Andrea L Gibson

Richard C Huard

Nicolaas C Gey

Van Pittius

Claudio Oliveira Luiz

Jeffrey Lazzarini

Natalia Driscoll

Thierry Kurepina

Christophe Zozio

Silvana Miranda Sola

Afranio L Spindola

Kritski

Nicolaas C Gey Van Pittius

Oliveira Lazzarini

Jeffrey Driscoll

Natalia Kurepina

Thierry Zozio

Christophe Sola

Silvana Miranda Spindola

Afra ˆnio

L Kritski

Daniel Fitzgerald

Kristin Kremer

Helmi Mardassi

Poonam Chitale

Jessica Brinkworth

Dario Garcia De Viedma

Brigitte Gicquel

Jean W Pape

Dick Van Soolingen

Barry N Kreiswirth

Robin M Warren

Paul D Van Helden

Nalin Rastogi

Philip N Suffys

Jose Lapa

Silva

John L Ho
email: jlho@med.cornell.edu

Application of sensitive and specific molecular methods to uncover global dissemination of the major RDRio sublineage of the Latin american-mediterranean Mycobacterium tuberculosis spoligotype family

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

The Latin American-Mediterranean (LAM) family of Mycobacterium tuberculosis is believed to be the cause of ϳ15% of tuberculosis cases worldwide. Previously, we defined a prevalent sublineage of the LAM family in Brazil by a single characteristic genomic deletion designated RD Rio . Using the Brazilian strains, we pinpoint an Ag85C 103 single nucleotide polymorphism (SNP) (screened by restriction fragment length polymorphism [RFLP] analysis) that correctly identified all LAM family strains. Importantly, all RD Rio strains concomitantly possessed the RD174 deletion. These genetic signatures, along with a newly developed multiplex PCR for rapid differentiation between "wild-type" and RD Rio strains, were then used to analyze an international collection of M. tuberculosis strains. RD Rio M. tuberculosis was identified from four continents involving 11 countries. Phylogenetic analysis of the IS6110-RFLP patterns from representative RD Rio and LAM strains from Brazil, along with all representative clusters from a South African database, confirmed their genetic relatedness and transcontinental transmission. The Ag85C 103 SNP RFLP, as compared to results obtained using a PCR method targeting a LAM-restricted IS6110 element, correctly identified 99.8% of LAM spoligotype strains. Together, these tests were more accurate than spoligotyping at categorizing strains with indefinable spoligotypes and segregated true LAM strains from those with convergent spoligotypes. The fact that RD Rio strains were identified worldwide highlights the importance of this LAM family sublineage and suggests that this strain is a global threat that should be specifically targeted by public health resources. Our provision of simple and robust molecular methods will assist the evaluation of the LAM family and the RD Rio sublineage.

Mycobacterium tuberculosis is a successful obligate pathogen and the etiological agent of tuberculosis (TB). Worldwide, TB remains one of the leading causes of death from an infectious disease [START_REF] Corbett | The growing burden of tuberculosis: global trends and interactions with the HIV epidemic[END_REF][START_REF] Who | Global tuberculosis control: surveillance, planning, financing[END_REF].

Molecular typing, based on genetic markers, permits the rapid identification and species level identification of mycobacteria within the M. tuberculosis complex (MTC), as well as providing useful tools for examining the transmission and evolution of these microorganisms [START_REF] Brosch | A new evolutionary scenario for the Mycobacterium tuberculosis complex[END_REF][START_REF] Dale | Evolutionary relationships among strains of Mycobacterium tuberculosis with few copies of IS6110[END_REF][START_REF] Gagneux | Variable host-pathogen compatibility in Mycobacterium tuberculosis[END_REF][START_REF] Kremer | Comparison of methods based on different molecular epidemiological markers for typing of Mycobacterium tuberculosis complex strains: interlaboratory study of discriminatory power and reproducibility[END_REF][START_REF] Sola | Mycobacterium tuberculosis phylogeny reconstruction based on combined numerical analysis with IS1081, IS6110, VNTR, and DR-based spoligotyping suggests the existence of two new phylogeographical clades[END_REF]. One such technique, based on the amplification of several frequently observed deletion loci, can differentiate between the members of the MTC [START_REF] Huard | PCR-based method to differentiate the subspecies of the Mycobacterium tuberculosis complex on the basis of genomic deletions[END_REF][START_REF] Huard | Novel genetic polymorphisms that further delineate the phylogeny of the Mycobacterium tuberculosis complex[END_REF]. A study using this MTC PCR typing panel for the analysis of M. tuberculosis strains from Rio de Janeiro, Brazil, found that 30% of isolates would not amplify from the IS1561Ј locus [START_REF] Lazzarini | Discovery of a novel Mycobacterium tuberculosis lineage that is a major cause of tuberculosis in Rio de Janeiro, Brazil[END_REF]. These strains, designated RD Rio , were found to contain a chromosomal deletion of more than 26 kb. Further analysis of RD Rio strains with the molecular technique spoligotyping found that all RD Rio strains belonged to the Latin American-Mediterranean (LAM) spoligotype family [START_REF] Lazzarini | Discovery of a novel Mycobacterium tuberculosis lineage that is a major cause of tuberculosis in Rio de Janeiro, Brazil[END_REF].

Spoligotyping is based on the analysis of the direct repeat locus, which is comprised of directly repeated sequences interspersed with nonrepetitive spacer DNA [START_REF] Kamerbeek | Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology[END_REF]. This rapid PCRbased method allows the classification of strains into spoligotype families based on the presence or absence of spacer regions [START_REF] Sebban | A data-mining approach to spacer oligonucleotide typing of Mycobacterium tuberculosis[END_REF]. The evolution of the direct repeat locus is believed to be unidirectional, i.e., spacers are lost rather than gained [START_REF] Van Embden | Genetic variation and evolutionary origin of the direct repeat locus of Mycobacterium tuberculosis complex bacteria[END_REF]. Mechanisms thought to be responsible for this loss are the transposition of the insertion element IS6110 and the deletion of single or stretches of contiguous direct variant repeats through homologous recombination between neighboring or distant direct repeats [START_REF] Van Embden | Genetic variation and evolutionary origin of the direct repeat locus of Mycobacterium tuberculosis complex bacteria[END_REF]. The independent loss of similar spacer sets can lead to the convergent evolution of spoligotypes [START_REF] Warren | Microevolution of the direct repeat region of Mycobacterium tuberculosis: implications for interpretation of spoligotyping data[END_REF], since spoligotype families are defined predominantly by specific spacer patterns. This is therefore problematic since not all strains may truly represent their designated spoligotype family. Recently, other molecular markers have been utilized to define specific spoligotype families, such as the LAM-restricted IS6110 element [START_REF] Marais | Beijing and Haarlem genotypes are overrepresented among children with drug-resistant tuberculosis in the Western Cape Province of South Africa[END_REF] and an SNP in mgtC which can differentiate between Haarlem and non-Haarlem strains [START_REF] Alix | Identification of a Haarlem genotype-specific single nucleotide polymorphism in the mgtC virulence gene of Mycobacterium tuberculosis[END_REF].

