Latin American And Mediterranean Research Articles

Dual RNA Sequencing of Mycobacterium tuberculosis-Infected Human Splenic Macrophages Reveals a Strain-Dependent Host-Pathogen Response to Infection.

Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb), leading to pulmonary and extrapulmonary TB, whereby Mtb is disseminated to many other organs and tissues. Dissemination occurs early during the disease, and bacteria can be found first in the lymph nodes adjacent to the lungs and then later in the extrapulmonary organs, including the spleen. The early global gene expression response of human tissue macrophages and intracellular clinical isolates of Mtb has been poorly studied. Using dual RNA-seq, we have explored the mRNA profiles of two closely related clinical strains of the Latin American and Mediterranean (LAM) family of Mtb in infected human splenic macrophages (hSMs). This work shows that these pathogens mediate a distinct host response despite their genetic similarity. Using a genome-scale host–pathogen metabolic reconstruction to analyze the data further, we highlight that the infecting Mtb strain also determines the metabolic response of both the host and pathogen. Thus, macrophage ontogeny and the genetic-derived program of Mtb direct the host–pathogen interaction.

RDRioMycobacterium tuberculosis strains associated with isoniazid resistance in Northern Mexico

BackgroundIt has been established that the genomic background of Mycobacterium tuberculosis may influence disease progression, in particular for the Beijing family and the Latin American and Mediterranean (LAM)/RDRio strains. The purpose of this study was to evaluate the prevalence of the LAM/RDRio genotype in cases of tuberculosis from Mexico and their drug susceptibility profile. MethodsTwo hundred eighteen M. tuberculosis isolates were screened by 43-spacer spoligotyping. The LAM/RDRio genotype was confirmed by multiplex PCR, and the drug susceptibility testing was carried out in solid Löwenstein-Jensen media. ResultsAmong the LAM strains identified, 24 (63.1%) were confirmed as M. tuberculosis RDRio. All RDRio strains shared the RD174 deletion, that was associated with isoniazid resistance (p=0.0264). ConclusionsWe documented for the first time the isolation of the LAM/RDRio genotype in pulmonary cases of tuberculosis in Mexico, and we found resistance to the first-line anti-tuberculosis drug isoniazid in these strains.

Genomic-based surveillance reveals high ongoing transmission of multi-drug-resistant Mycobacterium tuberculosis in Southern Brazil

Genomic-based surveillance on the occurrence of drug resistance and its transmission dynamics has emerged as a powerful tool for the control of tuberculosis (TB). A whole-genome sequencing approach, phenotypic testing and clinical-epidemiological investigation were used to undertake a retrospective population-based study on drug-resistant (DR)-TB in Rio Grande do Sul, the largest state in Southern Brazil. The analysis included 305 resistant Mycobacterium tuberculosis strains sampled statewide from 2011 to 2014, and covered 75.7% of all DR-TB cases identified in this period. Lineage 4 was found to be predominant (99.3%), with high sublineage-level diversity composed mainly of 4.3.4.2 [Latin American and Mediterranean (LAM)/RD174], 4.3.3 (LAM/RD115) and 4.1.2.1 (Haarlem/RD182) sublineages. Genomic diversity was also reflected in resistance of the variants to first-line drugs. A large number of distinct resistance-conferring mutations, including variants that have not been reported previously in any other setting worldwide, and 22 isoniazid-monoresistant strains with mutations described as disputed in the rpoB gene but causing rifampicin resistance generally missed by automated phenotypic tests as BACTEC MGIT. Using a cut-off of five single nucleotide polymorphisms, the estimated recent transmission rate was 55.1%, with 168 strains grouped into 28 genomic clusters. The most worrying fact concerns multi-drug-resistant (MDR) strains, of which 73.4% were clustered. Different resistance profiles and acquisition of novel mutations intraclusters revealed important amplification of resistance in the region. This study described the diversity of M. tuberculosis strains, the basis of drug resistance, and ongoing transmission dynamics across the largest state in Southern Brazil, stressing the urgent need for MDR-TB transmission control state-wide.

