Characterization of the genetic diversity of Mycobacterium tuberculosis in São Paulo city, Brazil.

BACKGROUND: Tuberculosis 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). FINDINGS: Spoligotyping 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. CONCLUSIONS: Our 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.

Contribution of spoligotyping to the characterization of the population structure of Mycobacterium tuberculosis isolates in Portugal.

Tuberculosis is a major health problem in Portugal. To begin characterizing the population structure of Mycobacterium tuberculosis, spoligotyping was used for the systematic typing, through consecutive sampling, of patient isolates from the Amadora-Sintra area of Greater Lisbon. Distribution amongst major spoligotype families, including the Latin American Mediterranean (LAM), T, Haarlem and Beijing, was compared to that of the international spoligotype database SpolDB4 and to the European countries of traditional Portuguese immigration represented in SpolDB4. Spoligotypes from 665 isolates were analyzed and 97 shared international types (SITs) identified. In SpolDB4 Portugal is represented by part of the spoligotypes from this study explaining the reduced number of unidentified patterns. The importance of the LAM family, and especially of LAM1 and LAM9 sub-families that alone represented 38% of all the isolates in this study as compared to 8% relative to the European sub group, led us to believe that at least in this respect the population structure was closer to that of Africa and South America than to Europe. Spoligotypes characteristic of Portugal or Portuguese related settings were identified. These included SIT244 a T1 sub-family predominant in Portugal and Bangladesh, SIT64 a LAM 6 sub-family common to Portugal and Brazil, and SIT1106 a LAM 9 sub-family. These studies were the first in Portugal stressing the importance of monitoring the population structure of M. tuberculosis isolates, an important step towards gaining an understanding of tuberculosis and the dynamics of this disease.

In vitro effect of free and complexed indium(III) against Mycobacterium tuberculosis.

In mycobacteria, the study of inhibition by metal ions has been limited by the absence of suitable molecular vectors. Recently, we reported on the inhibitory activity of a family of chelators, macrocyclic compounds (MCC), against Mycobacterium tuberculosis. In this study equimolar concentrations of the free cations vanadium(IV), arsenic(III), iron(III), indium(III) and bismuth(III), and as 1:1 complexes with the MCC 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetra-acetic acid (TETA) were tested in vitro against M. tuberculosis using the Bactec 460 TB radiometric technology (Becton-Dickinson, MD, USA). Radiometric inhibition above 80% was obtained with free indium(III) and bismuth(III), and ranged from 80% to 99%, with the complexes of TETA with vanadium(IV), bismuth(III) and indium(III), in the order of increasing activity. The highest radiometric inhibition levels were obtained with the [In(TETA)]- complex, which caused drops of up to 4 log units in cellular viability. The minimal inhibitory concentration of this compound was evaluated at 3 microM.

Exploring Mycobacterium avium inhibition by macrocyclic compounds.

Derivatives of synthetic macrocyclic compounds, MCC, 12- and 14-membered tetraazamacrocycles with N-pendant arms, such as N-methyl (Mepy14), N-acetate (DOTA, TETA and ac3py14) and N-methylphosphonate (DOTP) groups, were investigated in terms of their in vitro activity against Mycobacterium avium and for intracellular clearance, using the murine macrophage cell line J-774. Perspective results on a laboratory strain, of opaque morphology, showed in vitro activity with varying inhibitory patterns from one compound to another. The most active compounds, such as TETA, presented N-acetate pendant arms. Inhibition levels of 90% and above were obtained at 50 mg/l. Inhibition was confirmed with both the free compound and its iron(III) complex for DOTP, Mepy14, ac3py14, and TETA. However, with DOTA, no inhibitory effect was observed for the iron(III) complex, suggesting that chelation was at the origin of the inhibitory effect or that the donor atoms of the ligand were strongly involved. Nevertheless, simple experiments indicated that ferric ion might not be responsible for this reversed activity. Intracellular activity using 50 mg/l of TETA confirmed in vitro results with the laboratory strain. Results expressed as relative growth (%), of the drug-containing samples compared to control samples ranged from 2 to 123% (growth promotion) with no apparent relationship between inhibitory activity and the colony morphology of the strains. These studies showed that the evaluation of synthetic macrocycles may be relevant in development of a new family of compounds for use against M. avium infections.

Update on the Spoligotypes of Mycobacterium tuberculosis complex isolates from the Fernando Fonseca Hospital (Amadora-Sintra, Portugal).

The present population study, from 1999 to 2003, has been based on the use of Spoligotyping in the genotyping of 452 isolates of the Mycobacterium tuberculosis complex from tuberculosis patients of the Fernando Fonseca Hospital. Spoligotypes were identified as "shared types" (STs) with the aid of an international database. Eleven rarely found STs, not identified in the database, grouped 8.4% of the isolates. Moreover, particular to Portugal, may be the predominance of STs identified in the database but not previously classified as genotypic families, such as ST244, ST150 and ST389, representing 13.3 % of the total. The identification of clinical isolates of M. africanum genotype Afri1 and of M. tuberculosis genotype CAS1 may confirm import of isolates of African and Asian origin. M. tuberculosis of the Beijing family was first reported by us as of 1999. Since then, the number of isolates at the Hospital has passed from one to five annually, representing 2.2% of the total and the tenth most predominant family in the present study. M. tuberculosis Beijing may correspond to an emerging problem in Portugal due to recent immigration from Eastern Europe and Asia. Other genotypes, ST150 and ST389, have shown increase, the significance of which is not clear. However, the relative frequencies of the predominant families LAM, T1 and Haarlem remained relatively stable. The present study confirms the genetic variability in Portugal of M. tuberculosis complex isolates. These studies may contribute to the definition of priorities in the national tuberculosis control programs.