Genotyping using single nucleotide polymorphisms (SNPs) has been applied to phylogenetically map the evolution of M. tuberculosis [START_REF] Baker | Silent nucleotide polymorphisms and a phylogeny for Mycobacterium tuberculosis[END_REF][START_REF] Filliol | Global phylogeny of Mycobacterium tuberculosis based on single nucleotide polymorphism (SNP) analysis: insights into tuberculosis evolution, phylogenetic accuracy of other DNA fingerprinting systems, and recommendations for a minimal standard SNP set[END_REF][START_REF] Gutacker | Single-nucleotide polymorphismbased population genetic analysis of Mycobacterium tuberculosis strains from 4 geographic sites[END_REF][START_REF] Gutacker | Genome-wide analysis of synonymous single nucleotide polymorphisms in Mycobacterium tuberculosis complex organisms: resolution of genetic relationships among closely related microbial strains[END_REF][START_REF] Sreevatsan | Restricted structural gene polymorphism in the Mycobacterium tuberculosis complex indicates evolutionarily recent global dissemination[END_REF]. SNPs frequently observed in the genes katG and gyrA permit the classification of MTC members into one of three principal genetic groups (PGG). The identification of SNPs at multiple loci within the M. tuberculosis genome provided enough variation to subdivide M. tuberculosis into several phylogenetic groups [START_REF] Baker | Silent nucleotide polymorphisms and a phylogeny for Mycobacterium tuberculosis[END_REF][START_REF] Filliol | Global phylogeny of Mycobacterium tuberculosis based on single nucleotide polymorphism (SNP) analysis: insights into tuberculosis evolution, phylogenetic accuracy of other DNA fingerprinting systems, and recommendations for a minimal standard SNP set[END_REF][START_REF] Gutacker | Single-nucleotide polymorphismbased population genetic analysis of Mycobacterium tuberculosis strains from 4 geographic sites[END_REF][START_REF] Gutacker | Genome-wide analysis of synonymous single nucleotide polymorphisms in Mycobacterium tuberculosis complex organisms: resolution of genetic relationships among closely related microbial strains[END_REF]. These phylogenetic groupings proved to be concordant with the three PGG, as well as IS6110 restriction fragment length polymorphism (RFLP) data and spoligotyping families [START_REF] Bhanu | Predominace of a novel Mycobacterium tuberculosis genotype in the Delhi region of India[END_REF][START_REF] Filliol | Snapshot of moving and expanding clones of Mycobacterium tuberculosis and their global distribution assessed by spoligotyping in an international study[END_REF][START_REF] Van Soolingen | Predominance of a single genotype of Mycobacterium tuberculosis in countries of east Asia[END_REF]. The LAM family, to which RD Rio strains belong, is found within PGG2 and in phylogenetic cluster VI according to Gutacker et al. [START_REF] Gutacker | Single-nucleotide polymorphismbased population genetic analysis of Mycobacterium tuberculosis strains from 4 geographic sites[END_REF][START_REF] Gutacker | Genome-wide analysis of synonymous single nucleotide polymorphisms in Mycobacterium tuberculosis complex organisms: resolution of genetic relationships among closely related microbial strains[END_REF].

In the present study, we validated a multiplex PCR method for the classification of RD Rio and "wild-type" non-RD Rio (WT) strains using a collection of previously characterized clinical M. tuberculosis isolates from Rio de Janeiro, Brazil [START_REF] Lazzarini | Discovery of a novel Mycobacterium tuberculosis lineage that is a major cause of tuberculosis in Rio de Janeiro, Brazil[END_REF]. This collection was then analyzed for the presence of an SNP previously identified at codon 103 of the gene encoding Ag85C (Rv0129c) [START_REF] Musser | of Mycobacterium tuberculosis host immune system protein targets: evidence of limited selective pressure[END_REF]. The Ag85C 103 SNP was found to be LAM lineage specific. A global collection of M. tuberculosis isolates was also examined to determine the worldwide distribution of RD Rio and the Ag85C 103 SNP. Supplemental to this, these isolates were examined for the presence or absence of the LAM-restricted IS6110 element as well as that of the region-of-difference (RD) loci within the pks15/1 gene (27) and RD174, which are markers for, respectively, the purported major Euro-American lineage of M. tuberculosis defined by Gagneux et al. and its West African sublineage [START_REF] Gagneux | Variable host-pathogen compatibility in Mycobacterium tuberculosis[END_REF]. Each of these phylogenetically informative markers was identified in M. tuberculosis strains from multiple worldwide locales linking them all to a common progenitor in the MTC evolutionary tree.

MATERIALS AND METHODS

Bacterial strains.

A previously characterized cohort of M. tuberculosis isolates [START_REF] Lazzarini | Discovery of a novel Mycobacterium tuberculosis lineage that is a major cause of tuberculosis in Rio de Janeiro, Brazil[END_REF] was analyzed in the present study. Briefly, these isolates were randomly selected from a repository of mycobacterial cultures at the Federal University of Rio de Janeiro, Rio de Janeiro, Brazil, between January 2002 and August 2003. Cell lysates of each isolate had previously been prepared as previously described [START_REF] Lazzarini | Discovery of a novel Mycobacterium tuberculosis lineage that is a major cause of tuberculosis in Rio de Janeiro, Brazil[END_REF]. A patient previously identified as having a mixed infection in the Rio de Janeiro study was also included. From this patient isolate, single colonies were previously extracted, and two isolates from each genotype were subsequently analyzed. In addition, M. tuberculosis isolates (as DNA/cell lysates) were sent from 20 countries covering five continents (as detailed in Table 1) to the Cornell Weill Medical Center for analysis. These strains were provided as a nonrepresentative sample, with a bias toward LAM strains, since it was previously ascertained that RD Rio M. tuberculosis strains were members of the LAM family [START_REF] Lazzarini | Discovery of a novel Mycobacterium tuberculosis lineage that is a major cause of tuberculosis in Rio de Janeiro, Brazil[END_REF] and the Ag85C 103 SNP appeared to be linked to LAM strains. It was therefore logical to select more strains belonging to this lineage to discern its sensitivity as a marker for the LAM family. All isolates and their corresponding molecular genotyping results are listed in Table S1 in the supplemental material. The following M. tuberculosis strains and MTC members were also analyzed to determine whether they contained the Ag85C 103 SNP (described below): M. tuberculosis H37Rv, M. tuberculosis CDC1551, M. bovis (ATCC 29210), M. bovis BCG Japan (ATCC 35737), M. africanum I (strain AF0271), and M. microti (strain 15496) [START_REF] Huard | PCR-based method to differentiate the subspecies of the Mycobacterium tuberculosis complex on the basis of genomic deletions[END_REF][START_REF] Huard | Novel genetic polymorphisms that further delineate the phylogeny of the Mycobacterium tuberculosis complex[END_REF].

Multiplex PCR analysis. A rapid multiplex PCR was developed in the present study to distinguish between WT and RD Rio strains. Previously described primers, used in single PCRs, were applied to develop the multiplex [START_REF] Huard | PCR-based method to differentiate the subspecies of the Mycobacterium tuberculosis complex on the basis of genomic deletions[END_REF][START_REF] Huard | Novel genetic polymorphisms that further delineate the phylogeny of the Mycobacterium tuberculosis complex[END_REF][START_REF] Lazzarini | Discovery of a novel Mycobacterium tuberculosis lineage that is a major cause of tuberculosis in Rio de Janeiro, Brazil[END_REF], and are detailed in Table S1 in the supplemental material. These primers either a The number of M. tuberculosis isolates is not a representative sample of each country or region. With the exception of Brazil and Haiti, the isolates were enriched for LAM spoligotypes.

b RJ, Rio de Janerio; MG, Minas Gerais. The numbers for the Rio de Janeiro M. tuberculosis isolates were as published previously [START_REF] Lazzarini | Discovery of a novel Mycobacterium tuberculosis lineage that is a major cause of tuberculosis in Rio de Janeiro, Brazil[END_REF]. The data for three isolates from the United States, three isolates from The Netherlands, and one isolate from the Republic of Djibouti were also taken from previously published sources [START_REF] Huard | Novel genetic polymorphisms that further delineate the phylogeny of the Mycobacterium tuberculosis complex[END_REF][START_REF] Lazzarini | Discovery of a novel Mycobacterium tuberculosis lineage that is a major cause of tuberculosis in Rio de Janeiro, Brazil[END_REF]. *, one isolate from each of these countries, except Spain and Chile, from which two isolates were obtained for analysis, was from Kremer et al. [START_REF] Kremer | Comparison of methods based on different molecular epidemiological markers for typing of Mycobacterium tuberculosis complex strains: interlaboratory study of discriminatory power and reproducibility[END_REF]. target IS1561Ј, which is internal to the RD Rio deleted region and so a marker for WT M. tuberculosis (530 bp), or flank the RD Rio locus and bridge the deletion in RD Rio strains (1,175 bp). Each PCR mix contained 25 l of High Fidelity PCR Master Mix (Roche Diagnostics, Indianapolis, IN), 2.5 l of dimethyl sulfoxide, 1 l of each primer (20 M), and 2.5 l of bacterial lysate and was adjusted to a final volume of 50 l with sterile distilled water. Amplification was performed in a Perkin-Elmer 9700 Thermocycler (PE Applied Biosystems, Foster City, CA) for 5 min at 95°C, followed by 45 cycles of 1 min at 95°C, 1 min at 60°C, and 4 min at 72°C, with a final extension cycle of 10 min at 72°C. When purified DNA samples were used, the number of cycles was reduced to 30. The amplified products were electrophoretically fractionated in a 1.5% agarose gel together with a 100-bp ladder (Invitrogen, Carlsbad, CA) and visualized by transillumination and ethidium bromide staining. The image was captured by using a Nighthawk Imaging System and Quality One Software (PDI, Inc., New York, NY).

