Frequency of first and second-line drug resistance-associated mutations among resistant Mycobacterium tuberculosis clinical isolates from São Paulo, Brazil

BACKGROUND Tuberculosis (TB) is an infectious disease caused by the bacterium Mycobacterium tuberculosis, and the number of new cases of multidrug resistant TB (MDR-TB), pre extensively drug-resistant TB (pre-XDR-TB) and extensively drug-resistant TB (XDR-TB) has increased considerably worldwide. OBJECTIVES Herein, using 156 M. tuberculosis isolates from 106 patients previously classified as MDR or pre-XDR or XDR isolates, we investigated the genetic mutation profiles associated with phenotypic resistances in patients with MDR-TB, pre-XDR-TB and XDR-TB, treatment outcomes and resistance evolution. METHODS Molecular analyses were performed by partial sequencing of the rpoB, katG, gyrA, gyrB, rrs genes and analysis of the fabG-inhA promoter region. Clinical, epidemiologic and demographic data were obtained from the TB Notification database system of São Paulo (TB-WEB) and the Information System for Special Tuberculosis Treatments (SITE-TB). FINDINGS Drug resistance was attributed to previously known mutations and a novel Asp449Val mutation in gyrB was observed in four isolates from the same patient. Ten patients had more than one isolate evaluated and eight of these patients displayed resistance progression. MAIN CONCLUSIONS The present study is the first to report the frequency of mutations related to second-line drug resistance in MDR-TB, pre-XDR-TB and XDR-TB isolates. The results could lead to the improvement of available technologies for the rapid detection of drug resistant TB.

Rapid tests for the detection of the Mycobacterium abscessus subsp. bolletii strain responsible for an epidemic of surgical-site infections in Brazil

A single strain of Mycobacterium abscessus subsp. bolletii, characterised by a particular rpoB sequevar and two highly related pulsed field gel electrophoresis patterns has been responsible for a nationwide outbreak of surgical infections in Brazil since 2004. In this study, we developed molecular tests based on polymerase chain reaction restriction-enzyme analysis (PRA) and sequencing for the rapid identification of this strain. Sequences of 15 DNA regions conserved in mycobacteria were retrieved from GenBank or sequenced and analysed in silico. Single nucleotide polymorphisms specific to the epidemic strain and located in enzyme recognition sites were detected in rpoB, the 3' region of the 16S rDNA and gyrB. The three tests that were developed, i.e., PRA-rpoB, PRA-16S and gyrB sequence analysis, showed 100%, 100% and 92.31% sensitivity and 93.06%, 90.28% and 100% specificity, respectively, for the discrimination of the surgical strain from other M. abscessus subsp. bolletii isolates, including 116 isolates from 95 patients, one environmental isolate and two type strains. The results of the three tests were stable, as shown by results obtained for different isolates from the same patient. In conclusion, due to the clinical and epidemiological importance of this strain, these tests could be implemented in reference laboratories for the rapid preliminary diagnosis and epidemiological surveillance of this epidemic strain.

Effects of tissue handling and processing steps on PCR for detection of Mycobacterium tuberculosis in formalin-fixed paraffin-embedded samples / Efeitos das etapas de tratamento e processamento do tecido na PCR para detecção de Mycobacterium tuberculosis em amostras fixadas em formalina e incluídas em parafina

Development and standardization of reliable methods for detection of Mycobacterium tuberculosis in clinical samples is an important goal in laboratories throughout the world. In this work, lung and spleen fragments from a patient who died with the diagnosis of miliary tuberculosis were used to evaluate the influence of the type of fixative as well as the fixation and paraffin inclusion protocols on PCR performance in paraffin embedded specimens. Tissue fragments were fixed for four h to 48 h, using either 10 percent non-buffered or 10 percent buffered formalin, and embedded in pure paraffin or paraffin mixed with bee wax. Specimens were submitted to PCR for amplification of the human beta-actin gene and separately for amplification of the insertion sequence IS6110, specific from the M. tuberculosis complex. Amplification of the beta-actin gene was positive in all samples. No amplicons were generated by PCR-IS6110 when lung tissue fragments were fixed using 10 percent non-buffered formalin and were embedded in paraffin containing bee wax. In conclusion, combined inhibitory factors interfere in the detection of M. tuberculosis in stored material. It is important to control these inhibitory factors in order to implement molecular diagnosis in pathology laboratories.