RDRioMycobacterium tuberculosis strains associated with isoniazid resistance in Northern Mexico

BackgroundIt has been established that the genomic background of Mycobacterium tuberculosis may influence disease progression, in particular for the Beijing family and the Latin American and Mediterranean (LAM)/RDRio strains. The purpose of this study was to evaluate the prevalence of the LAM/RDRio genotype in cases of tuberculosis from Mexico and their drug susceptibility profile. MethodsTwo hundred eighteen M. tuberculosis isolates were screened by 43-spacer spoligotyping. The LAM/RDRio genotype was confirmed by multiplex PCR, and the drug susceptibility testing was carried out in solid Löwenstein-Jensen media. ResultsAmong the LAM strains identified, 24 (63.1%) were confirmed as M. tuberculosis RDRio. All RDRio strains shared the RD174 deletion, that was associated with isoniazid resistance (p=0.0264). ConclusionsWe documented for the first time the isolation of the LAM/RDRio genotype in pulmonary cases of tuberculosis in Mexico, and we found resistance to the first-line anti-tuberculosis drug isoniazid in these strains.

Genetic Diversity and Molecular Epidemiology of Mycobacterium tuberculosis in Roraima State, Brazil.

National border areas are special places for the spread of Mycobacterium tuberculosis (MTB). These regions concentrate vulnerable populations and constant population movements. Understanding the dynamics of the transmission of MTB is fundamental to propose control measures and to monitor drug resistance. We conducted a population-based prospective study of tuberculosis (TB) to evaluate molecular characteristics of MTB isolates circulating in Roraima, a state on the border of Venezuela and Guyana. Eighty isolates were genotyped by IS6110-RFLP (restriction fragment length polymorphism), spoligotyping, and 24-locus mycobacterial interspersed repetitive unit-variable number of repeats tandem (MIRU-VNTR). Drug susceptibility tests were performed by using the proportion method and GeneXpert® MTB/RIF (Cepheid, Sunnyvale, CA). Isolates showing a phenotypic resistance profile were submitted to polymerase chain reaction (PCR) and sequencing. Spoligotyping showed 40 distinct patterns with a high prevalence of Latin-American and Mediterranean (LAM), Haarlem (H), and the "ill-defined" T clades. Mycobacterial interspersed repetitive unit -VNTR and IS6110-RFLP showed clustering rates of 21.3% and 30%, respectively. Drug resistance was detected in 11 (15.1%) isolates, and all were found to have primary resistance; among these, six (8.2%) isolates were streptomycin mono-resistant, four (5.4%) isoniazid mono-resistant, and one (1.3%) multidrug resistant. This is the first study on the molecular epidemiology and drug resistance profile of MTB from Roraima. Herein, we describe high diversity of genetic profiles circulating in this region that may be driven by the introduction of new strain types because of large population flow in this region. In summary, our results showed that analyses of these circulating strains can contribute to a better understanding of TB epidemiology in the northern Brazilian border and be useful to establish public health policies on TB prevention.

Clonal expansion across the seas as seen through CPLP-TB database: A joint effort in cataloguing Mycobacterium tuberculosis genetic diversity in Portuguese-speaking countries

Tuberculosis (TB) remains a major health problem within the Community of Portuguese Language Speaking Countries (CPLP). Despite the marked variation in TB incidence across its member-states and continued human migratory flux between countries, a considerable gap in the knowledge on the Mycobacterium tuberculosis population structure and strain circulation between the countries still exists. To address this, we have assembled and analysed the largest CPLP M. tuberculosis molecular and drug susceptibility dataset, comprised by a total of 1447 clinical isolates, including 423 multidrug-resistant isolates, from five CPLP countries. The data herein presented reinforces Latin American and Mediterranean (LAM) strains as the hallmark of M. tuberculosis populational structure in the CPLP coupled with country-specific differential prevalence of minor clades. Moreover, using high-resolution typing by 24-loci MIRU-VNTR, six cross-border genetic clusters were detected, thus supporting recent clonal expansion across the Lusophone space.To make this data available to the scientific community and public health authorities we developed CPLP-TB (available at http://cplp-tb.ff.ulisboa.pt), an online database coupled with web-based tools for exploratory data analysis. As a public health tool, it is expected to contribute to improved knowledge on the M. tuberculosis population structure and strain circulation within the CPLP, thus supporting the risk assessment of strain-specific trends.

Genetic Structuration, Demography and Evolutionary History of Mycobacterium tuberculosis LAM9 Sublineage in the Americas as Two Distinct Subpopulations Revealed by Bayesian Analyses.