Spoligotyping and pncA polymorphism: a two step scenario for Mycobacterium bovis diagnosis in Portugal.

The differential diagnosis of Mycobacterium bovis is important in the control of transmission to the human population and also for treatment since M. bovis is naturally resistant to pyrazinamide. Eleven clinical isolates from the Fernando Fonseca Hospital with Spoligotypes indicative of M. bovis, through the absence of spacers 39-43 but that also counted with the absence of spacer 38, were analyzed. For the identification of these strains, the phenotypic analysis of pyrazinamide resistance and study of the polymorphisms of the pncA and gyrB genes were carried out. The study of the pncA polymorphism revealed that the strains analyzed did not contain the M. bovis specific mutation. In relation the gyrB polymorphisms, using the GenoTypeO MTBC kit, the strains were identified as belonging to the group M. tuberculosis, M. africanum subtipo II e M. canetti. The present investigation enabled us to define new genotypes on which future bacteriological studies should be based. Amongst these the study of the pncA polymorphism was considered important due to the immediate practical implications for the clinician. Evaluating transmission and defining groups of risk is an objective for which support from the veterinary services is considered relevant.

[Molecular identification using Spoligotyping of strains from the Mycobacterium tuberculosis complex isolated from the Hospital Fernando Fonseca]. / Identificação molecular pelo método de Spoligotyping de estirpes do complexo Mycobacterium tuberculosis isoladas no Hospital Fernando Fonseca.

Spoligotyping was used in the genotyping of 219 isolates of the Mycobacterium tuberculosis complex, from patients of the Hospital Fernando Fonseca. This technique, based on PCR methodology, analyses a region of the chromosome specific of the Mycobacterium tuberculosis complex, the DR locus (Direct Repeat). With the aid of an international database, we showed that the predominant Spoligotypes belonged to the LAM family (Latino-American Mediterranean), 29.2 %. The LAM 9 family, with 12.3 %, left us attentive to the possible import of the disease through populations from South America, were it has been frequently identified. The genotypic families T1 and Haarlem, with 6.4 % and 8.7 % respectively, represented a frequency typical to Europe. The Beijing family, with 1.4 %, may represent an emerging problem in our country due to recent immigration of Asian and Eastern European populations. Isolates with a Spoligotype of the M. bovis type were found at a high percentage, 3.7 %. In Europe, this infection is extremely rare suggesting the result may not be due to M. bovis infection but to M. bovis BCG (due to vaccination or eventual recombinant BCG based therapies), or M. africanum (due to the proximity of the two species). A high percentage of the Spoligotypes were not identified by the database, 21.4 %. This is the first study of this type amongst us and may be the starting point for the creation of a data base with important consequences on the national program against tuberculosis.

Serological screening for tuberculosis in the community: an evaluation of the Mycodot procedure in an African population with high HIV-2 prevalence (Republic of Guinea-Bissau).

The immuno-dot-blot assay MycoDot, which detects lipoarabinomannan (LAM) antibodies, was evaluated for the serological diagnosis of active pulmonary tuberculosis in patients in a rural community in the Republic of Guinea-Bissau. Sera from 269 adults (age > 15) and 33 children (age < 5) were assayed for antibodies in a blind manner and the results compared to the clinical status of tuberculosis. The assay had a specificity and a sensitivity of 92.4% and 63.0% respectively, when applied to the adult population. In HIV-2 infected individuals (27/269), the specificity and sensitivity of the assay were similar, 94.7% and 62.5% respectively. The assay did not provide high sensitivity for the diagnosis of tuberculosis in children. Sera from patients with leprosy cross-reacted with the antigen of the assay. It is concluded that this easily performed assay may be useful for the presumptive diagnosis of tuberculosis in adult populations in rural areas of developing countries where routine screening is not readily available.

Serological screening for tuberculosis in the community: an evaluation of the Mycodot procedure in an African population with high HIV-2 prevalence (Republic of Guinea-Bissau)

The immuno-dot-blot assay MycoDot, which detects lipoarabinomannan (LAM) antibodies, was evaluated for the serological diagnosis of active pulmonary tuberculosis in patients in a rural community in the Republic of Guinea-Bissau. Sera from 269 adults (age > 15) and 33 children (age < 5) were assayed for antibodies in a blind manner and the results compared to the clinical status of tuberculosis. The assay had a specificity and a sensitivity of 92.4 per cent and 63.0 per cent respectively, when applied to the adult population. In HIV-2 infected individuals (27/269), the specificity and sensitivity of the assay were similar, 94.7 per cent and 62.5 per cent respectively. The assay did not provide high sensitivity for the diagnosis of tuberculosis in children. Sera from patients with leprosy cross-reacted with the antigen of the assay. It is concluded that this easily performed assay may be useful for the presumptive diagnosis of tuberculosis in adult populations in rural areas of developing countries where routine screening is not readily available.