O desenvolvimento e a padronização de métodos confiáveis para a detecção de Mycobacterium tuberculosis em amostras clínicas é um objetivo importante nos laboratórios de todo o mundo. Neste trabalho, fragmentos de pulmão e baço de paciente que morreu com o diagnóstico de tuberculose miliar foram usados para avaliar a influência do tipo de fixador e dos protocolos de fixação e inclusão em parafina na performance da PCR. Fragmentos de tecido foram fixados por quatro h a 48 h, usando formalina não tamponada a 10 por cento ou formalina tamponada a 10 por cento e incluídos em parafina pura ou misturada a cera de abelha. As amostras foram submetidas a PCR para amplificação do gene da beta-actina humana e, separadamente, para amplificação da sequência de inserção IS6110, específica do complexo M. tuberculosis. O resultado da amplificação do gene da beta-actina foi positivo em todas as amostras. Não foram gerados amplicons na PCR-IS6110 em amostras de tecido pulmonar fixadas usando formalina não tamponada a 10 por cento e incluídas em parafina com cera de abelha. Em conclusão, fatores inibitórios combinados interferiram na detecção de M. tuberculosis em material de arquivo. É importante controlar estes fatores inibitórios para poder implementar o diagnóstico molecular em laboratórios de patologia.

Identification of non-tuberculous mycobacteria from the Central Public Health Laboratory from Mato Grosso do Sul and analysis of clinical relevance / Identificação de micobactérias não-tuberculosas do Laboratório Central de Saúde Pública de Mato Grosso de Sul e análise de dados clínicos dos pacientes

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.

Micobactérias não-tuberculosas isoladas no Laboratório Central de Saúde Pública de Mato Grosso do Sul em 2003 e 2004 foram identificadas usando métodos fenotípicos convencionais (TI) e PCR-Restriction Enzyme Analysis (PRA) tendo o gene hsp65 como alvo (PRA-hsp65). Em 15 dos 32 isolados analisados os resultados obtidos com ambos métodos foram concordantes, sendo 8 Mycobacterium avium, 3 M. fortutium, 1 M. kansasii, 1 M. flavescens, 1 M. peregrinum e 1 Nocardia brasiliensis. TI de 12 isolados não foi conclusiva. Perfis não descritos de PRA-hsp65 foram observados com 11 isolados. Dados dos prontuários médicos foram avaliados para inferir a relevância clínica dos isolados.

Comparison of methods for mycobacteria isolation from swine feces

Swine mycobacteriosis is an important cause of carcass condemnation at abattoirs. One of the best ways to recognize the etiologic agent involved, in live animals, is the fecal isolation, as 94 percent of the lesions are located in the digestive tract. Therefore, the goal of the present study was to compare the performance of four decontamination methods followed by inoculation in three different culture media, totalizing twelve procedures of mycobacteria search from swine fecal samples experimentally contaminated. The swine feces were artificially contaminated with 0.02 g of Mycobacterium avium, PIG-B strain, and subjected to mycobacteria isolation trial. The protocols used were: 1) modified Petroff or basic method; 2) modified Lowenstein-Jensen or acidic method; 3) modified Petroff or basic method with re-suspension in Amphotericin B; 4) modified Lowenstein-Jensen or acid method with re-suspension in Amphotericin B, followed by inoculation in Petragnani, Lowenstein-Jensen and Lowenstein-Jensen medium with antibiotics (Penicillin G and Nalidixic acid). There was a difference (p<0.05) between the mycobacterial recovery percentages from swine feces. The acid method with re-suspension in Amphotericin B solution and inoculation in Lowenstein-Jensen medium with antibiotics showed the best results (87 percent of mycobacteria recovery).

As micobacterioses suínas são responsáveis por condenações de carcaças em abatedouro e uma das melhores formas de se conhecer os agentes envolvidos nos animais vivos é o isolamento a partir das fezes, pois em 94 por cento das vezes, as lesões localizam-se no trato digestivo. Assim sendo, o presente estudo teve por objetivo comparar o desempenho de quatro métodos de descontaminação com semeadura em três diferentes meios de cultura, totalizando doze procedimentos na pesquisa de micobactérias a partir de amostras de fezes de suínos contaminadas experimentalmente. Amostras de fezes de suínos foram contaminadas artificialmente com 0,02g de Mycobacterium avium, estirpe de PIG-B, e submetidas à tentativa de isolamento de micobactérias, utilizando-se os seguintes protocolos de descontaminação: 1) Petroff modificado ou método básico; 2) Lowenstein-Jensen modificado ou método ácido; 3) Petroff modificado ou método básico e ressuspensão com anfotericina B; 4) Lowenstein-Jensen modificado ou método ácido e ressuspensão com anfotericina B; com subseqüente semeadura em meios de Petragnani, Lowenstein-Jensen e Lowenstein-Jensen com antibióticos (Penicilina G e Ácido nalidíxico). Houve diferença entre os percentuais de recuperação de micobactérias a partir das fezes de suínos (p<0,05) e o método ácido com ressuspensão em solução de anfotericina B e semeadura em meio de Lowenstein-Jensen com antibióticos apresentou os melhores resultados (87 por cento de recuperação de micobactérias).

