Marta Herranz

Whole Genome Sequencing Analysis of Intrapatient Microevolution in Mycobacterium tuberculosis: Potential Impact on the Inference of Tuberculosis Transmission

BACKGROUND It has been accepted that the infection by Mycobacterium tuberculosis (M. tuberculosis) can be more heterogeneous than considered. The emergence of clonal variants caused by microevolution events leading to population heterogeneity is a phenomenon largely unexplored. Until now, we could only superficially analyze this phenomenon by standard fingerprinting (RFLP and VNTR). METHODS In this study we applied whole genome sequencing for a more in-depth analysis of the scale of microevolution both at the intrapatient and interpatient scenarios. RESULTS We found that the amount of variation accumulated within a patient can be as high as that observed between patients along a chain of transmission. Intrapatient diversity was found both at the extrapulmonary and respiratory sites, meaning that this variability can be transmitted and impact on the inference of transmission events. One of the events studied allowed us to track for a single strain the complete process of (i) interpatient microevolution, (ii) intrapatient respiratory variation, and (iii) isolation of different variants at different infected sites of this patient. CONCLUSIONS Our study adds new data to the understanding of variability in M. tuberculosis in a wide clinical scenario and alerts about the difficulties of establishing thresholds to differentiate relatedness in M. tuberculosis with epidemiological purposes.

Evaluation of the new advanced 15-loci MIRU-VNTR genotyping tool in Mycobacterium tuberculosis molecular epidemiology studies

BackgroundDuring the last few years, PCR-based methods have been developed to simplify and reduce the time required for genotyping Mycobacterium tuberculosis (MTB) by standard approaches based on IS6110-Restriction Fragment Length Polymorphism (RFLP). Of these, MIRU-12-VNTR (Mycobacterial interspersed repetitive units- variable number of tandem repeats) (MIRU-12) has been considered a good alternative. Nevertheless, some limitations and discrepancies with RFLP, which are minimized if the technique is complemented with spoligotyping, have been found. Recently, a new version of MIRU-VNTR targeting 15 loci (MIRU-15) has been proposed to improve the MIRU-12 format.ResultsWe evaluated the new MIRU-15 tool in two different samples. First, we analyzed the same convenience sample that had been used to evaluate MIRU-12 in a previous study, and the new 15-loci version offered higher discriminatory power (Hunter-Gaston discriminatory index [HGDI]: 0.995 vs 0.978; 34.4% of clustered cases vs 57.5%) and better correlation (full or high correlation with RFLP for 82% of the clusters vs 47%). Second, we evaluated MIRU-15 on a population-based sample and, once again, good correlation with the RFLP clustering data was observed (for 83% of the RFLP clusters). To understand the meaning of the discrepancies still found between MIRU-15 and RFLP, we analyzed the epidemiological data for the clustered patients. In most cases, splitting of RFLP-clustered patients by MIRU-15 occurred for those without epidemiological links, and RFLP-clustered patients with epidemiological links were also clustered by MIRU-15, suggesting a good epidemiological background for clustering defined by MIRU-15.ConclusionThe data obtained by MIRU-15 suggest that the new design is very efficient at assigning clusters confirmed by epidemiological data. If we add this to the speed with which it provides results, MIRU-15 could be considered a suitable tool for real-time genotyping.

Systematic Survey of Clonal Complexity in Tuberculosis at a Populational Level and Detailed Characterization of the Isolates Involved

ABSTRACT Clonally complex infections by Mycobacterium tuberculosis are progressively more accepted. Studies of their dimension in epidemiological scenarios where the infective pressure is not high are scarce. Our study systematically searched for clonally complex infections (mixed infections by more than one strain and simultaneous presence of clonal variants) by applying mycobacterial interspersed repetitive-unit (MIRU)–variable-number tandem-repeat (VNTR) analysis to M. tuberculosis isolates from two population-based samples of respiratory (703 cases) and respiratory-extrapulmonary (R+E) tuberculosis (TB) cases (71 cases) in a context of moderate TB incidence. Clonally complex infections were found in 11 (1.6%) of the respiratory TB cases and in 10 (14.1%) of those with R+E TB. Among the 21 cases with clonally complex TB, 9 were infected by 2 independent strains and the remaining 12 showed the simultaneous presence of 2 to 3 clonal variants. For the 10 R+E TB cases with clonally complex infections, compartmentalization (different compositions of strains/clonal variants in independent infected sites) was found in 9 of them. All the strains/clonal variants were also genotyped by IS6110-based restriction fragment length polymorphism analysis, which split two MIRU-defined clonal variants, although in general, it showed a lower discriminatory power to identify the clonal heterogeneity revealed by MIRU-VNTR analysis. The comparative analysis of IS6110 insertion sites between coinfecting clonal variants showed differences in the genes coding for a cutinase, a PPE family protein, and two conserved hypothetical proteins. Diagnostic delay, existence of previous TB, risk for overexposure, and clustered/orphan status of the involved strains were analyzed to propose possible explanations for the cases with clonally complex infections. Our study characterizes in detail all the clonally complex infections by M. tuberculosis found in a systematic survey and contributes to the characterization that these phenomena can be found to an extent higher than expected, even in an unselected population-based sample lacking high infective pressure.

