Alice da Silva;Virgilio, Melissa Curcio;Giampaglia, Carmen Maria Saraiva;Chimara, Erica;Ferrazoli, Lucilaine;
Jornal Brasileiro de Patologia e Medicina Laboratorial , 2005, DOI: 10.1590/S1676-24442005000100003
Abstract: introduction: mycobacterium genus includes species of m. tuberculosis complex and others called non-tuberculous mycobacteria (ntm). so far, more than 100 species of ntm have been described. the objectives of this study were to estimate the ntm species diversity among strains isolated in s?o paulo state from 1991 to 1997, before the expansion of antiretroviral therapy, and to evaluate the rate of cases that fall under diagnostic criteria for bacteriological diagnosis of ntm infections. material and methods: 1,892 strains were isolated from sterile and non-sterile sites of 1,248 patients. results: 1,199 (96%) were identified as m. avium complex (mac), m. kansasii, m. chelonae, m. fortuitum, m. szulgai, m. xenopi, m. marinum, m. gordonae, m. terrae or m. nonchromogenicum whereas 49 (3.9%) couldn't be identified up to species. forty-seven (7,8%) cases from pulmonary sites were confirmed based on strains isolated from three or more samples from the same patient and 67 (34%) patients had ntm isolated from sterile sites, which confirmed the bacteriological diagnosis. conclusions: mac and m. kansasii were the most frequently especies isolated in s?o paulo state. national recomendations for the diagnosis and treatment of ntm infections would be useful for correct management of ntm infections.
Estudo descritivo da freqüência de micobactérias n?o tuberculosas na Baixada Santista (SP)
Zamarioli, Liliana Aparecida;Coelho, Andréa Gobetti Vieira;Pereira, Clemira Martins;Nascimento, Ana Carolina Chiou;Ueki, Suely Yoko Mizuka;Chimara, Erica;
Jornal Brasileiro de Pneumologia , 2008, DOI: 10.1590/S1806-37132008000800008
Abstract: objective: the present study aims at describing the frequency of nontuberculous mycobacteria (ntm) species identified through laboratory testing of samples collected from non-sterile sites (sputum), as well as its frequency in hiv-infected and non-hiv-infected individuals in the baixada santista region of the state of s?o paulo, brazil, in the period from 2000 to 2005. methods: retrospective analysis of sputum smear microscopy results and culture was conducted based on the records on file at the instituto adolfo lutz-santos, the regional tuberculosis laboratory. results: we analyzed 194 ntm strains isolated from 125 individuals, of whom 73 (58.4%) were hiv-negative and 52 (41.6%) were hiv-positive. thirteen different species were identified: mycobacterium kansasii; m. avium complex; m. fortuitum; m. peregrinum; m. gordonae; m. terrae; m. nonchromogenicum; m. intracellulare; m. flavescens; m. bohemicum; m. chelonae; m. shimoidei; and m. lentiflavum. in 19.2% of the cases, the bacteriological diagnosis was confirmed by isolation of the same species in at least two consecutive samples. conclusions: our results show the importance of including systematic identification of ntm in the laboratory routine, and that its integration into the clinical routine could improve the characterization of the disease, thereby informing the planning of effective control measures in specific populations, such as individuals presenting tuberculosis/hiv co-infection.
