Epidemiologia clássica e molecular da tuberculose pulmonar em pacientes da região norte de Minas Gerais

Pós-graduação em Biociências e Biotecnologia Aplicadas à Farmácia - FCFAR

First Baseline of Circulating Genotypic Lineages of Mycobacterium tuberculosis in Patients from the Brazilian Borders with Argentina and Paraguay

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Diversidade clínica, epidemiológica e Genética do Mycobacterium tuberculosis na região Noroeste paulista

Pós-graduação em Genética - IBILCE

Caracterização genética e distribuição espacial dos isolados de Mycobacterium leprae em região de baixa prevalência no Brasil

Pós-graduação em Doenças Tropicais - FMB

Mycobacterium tuberculosis transmission in a country with low tuberculosis incidence: role of immigration and HIV infection

Immigrants from high-burden countries and HIV-coinfected individuals are risk groups for tuberculosis (TB) in countries with low TB incidence. Therefore, we studied their role in transmission of Mycobacterium tuberculosis in Switzerland. We included all TB patients from the Swiss HIV Cohort and a sample of patients from the national TB registry. We identified molecular clusters by spoligotyping and mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) analysis and used weighted logistic regression adjusted for age and sex to identify risk factors for clustering, taking sampling proportions into account. In total, we analyzed 520 TB cases diagnosed between 2000 and 2008; 401 were foreign born, and 113 were HIV coinfected. The Euro-American M. tuberculosis lineage dominated throughout the study period (378 strains; 72.7%), with no evidence for another lineage, such as the Beijing genotype, emerging. We identified 35 molecular clusters with 90 patients, indicating recent transmission; 31 clusters involved foreign-born patients, and 15 involved HIV-infected patients. Birth origin was not associated with clustering (adjusted odds ratio [aOR], 1.58; 95% confidence interval [CI], 0.73 to 3.43; P = 0.25, comparing Swiss-born with foreign-born patients), but clustering was reduced in HIV-infected patients (aOR, 0.49; 95% CI, 0.26 to 0.93; P = 0.030). Cavitary disease, male sex, and younger age were all associated with molecular clustering. In conclusion, most TB patients in Switzerland were foreign born, but transmission of M. tuberculosis was not more common among immigrants and was reduced in HIV-infected patients followed up in the national HIV cohort study. Continued access to health services and clinical follow-up will be essential to control TB in this population.

"Pseudo-Beijing": evidence for convergent evolution in the direct repeat region of Mycobacterium tuberculosis

Background Mycobacterium tuberculosis has a global population structure consisting of six main phylogenetic lineages associated with specific geographic regions and human populations. One particular M. tuberculosis genotype known as “Beijing” has repeatedly been associated with drug resistance and has been emerging in some parts of the world. “Beijing” strains are traditionally defined based on a characteristic spoligotyping pattern. We used three alternative genotyping techniques to revisit the phylogenetic classification of M. tuberculosis complex (MTBC) strains exhibiting the typical “Beijing” spoligotyping pattern. Methods and Findings MTBC strains were obtained from an ongoing molecular epidemiological study in Switzerland and Nepal. MTBC genotyping was performed based on SNPs, genomic deletions, and 24-loci MIRU-VNTR. We identified three MTBC strains from patients originating from Tibet, Portugal and Nepal which exhibited a spoligotyping patterns identical to the classical Beijing signature. However, based on three alternative molecular markers, these strains were assigned to Lineage 3 (also known as Delhi/CAS) rather than to Lineage 2 (also known as East-Asian lineage). Sequencing of the RD207 in one of these strains showed that the deletion responsible for this “Pseudo-Beijing” spoligotype was about 1,000 base pairs smaller than the usual deletion of RD207 in classical “Beijing” strains, which is consistent with an evolutionarily independent deletion event in the direct repeat (DR) region of MTBC. Conclusions We provide an example of convergent evolution in the DR locus of MTBC, and highlight the limitation of using spoligotypes for strain classification. Our results indicate that a proportion of “Beijing” strains may have been misclassified in the past. Markers that are more phylogenetically robust should be used when exploring strain-specific differences in experimental or clinical phenotypes.

