Revealing hidden clonal complexity in Mycobacterium tuberculosis infection by qualitative and quantitative improvement of sampling

AbstractThe analysis of microevolution events, its functional relevance and impact on molecular epidemiology strategies, constitutes one of the most challenging aspects of the study of clonal complexity in infection by Mycobacterium tuberculosis. In this study, we retrospectively evaluated whether two improved sampling schemes could provide access to the clonal complexity that is undetected by the current standards (analysis of one isolate from one sputum). We evaluated in 48 patients the analysis by mycobacterial interspersed repetitive unit–variable number tandem repeat of M. tuberculosis isolates cultured from bronchial aspirate (BAS) or bronchoalveolar lavage (BAL) and, in another 16 cases, the analysis of a higher number of isolates from independent sputum samples. Analysis of the isolates from BAS/BAL specimens revealed clonal complexity in a very high proportion of cases (5/48); in most of these cases, complexity was not detected when the isolates from sputum samples were analysed. Systematic analysis of isolates from multiple sputum samples also improved the detection of clonal complexity. We found coexisting clonal variants in two of 16 cases that would have gone undetected in the analysis of the isolate from a single sputum specimen. Our results suggest that analysis of isolates from BAS/BAL specimens is highly efficient for recording the true clonal composition of M. tuberculosis in the lungs. When these samples are not available, we recommend increasing the number of isolates from independent sputum specimens, because they might not harbour the same pool of bacteria. Our data suggest that the degree of clonal complexity in tuberculosis has been underestimated because of the deficiencies inherent in a simplified procedure

Differences in the robustness of clusters involving the Mycobacterium tuberculosis strains most frequently isolated from immigrant cases in Madrid

AbstractTuberculosis cases infected by the same Mycobacterium tuberculosis (MTB) strain are considered to be clustered and involved in a transmission chain. Large clusters are assumed to represent active transmission chains in a population. In the present study, we focused on the analysis of large clusters defined by IS6110-restriction fragment length polymorphism (RFLP) typing in the immigrant population in Madrid. We identified 12 large clusters (involving 43% of the isolates) comprising 4–23 representatives. We proposed a gradient of epidemiological certainty for these large clusters. For a cluster to be considered robust and a good indicator of recent transmission, the MTB strain involved should not have been identified in a geographically and epidemiologically unrelated population and the cluster had to be re-confirmed by another highly discriminative molecular marker (MIRU-VNTR). The clusters that we discovered were classified into three categories: high, intermediate and low expected epidemiological value. In the largest cluster in the study (cluster M6; 23 representatives), failures by both criteria were identified: the representative seven-band RFLP pattern was also the most prevalent in the unrelated population (25 cases) and the cluster was fully split by MIRU-15, suggesting a lack of epidemiological value. The RFLP pattern representative of this cluster was also identified in 64 isolates from five countries in the Latin American genotype database, and again proved to be heterogeneous according to the MIRU-15 analysis. Specific analysis of large clusters, combined with the application of criteria for evaluating their robustness, could help identify uninformative clusters and target epidemiological resources towards those clusters with higher expected epidemiological value

Characterization of Mycobacterium tuberculosis Beijing isolates from the Mediterranean area

<p>Abstract</p> <p>Background</p> <p>The Beijing lineage of <it>Mycobacterium tuberculosis </it>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.</p> <p>Results</p> <p>Only 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 IS<it>6110 </it>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.</p> <p>Conclusions</p> <p>The 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.</p

A non-canonical mismatch repair pathway in prokaryotes

Mismatch repair (MMR) is a near ubiquitous pathway, essential for the maintenance of genome stability. Members of the MutS and MutL protein families perform key steps in mismatch correction. Despite the major importance of this repair pathway, MutS–MutL are absent in almost all Actinobacteria and many Archaea. However, these organisms exhibit rates and spectra of spontaneous mutations similar to MMR-bearing species, suggesting the existence of an alternative to the canonical MutS–MutL-based MMR. Here we report that Mycobacterium smegmatis NucS/EndoMS, a putative endonuclease with no structural homology to known MMR factors, is required for mutation avoidance and anti-recombination, hallmarks of the canonical MMR. Furthermore, phenotypic analysis of naturally occurring polymorphic NucS in a M. smegmatis surrogate model, suggests the existence of M. tuberculosis mutator strains. The phylogenetic analysis of NucS indicates a complex evolutionary process leading to a disperse distribution pattern in prokaryotes. Together, these findings indicate that distinct pathways for MMR have evolved at least twice in nature

Genomic Diversity of Mycobacterium tuberculosis Complex Strains in Cantabria (Spain), a Moderate TB Incidence Setting