Tuberculosis (TB) remains broadly present in the Americas despite intense global efforts for its control and elimination. Starting from a large dataset comprising spoligotyping (n = 21183 isolates) and 12-loci MIRU-VNTRs data (n = 4022 isolates) from a total of 31 countries of the Americas (data extracted from the SITVIT2 database), this study aimed to get an overview of lineages circulating in the Americas. A total of 17119 (80.8%) strains belonged to the Euro-American lineage 4, among which the most predominant genotypic family belonged to the Latin American and Mediterranean (LAM) lineage (n = 6386, 30.1% of strains). By combining classical phylogenetic analyses and Bayesian approaches, this study revealed for the first time a clear genetic structuration of LAM9 sublineage into two subpopulations named LAM9C1 and LAM9C2, with distinct genetic characteristics. LAM9C1 was predominant in Chile, Colombia and USA, while LAM9C2 was predominant in Brazil, Dominican Republic, Guadeloupe and French Guiana. Globally, LAM9C2 was characterized by higher allelic richness as compared to LAM9C1 isolates. Moreover, LAM9C2 sublineage appeared to expand close to twenty times more than LAM9C1 and showed older traces of expansion. Interestingly, a significant proportion of LAM9C2 isolates presented typical signature of ancestral LAM-RDRio MIRU-VNTR type (224226153321). Further studies based on Whole Genome Sequencing of LAM strains will provide the needed resolution to decipher the biogeographical structure and evolutionary history of this successful family.

Prevalence of Mycobacterium tuberculosis LAM family in the worldwide population: A systematic review and meta-analysis

BackgroundTuberculosis (TB) disease caused by Mycobacterium tuberculosis (MTB) is still an important cause of morbidity and mortality. Latin American and Mediterranean (LAM) is one of a family of phylogenic MTB, and its name is derived from the geographical area that has been isolated. The objective of this study is to determine the prevalence of MTB LAM family worldwide by meta-analysis and a systematic review. MethodsData sources for this study are comprised of 70 original articles (2001–2013) that were published in the literature in several databases, including PubMed, Science Direct, Google Scholar, Biological abstracts, ISI Web of Knowledge and IranMedex. Data were analyzed using meta-analysis (Meta R, Version 2.13 package [p<0.10], CI: 95%). ResultsThe highest and lowest occurrence rate of the LAM family in MTB was in Europe and Asia. Totally, the prevalence of the LAM family in Europe, Africa, America and Asia was 32.10%, 29.20%, 14.4% and 0.2%, respectively. ConclusionsThis study shows that MTB LAM family is prevalent in European countries. LAM sub-lineage is a major focus of studies carried out in different countries. Proper technique for prevention of transmission of MTB is necessary.

First baseline of circulating genotypic lineages of Mycobacterium tuberculosis in patients from the brazilian borders with Argentina and Paraguay.

BackgroundAt the triple border Brazil/Paraguay/Argentina there is easy mobility from one city to another for economic and tourism activities. This constant and fast population mobility is mainly to visit Iguazu Falls, in the Iguazu River, on the border of the Brazilian state of Paraná and the Argentina. As the incidence of tuberculosis is high in this setting, our study aimed to establish a first baseline of circulating genotypic lineages of Mycobacterium tuberculosis.Methodology/Principal FindingsThis study included 120 patients from 10 cities in southwestern Paraná, Brazil with pulmonary symptoms, from July 2009 to July 2011. Information about sex, age, clinical features and address was collected by reviewing the national tuberculosis notification database. Of these, 96 (80%) isolates were identified as M. tuberculosis and 22 (22.9%) were drug resistant (20, 20.8% INH mono-resistant and 2, 2.1% multidrug-resistant). All isolates were subjected to genotyping by Spoligotyping and MIRU-VNTR typing. The distribution of the isolates analyzed by spoligotyping revealed 30 distinct patterns. The four mainly detected clades were Latin American and Mediterranean (LAM), ill-defined T, Haarlem (H) and S. The MIRU-VNTR showed 85 distinct patterns. Spoligotyping combined to MIRU-VNTR allowed 90 distinct patterns.Conclusions/SignificanceOur study demonstrated that there is significant molecular diversity in circulating M. tuberculosis, with predominance of the LAM and T clades in cities of southwestern Paraná, Brazil, bordering Argentina and Paraguay.