Advanced Survey of Tuberculosis Transmission in a Complex Socioepidemiologic Scenario with a High Proportion of Cases in Immigrants

BACKGROUND An increase in the incidence of tuberculosis (TB) in immigrants has changed the socioepidemiologic scenario in Spain. It is generally assumed that TB in immigrants is the result of importation of infection, but the role of recent transmission is rarely considered. Standard contact tracing is not suitable for the survey of transmission in this complex scenario. METHODS During the study period (2003-2006), we genotyped 356 (90.4%) of 394 isolates from patients with microbiologically confirmed TB in Almería, the province with the highest percentage of TB cases among immigrants in Spain. The epidemiologic survey of TB transmission was performed by active data collection using standardized interviews of the patients with TB and subsequent interviews of the clustered patients (who were clustered on the basis of the restriction fragment-length polymorphism types of their isolates) to identify transmission locations (supported by nominal and/or photographic recognition by the clustered patients). RESULTS Of all 356 genotyped isolates, 131 (36.8%) were clustered, suggesting recent transmission. The difference between the clustering rate for immigrants (32.8%) and that for native patients (41.6%) was not statistically significant (P = .087); of the 45 clusters, 15 (33.3%) involved only immigrants, 17 (37.8%) involved only autochthonous patients, and 13 (28.9%) involved both immigrants and autochthonous patients. The advanced system to investigate the clustered patients succeeded in detecting links in 10 of the 12 clusters that involved >4 patients, whereas the conventional approach, based on contact tracing, could detect links in only 2 clusters. CONCLUSIONS Recent transmission among immigrants and transmission permeability between the immigrant and autochthonous populations were found. Epidemiologic strategies that combine universal genotyping and refined surveys of the clustered patients are needed to investigate transmission patterns in complex scenarios.

Isolation of Mycobacterium tuberculosis Strains with a Silent Mutation in rpoB Leading to Potential Misassignment of Resistance Category

ABSTRACT Our study provides an alert regarding the transmission of rifampin-susceptible strains of Mycobacterium tuberculosis with a silent substitution in codon 514 of rpoB. Among 1,450 cases, we identified 12 isolates sharing this mutation and related restriction fragment length polymorphism (RFLP) types. The mutation impaired hybridization with the wild-type probes in three independent commercial assays, which could lead to misassignment of resistance.

Characterization of Microevolution Events in Mycobacterium tuberculosis Strains Involved in Recent Transmission Clusters

ABSTRACT Under certain circumstances, it is possible to identify clonal variants of Mycobacterium tuberculosis infecting a single patient, probably as a result of subtle genetic rearrangements in part of the bacillary population. We systematically searched for these microevolution events in a different context, namely, recent transmission chains. We studied the clustered cases identified using a population-based universal molecular epidemiology strategy over a 5-year period. Clonal variants of the reference strain defining the cluster were found in 9 (12%) of the 74 clusters identified after the genotyping of 612 M. tuberculosis isolates by IS6110 restriction fragment length polymorphism analysis and mycobacterial interspersed repetitive units–variable-number tandem repeat typing. Clusters with microevolution events were epidemiologically supported and involved 4 to 9 cases diagnosed over a 1- to 5-year period. The IS6110 insertion sites from 16 representative isolates of reference and microevolved variants were mapped by ligation-mediated PCR in order to characterize the genetic background involved in microevolution. Both intragenic and intergenic IS6110 locations resulted from these microevolution events. Among those cases of IS6110 locations in intergenic regions which could have an effect on the regulation of adjacent genes, we identified the overexpression of cytochrome P450 in one microevolved variant using quantitative real-time reverse transcription-PCR. Our results help to define the frequency with which microevolution can be expected in M. tuberculosis transmission chains. They provide a snapshot of the genetic background of these subtle rearrangements and identify an event in which IS6110-mediated microevolution in an isogenic background has functional consequences.