Ag85C PCR analysis and restriction enzyme digest of Ag85C amplicons. A region of the Ag85C gene previously found to contain a silent SNP (GAG to GAA) at codon 103 (29) was targeted. Primers were designed by using the Lasergene program (DNASTAR, Inc., Madison, WI), and are detailed in Table S1 in the supplemental material. The PCR mix and cycle conditions used for the multiplex PCR were also used to amplify the Ag85C region. The amplified products (519 bp) were also visualized as described above. All Ag85C amplicons were digested by using the restriction enzyme MnlI (New England Biolabs, Ipswich, MA) according to the manufacturer's instructions. The digested products were then separated on a 4% agarose gel containing ethidium bromide and visualized over UV light. Each gel also contained a 100-bp ladder to measure the size of the digested products. The presence of the SNP results in the loss of one of three restriction enzyme digestion sites and is visualized by the presence of a 461-bp band and a second band of 58 bp (which was not always detected using the gel method). An absence of the SNP is seen as three bands of 365, 96, and 58 bp (which also was not always visible on the gel). The protocol was therefore internally controlled for product digestion of the 519-bp amplicon.

Sequence analysis. DNA sequencing was performed by the Cornell University BioResource Center (Ithaca, NY [http://www.brc.cornell.edu]), using a BigDye terminator kit (PE Applied Biosystems) and analyzed by using an ABI 3700 DNA sequencer. The primers used for PCR amplification were also used for the sequencing reaction, with the exception of the reverse RD Rio sequencing primer, where an internal reverse primer closer to the site of deletion was used (detailed in Table S1 in the supplemental material).

Analysis of pks15/1 and RD174. A 7-bp deletion in the pks15/1 gene ( 27) and RD174 are markers that have been previously reported [START_REF] Gagneux | Variable host-pathogen compatibility in Mycobacterium tuberculosis[END_REF]. Detailed in Table S1 in the supplemental material are the primers used to amplify pks15/1 region and the RD174 (forward flanking, internal forward [internal to RD174], and reverse flanking primers). The PCR conditions used to amplify both loci were the same as given above. Once the specific pks15/1 site was amplified, sequence analysis (detailed above) was performed to determine whether the 7-bp deletion common to PGG2 and PGG3 strains was present. For RD174 analysis, the amplified products were analyzed on a 1.5% agarose gel. A 300-bp band indicated an intact locus, whereas a 500-bp band indicated the locus was deleted.

Analysis of the LAM-restricted IS6110 element. PCR primers that identify the presence or absence of an IS6110 insertion element unique to all LAM strains (26) were used to amplify all discrepant isolates (Table S1 in the supplemental material). Briefly, a 205-bp band indicates a LAM strain by the presence of an IS6110 element, whereas a 141-bp band indicates a non-LAM strain lacking the IS6110 element.

Statistical analysis. Statistical analysis was performed by using Prism 4 (GraphPad Software, Inc., San Diego, CA). Categorical variables were compared by a two-tailed Fisher exact test. A P value of Յ0.05 was considered significant.

RESULTS

RD Rio multiplex PCR development. In our previous study, patient isolates were all characterized by using single PCRs containing primer pairs specific to either RD Rio or WT strains [START_REF] Lazzarini | Discovery of a novel Mycobacterium tuberculosis lineage that is a major cause of tuberculosis in Rio de Janeiro, Brazil[END_REF]. Since this is time-and resource-consuming, we developed a more rapid multiplex PCR protocol to differentiate RD Rio and WT M. tuberculosis. A subset of isolates from our previous Rio de Janeiro study (20 WT strains, 20 RD Rio strains and one mixed sample) was reevaluated to validate the protocol. In all cases, the expected amplicon size(s) was observed, i.e., 530 bp for WT strains, 1,175 bp for RD Rio strains, and a double band for the mixed infection sample (Fig. 1A).

Identification and evaluation of the Ag85C 103 SNP. The Rio de Janeiro collection was screened for the Ag85C 103 SNP (29), using a restriction enzyme digest approach. A total of 306/310 patient isolates (90 RD Rio M. tuberculosis and 216 WT patient isolates) and 1 isolate from a patient with a mixed infection, from which WT and RD Rio colonies were previously extracted (four isolates in total), were available for analysis. Overall, 180 of 306 (58.8%) patient isolates contained the Ag85C 103 SNP. A total of 20 isolates were selected, 10 for each digestion pattern, and sequenced to confirm for mutant and WT isolates. The sequencing results were concordant with the Ag85C 103 digest results (data not shown). Figure 1B illustrates examples of the Ag85C 103 restriction digest patterns. Overall, all RD Rio isolates analyzed contained the SNP (n ϭ 90), whereas less than half of the WT (43.5%) strains did. From the patient with the mixed infection, the RD Rio clones both harbored the polymorphism, while the two WT isolates did not. When the spoligotyping profiles of the Rio de Janeiro strains were compared (n ϭ 293), all LAM strains (n ϭ 161) were found to contain Ag85C 103 , whereas the majority of WT non-LAM strains (92.4%) did not (P Ͻ 0.0001; two-tailed Fisher exact test, Table 2). A total of 10 non-LAM isolates, as defined by spoligotyping, contained the Ag85C 103 SNP. These isolates were sequenced, and the results were found to be concordant with the Ag85C 103 digest results (data not shown). These discrepant strains were later analyzed by using the LAM IS6110-specific PCR assay, the results of which are detailed below.

RD174 and pks15/1 7-bp deletion PCR Analysis. Gagneux et al. [START_REF] Gagneux | Variable host-pathogen compatibility in Mycobacterium tuberculosis[END_REF] constructed a phylogenetic tree based on the presence or absence of common long sequence polymorphisms (LSPs) found in M. tuberculosis strains and used these RD loci to construct an evolutionary tree. To determine whether RD174 was a marker for LAM and/or RD Rio strains, we evaluated the Rio de Janeiro strain collection for this LSP. Remarkably, RD174 was deleted in all RD Rio strains but remained intact in all WT strains, including WT LAM strains (Fig. 1C). This suggests that RD174 is in fact a parallel marker for the RD Rio genotype. The RD174 polymorphism is believed to be at the node of a sub-branch of the Euro-American lineage, defined by the pks15/1 7-bp deletion. Therefore, we tested a selection of strains from the Rio de Janeiro collection to confirm that isolates harboring the RD174 polymorphism did belong to this lineage and also to ascertain whether the Euro-American lineage contained both LAM and non-LAM strains. A total of 10 RD Rio isolates, 15 WT LAM isolates, and 13 WT non-LAM isolates, representing ca. 10% of each group from Rio de Janeiro, were analyzed. All LAM strains including RD Rio iso-lates (PGG2) were found to possess the 7-bp pks15/1 deletion, as did the non-LAM strains belonging to PGG2 and PGG3 (data stated herein). The only strains not containing the pks15/1 deletion were two W/Beijing strains and an EAI strain, both of which are PGG1 isolates, confirming that the pks15/1 7-bp deletion is PGG2 and PGG3 restricted [START_REF] Huard | Novel genetic polymorphisms that further delineate the phylogeny of the Mycobacterium tuberculosis complex[END_REF].