Long-term molecular surveillance of multidrug-resistant tuberculosis in Spain

The data presented here span 11years (1998–2008) of monitoring of multidrug-resistant tuberculosis (MDR-TB) clustering through molecular typing techniques in Spain. The molecular and epidemiological data of 480 multidrug-resistant Mycobacterium tuberculosis complex isolates were analyzed.Thirty-one clusters involving 157 (32.7%) patients were identified. The proportion of immigrants increased substantially over the study period reaching 65% in 2008; however, the clustering rate remained stable indicating that local transmission was little influenced by imported MDR-TB. The three major clusters respond to the persistence of two autochthonous strains throughout the study period and an extensively drug-resistant (XDR) Mycobacterium bovis outbreak with only two cases was reported since 2002.Molecular and epidemiological evidence for the importation of new strains and their spread within the community was found. Immigrant-only clusters most often grouped patients infected abroad with strains belonging to rare spoligotypes. Conversely, widespread spoligotypes of the Latin-American and Mediterranean (LAM) and Haarlem families were responsible for the majority of the MDR-TB local transmission.The demonstration of clusters spanning several Spanish regions that have been ongoing throughout the study period makes it advisable to maintain a continuous molecular surveillance in order to monitor the spread of MDR-TB.

Mycobacterium tuberculosisComplex Genotype Diversity and Drug Resistance Profiles in a Pediatric Population in Mexico

The aim of this study was to determine the frequency of drug resistance and the clonality of genotype patterns in M. tuberculosis clinical isolates from pediatric patients in Mexico (n = 90 patients from 19 states; time period—January 2002 to December 2003). Pulmonary disease was the most frequent clinical manifestation (71%). Children with systemic tuberculosis (TB) were significantly younger compared to patients with localized TB infections (mean 7.7 ± 6.2 years versus 15 ± 3.4 years P = 0.001). Resistance to any anti-TB drug was detected in 24/90 (26.7%) of the isolates; 21/90 (23.3%) and 10/90 (11.1%) were resistant to Isoniazid and Rifampicin, respectively, and 10/90 (11.1%) strains were multidrug-resistant (MDR). Spoligotyping produced a total of 55 different patterns; 12/55 corresponded to clustered isolates (n = 47, clustering rate of 52.2%), and 43/55 to unclustered isolates (19 patterns were designated as orphan by the SITVIT2 database). Database comparison led to designation of 36 shared types (SITs); 32 SITs (n = 65 isolates) matched a preexisting shared type in SITVIT2, whereas 4 SITs (n = 6 isolates) were newly created. Lineage classification based on principal genetic groups (PGG) revealed that 10% of the strains belonged to PGG1 (Bovis and Manu lineages). Among PGG2/3 group, the most predominant clade was the Latin-American and Mediterranean (LAM) in 27.8% of isolates, followed by Haarlem and T lineages. The number of single drug-resistant (DR) and multidrug-resistant (MDR-TB) isolates in this study was similar to previously reported in studies from adult population with risk factors. No association between the spoligotype, age, region, or resistance pattern was observed. However, contrary to a study on M. tuberculosis spoligotyping in Acapulco city that characterized a single cluster of SIT19 corresponding to the EAI2-Manila lineage in 70 (26%) of patients, not a single SIT19 isolate was found in our pediatric patient population. Neither did we find any shared type belonging to the EAI family which represents ancestral PGG1 strains within the M. tuberculosis complex. We conclude that the population structure of pediatric TB in our setting is different from the one prevailing in adult TB patient population of Guerrero.

Spoligotype-Based Comparative Population Structure Analysis of Multidrug-Resistant and Isoniazid-Monoresistant Mycobacterium tuberculosis Complex Clinical Isolates in Poland

