BackgroundTuberculosis is a major health problem in São Paulo, Brazil, which is the most populous and one of the most cosmopolitan cities in South America. To characterize the genetic diversity of Mycobacterium tuberculosis in the population of this city, the genotyping techniques of spoligotyping and MIRU were applied to 93 isolates collected in two consecutive years from 93 different tuberculosis patients residing in São Paulo city and attending the Clemente Ferreira Institute (the reference clinic for the treatment of tuberculosis).FindingsSpoligotyping generated 53 different spoligotype patterns. Fifty-one isolates (54.8%) were grouped into 13 spoligotyping clusters. Seventy- two strains (77.4%) showed spoligotypes described in the international databases (SpolDB4, SITVIT), and 21 (22.6%) showed unidentified patterns. The most frequent spoligotype families were Latin American Mediterranean (LAM) (26 isolates), followed by the T family (24 isolates) and Haarlem (H) (11 isolates), which together accounted for 65.4% of all the isolates. These three families represent the major genotypes found in Africa, Central America, South America and Europe. Six Spoligo-International-types (designated SITs by the database) comprised 51.8% (37/72) of all the identified spoligotypes (SIT53, SIT50, SIT42, SIT60, SIT17 and SIT1). Other SITs found in this study indicated the great genetic diversity of M. tuberculosis, reflecting the remarkable ethnic diversity of São Paulo city inhabitants. The MIRU technique was more discriminatory and did not identify any genetic clusters with 100% similarity among the 93 isolates. The allelic analysis showed that MIRU loci 26, 40, 23 and 10 were the most discriminatory. When MIRU and spoligotyping techniques were combined, all isolates grouped in the 13 spoligotyping clusters were separated.ConclusionsOur data indicated the genomic stability of over 50% of spoligotypes identified in São Paulo and the great genetic diversity of M. tuberculosis isolates in the remaining SITs, reflecting the large ethnic mix of the São Paulo city inhabitants. The results also indicated that in this city, M. tuberculosis isolates acquired drug resistance independently of genotype and that resistance was more dependent on the selective pressure of treatment failure and the environmental circumstances of patients.

Rhodococcus equi has emerged as an opportunistic pathogen associated with pulmonary, invasive or systemic infections in immunocompromised patients. We report the identification of 51 R. equi isolates found in sputum samples of 546 individuals suspected to have pulmonary tuberculosis in two Public Health Hospital Units in Brazil.The epidemiology of R. equi infection as well as the phenotypic identification and drug susceptibility profile of isolates are described in this paper.Key words: Rhodococcus equi -identification -tuberculosis -antimicrobial profile -epidemiologyThe genus Rhodococcus belongs to the group of nocardioform, Gram-positive rods containing mycolic acids in the cell wall; this group also includes the genera My� cobacterium, Nocardia, Corynebacterium, Dietzia, Gor� donia, Millisia, Segniliparus, Skermania, Tsukamurella and Williamsia (Soddell et al. 2006a, b, Tsitko et al. 2006). The genus Rhodococcus was discovered by Zopf in 1891 (Goodfellow & Alderson 1977) and comprises 30 species, of which Rhodococcus equi is considered the most opportunistic pathogen in mammals, including humans (Meijer & Prescott 2004). The first case of human infection was reported in 1967 in a patient presenting with a pulmonary abscess. The two first cases that occurred in Brazil were reported by Severo et al. (2001). However, with the AIDS epidemic, the number of patients infected by R. equi has grown (Roda et al. 2009). R. equi also causes infection in patients with lymphoma, chronic renal failure, alcoholism, lung cancer, leukaemia, diabetes mellitus and other immunodeficient syndromes. Some cases have been reported in which the infection can also occur in immunocompetent hosts (von Bargen & Haas 2009).When incubated aerobically at 37°C, R. equi grows efficiently in the majority of nonselective culture media, including media used in mycobacteria isolation and produces irregular, smooth and mucoid colonies that turn a shade of salmon pink to yellow after a week of growth (Prescott 1991). R. equi appears coccoid on stained smears of clinical specimens, especially purulent material and tissue (obtained by biopsy, during surgery and upon autopsy). However, long rods have been reported in clinical specimens isolated from blood, sputum and bronchial lavage fluid In general, R. equi is biochemically non-reactive, has no proteolytic activity and fails to oxidize or ferment carbohydrates, with the exception of glucose, which is oxidized by R. equi in 14 days. R. equi is strictly aerobic, catalase positive, oxidase negative and mostly urease positive. R. equi produces soluble "equi factors" that are associated with phospholipase and cholesterol oxidase activity and interact with phospholipase D of Listeria ivanovii to induce complete haemolysis of sheep erythrocytes (Prescott 1991).The Laboratory of Mycobacteriology at the Adolfo Lutz Institute in Ribeirão Preto (SP, Brazil) has frequently isolated partial acid-fast bacteria from sputum of patients suspected to have pulmonary tuberculosis. Considering the emerging clinical im...

Starting with 257 outpatients attending the specialized health service for tuberculosis (TB) between 2002 and 2006 in Araraquara, an agro-industrial area with low tuberculosis (TB) incidence in São Paulo state, Brazil, positive mycobacterial cultures were obtained in 130 cases, of which 121 were confirmed as Mycobacterium tuberculosis complex. This report assesses the genetic diversity observed on 69.42% (n=84) of the clinical isolates, for which both spoligotyping and 12-loci MIRU typing data were fully interpretable. In order to monitor changes in the population dynamics of circulating M. tuberculosis strains over time, spoligotypes were compared from this study (n=84) with an earlier study from 1998 to 2001 (n=70 strains); and these two datasets from low-incidence Araraquara area were also compared with a 2-year cohort in the nearby higher-incidence São Paulo city area from 2006 to 2008 (n=93). The results obtained showed that with 58.3% (49/84) of the strains, the Latin-American-Mediterranean (LAM) was the predominant lineage in the present follow-up study; major patterns being SIT42/LAM9 11.9% (10/84), and SIT20/LAM1 10.7% (9/84). As compared with the 1998-2001 period when 40% (28/70) of the isolates belonged to the ill-defined T family, it was replaced by LAM strains between 2002 and 2006 with a visible shift to a population structure characteristic of the metropolitan São Paulo city. Further typing of the follow-up isolates from 2002 to 2006 using 12 loci MIRUs in conjunction with conventional epidemiology did not link this population structure shift to an increase in ongoing transmission or drug-resistance. Instead, it is most probably linked to movements of the important migrant community of Araraquara to higher TB incidence metropolitan areas such as São Paulo city. This is of particular concern owing to the increment in the global burden of LAM strains and the recent association of certain LAM sublineages with multidrug- and extensively drug-resistant TB. These observations suggest the need for further molecular monitoring of the TB population structure and the evaluation of transmission trends amongst migrant workers and other risk groups, such as persons in homeless shelters, in correctional facilities, drug users, and those with HIV infection, etc.

We report a comparative study of two DNA extraction techniques, thermolysis and chemical lysis (CTAB), for molecular identification and genotyping of M. tuberculosis. Forty DNA samples were subjected to PCR and the results demonstrated that with thermolysis it is possible to obtain useful data that enables fast identification and genotyping