Bacillus anthracis: Molecular taxonomy, population genetics, phylogeny and patho-evolution

Bacillus anthracis, the etiological agent of anthrax, manifests a particular bimodal lifestyle. This bacterial species alternates between short replication phases of 20-40 generations that strictly require infection of the host, normally causing death, interrupted by relatively long, mostly dormant phases as spores in the environment. Hence, the B. anthracis genome is highly homogeneous. This feature and the fact that strains from nearly all parts of the world have been analysed for canonical single nucleotide polymorphisms (canSNPs) and variable number tandem repeats (VNTRs) has allowed the development of molecular epidemiological and molecular clock models to estimate the age of major diversifications in the evolution of B. anthracis and to trace the global spread of this pathogen, which was mostly promoted by movement of domestic cattle with settlers and by international trade of contaminated animal products. From a taxonomic and phylogenetic point of view, B. anthracis is a member of the Bacillus cereus group. The differentiation of B. anthracis from B. cereus sensu strict, solely based on chromosomal markers, is difficult. However, differences in pathogenicity clearly differentiate B. anthracis from B. cereus and are marked by the strict presence of virulence genes located on the two virulence plasmids pXO1 and pXO2, which both are required by the bacterium to cause anthrax. Conversely, anthrax-like symptoms can also be caused by organisms with chromosomal features that are more closely related to B. cereus, but which carry these virulence genes on two plasmids that largely resemble the B. anthracis virulence plasmids. (C) 2011 Elsevier B.V. All rights reserved.

Splitting of a prevalent Mycobacterium bovis spoligotype by variable-number tandem-repeat typing reveals high heterogeneity in an evolving clonal group

Mycobacterium bovis populations in countries with persistent bovine tuberculosis usually show a prevalent spoligotype with a wide geographical distribution. This study applied mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing to a random panel of 115 M. bovis isolates that are representative of the most frequent spoligotype in the Iberian Peninsula, SB0121. VNTR typing targeted nine loci: ETR-A (alias VNTR2165), ETR-B (VNTR2461), ETR-D (MIRU4, VNTR580), ETR-E (MIRU31, VNTR3192), MIRU26 (VNTR2996), QUB11a (VNTR2163a), QUB11b (VNTR2163b), QUB26 (VNTR4052), and QUB3232 (VNTR3232). We found a high degree of diversity among the studied isolates (discriminatory index [D] = 0.9856), which were split into 65 different MIRU-VNTR types. An alternative short-format MIRU-VNTR typing targeting only the four loci with the highest variability values was found to offer an equivalent discriminatory index. Minimum spanning trees using the MIRU-VNTR data showed the hypothetical evolution of an apparent clonal group. MIRU-VNTR analysis was also applied to the isolates of 176 animals from 15 farms infected by M. bovis SB0121; in 10 farms, the analysis revealed the coexistence of two to five different MIRU types differing in one to six loci, which highlights the frequency of undetected heterogeneity.

Standard genotyping overestimates transmission of Mycobacterium tuberculosis among immigrants in a low incidence country.

Immigrants from high tuberculosis (TB) incidence regions are a risk group for TB in low-incidence countries such as Switzerland. In a previous analysis of a nationwide collection of 520 Mycobacterium tuberculosis isolates from 2000-2008, we identified 35 clusters comprising 90 patients based on standard genotyping (24-loci MIRU-VNTR and spoligotyping). Here, we used whole genome sequencing (WGS) to revisit these transmission clusters. Genome-based transmission clusters were defined as isolate pairs separated by ≤12 single nucleotide polymorphisms (SNPs). WGS confirmed 17/35 (49%) MIRU-VNTR clusters; the other 18 clusters contained pairs separated by >12 SNPs. Most transmission clusters (3/4) of Swiss-born patients were confirmed by WGS, as opposed to 25% (4/16) of clusters involving only foreign-born patients. The overall clustering proportion using standard genotyping was 17% (90 patients, 95% confidence interval [CI]: 14-21%), but only 8% (43 patients, 95% CI: 6-11%) using WGS. The clustering proportion was 17% (67/401, 95% CI: 13-21%) using standard genotyping and 7% (26/401, 95% CI: 4-9%) using WGS among foreign-born patients, and 19% (23/119, 95% CI: 13-28%) and 14% (17/119, 95% CI: 9-22%), respectively, among Swiss-born patients. Using weighted logistic regression, we found weak evidence for an association between birth origin and transmission (aOR 2.2, 95% CI: 0.9-5.5, comparing Swiss-born patients to others). In conclusion, standard genotyping overestimated recent TB transmission in Switzerland when compared to WGS, particularly among immigrants from high TB incidence regions, where genetically closely related strains often predominate. We recommend the use of WGS to identify transmission clusters in low TB incidence settings.