The spoligotyping-based population structure of multidrug-resistant (MDR) Mycobacterium tuberculosis strains isolated in Poland (n = 46), representing all culture-positive MDR tuberculosis (MDR-TB) cases, was compared to that of isoniazid (INH)-monoresistant strains (n = 71) isolated in 2004. The latter data set from a previous study (E. Augustynowicz-Kopeć, T. Jagielski, and Z. Zwolska, J. Clin. Microbiol. 2008, 46:4041-4044) represented 87% of all INH-monoresistant strains. The clustering rates and genotypic-diversity indexes for the 2 subpopulations were not significantly different (P = 0.05). The results were entered in the SITVIT2 database to assign specific shared type designations, corresponding genotypic lineages, and geographical distributions and compared to available data from neighboring countries (Germany, n = 704; Czech Republic, n = 530; Sweden, n = 379; Kaliningrad, Russia, n = 90) and strains from previous studies in Poland (n = 317). MDR strains resulted in 27 patterns (20 unique strains within the study and 7 clusters containing 2 to 6 isolates per cluster with a clustering rate of 56.5%) and belonged to the following genotypic lineages: ill-defined T family (28.3%), Haarlem (17.4%), Latin American and Mediterranean (LAM) (13%), Beijing (8.7%), S family (4.35%), and the X clade (2.17%). Comparison of the genetic structure of the MDR strains with that of INH-monoresistant strains showed that a total of 9 patterns were shared by both groups; these represented 1/3 of the MDR strains and 2/3 of the INH-monoresistant strains. Interestingly, 76.1% of the MDR isolates and 71.8% of the INH-resistant isolates yielded spoligotypes that were previously reported from Poland. The observation that nearly half of the spoligotypes identified among both MDR (48.1%) and INH-monoresistant (43.3%) M. tuberculosis isolates were present in Poland's neighboring countries suggested that a significant proportion of MDR and INH-resistant TB cases in Poland were caused by strains actively circulating in Poland or its neighbors. Our results corroborate the leading role of the T and Haarlem genotypes in the epidemiology of drug-resistant TB in Poland. Nevertheless, the LAM and Beijing family strains that infected, correspondingly, 13% and 9% of patients with MDR-TB were absent among the strains from patients with INH-monoresistant TB, suggesting that a proportion of MDR-TB cases in Poland are due to ongoing transmission of MDR clones exhibiting specific genotypes. Study of the population genetic relationships between MDR and INH-monoresistant strains by drawing minimum spanning trees showed that ill-defined T1 sublineage strains (1/3 of all INH-monoresistant strains), represented by its prototype, SIT53, constituted the central node of the tree, followed by strains belonging to the well-defined H3, H1, and S subgroups. However, the MDR group, in addition, contained LAM (n = 6) and Beijing (n = 4) lineage isolates. With the exception of the 4 Beijing lineage strains in the latter group and a single orphan isolate in the INH-monoresistant group, none of the remaining 112/117 isolates belonged to principal genetic group 1 (PGG1) in our study. Given the high rate of clustering and the near absence of immigrants in the study, the persistence of MDR-TB in Poland seems to result from active transmission of MDR strains within the autochthonous population, the bulk of it caused by evolutionarily recent tubercle bacilli.

Single nucleotide polymorphisms in cell wall biosynthesis-associated genes and phylogeny of Mycobacterium tuberculosis lineages

To investigate specific single nucleotide polymorphisms (SNPs) of different lineages of Mycobacterium tuberculosis, cell wall biosynthesis-associated genes encoding antigen 85 complex (fbpA, fbpB, and fbpC) and mannosyltransferase (pimB) were analyzed. Genetically diversified and predominant M. tuberculosis and Mycobacterium bovis genotypes identified in Taiwan (26 Beijing and 44 non-Beijing) were included in the study. Sequence analyses revealed that nine novel SNPs were found within main lineages of M. tuberculosis complex, including East-African-Indian (EAI), Beijing, Central-Asian (CAS), Bovis, and one lineage containing Latin American and Mediterranean (LAM), Haarlem and T. Specifically, a SNP in pimB codon 270 was identified in EAI, fbpA codon 156 in ancestral Beijing, fbpB codon 238 in modern Beijing, fbpA codon 4 and fbpC codon 158 in CAS, fbpA codon 311 in M. bovis and an additional SNP in fbpB codon 140 in M. bovis-BCG, and pimB codon 107 in other spoligotypes lineages including an additional SNP in fbpC codon 103 in LAM. In addition, we proved that isolates with spoligotype shared type (ST) no. 523 (carrying all 43 spacers), designated as unknown lineage in an international spoligotyping database (SpolDB4), belong to an early ancestral Beijing sublineage. Accordingly, a phylogenetic tree was constructed using those SNPs, and an evolutionary hypothesis for lineages of M. tuberculosis was proposed. These novel lineage-specific SNPs will be informative genetic markers in molecular epidemiological and evolutionary studies of M. tuberculosis.