Isolamento de micobactérias n?o-tuberculosas em S?o José do Rio Preto entre 1996 e 2005
Pedro, Heloisa da Silveira Paro;Pereira, Maria Izabel Ferreira;Goloni, Maria do Rosário Assad;Ueki, Suely Yoko Mizuka;Chimara, Erica;
Jornal Brasileiro de Pneumologia , 2008, DOI: 10.1590/S1806-37132008001100010
Abstract: objective: to study the incidence of nontuberculous mycobacteria and the range of species isolated between 1996 and 2005 at a regional branch of the adolfo lutz institute-located in the city of s?o josé do rio preto, brazil-and to show the importance of laboratory testing. methods: mycobacteria were isolated from pulmonary and extrapulmonary specimens and identified through phenotyping and molecular methods (polymerase chain reaction-restriction enzyme analysis). results: we isolated 317 nontuberculous mycobacterium strains: mycobacterium avium complex, 182 (57.4%); m. gordonae, 33 (10.4%); m. fortuitum, 25 (7.9%); m. chelonae, 8 (2.5%); m. terrae complex, 8(2.5%); m.kansasii, 7 (2.2%); and less frequent species, 54 (17%). during this period, 72 cases (33.3%) were characterized as mycobacteriosis, according to bacteriological criteria established by the american thoracic society in 2007. of those 72 cases, 56 were attributed to m.avium complex. of those 56, 29 (51.8%) were characterized as disseminated disease. six cases were attributed to m. fortuitum, 3 to m. gordonae, 2 to m. chelonae, 1 to m. abscessus, 1 to m. kansasii, 1 to m. intracellulare, 1 to m. malmoense and 1 to mycobacterium ssp. conclusions: these results show the importance of the bacteriological diagnosis, since identification of the species enables early and appropriate treatment.
Cord factor detection and macroscopic evaluation of mycobacterial colonies: an efficient combined screening test for the presumptive identification of Mycobacterium tuberculosis complex on solid media
Sime?o, Fernanda Cristina dos Santos;Chimara, Erica;Oliveira, Rosangela Siqueira;Yamauchi, Jonas Umeoka;Latrilha, Fábio Oliveira;Telles, Maria Alice da Silva;
Jornal Brasileiro de Pneumologia , 2009, DOI: 10.1590/S1806-37132009001200008
Abstract: objective: the rapid differentiation between mycobacterium tuberculosis and nontuberculous mycobacteria is fundamental for patients co-infected with tuberculosis and hiv. to that end, we use two methods in our laboratory: detection of cord factor and pcr-restriction enzyme analysis (pra). the objective of this study was to evaluate the accuracy of a screening test on solid medium as a rapid method for the presumptive identification of m. tuberculosis complex, considering costs and turnover time. methods: a total of 152 strains were submitted to a combined screening test, consisting of the detection of cord factor under microscopy (ziehl-neelsen staining) and evaluation of the macroscopic aspect of colonies, as well as to pra, which was used as the gold standard. costs were estimated by calculating the price of all of the materials needed for each test. results: the overall accuracy of cord factor detection alone was 95.4% (95% ci: 90.7-98.1%), and that of the combined screening test was 99.3% (95% ci: 96.4-100%). cord factor detection costs us$ 0.25, whereas the pra costs us$ 7.00. results from cord factor detection are ready in 2 days, whereas pra requires 4 days to yield results. conclusions: the presumptive identification of m. tuberculosis using the macroscopic evaluation of colonies combined with cord factor detection under microscopy is a simple, rapid and inexpensive test. we recommend the combined screening test to rapidly identify m. tuberculosis in resource-poor settings and in less well-equipped laboratories while awaiting a definite identification by molecular or biochemical methods.
Molecular characterization of Mycobacterium kansasii isolates in the State of S?o Paulo between 1995-1998
Chimara, Erica;Giampaglia, Carmen Maria Saraiva;Martins, Maria Concei??o;Telles, Maria Alice da Silva;Ueki, Suely Yoko Mizuka;Ferrazoli, Lucilaine;
Memórias do Instituto Oswaldo Cruz , 2004, DOI: 10.1590/S0074-02762004000700013
Abstract: mycobacterium kansasii is the most common cause of pulmonary nontuberculous mycobacteria infection and classical identification of this pathogen needs a time consuming phenotypic tests. polymerase chain reaction-restriction fragment lenght polymorphism analysis (pra) of the gene enconding for the 65kda heat shock (hsp65) protein offers an easy, rapid, and inexpensive procedure to identify and subtype m. kansasii isolates. in the present study, we performed a retrospective analysis of patients who had mycobacteria identified on the basis of phenotypic tests by means of a review of database at mycobacteria laboratory of the instituto adolfo lutz in the period 1995-1998. a total of 9381 clinical isolates were analyzed of which 7777 (82.9%) were identified as m. tuberculosis complex and 1604 (17.1%) as nontuberculous mycobacteria. of the 296 m. kansasii isolates, 189 (63.8%) isolates obtained from 119 patients were viable and were analyzed by pra-hsp65. hundred eight two (98.9%) were classified as m. kansasii type i. two isolates were classified as type ii and iii and five isolates were characterized as other mycobacterium species. clinical isolates of m. kansasii in the state of s?o paulo was almost exclusively subtype i regardless of hiv status.