Deep stromal mycobacterial keratitis: viable bacteria after six months of treatment: case report and literature review

O objetivo do caso é descrever a presença de micobactérias viáveis em pacientes com ceratite, 6 meses após tratamento intensivo. A identificação de espécies, foi efetuada usando método PRA (polymerase chain reaction seguida pela restriction endonuclease analysis). Clonalidade foi avaliada pelos métodos RAPD (randomly amplified polymorphic DNA) e ERIC-PCR (enterobacterial repetitive intergenic consensus - polymerase chain reaction). Paciente refere trauma com corpo estranho metálico há 3 semanas. A cultura da córnea revelou Mycobacterium abscessus. Após 6 meses de tratamento tópico e sistêmico, paciente apresentava-se sem inflamação, sendo considerado clinicamente curado. Realizou-se então, uma ceratoplastia penetrante com intuitos ópticos. A cultura da córnea transplantada revelou micobactérias de mesma origem clonal. O achado mais interessante neste relato, foi a positividade da cultura da córnea transplantada após 6 meses de intenso tratamento específico. Ao nosso conhecimento, esse é o primeiro caso relatado na literatura mostrando essa possibilidade em tratamento de ceratites por micobactérias. Assim, os pacientes com ceratite por Mycobacterium abscessus podem apresentar bactérias viáveis após longo tempo de tratamento específico e precisam ser seguidos cuidadosamente por um longo período de tempo.

Mycobacterium tuberculosis complex differentiation using gyrB-restriction fragment length polymorphism analysis

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, São 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.

Avaliaçäo da virulência em hamsters (Mesocricetus auratus) de estirpes de Mycobacterium avium presentes na populaçäo de suínos do sul do Brasil / Evaluation of the virulence in hamsters (Mesocricetus auratus) of Mycobacterium avium strains from the swine population of the south of Brazil

Tendo sido comprovada a existência de quatro famílias molecularmente distintas de M. avium (PIG-A, B, C e D) circulando em suínos da regiäo sul do Brasil, e havendo dúvidas a respeito da importância da transmissäo horizontal como mecanismo de manutençäo da doença, o presente teve por objetivo estudar a virulência dessas estirpes, informaçäo importante para o aperfeiçoamento dos métodos de controle. Uma estirpe representante de cada família foi inoculada pela via intra-peritoneal em 48 hamsters com uma dose de 30.000 U.F.C. por animal. Após 2, 13, 26 e 40 dias da inoculaçäo (T1 a T4), 12 hamsters inoculados de cada família foram anestesiados, sacrificados e os agentes foram quantificados no fígado, baço e pulmäo. Os resultados foram expressos em número de U.F.C./g de órgäo. A presença das estirpes foi pesquisada no sangue e também foram realizados exames histológicos. As estirpes PIG-A, B, C e D induziram a formaçäo de lesöes granulomatosas no fígado e baço a partir do segundo dia pós-inoculaçäo e disseminaram-se pela via hemática, alcançando os pulmöes. O baço sempre apresentou maiores contagens de U.F.C., seguido pelo figado e pulmöes. Diferenças entre as estirpes foram constatadas através de análises das contagens de U.F.C de baço (T1: p<0,001; T2: p<0,001; T3: p<0,001 e T4: p<0,001), permitindo a construçäo da seguinte escala de virulência: PIG-B> PIG-A> PIG-D> PIG-C

Discrimination of members of the Mycobacterium avium complex by polymerase chain reaction

Mycobacterium avium complex (MAC) species cannot be discriminated by the usual methods of biochemical identification of mycobacteria. This study showed that amplification by PCR of DT1 and DT6, two single copy sequences identified in the genome of M. avium serotype 2, the insertion sequence IS1245, found to be consistently present in M. avium strains and the heat-shock protein gene hsp65, followed by restriction polymorphism analysis, are rapid and accurate tests for the differentiation of the species M. avium, M. intracellulare, and M. scrofulaceum.