Detección, caracterización y análisis funcional de la complejidad clonal en la tuberculosis humana y bovina

Las herramientas de genotipado en tuberculosis fueron desarrolladas inicialmente para la realización de estudios epidemiológicos. Sin embargo han permitido asimismo desvelar la complejidad clonal existente en las infecciones causadas por Mycobacterium tuberculosis (MTB) y Mycobacterium bovis (M. bovis), poniendo así en cuestión la asunción de que cada episodio de tuberculosis (TB) estuviera causado por una única cepa. De este modo se comenzaron a describir situaciones de coinfección por más de una cepa (infección mixta) o bien de presencia simultánea de variantes clonales (infección policlonal), pudiendo estas, además, ofrecer una distribución heterogénea de las mismas en los diferentes tejidos infectados (infección compartimentalizada). Sin embargo, son pocos los estudios existentes entorno a estos fenómenos, y los que se han realizado atienden a la mera descripción de casos anecdóticos o al estudio de estos eventos en poblaciones en donde la incidencia de TB es alta. Así, el primer objetivo de esta tesis se centró en dimensionar la complejidad clonal existente en las infecciones por MTB en una población no seleccionada, en un entorno de moderada incidencia, donde las expectativas de encontrar la citada complejidad eran escasas. Mediante el análisis por RFLP-IS6110 y MIRU-VNTR se detectaron infecciones complejas en 11 pacientes con TB pulmonar (1,6%) y en 10 pacientes con TB pulmonar y extrapulmonar (14,1%). De estos 21 casos, 9 correspondieron a infecciones mixtas y 12 a infecciones policlonales. Por último, en 9 casos (5 pacientes con infección mixta y 4 con infección policlonal) se documentó la compartimentalización de la infección. Tras la descripción sistemática de los casos de TB con infecciones complejas, nos centramos en el estudio de una de sus modalidades, la infección policlonal. En concreto decidimos abordar la evaluación del posible significado funcional que pudiera conllevar la adquisición de las sutiles reorganizaciones genéticas identificadas entre variantes clonales, que surgen como resultado de eventos de microevolución...

Microvariation at the human D1S80 locus

Microvariant allelic polymorphisms have been known since 1966 when Harris, Hubby and Lewontin described the huge store of genetic variation detectable at the polypeptide level. Later Jeffreys used MVR (minisatellite variant repeat) analysis to describe the variation hidden within minisatellite VNTRs and to propose a mutational mechanism.^ The questions I have asked follow these traditions: (1) How much microvariant polymorphism exists at the discrete allele minisatellite D1S80 locus? (2) Do alleles or groups of alleles associate randomly with the flanking markers to form haplotypes? (3) What mechanisms might explain mutations at this locus? What are the phylogenetic relationships among the alleles?^ The minisatellite locus D1S80 (1p35-36), GenBank sequence (Accession # D28507), is a highly polymorphic Variable Number of Tandem Repeat (VNTR) based on a 16 base core. D1S80 alleles are electrophoretically separable into discontinuous sets of equivalent length alleles. Sequence variation or minor length variation within these classes was expected: I have sought to determine the nature of this microvariant heterogeneity by sequencing nominal and variant alleles.^ Alleles were analyzed by Single-Strand Conformation Polymorphism (SSCP) analysis. Sequences were determined to ascertain whether sequence variation or size variation is the major cause of altered electrophoretic migration of microvariant D1S80 alleles. Twenty three alleles from 14 previously typed individuals were sequenced. The individuals were from African American, Caucasian, or Hispanic databases.^ A Tsp509 I restriction site, previously reported as a Hinf I flanking polymorphism, and a 3$\sp\prime$ flanking region BsoF I restriction site polymorphism were identified. There appears to be a strong association of the 5$\sp\prime$ flanking region Hinf I(+) and Tsp509 I(-) site and the 3$\sp\prime$ flanking region BsoF I(-) site with the 18 allele, while the 24 tends to be associated with the Hinf I(-), Tsp509 I(+) and BsoF I(+) sites.^ The general conclusion for this locus is clearly the closer you look, the more you find. D1S80 allelic polymorphisms are primarily due to variation in the number of repeat units and to sequence variation among repeats. The sequenced based gene tree depicts two major classes of alleles which conform to the two most common alleles, reflecting either equivalent age or population size bottlenecks. ^

Long-term assessment of wild boar harvesting and cattle removal for bovine tuberculosis control in free ranging populations