RD Rio genotype evaluation of global LAM strains. An international collection of M. tuberculosis strains (total of 851 isolates [see Materials and Methods]) was analyzed by multiplex PCR to determine whether RD Rio M. tuberculosis has disseminated outside of Rio de Janeiro. Overall, the RD Rio genotype was identified in isolates from 11 of the 20 countries represented, showing that RD Rio strains are circulating internationally and are not restricted to Brazil. Table 1 shows the classification of isolates by genotype for each geographical area. As well as identifying RD Rio M. tuberculosis in a second state of Brazil (Minas Gerais, where 36% of the provided strains were RD Rio ), RD Rio was also identified in Ecuador and French Guiana, both South American countries, and in North America (i.e., the United States). Moreover, RD Rio M. tuberculosis was observed in all three Caribbean islands studied (Guadeloupe, Haiti, and Martinique). Of great importance was the fact that RD Rio strains were also identified outside of the Americas, in Europe (The Netherlands and Spain) and in Africa (South Africa and the Republic of Djibouti). These results clearly demonstrate that RD Rio M. tuberculosis has indeed disseminated globally. To provide additional support that the RD Rio and LAM strains from Brazil were related to those found elsewhere, we performed IS6110 RFLP phylogenetic analysis of isolates from the Rio de Janeiro collection previously studied by IS6110 RFLP together with one isolate from each cluster (strain/RFLP signature) from a South African database (data not shown). The genetic relatedness of LAM strains and specifically RD Rio strains from Rio de Janeiro with their counterparts from South Africa were confirmed by IS6110 RFLP phylogenetic analysis (see Fig. S1 in the supplemental material).

Ag85C 103 SNP correlates with LAM-restricted IS6110 insertion PCR in analysis of global LAM strains. The M. tuberculosis isolates from each country were examined for the Ag85C 103 SNP, and the results were compared to their spoligotypes (Table 2; the spoligopatterns and spoligotypes designation assigned by SpolDB4 are presented in Table S2 in the supplemental material). In almost all cases, LAM strains contained the Ag85C 103 SNP (540 of 547 [98.7%]), while the majority of non-LAM strains did not contain this SNP (212 of 240 [88.3%]) (P Յ 0.0001; two-tailed Fisher exact test). As previously observed in the Rio de Janeiro cohort, all RD Rio strains contained the SNP. At first, this suggested that the Ag85C 103 SNP was not as sensitive or specific as previously seen in the Rio de Janeiro cohort. Overall, 7 LAM strains without the Ag85C 103 SNP and 28 non-LAM strains with the SNP were observed. Possible explanations for these two scenarios may be the convergent evolution of spoligotypes and/or the presence of SNP-containing ancestral progenitor strains. To further explore these discrepant strains, we analyzed them for a site-specific IS6110-element that is unique to LAM strains [START_REF] Marais | Beijing and Haarlem genotypes are overrepresented among children with drug-resistant tuberculosis in the Western Cape Province of South Africa[END_REF]. Interestingly, 27 of 28 non-LAM strains (according to spoligotyping) with the Ag85C 103 SNP were found to be LAM when analyzed with the LAM-specific IS6110 PCR, in- a The numbers in parentheses differ from those in Table 1 because the spoligotype pattern was not available, the spoligotype pattern was not definable (see Table 3), or the isolate appeared to be a mixed sample according to at least one of the molecular tests and thus the spoligotype was not reliable. The mixed isolate from Rio de Janeiro that was separated into single colonies was included in the analysis.

dicating that these strains are of the LAM lineage. Of the seven LAM strains (according to spoligotyping) without the Ag85C 103 SNP, six strains were PCR amplified as non-LAM, indicating that the spoligotypes of these strains were the result of convergence. Therefore, this left only two discrepant strains, a LAM strain without the Ag85C 103 SNP and a non-LAM strain with the SNP, resulting in a sensitivity and specificity rate for the Ag85C 103 SNP of 99.8 and 99.6%, respectively.

From the international collection, an additional 37 M. tuberculosis isolates had spoligotypes that could not be segregated into LAM or non-LAM groups (Table 3). Although these strains had spacers 21 to 24 and spacers 33 to 36 deleted, some of the surrounding spacers were also missing, thus preventing accurate clade designation. To confirm whether these isolates belonged to the LAM lineage, all were analyzed for the LAMspecific IS6110 PCR and segregated into LAM/non-LAM groups according to the presence or absence of this element (Table 3). The Ag85C 103 SNP results were 100% concordant to the IS6110 PCR results. Importantly, this shows that the Ag85C 103 SNP is an accurate marker for the differentiation of LAM/non-LAM strains and particularly useful when a spoligotype pattern is indistinguishable.

In addition to the international M. tuberculosis strain collection, several other MTC isolates were examined for the Ag85C 103 SNP. It was found to be absent from M. tuberculosis H37Rv and M. tuberculosis CDC1551, as well as representative strains from other MTC members, namely, M. africanum I, M. microti, M. bovis, and M. bovis BCG (Japan).

RD Rio deletion correlates with RD174. The international strain collection was also analyzed for RD174 by PCR to confirm that this polymorphism was indeed a marker for RD Rio strains. All RD Rio strains had RD174 deleted, and all WT strains had this region intact (n ϭ 848). Of note, the data indicated that four patients had mixed strain M. tuberculosis infections that included an RD Rio M. tuberculosis strain, as has been noted previously [START_REF] Lazzarini | Discovery of a novel Mycobacterium tuberculosis lineage that is a major cause of tuberculosis in Rio de Janeiro, Brazil[END_REF]. One isolate from Minas Gerais and one from South Africa that were found to be mixed by the multiplex PCR and also had a double band corresponding to both an intact and a deleted RD174 region. The remaining two mixed isolates (according to the multiplex PCR), one from South Africa and one from Minas Gerais, were not mixed by RD174, suggesting that the multiplex RD Rio PCR may be more sensitive at identifying mixed infections that include these strains than the use of RD174. Since only one sample from each of the above mixed isolates was evaluated, the possibility of laboratory cross-contamination could not be excluded.

DISCUSSION

This study aimed to identify specific markers able to define the LAM family lineage and one of its major sublineages, RD Rio , and to develop a multiplex PCR to rapidly identify RD Rio strains. The newly developed multiplex PCR was then utilized to reveal that the RD Rio sublineage was not just a problem localized to Rio de Janeiro in Brazil, where it was first identified, but that it is found globally.

We identified a synonymous SNP in the gene for Ag85C that was linked to LAM strains. Initially, the Ag85C 103 SNP did not appear to be a particularly accurate marker for LAM strains compared to spoligotyping. Initially, the sensitivity of this marker by comparison to spoligotyping was 98.7%, while the specificity was 88.3% in our global collection. However, further analysis of the discrepant isolates based on the presence or absence of an IS6110 element unique to LAM strains proved that the sole use of spoligotype patterns are not altogether reliable. As previously observed, the loss of these spacers can lead to the convergent evolution of strains [START_REF] Warren | Microevolution of the direct repeat region of Mycobacterium tuberculosis: implications for interpretation of spoligotyping data[END_REF], since spoligotype families are mainly defined by specific spacer patterns. We observed this phenomenon in seven strains that had "LAM" spoligotypes but did not contain Ag85C 103 . All but one strain lacked the LAM-specific IS6110 element, proving that these strains were not true LAM strains. Conversely, the 28 "non-LAM" discrepant strains containing Ag85C 103 could not be explained by convergent evolution, considering that spacers are lost rather than gained. IS6110 PCR analysis found that b That is, the definition given by the international spoligotyping database (SpolDB4; Brudey et al. [START_REF] Brudey | Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology[END_REF]), which tentatively classifies spoligotypes into clades. c Defined by LAM-specific IS6110 PCR. d NA, not available. e The same isolate. f The same isolates. g The final spoligotype is the LAM9 reference spoligotype.

