Identification of clonal complexes of Mycobacterium bovis in Brazil.

Bovine tuberculosis is a disease that is widely distributed around the world. Its causative agent, Mycobacterium bovis, has characteristics of a microorganism with clonal multiplication in populations with no evidence of genetic exchange between strains, and, consequently, a group of strains can be identified as descending from a common ancestor. The aim of this study was to investigate the clonal complexes of M. bovis isolated from samples of lesions suggestive of bovine tuberculosis collected from slaughterhouses in various states of Brazil between 2006 and 2012. Ninety samples were analyzed, and it was found that 14.4% belonged to the clonal complex European1 and 81.1% to the clonal complex European2, while 4.65% were not identified as any of the four known complexes.

Risk factors for human Mycobacterium bovis infections in an urban area of Brazil.

BACKGROUND: The World Health Organization (WHO) has classified human zoonotic tuberculosis (TB) due to Mycobacterium bovis as a neglected issue in the developing world. In a recent cross-sectional study in Brazil, three of 189 TB patients presented with a coinfection of M. bovis and M. tuberculosis and were selected as cases for this study. OBJECTIVE: The aim was to evaluate risk factors (RF) for zoonotic TB in an urban area of Brazil in order to guide preventive programmes. METHODS: A matched case-control study was carried out nested within a cross-sectional study. For each of the three cases, 14 age- and sex-matched controls (TB due to M. tuberculosis) were selected. FINDINGS: Zoonotic potential exposures (ZE) and extrapulmonary TB (EPTB) were independently associated with zoonotic TB in multivariate analyses. CONCLUSIONS: ZE by occupation and consumption of raw milk and derivative products that place individuals in direct and indirect contact with animals and their excretions/secretions increase the risk for zoonotic TB in Brazil, especially among those with EPTB. Therefore, measures such as efficient control of bovine TB, distribution of pasteurised milk and its derivative products, and the diagnosis and monitoring of zoonotic TB in humans are essential steps, especially in developing countries where bovine TB is enzootic, and further studies are necessary.

Risk factors for human Mycobacterium bovis infections in an urban area of Brazil

BACKGROUND The World Health Organization (WHO) has classified human zoonotic tuberculosis (TB) due to Mycobacterium bovis as a neglected issue in the developing world. In a recent cross-sectional study in Brazil, three of 189 TB patients presented with a coinfection of M. bovis and M. tuberculosis and were selected as cases for this study. OBJECTIVE The aim was to evaluate risk factors (RF) for zoonotic TB in an urban area of Brazil in order to guide preventive programmes. METHODS A matched case-control study was carried out nested within a cross-sectional study. For each of the three cases, 14 age- and sex-matched controls (TB due to M. tuberculosis) were selected. FINDINGS Zoonotic potential exposures (ZE) and extrapulmonary TB (EPTB) were independently associated with zoonotic TB in multivariate analyses. CONCLUSIONS ZE by occupation and consumption of raw milk and derivative products that place individuals in direct and indirect contact with animals and their excretions/secretions increase the risk for zoonotic TB in Brazil, especially among those with EPTB. Therefore, measures such as efficient control of bovine TB, distribution of pasteurised milk and its derivative products, and the diagnosis and monitoring of zoonotic TB in humans are essential steps, especially in developing countries where bovine TB is enzootic, and further studies are necessary.

Validation of a real-time PCR assay for the molecular identification of Mycobacterium tuberculosis

Mycobacterium tuberculosis is the major cause of tuberculosis in humans. This bacillus gained prominence with the occurrence of HIV, presenting itself as an important opportunistic infection associated with acquired immunodeficiency syndrome (AIDS). The current study aimed to develop a real-time PCR using Eva Green technology for molecular identification of M. tuberculosis isolates. The primers were designed to Rv1510 gene. Ninety nine samples of M. tuberculosis and sixty samples of M. bovis were tested and no sample of the bovine bacillus was detected by the qPCR. Statistical tests showed no difference between the qPCR and biochemical tests used to identify the Mycobacterium tuberculosis. The correlation between tests was perfect with Kappa index of 1.0 (p < 0.001, CI = 0.84 - 1.0). The diagnostic sensitivity and specificity were 100% (CI = 95.94% - 100%) and 100% (CI = 93.98% - 100%). This qPCR was developed with the goal of diagnosing the bacillus M. tuberculosis in samples of bacterial suspension. TB reference laboratories (health and agriculture sectors), public health programs and epidemiological studies probably may benefit from such method.