Molecular Evolutionary History of Tubercle Bacilli Assessed by Study of the Polymorphic Nucleotide within the Nitrate Reductase ( narGHJI ) Operon Promoter

A well-characterized collection of Mycobacterium tuberculosis complex (MTC) isolates, representing all known subspecies as well as some relevant genotypic families of M. tuberculosis, was analyzed for the newly discovered narGHJI -215 C-to-T promoter single-nucleotide polymorphism (SNP). This point mutation has been shown in earlier studies to be responsible for the differential nitrate reductase activity of M. tuberculosis versus M. bovis. As previously defined by the presence or the absence of the TbD1 genetic locus, the group included both the "modern" W-Beijing, Haarlem, and Central-Asian1 (CAS1) families as well as the "ancestral" East-African-Indian (EAI) clade. Interestingly, among "modern" M. tuberculosis isolates, those previously classified as Principal Genetic Group 1 (PGG1) organisms by katG463-gyrA95 polymorphism analysis did not present the two-banded narGHJI restriction fragment length polymorphism analysis of PCR products pattern common to the other PGG1 MTC members, including the "ancestral" M. tuberculosis isolates. Instead, they showed a one-banded pattern, aligning them with other evolutionarily recent M. tuberculosis isolates of the PGG2 and PGG3 groups, such as Haarlem, Latin-American and Mediterranean (LAM), and X families. The presence of a nitrate reductase producer phenotype in "Mycobacterium canettii" and some "ancestral" M. tuberculosis isolates, despite a two-band -215C genotype, argues in favor of an alternate mechanism to explain the differential nitrate reductase activity of certain PGG1 subspecies of the MTC. Overall, these findings may help to establish the precise evolutionary history of important genotype families such as W-Beijing and suggest that the -215T genotype may have contributed the virulence, spread, and evolutionary success of "modern" M. tuberculosis strains compared to the remaining MTC organisms.

Data mining of Mycobacterium tuberculosis complex genotyping results using mycobacterial interspersed repetitive units validates the clonal structure of spoligotyping-defined families

Recently, a combination of spoligotyping and bioinformatics was proposed as a potential tool for defining major circulating clades of tuberculosis bacilli. In the present study, we attempted to validate the above mentioned classification using a new high-throughput marker, named mycobacterial interspersed repetitive units (MIRUs). Using 12 MIRU loci and spoligotyping, we performed data mining of results on clinical isolates of the Mycobacterium tuberculosis complex representative of global mycobacterial allelic diversity. Knowledge rules permitting automatic labeling of major M. tuberculosis families were defined. Using this strategy, MIRU 24 appeared to be most appropriate for classifying our dataset. The Bovis family was shown to be perfectly classified by a maximum of 3 MIRUs, followed by Africanum and East African Indian (EAI) families by 4 MIRUs, the Beijing family by 6 MIRUs, Haarlem and X families by 8 MIRUs, the T family by 9, and the Latin-American and Mediterranean (LAM) family by 10 MIRUs. Considering the hierarchy of family divergence, our results corroborate a recent suggestion that EAI is the ancestral family followed by Africanum and Bovis. On the other hand, T, X, LAM and Haarlem families appear to be of more recent evolution. These results indicate that data mining of MIRUs is a valuable new tool for analyzing the evolutionary dynamics of the M. tuberculosis complex, and for monitoring an infectious disease such as tuberculosis.

Data mining of Mycobacterium tuberculosis complex genotyping results using mycobacterial interspersed repetitive units validates the clonal structure of spoligotyping-defined families

Recently, a combination of spoligotyping and bioinformatics was proposed as a potential tool for defining major circulating clades of tuberculosis bacilli. In the present study, we attempted to validate the above mentioned classification using a new high-throughput marker, named mycobacterial interspersed repetitive units (MIRUs). Using 12 MIRU loci and spoligotyping, we performed data mining of results on clinical isolates of the Mycobacterium tuberculosis complex representative of global mycobacterial allelic diversity. Knowledge rules permitting automatic labeling of major M. tuberculosis families were defined. Using this strategy, MIRU 24 appeared to be most appropriate for classifying our dataset. The Bovis family was shown to be perfectly classified by a maximum of 3 MIRUs, followed by Africanum and East African Indian (EAI) families by 4 MIRUs, the Beijing family by 6 MIRUs, Haarlem and X families by 8 MIRUs, the T family by 9, and the Latin-American and Mediterranean (LAM) family by 10 MIRUs. Considering the hierarchy of family divergence, our results corroborate a recent suggestion that EAI is the ancestral family followed by Africanum and Bovis. On the other hand, T, X, LAM and Haarlem families appear to be of more recent evolution. These results indicate that data mining of MIRUs is a valuable new tool for analyzing the evolutionary dynamics of the M. tuberculosis complex, and for monitoring an infectious disease such as tuberculosis.