these apparent "non-LAM" strains were in fact true LAM lineage strains since 27 of 28 possessed the LAM-specific IS6110 element. Overall, the sensitivity and specificity of the Ag85C 103 SNP were 99.8 and 99.6%, respectively. Although an isolate with a LAM spoligotype but without Ag85C 103 could be considered as an example of convergent evolution, i.e., not a true LAM strain, the single discrepant isolate was confirmed as a LAM strain by the presence of the LAM-specific IS6110 element. Conceptually, the insertion of this IS6110 element and the Ag85C 103 mutation are unlikely to have occurred simultaneously, and so it is reasonable to conclude that one polymorphism predates the other. In this case one could argue that the IS6110 insertion occurred first; however, one Ag85C 103 -positive non-LAM strain (confirmed by IS6110 PCR) was observed, which would argue the converse. We cannot at present be certain in what order these polymorphisms arose, but hopefully further genetic analyses of these strains will settle the issue. Interestingly, to date there has been scant evidence of horizontal genetic exchange in M. tuberculosis, although data support that it has occurred in the MTC progenitor species "Mycobacterium canettii" (prototuberculosis) [START_REF] Brisse | A re-evaluation of M. prototuberculosis": continuing the debate[END_REF][START_REF] Supply | Linkage disequilibrium between minisatellite loci supports clonal evolution of Mycobacterium tuberculosis in a high tuberculosis incidence area[END_REF]. Given that mixed strain infections appear to be more common than previously assumed, one or the other of the above discordant stains may well represent a rare example of just such a genomic homogenizing event in M. tuberculosis.

In some instances the spoligotype pattern was indefinable and could not be designated as a LAM or a non-LAM strain. Both the Ag85C 103 SNP and the LAM-specific IS6110 PCR were able to differentiate these isolates into LAM and non-LAM strains, proving that spoligotyping alone is not always the best method for defining LAM strains. As we have demonstrated, Ag85C 103 SNP analysis can also define LAM family strains that are misclassified by spoligotyping (i.e., have a non-LAM spoligotype pattern). Ag85C 103 SNP analysis is not a replacement for spoligotyping, since the latter can both define and differentiate between LAM family strains. However, it is relatively cheap compared to spoligotyping and does not require any specialized equipment. The Ag85C 103 SNP PCR assay could therefore serve as a handy postspoligotyping confirmatory tool in epidemiological studies and/or in resource poor settings as a screening method for LAM strains to limit the number of isolates requiring full characterization with spoligotyping.

Specific SNPs have been linked to each of the MTC species and various tubercle bacilli, as well as many of the M. tuberculosis lineages [START_REF] Alix | Identification of a Haarlem genotype-specific single nucleotide polymorphism in the mgtC virulence gene of Mycobacterium tuberculosis[END_REF][START_REF] Baker | Silent nucleotide polymorphisms and a phylogeny for Mycobacterium tuberculosis[END_REF][START_REF] Filliol | Global phylogeny of Mycobacterium tuberculosis based on single nucleotide polymorphism (SNP) analysis: insights into tuberculosis evolution, phylogenetic accuracy of other DNA fingerprinting systems, and recommendations for a minimal standard SNP set[END_REF][START_REF] Goh | Molecular evolutionary history of tubercle bacilli assessed by study of the polymorphic nucleotide within the nitrate reductase (narGHJI) operon promoter[END_REF][START_REF] Huard | Novel genetic polymorphisms that further delineate the phylogeny of the Mycobacterium tuberculosis complex[END_REF][START_REF] Sreevatsan | Restricted structural gene polymorphism in the Mycobacterium tuberculosis complex indicates evolutionarily recent global dissemination[END_REF][START_REF] Victor | Molecular characteristics and global spread of Mycobacterium tuberculosis with a western cape F11 genotype[END_REF], thereby providing consistent phylogenetic markers and allowing inter-and intraspecies segregation with relative ease. For example, the "M. canettii" hsp65 631 SNP differentiates it from the rest of the MTC [START_REF] Huard | Novel genetic polymorphisms that further delineate the phylogeny of the Mycobacterium tuberculosis complex[END_REF]. Within M. tuberculosis lineages, a single SNP in mgtC was found to differentiate between Haarlem and non-Haarlem strains of M. tuberculosis (1), while an SNP in rrs 491 was specifically associated with the F11 (LAM3) clade, a dominant M. tuberculosis lineage in the Western Cape of South Africa [START_REF] Victor | Molecular characteristics and global spread of Mycobacterium tuberculosis with a western cape F11 genotype[END_REF]. Genome SNP analysis has previously been used to classify M. tuberculosis into phylogenetic lineages [START_REF] Baker | Silent nucleotide polymorphisms and a phylogeny for Mycobacterium tuberculosis[END_REF][START_REF] Filliol | Global phylogeny of Mycobacterium tuberculosis based on single nucleotide polymorphism (SNP) analysis: insights into tuberculosis evolution, phylogenetic accuracy of other DNA fingerprinting systems, and recommendations for a minimal standard SNP set[END_REF][START_REF] Gutacker | Single-nucleotide polymorphismbased population genetic analysis of Mycobacterium tuberculosis strains from 4 geographic sites[END_REF]. One such tree divided M. tuberculosis into nine distinct lineages; LAM strains and a subset of T strains segregated into SNP cluster VI [START_REF] Gutacker | Single-nucleotide polymorphismbased population genetic analysis of Mycobacterium tuberculosis strains from 4 geographic sites[END_REF]. The Ag85C 103 SNP was not specific to all strains in cluster VI since several "T" strains from our USA collection that were known to belong to cluster VI did not contain the Ag85C 103 SNP (data not shown). Nonetheless, the identification of a single SNP that is specifically associated with the LAM family lineage (LAM strains comprise ϳ80% of cluster VI isolates) could potentially be utilized as an indicator to identify cluster VI strains. As such, this test would reduce the amount of SNP analysis required to characterize this major phylogenetic lineage. In addition, since we provide a restriction digest approach for the Ag85C 103 SNP, the need for more costly DNA sequence analysis would be eliminated.

LSPs have also been used to define both MTC members and M. tuberculosis lineages [START_REF] Brosch | A new evolutionary scenario for the Mycobacterium tuberculosis complex[END_REF][START_REF] Gagneux | Variable host-pathogen compatibility in Mycobacterium tuberculosis[END_REF][START_REF] Hirsh | Stable association between strains of Mycobacterium tuberculosis and their human host populations[END_REF][START_REF] Huard | Novel genetic polymorphisms that further delineate the phylogeny of the Mycobacterium tuberculosis complex[END_REF]. Gagneux et al. [START_REF] Gagneux | Variable host-pathogen compatibility in Mycobacterium tuberculosis[END_REF] developed a global phylogenetic tree based on several LSPs, identified through whole-genome screening, and found that geographical regions are associated with certain lineages. Using markers from this tree we linked RD Rio M. tuberculosis to the West African sublineage of the major Euro-American lineage [START_REF] Gagneux | Variable host-pathogen compatibility in Mycobacterium tuberculosis[END_REF]. Our study demonstrated that RD174 was specific for RD Rio M. tuberculosis and not observed in any other LAM strains. In addition, the major branch of the Euro-American lineage is defined by a 7-bp deletion in a polyketide synthase gene (pks15/1). Interestingly, the pks15/1 deletion was previously found to be synonymous with the SNP at katG 463 CGG; therefore, the Euro-American lineage consists of PGG2 and PGG3 M. tuberculosis strains [START_REF] Huard | Novel genetic polymorphisms that further delineate the phylogeny of the Mycobacterium tuberculosis complex[END_REF][START_REF] Marmiesse | Macro-array and bioinformatic analyses reveal mycobacterial 'core' genes, variation in the ESAT-6 gene family and new phylogenetic markers for the Mycobacterium tuberculosis complex[END_REF] and not PGG1/ancestral strains. Our data concurred with this observation.

