Assessment of bovine tuberculosis risk factors based on nationwide molecular epidemiology

This assessment aimed to elaborate a statistical nationwide model for analyzing the space-time dynamics of bovine tuberculosis in search of potential risk factors that could be used to better target surveillance measures. A database comprising Mycobacterium bovis molecular profiles from all isolates obtained from Belgian outbreaks during the 1995-to-2006 period (n = 415) allowed the identification of a predominant spoligotype (SB0162). Various databases compiling 49 parameters to be tested were queried using a multiple stepwise logistic regression to assess bovine tuberculosis risk factors. Two isolate datasets were analyzed: the first included all Mycobacterium bovis isolates, while the second included only data related to the SB0162 type strain. When all Mycobacterium bovis isolates were included in the model, several risk factors were identified: history of bovine tuberculosis in the herd (P < 0.001), proximity of an outbreak (P < 0.001), cattle density (P < 0.001), and annual amplitude of mean middle-infrared temperature (P < 0.001). The approach restricted to the predominant SB0162 type strain additionally highlighted the proportion of movements from an infected area during the current year as a main risk factor (P = 0.009). This study identified several risk factors for bovine tuberculosis in cattle, highlighted the usefulness of molecular typing in the study of bovine tuberculosis epidemiology, and suggests a difference of behavior for the predominant type strain. It also emphasizes the role of animals' movements in the transmission of the disease and supports the importance of controlling trade movements

The use of microbead-based spoligotyping for Mycobacterium tuberculosis complex to evaluate the quality of the conventional method: Providing guidelines for Quality Assurance when working on membranes

Contains fulltext : 124321.pdf (publisher's version ) (Open Access)BACKGROUND: The classical spoligotyping technique, relying on membrane reverse line-blot hybridization of the spacers of the Mycobacterium tuberculosis CRISPR locus, is used world-wide (598 references in Pubmed on April 8th, 2011). However, until now no inter-laboratory quality control study had been undertaken to validate this technique. We analyzed the quality of membrane-based spoligotyping by comparing it to the recently introduced and highly robust microbead-based spoligotyping. Nine hundred and twenty-seven isolates were analyzed totaling 39,861 data points. Samples were received from 11 international laboratories with a worldwide distribution. METHODS: The high-throughput microbead-based Spoligotyping was performed on CTAB and thermolyzate DNA extracted from isolated Mycobacterium tuberculosis complex (MTC) strains coming from the genotyping participating centers. Information regarding how the classical Spoligotyping method was performed by center was available. Genotype discriminatory analyses were carried out by comparing the spoligotypes obtained by both methods. The non parametric U-Mann Whitney homogeneity test and the Spearman rank correlation test were performed to validate the observed results. RESULTS: Seven out of the 11 laboratories (63%), perfectly typed more than 90% of isolates, 3 scored between 80-90% and a single center was under 80% reaching 51% concordance only. However, this was mainly due to discordance in a single spacer, likely having a non-functional probe on the membrane used. The centers using thermolyzate DNA performed as well as centers using the more extended CTAB extraction procedure. Few centers shared the same problematic spacers and these problematic spacers were scattered over the whole CRISPR locus (Mostly spacers 15, 14, 18, 37, 39, 40). CONCLUSIONS: We confirm that classical spoligotyping is a robust method with generally a high reliability in most centers. The applied DNA extraction procedure (CTAB or thermolyzate) did not affect the results in this study. However performance was center-dependent, suggesting that training is a key component in quality assurance of spoligotyping. Overall, no particular spacer yielded a higher degree of deviating results, suggesting that errors occur randomly either in the process of re-using membranes, or during the reading of the results and transferring of data from the film to a digital file. Last, the performance of the microbead-based method was excellent as previously shown by Cowan et al. (J. Clin. Microbiol. 2004) and Zhang et al. (J. Med. Microbiol. 2009) and demonstrated the proper detection of spacer 15 that is known to occasionally give weak signals in the classical spoligotyping

Primary psoas muscle abscess due to Mycobacterium xenopi.

SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Systematic analysis of pyrazinamide-resistant spontaneous mutants and clinical isolates of Mycobacterium tuberculosis.

&lt;p&gt;Pyrazinamide (PZA) is a first-line antitubercular drug known for its activity against persistent Mycobacterium tuberculosis bacilli. We set out to systematically determine the PZA susceptibility profiles and mutations in the pyrazinamidase (pncA) gene of a collection of multidrug-resistant tuberculosis (MDR-TB) clinical isolates and PZA-resistant (PZA(r)) spontaneous mutants. The frequency of acquired resistance to PZA was determined to be 10(-5) bacilli in vitro. Selection at a lower concentration of PZA yielded a significantly larger number of spontaneous mutants. The methodical approach employed allowed for determination of the frequency of the PZA(r) phenotype correlated with mutations in the pncA gene, which was 87.5% for the laboratory-selected spontaneous mutants examined in this study. As elucidated by structural analysis, most of the identified mutations were foreseen to affect protein activity through either alteration of an active site residue or destabilization of protein structure, indicating some preferential mutation site rather than random scattering. Twelve percent of the PZA(r) mutants did not have a pncA mutation, strongly indicating the presence of at least one other mechanism(s) of PZA(r).&lt;/p&gt;</p

Faster identification of mycobacteria using gas liquid and thin layer chromatography

