Background: Using phylogenetic approaches, the expectation that parallel cladogenesis should occur between parasites and hosts has been validated in some studies, but most others provided evidence for frequent host shifts. Here we examine the evolutionary history of the association between Microbotryum fungi that cause anther smut disease and their Caryophyllaceous hosts. We investigated the congruence between host and parasite phylogenies, inferred cospeciation events and host shifts, and assessed whether geography or plant ecology could have facilitated the putative host shifts identified. For cophylogeny analyses on microorganisms, parasite strains isolated from different host species are generally considered to represent independent evolutionary lineages, often without checking whether some strains actually belong to the same generalist species. Such an approach may mistake intraspecific nodes for speciation events and thus bias the results of cophylogeny analyses if generalist species are found on closely related hosts. A second aim of this study was therefore to evaluate the impact of species delimitation on the inferences of cospeciation. Results: We inferred a multiple gene phylogeny of anther smut strains from 21 host plants from several geographic origins, complementing a previous study on the delimitation of fungal species and their host specificities. We also inferred a multi-gene phylogeny of their host plants, and the two phylogenies were compared. A significant level of cospeciation was found when each host species was considered to harbour a specific parasite strain, i.e. when generalist parasite species were not recognized as such. This approach overestimated the frequency of cocladogenesis because individual parasite species capable of infecting multiple host species (i.e. generalists) were found on closely related hosts. When generalist parasite species were appropriately delimited and only a single representative of each species was retained, cospeciation events were not more frequent than expected under a random distribution, and many host shifts were inferred. Current geographic distributions of host species seemed to be of little relevance for understanding the putative historical host shifts, because most fungal species had overlapping geographic ranges. We did detect some ecological similarities, including shared pollinators and habitat types, between host species that were diseased by closely related anther smut species. Overall, genetic similarity underlying the host-parasite interactions appeared to have the most important influence on specialization and host-shifts: generalist multi-host parasite species were found on closely related plant species, and related species in the Microbotryum phylogeny were associated with members of the same host clade. Conclusion: We showed here that Microbotryum species have evolved through frequent host shifts to moderately distant hosts, and we show further that accurate delimitation of parasite species is essential for interpreting cophylogeny studies.

Analysis of genome sequencing data from >100,000 genomes of Mycobacterium tuberculosis complex using TB-Annotator software revealed a previously unknown lineage, proposed name L10, in central Africa. Phylogenetic reconstruction suggests L10 could represent a missing link in the evolutionary and geographic migration histories of M. africanum.

Tuberculosis (TB) remains a public health problem in Madagascar. A crucial element of TB control is the development of an easy and rapid method for the orientation of TB control strategies in the country. Our main objective was to develop a TB spatial hotspot identification method by combining spatial analysis and TB genotyping method in Antananarivo. Sputa of new pulmonary TB cases from 20 TB diagnosis and treatment centers (DTCs) in Antananarivo were collected from August 2013 to May 2014 for culture. Mycobacterium tuberculosis complex (MTBC) clinical isolates were typed by spoligotyping on a Luminex® 200 platform. All TB patients were respectively localized according to their neighborhood residence and the spatial distribution of all pulmonary TB patients and patients with genotypic clustered isolates were scanned respectively by the Kulldorff spatial scanning method for identification of significant spatial clustering. Areas exhibiting spatial clustering of patients with genotypic clustered isolates were considered as hotspot TB areas for transmission. Overall, 467 new cases were included in the study, and 394 spoligotypes were obtained (84.4%). New TB cases were distributed in 133 of the 192 Fokontany (administrative neighborhoods) of Antananarivo (1 to 15 clinical patients per Fokontany) and patients with genotypic clustered isolates were distributed in 127 of the 192 Fokontany (1 to 13 per Fokontany). A single spatial focal point of epidemics was detected when ignoring genotypic data (p = 0.039). One Fokontany of this focal point and three additional ones were detected to be spatially clustered when taking genotypes into account (p < 0.05). These four areas were declared potential TB transmission hotspots in Antananarivo and will be considered as priority targets for surveillance in the future. This method, combining spatial analysis and TB genotyping will now be used for further focused clinical and epidemiological studies in Madagascar and will allow better TB control strategies by public health authorities.

Background: Cambodia is among the 22 high-burden TB countries, and has one of the highest rates of TB in South-East Asia. This study aimed to describe the genetic diversity among clinical Mycobacterium tuberculosis complex (MTC) isolates collected in Cambodia and to relate these findings to genetic diversity data from neighboring countries. Methods: We characterized by 24 VNTR loci genotyping and spoligotyping 105 Mycobacterium tuberculosis clinical isolates collected between 2007 and 2008 in the region of Phnom-Penh, Cambodia, enriched in multidrug-resistant (MDR) isolates (n = 33). Results: Classical spoligotyping confirmed that the East-African Indian (EAI) lineage is highly prevalent in this area (60%-68% respectively in whole sample and among non-MDR isolates). Beijing lineage is also largely represented (30% in whole sample, 21% among non-MDR isolates, OR = 4.51, CI_95% [1.77, 11.51]) whereas CAS lineage was absent. The 24 loci MIRU-VNTR typing scheme distinguished 90 patterns with only 13 multi-isolates clusters covering 28 isolates. The clustering of EAI strains could be achieved with only 8 VNTR combined with spoligotyping, which could serve as a performing, easy and cheap genotyping standard for this family. Extended spoligotyping suggested relatedness of some unclassified "T1 ancestors" or "Manu" isolates with modern strains and provided finer resolution. Conclusions: The genetic diversity of MTC in Cambodia is driven by the EAI and the Beijing families. We validate the usefulness of the extended spoligotyping format in combination with 8 VNTR for EAI isolates in this region.

Infra-species taxonomy is a prerequisite to compare features such as virulence in different pathogen lineages. Mycobacterium tuberculosis complex taxonomy has rapidly evolved in the last 20 years through intensive clinical isolation, advances in sequencing and in the description of fast-evolving loci (CRISPR and MIRU-VNTR). On-line tools to describe new isolates have been set up based on known diversity either on CRISPRs (also known as spoligotypes) or on MIRU-VNTR profiles. The underlying taxonomies are largely concordant but use different names and offer different depths. The objectives of this study were 1) to explicit the consensus that exists between the alternative taxonomies, and 2) to provide an on-line tool to ease classification of new isolates. Genotyping (24-VNTR, 43-spacers spoligotypes, IS6110-RFLP) was undertaken for 3,454 clinical isolates from the Netherlands (2004-2008). The resulting database was enlarged with African isolates to include most human tuberculosis diversity. Assignations were obtained using TB-Lineage, MIRU-VNTRPlus, SITVITWEB and an algorithm from Borile et al. By identifying the recurrent concordances between the alternative taxonomies, we proposed a consensus including 22 sublineages. Original and consensus assignations of the all isolates from the database were subsequently implemented into an ensemble learning approach based on Machine Learning tool Weka to derive a classification scheme. All assignations were reproduced with very good sensibilities and specificities. When applied to independent datasets, it was able to suggest new sublineages such as pseudo-Beijing. This Lineage Prediction tool, efficient on 15-MIRU, 24-VNTR and spoligotype data is available on the web interface “TBminer.” Another section of this website helps summarizing key molecular epidemiological data, easing tuberculosis surveillance. Altogether, we successfully used Machine Learning on a large dataset to set up and make available the first consensual taxonomy for human Mycobacterium tuberculosis complex. Additional developments using SNPs will help stabilizing it.

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. 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. 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). 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.