Draft Genome Sequence of Mycobacterium bovis Strain AN5, Used for Production of Purified Protein Derivative.

Mycobacterium bovis strain AN5 has been used to produce purified protein derivative (PPD) for the intradermal test for bovine tuberculosis since it was introduced in 1948. This work reports the draft genome sequence of M. bovis AN5, which is used for the production of bovine PPD in Brazil, as well as comparisons to other strains of M. bovis and Mycobacterium tuberculosis.

Validation of a real-time PCR assay for the molecular identification of Mycobacterium tuberculosis.

Mycobacterium tuberculosis is the major cause of tuberculosis in humans. This bacillus gained prominence with the occurrence of HIV, presenting itself as an important opportunistic infection associated with acquired immunodeficiency syndrome (AIDS). The current study aimed to develop a real-time PCR using Eva Green technology for molecular identification of M. tuberculosis isolates. The primers were designed to Rv1510 gene. Ninety nine samples of M. tuberculosis and sixty samples of M. bovis were tested and no sample of the bovine bacillus was detected by the qPCR. Statistical tests showed no difference between the qPCR and biochemical tests used to identify the Mycobacterium tuberculosis. The correlation between tests was perfect with Kappa index of 1.0 (p < 0.001, CI = 0.84 - 1.0). The diagnostic sensitivity and specificity were 100% (CI = 95.94% - 100%) and 100% (CI = 93.98% - 100%). This qPCR was developed with the goal of diagnosing the bacillus M. tuberculosis in samples of bacterial suspension. TB reference laboratories (health and agriculture sectors), public health programs and epidemiological studies probably may benefit from such method.

Tuberculosis patients co-infected with Mycobacterium bovis and Mycobacterium tuberculosis in an urban area of Brazil.

In this cross-sectional study, mycobacteria specimens from 189 tuberculosis (TB) patients living in an urban area in Brazil were characterised from 2008-2010 using phenotypic and molecular speciation methods (pncA gene and oxyR pseudogene analysis). Of these samples, 174 isolates simultaneously grew on Löwenstein-Jensen (LJ) and Stonebrink (SB)-containing media and presented phenotypic and molecular profiles of Mycobacterium tuberculosis, whereas 12 had molecular profiles of M. tuberculosis based on the DNA analysis of formalin-fixed paraffin wax-embedded tissue samples (paraffin blocks). One patient produced two sputum isolates, the first of which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, and the second of which only grew on SB media and presented phenotypic profiles of Mycobacterium bovis. One patient provided a bronchial lavage isolate, which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, but had molecular profiles of M. bovis from paraffin block DNA analysis, and one sample had molecular profiles of M. tuberculosis and M. bovis identified from two distinct paraffin blocks. Moreover, we found a low prevalence (1.6%) of M. bovis among these isolates, which suggests that local health service procedures likely underestimate its real frequency and that it deserves more attention from public health officials.