Gas liquid chromatography (GLC) and thin layer chromatography (TLC) analysis of cell wall content was used for identification of mycobacteria isolated in primary cultures. GLC permitted determination of the fatty acid and alcohol profiles of Mycobacterium simiae and Mycobacterium marinum and detection of a peak in Mycobacterium ulcerans formerly described for Mycobacterium malmoense. Using the data obtained to fill some of the gaps in the dichotomic trees of Tisdall et al. and Jantzen et al., GLC analysis allowed full identification of 8 of 22 mycobacterial species after 24 hours. The other 14 species could be divided into four groups on the basis of similar findings on GLC. TLC was used for full identification of three species. The identification results of conventional methods were concordant with those of GLC and TLC in 161 of 169 strains (93%) representing 21 different species. Using primarily chromatography for analysis of cell wall content, and in the case of some species complementary biochemical tests, the identification procedure could be shortened to a maximum of three days after primary culture

Molecular epidemiology of Mycobacterium tuberculosis complex in Brussels, 2010-2013

The tuberculosis (TB) incidence rate in Brussels-Capital Region is 3-fold higher than in Belgium as a whole. Eight years after the realization of initial prospective population-based molecular epidemiology investigations in this Region, a similar study over the period 2010-2013 was conducted. TB strains isolated from 945 patients were submitted to genotyping by standardized 24-locus-MIRU-VNTR typing and spoligotyping. The phylogenetic analysis showed that the LAM (16.7%) and Haarlem (15.7%) branches are the two most prevalent TB lineages circulating in Brussels. Analysis of the MDR subgroup showed an association with Beijing strains (39.9%) and patients native of Eastern Europe (40.7%). Genotyping detected 113 clusters involving 321 patients, giving a recent transmission index of 22.9%. Molecular-guided epidemiological investigations and routine surveillance activities revealed family transmission or social contact for patients distributed over 34 clusters. Most of the patients were foreign-born (75.7%). However, cluster analysis revealed only limited transnational transmission. Comparison with the previous study shows a stable epidemiological situation except for the mean age difference between Belgian-born and foreign-born patients which has disappeared. This study confirms that molecular epidemiology has become an important determinant for TB control programs. However, sufficient financial means need to be available to perform all required epidemiological investigations. © 2017 Vluggen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Evaluation of the epidemiological relevance of variable-number tandem-repeat genotyping of Mycobacterium bovis and comparison of the method with IS6110 restriction fragment lenght polymorphism analysis and spoligotyping

Sources of Mycobacterium bovis contamination remain unclear for many cases of animal and human disease. A major limitation is the lack of sufficiently informative or epidemiologically well evaluated molecular methods for typing. Here, we report an evaluation of a high-throughput method based on 29 mycobacterial interspersed repetitive unit-variable-number tandem-repeat (MIRU-VNTR) loci to genotype 127 M. bovis isolates from cattle from 77 different Belgian farms, representative of a nationwide collection obtained from 1995 to 2003. MIRU-VNTR stability was demonstrated by analyzing a series of 74 isolates in total, obtained from different animals from a single farm or from different farms with an identified epidemiological link. The genotyping results and the genotypic diversity (h) were compared with those obtained by IS6110 restriction fragment length polymorphism (RFLP) analysis and spoligotyping. Among 68 isolates with no known epidemiological link, MIRU-VNTR typing discriminated better than either RFLP analysis or spoligotyping, [corrected] taken individually (32 versus 16 and 17 genotypes; h = 0.91 versus 0.73 and 0.85, respectively) or in combination (32 versus 28 genotypes; h = 0.91 versus 0.92). Maximal resolution was already achieved with a subset of 9 loci. The observed congruence of the genetic relationships based on IS6110 RFLP analysis, spoligotyping, and MIRU-VNTR markers is consistent with a clonal population structure of M. bovis. These results support MIRU-VNTR typing as a convenient and discriminatory technique for analysis of the population structure of M. bovis in much greater detail and for addressing some still unresolved issues in the epidemiology of the pathogen</p

From multidrug- to extensively drug-resistant tuberculosis: upward trends as seen from a 15-year nationwide study.

&lt;p&gt;&lt;b&gt;BACKGROUND: &lt;/b&gt;Emergence of extensively drug-resistant tuberculosis (XDR-TB) represents an enormous challenge to Public Health globally.&lt;/p&gt;&lt;p&gt;&lt;b&gt;METHODS: &lt;/b&gt;Progression towards XDR-TB was investigated in Belgium, a country with a typically low TB incidence, by analyzing the magnitude, characteristics, and treatment success of multidrug-resistant tuberculosis (MDR-TB) through a population-based study from 1994 to 2008.&lt;/p&gt;&lt;p&gt;&lt;b&gt;RESULTS: &lt;/b&gt;Among the 174 MDR-TB patients, 81% were foreign-born, 48% of these being asylum seekers. Although the number of MDR-TB patients remained stable through the study period at around 15 new cases annually, frequencies of resistance of the patients&#039; first MDR-TB isolate to second-line drugs increased, as well as the total number of antibiotics it was resistant to (p&lt;0.001). XDR-TB cases were detected from 2002 onwards. For 24 patients, additional resistance to several second-line drugs was acquired during treatment. Molecular-guided investigations indicated little to no contribution of in-country clonal spread or exogenous re-infection. The increase of pre-XDR and XDR cases could be attributed to rising proportions of patients from Asia and Central and Eastern Europe (p&lt;0.001) and an increase in the isolation of Beijing strains in these groups (p&lt;0.001). Despite augmented resistance, the treatment success rate improved from 63.0% to 75.8% (p = 0.080) after implementation in 2005 of improved surveillance measures and therapeutic access.&lt;/p&gt;&lt;p&gt;&lt;b&gt;CONCLUSIONS: &lt;/b&gt;Increasing severity in drug resistance patterns leading to more XDR- and &quot;panresistant&quot; TB cases in a country with a low TB incidence like Belgium represents a strong alert on worsening situations in other world regions and requires intense public health measures.&lt;/p&gt;</p