Snapshot of moving and expanding clones of Mycobacterium tuberculosis and their global distribution assessed by spoligotyping in an international study.

The present update on the global distribution of Mycobacterium tuberculosis complex spoligotypes provides both the octal and binary descriptions of the spoligotypes for M. tuberculosis complex, including Mycobacterium bovis, from >90 countries (13,008 patterns grouped into 813 shared types containing 11,708 isolates and 1,300 orphan patterns). A number of potential indices were developed to summarize the information on the biogeographical specificity of a given shared type, as well as its geographical spreading (matching code and spreading index, respectively). To facilitate the analysis of hundreds of spoligotypes each made up of a binary succession of 43 bits of information, a number of major and minor visual rules were also defined. A total of six major rules (A to F) with the precise description of the extra missing spacers (minor rules) were used to define 36 major clades (or families) of M. tuberculosis. Some major clades identified were the East African-Indian (EAI) clade, the Beijing clade, the Haarlem clade, the Latin American and Mediterranean (LAM) clade, the Central Asian (CAS) clade, a European clade of IS6110 low banders (X; highly prevalent in the United States and United Kingdom), and a widespread yet poorly defined clade (T). When the visual rules defined above were used for an automated labeling of the 813 shared types to define nine superfamilies of strains (Mycobacterium africanum, Beijing, M. bovis, EAI, CAS, T, Haarlem, X, and LAM), 96.9% of the shared types received a label, showing the potential for automated labeling of M. tuberculosis families in well-defined phylogeographical families. Intercontinental matches of shared types among eight continents and subcontinents (Africa, North America, Central America, South America, Europe, the Middle East and Central Asia, and the Far East) are analyzed and discussed.

Genotyping of the Mycobacterium tuberculosis complex using MIRUs: association with VNTR and spoligotyping for molecular epidemiology and evolutionary genetics

The recent introduction of molecular methods has gained increased acceptance as a powerful tool for epidemiology and phylogeny of tuberculosis (TB). In this investigation, the efficiency of molecular typing using mycobacterial interspersed repetitive units (MIRUs) was assessed on a set of 116 Mycobacterium tuberculosis complex clinical isolates from 11 different geographic origins. The results obtained were compared with spoligotyping and variable number of tandem DNA repeats (VNTRs) typing data. Eighty-nine different MIRU profiles were obtained on the sample studied. Spoligotyping- or VNTR-defined clusters were split into subclusters by MIRU typing. Conversely, almost all of the clinical isolates clustered by MIRUs were shown to belong to spoligotyping-based defined clusters. The calculation of the discriminative power by the Hunter–Gaston index (HGI) for VNTR, spoligotyping and MIRU typing gave the values of, respectively, 0.959, 0.965 and 0.988, showing the high discriminative power of the MIRUs. The allelic diversity of the sample was calculated for each of the MIRU–VNTR loci; five MIRU loci (MIRU nos. 10, 23, 26, 31 and 40) were “highly discriminant”, four (MIRU nos. 4, 16, 24 and 39) were “moderately discriminant”, and three (MIRU nos. 2, 20 and 27) were “poorly discriminant”. Among the three complementary VNTRs (exact tandem repeats ETR-A, ETR-B and ETR-C), ETR-A was the most discriminant locus. A combined numerical analysis of spoligotyping, VNTR and MIRU typing results partly corroborated a recently hypothesized evolutionary scenario for the M. tuberculosis complex. M. canettii would be the first branch to have diverged from a common M. tuberculosis complex ancestor. The East-African Indian (EAI) clade could be the first family to have diverged thereafter. A third branching separated a M. africanum– M. bovis clade, followed by a node separating Beijing versus non-Beijing M. tuberculosis. The Beijing clade was distinct from the Central Asian 1 (CAS1) family. Among non-Beijing strains, branches such as the Latin-American and Mediterranean (LAM), X and Haarlem clades diverged later. In conclusion, the results obtained show the congruence between clades defined by spoligotyping, and MIRU–VNTR, and underline the potential of these methods for M. tuberculosis phylogeny reconstruction. We also conclude that MIRU typing is a very promising method that may be used in a “two PCR-based” genotyping strategy, in conjunction to conventional epidemiological investigations.