Tuberculosis patients co-infected with Mycobacterium bovis and Mycobacterium tuberculosis in an urban area of Brazil

In this cross-sectional study, mycobacteria specimens from 189 tuberculosis (TB) patients living in an urban area in Brazil were characterised from 2008-2010 using phenotypic and molecular speciation methods (pncA gene and oxyR pseudogene analysis). Of these samples, 174 isolates simultaneously grew on Löwenstein-Jensen (LJ) and Stonebrink (SB)-containing media and presented phenotypic and molecular profiles of Mycobacterium tuberculosis, whereas 12 had molecular profiles of M. tuberculosis based on the DNA analysis of formalin-fixed paraffin wax-embedded tissue samples (paraffin blocks). One patient produced two sputum isolates, the first of which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, and the second of which only grew on SB media and presented phenotypic profiles of Mycobacterium bovis. One patient provided a bronchial lavage isolate, which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, but had molecular profiles of M. bovis from paraffin block DNA analysis, and one sample had molecular profiles of M. tuberculosis and M. bovis identified from two distinct paraffin blocks. Moreover, we found a low prevalence (1.6%) of M. bovis among these isolates, which suggests that local health service procedures likely underestimate its real frequency and that it deserves more attention from public health officials.

Spoligotyping and variable number tandem repeat analysis of Mycobacterium bovis isolates from cattle in Brazil.

We performed spoligotyping and 12-mycobacterial interspersed repetitive unit-variable number tandem repeats (MIRU-VNTRs) typing to characterise Mycobacterium bovis isolates collected from tissue samples of bovines with lesions suggestive for tuberculosis during slaughter inspection procedures in abattoirs in Brazil. High-quality genotypes were obtained with both procedures for 61 isolates that were obtained from 185 bovine tissue samples and all of these isolates were identified as M. bovis by conventional identification procedures. On the basis of the spoligotyping, 53 isolates were grouped into nine clusters and the remaining eight isolates were unique types, resulting in 17 spoligotypes. The majority of the Brazilian M. bovis isolates displayed spoligotype patterns that have been previously observed in strains isolated from cattle in other countries. MIRU-VNTR typing produced 16 distinct genotypes, with 53 isolates forming eight of the groups, and individual isolates with unique VNTR profiles forming the remaining eight groups. The allelic diversity of each VNTR locus was calculated and only two of the 12-MIRU-VNTR loci presented scores with either a moderate (0.4, MIRU16) or high (0.6, MIRU26) discriminatory index (h). Both typing methods produced similar discriminatory indexes (spoligotyping h = 0.85; MIRU-VNTR h = 0.86) and the combination of the two methods increased the h value to 0.94, resulting in 29 distinct patterns. These results confirm that spoligotyping and VNTR analysis are valuable tools for studying the molecular epidemiology of M. bovis infections in Brazil.

Spoligotyping and variable number tandem repeat analysis of Mycobacterium bovis isolates from cattle in Brazil

We performed spoligotyping and 12-mycobacterial interspersed repetitive unit-variable number tandem repeats (MIRU-VNTRs) typing to characterise Mycobacterium bovis isolates collected from tissue samples of bovines with lesions suggestive for tuberculosis during slaughter inspection procedures in abattoirs in Brazil. High-quality genotypes were obtained with both procedures for 61 isolates that were obtained from 185 bovine tissue samples and all of these isolates were identified as M. bovis by conventional identification procedures. On the basis of the spoligotyping, 53 isolates were grouped into nine clusters and the remaining eight isolates were unique types, resulting in 17 spoligotypes. The majority of the Brazilian M. bovis isolates displayed spoligotype patterns that have been previously observed in strains isolated from cattle in other countries. MIRU-VNTR typing produced 16 distinct genotypes, with 53 isolates forming eight of the groups, and individual isolates with unique VNTR profiles forming the remaining eight groups. The allelic diversity of each VNTR locus was calculated and only two of the 12-MIRU-VNTR loci presented scores with either a moderate (0.4, MIRU16) or high (0.6, MIRU26) discriminatory index (h). Both typing methods produced similar discriminatory indexes (spoligotyping h = 0.85; MIRU-VNTR h = 0.86) and the combination of the two methods increased the h value to 0.94, resulting in 29 distinct patterns. These results confirm that spoligotyping and VNTR analysis are valuable tools for studying the molecular epidemiology of M. bovis infections in Brazil.