Wild boar is a recognized reservoir of bovine tuberculosis (TB) in the Mediterranean ecosystems, but information is scarce outside of hotspots in southern Spain. We describe the first high-prevalence focus of TB in a non-managed wild boar population in northern Spain and the result of eight years of TB management. Measures implemented for disease control included the control of the local wild boar population through culling and stamping out of a sympatric infected cattle herd. Post-mortem inspection for detection of tuberculosis-like lesions as well as cultures from selected head and cervical lymph nodes was done in 745 wild boar, 355 Iberian ibexes and five cattle between 2004 and 2012. The seasonal prevalence of TB reached 70% amongst adult wild boar and ten different spoligotypes and 13 MIRU-VNTR profiles were detected, although more than half of the isolates were included in the same clonal complex. Only 11% of infected boars had generalized lesions. None of the ibexes were affected, supporting their irrelevance in the epidemiology of TB. An infected cattle herd grazed the zone where 168 of the 197 infected boars were harvested. Cattle removal and wild boar culling together contributed to a decrease in TB prevalence. The need for holistic, sustained over time, intensive and adapted TB control strategies taking into account the multi-host nature of the disease is highlighted. The potential risk for tuberculosis emergence in wildlife scenarios where the risk is assumed to be low should be addressed.

Splitting of a prevalent Mycobacterium bovis spoligotype by variable-number tandem-repeat typing reveals high heterogeneity in an evolving clonal group

Mycobacterium bovis populations in countries with persistent bovine tuberculosis usually show a prevalent spoligotype with a wide geographical distribution. This study applied mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing to a random panel of 115 M. bovis isolates that are representative of the most frequent spoligotype in the Iberian Peninsula, SB0121. VNTR typing targeted nine loci: ETR-A (alias VNTR2165), ETR-B (VNTR2461), ETR-D (MIRU4, VNTR580), ETR-E (MIRU31, VNTR3192), MIRU26 (VNTR2996), QUB11a (VNTR2163a), QUB11b (VNTR2163b), QUB26 (VNTR4052), and QUB3232 (VNTR3232). We found a high degree of diversity among the studied isolates (discriminatory index [D] = 0.9856), which were split into 65 different MIRU-VNTR types. An alternative short-format MIRU-VNTR typing targeting only the four loci with the highest variability values was found to offer an equivalent discriminatory index. Minimum spanning trees using the MIRU-VNTR data showed the hypothetical evolution of an apparent clonal group. MIRU-VNTR analysis was also applied to the isolates of 176 animals from 15 farms infected by M. bovis SB0121; in 10 farms, the analysis revealed the coexistence of two to five different MIRU types differing in one to six loci, which highlights the frequency of undetected heterogeneity.

Occurrence of Mycobacterium avium subspecies paratuberculosis across host species and European countries with evidence for transmission between wildlife and domestic ruminants

BACKGROUND Mycobacterium avium subspecies paratuberculosis (Map) causes an infectious chronic enteritis (paratuberculosis or Johne's disease) principally of ruminants. The epidemiology of Map is poorly understood, particularly with respect to the role of wildlife reservoirs and the controversial issue of zoonotic potential (Crohn's disease). Genotypic discrimination of Map isolates is pivotal to descriptive epidemiology and resolving these issues. This study was undertaken to determine the genetic diversity of Map, enhance our understanding of the host range and distribution and assess the potential for interspecies transmission. RESULTS 164 Map isolates from seven European countries representing 19 different host species were genotyped by standardized IS900--restriction fragment length polymorphism (IS900-RFLP), pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphisms (AFLP) and mycobacterial interspersed repeat unit-variable number tandem repeat (MIRU-VNTR) analyses. Six PstI and 17 BstEII IS900-RFLP, 31 multiplex [SnaBI-SpeI] PFGE profiles and 23 MIRU-VNTR profiles were detected. AFLP gave insufficient discrimination of isolates for meaningful genetic analysis. Point estimates for Simpson's index of diversity calculated for the individual typing techniques were in the range of 0.636 to 0.664 but a combination of all three methods increased the discriminating power to 0.879, sufficient for investigating transmission dynamics. Two predominant strain types were detected across Europe with all three typing techniques. Evidence for interspecies transmission between wildlife and domestic ruminants on the same property was demonstrated in four cases, between wildlife species on the same property in two cases and between different species of domestic livestock on one property. CONCLUSION The results of this study showed that it is necessary to use multiple genotyping techniques targeting different sources of genetic variation to obtain the level of discrimination necessary to investigate transmission dynamics and trace the source of Map infections. Furthermore, the combination of genotyping techniques may depend on the geographical location of the population to be tested. Identical genotypes were obtained from Map isolated from different host species co-habiting on the same property strongly suggesting that interspecies transmission occurs. Interspecies transmission of Map between wildlife species and domestic livestock on the same property provides further evidence to support a role for wildlife reservoirs of infection.