Surprisingly, there are a limited number of studies focusing solely on LAM strains. Many studies have investigated W/Beijing strains predominantly since these strains are prevalent across Asia and also contributed greatly to the TB resurgence in the United States in the early 1990s [START_REF] Bifani | Global dissemination of the Mycobacterium tuberculosis W-Beijing family strains[END_REF][START_REF] Yang | Diversity of DNA fingerprints of Mycobacterium tuberculosis isolates in the United States[END_REF]. In comparison to LAM strains, which are responsible for ca. 15% of the world's TB cases (SpolDB4 [START_REF] Brudey | Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology[END_REF]), W/Beijing strains account for just ϳ10% of M. tuberculosis strains worldwide. Therefore, there is a need for more research efforts geared toward the LAM family. The development of a multiplex PCR enabled the rapid segregation of M. tuberculosis strains into WT and RD Rio genotypes. We found that TB caused by RD Rio M. tuberculosis is not restricted to Brazil or even the Americas but is found in four of the five continents examined. Although RD Rio was not identified in Asia, only two isolates in the present study were examined from this continent, and so further studies are needed to assess whether RD Rio M. tuberculosis is circulating in Asia as well. Studies have shown that W/Beijing and LAM strains are the major cause of TB in Russian prisons [START_REF] Drobniewski | Rifampin-and multidrug-resistant tuberculosis in Russian civilians and prison inmates: dominance of the Beijing strain family[END_REF][START_REF] Ignatova | Predominance of multidrug-resistant LAM and Beijing family strains among Mycobacterium tuberculosis isolates recovered from prison inmates in Tula Region, Russia[END_REF][START_REF] Shemyakin | Characterization of drug-resistant isolates of Mycobacterium tuberculosis derived from Russian inmates[END_REF]. Of the LAM strains, both LAM9 and LAM1 strains were major contributors. Our previous study found that these two LAM types were associated with the RD Rio genotype [START_REF] Lazzarini | Discovery of a novel Mycobacterium tuberculosis lineage that is a major cause of tuberculosis in Rio de Janeiro, Brazil[END_REF] and that LAM1 strains were exclusively found to be RD Rio M. tuberculosis. Although we did identify one WT LAM1 strain and two mixed infections with LAM1 spoligotypes, 48 of 51 (94.1%) of LAM1 strains in this study were nonetheless RD Rio M. tuberculosis. These data thus indirectly suggest that RD Rio may well be a significant cause of TB in Northern Asia, as well by virtue of the LAM1 prevalence in this region. This hypothesis, however, remains to be substantiated by evaluating Russian LAM strains.

Interestingly, two of the RD Rio M. tuberculosis strains isolated in Madrid, Spain, were from the most prevalent cluster of M. tuberculosis reported in that city, namely, strain 5 [START_REF] Martin | Molecular, epidemiological, and infectivity characterisation of a Mycobacterium tuberculosis strain prevalent in Madrid[END_REF].

Strain 5 (ST20 or LAM1) has an identical mycobacterial interspersed repetitive unit (MIRU) pattern to a hypothetical progenitor MIRU-type for RD Rio strains and a very similar RFLP pattern [START_REF] Lazzarini | Discovery of a novel Mycobacterium tuberculosis lineage that is a major cause of tuberculosis in Rio de Janeiro, Brazil[END_REF]. These Spanish RD Rio strains were isolated in 2002 and 2003, but strain 5 has remained the predominant strain in Madrid for the last 13 years, whereas other others strain types have declined. This suggests that RD Rio M. tuberculosis was circulating in Madrid in 1993 and possibly earlier.

As previously hypothesized [START_REF] Lazzarini | Discovery of a novel Mycobacterium tuberculosis lineage that is a major cause of tuberculosis in Rio de Janeiro, Brazil[END_REF], the persistence of the RD Rio sublineage may be attributed to either intrinsic genetic factors affording biological advantage for the tubercle bacilli or risk factors of the host population that sustain dissemination.

Taking into account that RD Rio has disseminated outside of the Americas and the fact that it is part of the LAM family, RD Rio strains could indeed be a significant global problem. Complementary to the presented genetic data, IS6110 RFLP phylogenetic analysis of isolates from the Rio de Janeiro collection ( 25), along with one isolate from each cluster (strain/ RFLP signature) from a South African database (data not shown), confirmed genetic relatedness; their all of the LAM strains from Rio de Janeiro fall within one of the RFLP families previously described in South Africa (30) (see Fig. S1 in the supplemental material). Moreover, the RD Rio strains from Rio de Janeiro grouped with the South African F9 and F13 families (LAM11 and LAM1, respectively) [START_REF] Streicher | Spoligotype signatures in the Mycobacterium tuberculosis complex[END_REF], which in the present study were shown to be of the RD Rio genotype. Recently, Chihota et al. ( 8) identified a group of strains (named the Southern Africa 1 or SAF1 family) that made up a predominant proportion of strains isolated in Zimbabwe (47.2%) and Zambia (65%) and which were shown to fall into the South African F9 family (and thus the RD Rio lineage). These findings further support the present genetic data and highlight the fact that RD Rio indeed appears to be a significant cause of TB worldwide. Our rapid PCR technique may be useful in the epidemiological tracking of the RD Rio genotype and in ascertaining the level of RD Rio M. tuberculosis circulating in a population. It is of great importance, when developing public health control strategies for TB, to understand the strains circulating not only on a regional scale but globally as well.

Recently, Flores et al. ( 14) assigned strains to the W/Beijing lineage based upon possession of the prototypic W/Beijing RD105 deletion, despite the absence of typical W/Beijing family spoligotype patterns. Likewise, in the present study, we identified several genetic signatures of the LAM family and its major RD Rio sublineage that allow us to propose to extend the genetic definition for a LAM lineage strain (as distinguished from the defining characteristics of a LAM spoligotype family strain). We classify the LAM lineage by (i) being PGG2, as well as possessing (ii) the 7-bp pks15/1 deletion, (iii) the SNP at Ag85C 103 , and (iv) the restricted site-specific IS6110 insertion element. On the other hand, LAM spoligotype family strains (within the LAM lineage, as defined by the above markers) exhibit prototypic spoligotype patterns with an absence of spacers 21 to 24 and spacers 33 to 36 [START_REF] Brudey | Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology[END_REF]. LAM RD Rio M. tuberculosis can be further characterized by the RD Rio and RD174 deletions. Now that we have highlighted the global problem of the LAM family and its prominent RD Rio sublineage and developed a series of simple genetic signatures for their characterization, we hope that research interests will fo-cus on this highly prevalent M. tuberculosis lineage and provide much-needed information regarding the bases of their success.