Background Tuberculosis (TB) control strategies are focused mainly on prevention, early diagnosis, compliance to treatment and contact tracing. The objectives of this study were to explore the frequency and risk factors of recent transmission of clinical isolates of Mycobacterium tuberculosis complex (MTBC) in Cantabria in Northern Spain from 2012 through 2013 and to analyze their clonal complexity for better understanding of the transmission dynamics in a moderate TB incidence setting. Methods DNA from 85 out of 87 isolates from bacteriologically confirmed cases of MTBC infection were extracted directly from frozen stocks and genotyped using the mycobacterial interspersed repetitive units-variable number tandem repeat (MIRU-VNTR) method. The MIRUVNTRplus database tool was used to identify clusters and lineages and to build a neighbor joining (NJ) phylogenetic tree. In addition, data were compared to the SITVIT2 database at the Pasteur Institute of Guadeloupe. Results The rate of recent transmission was calculated to 24%. Clustering was associated with being Spanish-born. A high prevalence of isolates of the Euro-American lineage was found. In addition, MIRU-VNTR profiles of the studied isolates corresponded to previously found MIRU-VNTR types in other countries, including Spain, Belgium, Great Britain, USA, Croatia, South Africa and The Netherlands. Six of the strains analyzed represented clonal variants. Conclusion Transmission of MTBC is well controlled in Cantabria. The majority of TB patients were born in Spain. The population structure of MTBC in Cantabria has a low diversity of major clonal lineages with the Euro-American lineage predominating

Molecular epidemiology, drug susceptibility and economic aspects of tuberculosis in mubende district, Uganda

<div><p>Background</p><p>Tuberculosis (TB) remains a global public health problem whose effects have major impact in developing countries like Uganda. This study aimed at investigating genotypic characteristics and drug resistance profiles of <i>Mycobacterium tuberculosis</i> isolated from suspected TB patients. Furthermore, risk factors and economic burdens that could affect the current control strategies were studied.</p><p>Methods</p><p>TB suspected patients were examined in a cross-sectional study at the Mubende regional referral hospital between February and July 2011. A questionnaire was administered to each patient to obtain information associated with TB prevalence. Isolates of <i>M. tuberculosis</i> recovered during sampling were examined for drug resistance to first line anti-TB drugs using the BACTEC-MGIT960^TMsystem. All isolates were further characterized using deletion analysis, spoligotyping and MIRU-VNTR analysis. Data were analyzed using different software; MIRU-VNTR <i>plus</i>, SITVITWEB, BioNumerics and multivariable regression models.</p><p>Results</p><p><i>M. tuberculosis</i> was isolated from 74 out of 344 patients, 48 of these were co-infected with HIV. Results from the questionnaire showed that previously treated TB, co-infection with HIV, cigarette smoking, and overcrowding were risk factors associated with TB, while high medical related transport bills were identified as an economic burden. Out of the 67 isolates that gave interpretable results, 23 different spoligopatterns were detected, nine of which were novel patterns. T2 with the sub types Uganda-I and Uganda-II was the most predominant lineage detected. Antibiotic resistance was detected in 19% and multidrug resistance was detected in 3% of the isolates.</p><p>Conclusion</p><p>The study detected <i>M. tuberculosis</i> from 21% of examined TB patients, 62% of whom were also HIV positive. There is a heterogeneous pool of genotypes that circulate in this area, with the T2 lineage being the most predominant. High medical related transport bills and drug resistance could undermine the usefulness of the current TB strategic interventions.</p></div

Spread of a SARS-CoV-2 variant through Europe in the summer of 2020.

Following its emergence in late 2019, the spread of SARS-CoV-21,2 has been tracked by phylogenetic analysis of viral genome sequences in unprecedented detail3–5. Although the virus spread globally in early 2020 before borders closed, intercontinental travel has since been greatly reduced. However, travel within Europe resumed in the summer of 2020. Here we report on a SARS-CoV-2 variant, 20E (EU1), that was identified in Spain in early summer 2020 and subsequently spread across Europe. We find no evidence that this variant has increased transmissibility, but instead demonstrate how rising incidence in Spain, resumption of travel, and lack of effective screening and containment may explain the variant’s success. Despite travel restrictions, we estimate that 20E (EU1) was introduced hundreds of times to European countries by summertime travellers, which is likely to have undermined local efforts to minimize infection with SARS-CoV-2. Our results illustrate how a variant can rapidly become dominant even in the absence of a substantial transmission advantage in favourable epidemiological settings. Genomic surveillance is critical for understanding how travel can affect transmission of SARS-CoV-2, and thus for informing future containment strategies as travel resumes. © 2021, The Author(s), under exclusive licence to Springer Nature Limited

Molecular epidemiology of Mycobacterium bovis in Cameroon

We describe the largest molecular epidemiological study of Bovine Tuberculosis (bTB) in a sub-Saharan African country with higher spatial resolution providing new insights into bTB. Four hundred and ninety-nine samples were collected for culture from 201 and 179 cattle with and without bTB-like lesions respectively out of 2,346 cattle slaughtered at Bamenda, Ngaoundere, Garoua and Maroua abattoirs between 2012-2013. Two hundred and fifty-five M. bovis were isolated, identified and genotyped using deletion analysis, Hain® Genotype MTBC, spoligotyping and MIRU-VNTR. African 1 was the dominant M. bovis clonal complex, with 97 unique genotypes including 19 novel spoligotypes representing the highest M. bovis genetic diversity observed in Africa to date. SB0944 and SB0953 dominated (63%) the observed spoligotypes. A third of animals with multiple lesions had multiple strain infections. Higher diversity but little evidence of recent transmission of M. bovis was more common in Adamawa compared to the North-West Region. The Adamawa was characterised by a high frequency of singletons possibly due to constant additions from an active livestock movement network compared to the North-West Region where a local expansion was more evident. The latter combined with population-based inferences suggest an unstable and stable bTB-endemic status in the North-West and Adamawa Regions respectively