Identification of non-tuberculous mycobacteria from the Central Public Health Laboratory from Mato Grosso do Sul and analysis of clinical relevance
Moraes, Paulo Ricardo de Souza;Chimara, Erica;Telles, Maria Alice da Silva;Ueki, Suely Yoko Misuka;Cunha, Eunice Atsuko Totumi;Honer, Michael Robin;Le?o, Sylvia Cardoso;
Brazilian Journal of Microbiology , 2008, DOI: 10.1590/S1517-83822008000200013
Abstract: non-tuberculous mycobacteria isolated at the central public health laboratory from mato grosso do sul in 2003 and 2004 were identified by conventional phenotypic methods (ti) and by pcr-restriction enzyme analysis (pra) using the hsp65 gene as target (pra-hsp65). with 15 of the 32 analysed isolates, results of both methods were concordant, being 8 mycobacterium avium, 3 m. fortutium, 1 m. kansasii, 1 m. flavescens, 1 m. peregrinum and 1 nocardia brasiliensis. ti of 12 isolates was inconclusive. novel pra-hsp65 patterns were observed with 11 isolates. medical data were evaluated for inference of clinical relevance of these isolates.
Monitoramento em cabine de seguran?a biológica: manipula??o de cepas e descontamina??o em um laboratório de micobactérias
Ueki, Suely Yoko Mizuka;Chimara, Erica;Yamauchi, Jonas Umeoka;Latrilha, Fábio de Oliveira;Sime?o, Fernanda Cristina dos Santos;Moniz, Letícia Lisboa;Giampaglia, Carmen Maria Saraiva;Telles, Maria Alice da Silva;
Jornal Brasileiro de Patologia e Medicina Laboratorial , 2008, DOI: 10.1590/S1676-24442008000400005
Abstract: objectives: to verify the evidence of aerosol formation during the manipulation of mycobacteria strains for susceptibility (st) and identification tests (it) as well as the decontamination effect of alcohol solution 70% and ultraviolet (uv) radiation in biological safety cabinets (bsc) after laboratory procedures. methods: one plate was exposed in a bsc during st and it procedures. afterwards, the bsc was cleaned and decontaminated with alcohol solution 70% and exposed to uv radiation for 15 minutes. after that, another plate was exposed for two hours, only with the bsc ventilation on. both plates were incubated at 37°c and observed for 30 days. the smears from the isolated colonies were stained with ziehl neelsen and gram techniques, and acid fast bacilli (afb) were identified by conventional methods. results: in 38 plates exposed during st, there was mycobacteria growth in 10 plates (26.3%), fungi in one (2.6%) and bacilli in two (5.3%). among those plates that presented mycobacteria growth, eight (80%) were identified as m. tuberculosis and two (20%) had inconclusive identification. even after decontamination with alcohol solution 70% and uv radiation, two plates presented fungi growth (5.3%) and other two presented cocci growth (5.3%). among 30 plates exposed during it procedures, there was mycobacteria growth in 10 of them (33.3%), fungi in two (6.6%), cocci in one (3.4%) and one (3.4%) mixed mycobacteria and another bacillus. no growth was observed when alcohol solution 70% and uv radiation were used for decontamination after it procedures. conclusion: during the procedures there was aerosol formation with mycobacteria, which was proved by mycobacteria growth on the exposed plates. not only should adequate laboratory techniques be respected to minimize aerosol formation, but professional expertise, the continuity of capacity programs and periodic bsc maintenance should also be observed.
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