In conclusion, our finding that RD Rio strains were identified worldwide highlights the importance of this LAM family sublineage. Limited IS6110 RFLP analysis confirms the genetic relatedness of LAM and RD Rio strains and cross-transmission between continents. The findings from this report furthermore demonstrate that the RD Rio strain is a global threat and should be specifically targeted by public health resources. Our provision in this report of simple and robust molecular methods should assist in the evaluation of the LAM family and the RD Rio sublineage. Health (NIH) grants R21 AI063147 and R21 AI063147 (to J. A.L.G. performed the majority of the laboratory work, codeveloped the restriction digest of Ag85C SNP analysis (with R.C.H.), posed and tested the hypotheses (with J.L.H. and R.C.H.), and wrote the manuscript. R.C.H. initiated the multiplex assays and Ag85C SNP evaluation and, together with J.L.H., gathered the international M. tuberculosis collection, as well as guided the presentation, provided input during preparation, and edited the manuscript. N.C.G.V.P. made the suggestion to cross-compare LAM-specific IS6110 PCR with the Ag85C SNP, provided critical input in the evaluation of strains with certain spoligopatterns, and performed the phylogenetic analysis of the IS6110 RFLP pattern for the present study. N.C.G.V.P., together with R.M.W. and P.D.V.H., provided the samples with spoligopattern and RFLP data from South Africa. L.C.O.L. assisted in some of the earlier genetic analysis, assisted in generating more DNA lysates from Rio de Janeiro needed for the present study, coordinated shipment of these samples to Cornell, provided input in some of the data interpretation, and edited the manuscript for reference order and format. J.D. performed spoligotyping on samples without data and repeated the spoligotyping on select strains. N.K. contributed to the phylogenetic analysis of the IS6110 RFLP pattern for this study and, together with B.N.K., provided strains with spoligotyped data from North America. T.Z., C.S., and N.R. provided samples with spoligotyped data from French Caribbean countries, and C.S. provided suggestions to improve the manuscript. S.M.S. provided the samples with spoligotyped data from Belo Horizonte, Brazil, which was performed by S.M.S. in France in the laboratory of B.G. A.L.K. assisted S.M.S. in organizing the data file of the Belo Horizonte collection. D.F. and J.W.P. provided samples from Haiti. K.K. and D.V.S. provided samples with spoligotyped data from The Netherlands. H.M. provided samples with spoligotyped data from Tunisia. P.C. and J.B. assisted in some of the genotyping. D.G.D.V. provided samples from Spain and perspective on the prevalent strains in the epidemiology of TB in Spain. P.N.S. supervised the spoligotyping of the Rio de Janeiro samples. J.R.L.S., together with J.L.H., supervised the laboratory and clinical studies in Rio de Janeiro and shipment of the DNA lysates to United States. J.L.H. posed the hypotheses, coordinated the international collaborations, provided the overall management of the project, guided data analyses and interpretation, provided input for presentation and discussions during manuscript preparation. and edited the manuscript.

FIG. 1 .

 1 FIG. 1. (A) RD Rio multiplex PCR. The presence of a 530-bp band indicates a WT strain, while the presence of an 1,175-bp band indicates an RD Rio strain. Lanes: 1, 100-bp ladder; 2, M. tuberculosis H37Rv; 3 and 4, WT LAM strains; 5 and 6, RD Rio LAM strains; 7, WT non-LAM strain (Haarlem 1); 8, a mixed (WT/RD Rio ) isolate. (B) Ag85C 103 SNP digest analysis. The presence of a 461-bp band indicates that the SNP is present, while 365-and 96-bp bands indicate the SNP is absent. Lanes 1 to 7 are described as in panel A. (C) RD174 PCR analysis. A 300-bp band is indicative of an intact region, while a 500-bp band indicates that RD174 has been deleted. Lanes 1 to 7 are as described in panel B.

  L.H.), NIH Fogarty International Center training grant (FICTG) (D43 TW00018), Innovative Approaches for TB Control in Brazil (Rio-ICOHRTA)-U2R TW006885 NIH, FIC (J.R.L.E.S., R. Chaisson, and J.L.H.), Haiti AIDS Research Training: Models to Implementation (Haiti-ICOHRTA)-U2R TW006901 (W. D. Johnson), a grant from the Coordenac ¸a ˜o de Aperfeicoamento de Pessoal de Nivel Superior (Ministry of Education of Brazil) from the Brazilian Ministry of Health (024/94 DST/AIDS), Fundac ¸a ˜o Universita ´ria Jose ´Bonifa ´cio/FUJB, the Brazilian Research Council/CNPq, the Brazilian Research Council/World Bank Millennium Institute of Science, the Programa de Apoio a Nu ´cleos de Excele ˆncia, and PAPES IV/FIOCRUZ (P.N.S. and J.R.L.E.S.), and a grant from the Laura Cook Hull Trust Fund (LCHTF) (W. D. Johnson). A.L.G. was supported in part by the LCHTF, and L.C.O.L was a FICTG and is now a Rio-ICOHRTA and a CNPq trainee. The study performed in N.R.'s laboratory was partially funded by the European Regional Development Fund, European Commission (ERDF/FEDER, A34-05). T.Z. received a Ph.D. fellowship awarded by the European Union and the Regional Council of Guadeloupe and the International Network of Pasteur Institutes. Funding for the Spanish isolates was from the Fondo de Investigaciones Sanitarias (FIS030654; FIS060882) and the Instituto de Salud Carlos III (CIBER Enfermedades Respiratorias CIBERES; CB06/06/ 0058).

TABLE 1 .

 1 Proportion of RD Rio M. tuberculosis isolates observed in sample sets from each country a

	Country b	No. of patient isolates	No. of strains (%) WT RD Rio	WT/RD Rio ratio (%)
	Brazil (RJ)	314	218 (69)	93 (30)	3 (1)
	South Africa	150	110 (73)	37 (25)	3 (2)
	Brazil (MG)	105	64 (61)	39 (37)	2 (2)
	The Netherlands	83	61 (73)	22 (27)	0
	United States*	67	34 (51)	33 (49)	0
	Tunisia	49	49 (100)	0	0
	Guadeloupe	26	2 (8)	24 (92)	0
	French Guiana	23	10 (43)	13 (57)	0
	Haiti	17	13 (76)	4 (24)	0
	Spain*	4	1 (25)	3 (75)	0
	Chile*	2	2 (100)	0	0
	Martinique	2	1 (50)	1 (50)	0
	Canada*	1	1 (100)	0	0
	Burundi*	1	1 (100)	0	0
	Mongolia*	1	1 (100)	0	0
	Tahiti*	1	1 (100)	0	0
	Honduras*	1	1 (100)	0	0
	Czech Republic	1	1 (100)	0	0
	Russia	1	1 (100)	0	0
	Ecuador*	1	0	1 (100)	0
	Republic of Djibouti	1	0	1 (100)	0
	Total	851	572 (67) 271 (32)	8 (1)

TABLE 2 .

 2 Division of LAM and non-LAM isolates (designated by spoligotyping) by the presence or absence of Ag85C 103 according to country of origin

		No. of LAM	No. of non-LAM
	Country of origin	isolates (%)	isolates (%)
	(no. of isolates) a	SNP	SNP	SNP	SNP
		present	absent	present	absent
	Brazil (RJ) (293)	161 (55)	0	10 (3)	122 (42)
	South Africa (145)	101 (70)	0	17 (12)	27 (18)
	Brazil (MG) (96)	62 (65)	0	1 (1)	33 (34)
	The Netherlands (73)	68 (93)	4 (6)	0	1 (1)
	United States (63)	50 (79)	2 (3)	0	11 (18)
	Tunisia (47)	44 (94)	0	0	3 (6)
	Guadeloupe (21)	19 (90)	0	0	2 (10)
	French Guiana (20)	20 (100)	0	0	0
	Haiti (15)	3 (20)	1 (7)	0	11 (73)
	Spain (3)	3 (100)	0	0	0
	Chile (2)	2 (100)	0	0	0
	Martinique (2)	2 (100)	0	0	0
	Ecuador (1)	1 (100)	0	0	0
	Mongolia (1)	1 (100)	0	0	0
	Tahiti (1)	1 (100)	0	0	0
	Honduras (1)	1 (100)	0	0	0
	Republic of Djibouti	1 (100)	0	0	0
	Czech Republic (1)	0	0	0	1 (100)
	Russia (1)	0	0	0	1 (100)
	Total (787)	540 (68)	7 (1)	28 (4)	212 (27)

TABLE 3 .

 3 Indistinguishable spoligotype patterns a That is, spoligotype patterns that could not be segregated into LAM or non-LAM strains.