The clonal composition of Mycobacterium tuberculosis in clinical specimens could be modified by culture

BACKGROUND The application of molecular tools has revealed that infection by Mycobacterium tuberculosis (MTB) is more complex than initially assumed. Genotyping is generally performed on cultures. However, there is no information about bacterial clonal complexity in clinical specimens or whether standard culture procedures can modify this complexity. METHODS An in vitro assay was performed to determine whether culture can modify the clonal complexity of the MTB population in clinical specimens. Pairs of MTB strains (10 pairs) or stain-positive sputa (4 pairs) were mixed in different volumetric proportions. The DNA extracted from the mixtures before and after culture was genotyped using mycobacterial interspersed repetitive unit-variable-number tandem repeat analysis to detect potential changes in the proportion of the mixed strains. RESULTS In 6/10 pairs of MTB strains and 2/4 pairs of sputa, marked changes were observed in clonal composition after culture, even in mixtures of strains differing in their drug-susceptibility patterns. In some cases, only one of the mixed strains was detected after culture. CONCLUSIONS The initial clonal composition in bacteriologically complex clinical specimens could be underestimated if genotyping analysis is performed after culture. Genotyping strategies aimed at analyzing clinical samples must be optimized to reveal the real dimension of clonal complexity in infection by MTB.

Characterization of Mycobacterium tuberculosis Beijing isolates from the Mediterranean area.

BackgroundThe Beijing lineage of Mycobacterium tuberculosis is causing concern due to its global distribution and its involvement in severe outbreaks. Studies focused on this lineage are mainly restricted to geographical settings where its prevalence is high, whereas those in other areas are scarce. In this study, we analyze Beijing isolates in the Mediterranean area, where this lineage is not prevalent and is mainly associated with immigrant cases.ResultsOnly 1% (N = 26) of the isolates from two population-based studies in Spain corresponded to Beijing strains, most of which were pan-susceptible and from Peruvian and Ecuadorian patients. Restriction fragment length polymorphism typing with the insertion sequence IS6110 identified three small clusters (2-3 cases). Mycobacterial interspersed repetitive unit-variable number tandem repeat typing (MIRU-15) offered low discriminatory power, requiring the introduction of five additional loci. A selection of the Beijing isolates identified in the Spanish sample, together with a sample of Beijing strains from Italy, to broaden the analysis context in the Mediterranean area, were assayed in an infection model with THP-1 cells. A wide range of intracellular growth rates was observed with only two isolates showing an increased intracellular replication, in both cases associated with contained production of TNF-α. No correlation was observed between virulence and the Beijing phylogenetic group, clustered/orphan status, or resistance. The Beijing strain responsible for extensive spread on Gran Canaria Island was also identified in Madrid, but did not lead to secondary cases and did not show high infectivity in the infection model.ConclusionsThe Beijing lineage in our area is a non-homogeneous family, with only certain highly virulent representatives. The specific characterization of Beijing isolates in different settings could help us to accurately identify the virulent representatives before making general assumptions about this lineage.

Transmission permeability of tuberculosis involving immigrants, revealed by a multicentre analysis of clusters

In recent years, the number of cases of tuberculosis (TB) among immigrants in Spain has increased markedly, and led to this analysis of the recent transmission patterns of TB in the immigrant population in Madrid. The countries from which the highest number of immigrant cases have been reported were Ecuador (21%), Romania (16%), Morocco (12%), Peru (11%) and Bolivia (9%). Fifty-one per cent of the cases were from South America. In a multicentre study (2004-2006), IS6110 restriction fragment length polymorphism and spoligotyping were used to genotype the Mycobacterium tuberculosis isolates from 632 immigrant cases from 47 countries. A total of 183 cases (29%) were grouped into 59 clusters, which are markers of potential transmission events. Most of the clusters (81%) included patients living in different healthcare districts, and 54% of the clusters were multinational. When a sample of 478 autochthonous cases was included, 53% of the clusters involving immigrants also included autochthonous cases. This study revealed marked transmission permeability among nationalities and between the immigrant and the autochthonous populations.

Real-Time Molecular Epidemiology of Tuberculosis by Direct Genotyping of Smear-Positive Clinical Specimens

ABSTRACT We applied MIRU-VNTR (mycobacterial interspersed repetitive-unit–variable-number tandem-repeat typing) to directly analyze the bacilli present in 61 stain-positive specimens from tuberculosis patients. A complete MIRU type (24 loci) was obtained for all but one (no amplification in one locus) of the specimens (98.4%), and the allelic values fully correlated with those obtained from the corresponding cultures. Our study is the first to demonstrate that real-time genotyping of Mycobacterium tuberculosis can be achieved, fully transforming the way in which molecular epidemiology techniques can be integrated into control programs.