				No. of isolates with
	Spoligotype pattern	No. of isolates	SpolDB4 designation b	the Ag85C 103 SNP:	No. of LAM or non-LAM isolates c
				Present	Absent
	ⅢⅢⅢⅢⅢⅢⅢⅢⅢⅢⅢⅢⅢⅢⅢⅢⅢⅢⅢⅢ▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫	2	U	2	0	2 LAM
	ⅢⅢⅢⅢⅢⅢⅢⅢⅢⅢ▫ⅢⅢⅢⅢⅢⅢⅢⅢⅢ▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫	1	N A d	1	0	1 LAM
	ⅢⅢⅢⅢⅢⅢⅢⅢⅢ▫▫ⅢⅢⅢⅢⅢⅢⅢⅢⅢ▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫	1	NA	1	0	1 LAM
	ⅢⅢⅢⅢⅢⅢⅢⅢ▫▫▫ⅢⅢⅢⅢⅢⅢⅢⅢ▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫ⅢⅢⅢⅢⅢ	4	U (LAM3)?	3	1 e	3 LAM, 1 non-LAM e
	ⅢⅢⅢⅢⅢⅢⅢⅢ▫▫▫ⅢⅢⅢ▫▫▫Ⅲ▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫ⅢⅢⅢⅢ	1	U	1	0	1 LAM
	ⅢⅢⅢⅢ▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫ⅢⅢⅢⅢⅢⅢⅢⅢ▫▫▫▫ⅢⅢⅢ▫ⅢⅢⅢ	7	T2 (LAM type?)	7	0	7 LAM
	▫▫▫▫ⅢⅢⅢⅢⅢⅢⅢ▫ⅢⅢⅢⅢⅢⅢ▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫ⅢⅢⅢⅢⅢⅢ	1	U	0	1	1 Non-LAM
	▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫ⅢⅢⅢⅢⅢⅢⅢⅢ▫▫▫▫ⅢⅢⅢⅢⅢⅢⅢ	15	LAM3/S convergent	5	10 f	5 LAM, 10 non-LAM f
	▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫ⅢⅢⅢⅢⅢⅢⅢⅢ▫▫▫▫ⅢⅢⅢ▫ⅢⅢⅢ	4	T2	0	4	3 non-LAM
	Ⅲ▫Ⅲ▫▫▫▫ⅢⅢⅢⅢⅢⅢ▫▫ⅢⅢⅢⅢⅢ▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫▫ⅢⅢⅢⅢⅢⅢ	1	NA	0	1	1 Non-LAM
	ⅢⅢⅢⅢⅢⅢⅢⅢⅢⅢⅢⅢⅢⅢⅢⅢⅢⅢⅢⅢ▫▫▫▫ⅢⅢⅢⅢⅢⅢⅢⅢ▫▫▫▫ⅢⅢⅢⅢⅢⅢⅢ g

a

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ACKNOWLEDGMENTS

We thank Maryse Timothee for the microbiological processing of the M. tuberculosis strains from Haiti and Scott Weisenberg, Hongxia Zhu, and Warren D. Johnson for their suggestions, support, and encouragement.

Funding for this project was provided by National Institutes of


Saad Mubeen
email: saad.mubeen@mdh.se

Jukka Mäki-Turja
email: jukka.maki-turja@mdh.se

John Lundbäck
email: john.lundback@arcticus-systems.com

Mattias Gålnander
email: mattias.galnander@arcticus-systems.com

Kurt-Lennart Lundbäck
email: kurt.lundback@arcticus-systems.com

Mikael Sjödin
email: mikael.sjodin@mdh.se

Bud Lawson

Harold " Bud
email: ‡bud@lawson.se

" Lawson

Academic-industrial Collaboration in the Vehicle Software Domain: Experiences and End-user Perspective

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

I. INTRODUCTION

A large share of innovation and customer value in modern vehicles comes from advanced computer-controlled functionality. With the increasing volume of such functionality, the vehicle software has tremendously increased in size and complexity in the past few years [START_REF] Broy | Engineering automotive software[END_REF], [START_REF] Charette | This Car Runs on Code[END_REF]. The distributed nature of this software over several Electronic Control Units (ECUs) further adds to its complexity. Moreover, the safety-critical nature of several vehicle functions puts real-time requirements on them. The developers of such functions are required to ensure that the functions are predictable, i.e., they behave in a timely manner when executed. The software complexity can be managed by using the tools that employ the principles of component-based software engineering [START_REF] Crnkovic | Building Reliable Component-Based Software Systems[END_REF], [START_REF] Henzinger | The Embedded Systems Design Challenge[END_REF] and modeldriven engineering [START_REF] Schmidt | Guest editor's introduction: Model-driven engineering[END_REF]. On the other hand, predictability of the functions is guaranteed by the tools that implement realtime schedulability analysis [START_REF] Audsley | Fixed priority pre-emptive scheduling: an historic perspective[END_REF], [START_REF] Mubeen | Support for end-to-end response-time and delay analysis in the industrial tool suite: Issues, experiences and a case study[END_REF], [START_REF] Henia | System level performance analysis -the symta/s approach[END_REF]. Such analysis can validate the timing requirements, without performing exhaustive testing, before the functions are deployed in the target platforms.

In this paper, we consider the case of one such tool chain: Rubus [START_REF]Rubus models, methods and tools[END_REF]. The Rubus tool chain supports model-and component-based development of vehicle software and its predictable execution on a real-time operating system (RTOS) that is certified to ISO 26262:2011 safety standard according to ASIL D. In this paper, we describe the experiences of various collaborators in the academic-industrial collaboration that has resulted in the development of the Rubus tool chain and its evolution based on the state-of-the-art research results, industrial needs and feedback from the end-users. One unique characteristic of the selected collaboration is that it offers a clear value chain from academia (mainly Mälardalen University); through tool developer (Arcticus Systems1 ); and finally, to the end users of the technology (e.g. Volvo Construction Equipment2 and BAE Systems Hägglunds3 ) as shown in Fig. 1. This figure shows the flow of information within the selected academic-industrial collaboration.

Volvo Construction Equipment

Fig. 1. Example of flows of information within the collaboration.

II. THE RUBUS TOOL CHAIN AND PERSPECTIVE OF THE

TOOL PROVIDER There is a large number of tool providers in the vehicle domain such as Arcticus Systems, Vector, Symta Vision, Systemite, Mentor Graphics, IBM, Arccore, ETAS, Continental, dSpace, Berner & Mattner, Fraunhofer, just to name a few. In this paper, we consider the case of the Rubus tool chain that is developed by Arcticus Systems in close collaboration with several academic and industrial partners. The main reasons for selecting Rubus are as follows. The software developed using Rubus has a small run-time foot print (timing and memory overhead) as compared to several other componentbased development technologies including AUTOSAR [START_REF]AUTOSAR Techincal Overview[END_REF]. While most of the tool chains and models need complementary tools from other vendors to support the software development at all abstraction levels of EAST-ADL [START_REF]EAST-ADL Domain Model Specification[END_REF], the Rubus tool chain is applicable to all the abstraction levels. A detailed comparison of the Rubus tool chain with other related models and tool chains is discussed in [START_REF] Mubeen | Communications-Oriented Development of Component-Based Vehicular Distributed Real-Time Embedded Systems[END_REF]. Arcticus Systems and their customers are relatively open in discussing the theories and details about their tools, use cases and experiences with the research community. Finally, the Rubus tool chain has been successfully used in the vehicle industry for about 20 years.

The main products in the Rubus tool chain include Rubus-ICE (Integrated component model Development Environment) and Rubus RTOS as portrayed in Fig. 2. A real-time application developed using Rubus-ICE can be executed on a variety of platforms, that is, various hardware and real-time operating systems. Rubus-ICE is utilized by several international companies including Volvo Construction Equipment, Volvo Group, BAE Systems Hägglunds, Elektroengine, BorgWarner, Hoerbiger and Knorr-Bremse in developing, simulating and implementing time-critical and non-time critical applications. Rubus provides for the implementation of the model-driven development concept by providing the following properties.

Confidential/proprietary information of Arcticus Systems AB is contained herein and may not be disclosed, displayed, used reproduced or copied without prior written consent. Failure to comply with this notice may result in liability for costs, damages or losses.

© 2015, Arcticus Systems AB. All rights reserved. Rubus® is a registered trademark of Arcticus Systems.

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• RCM -Viewpoint of the developer/development team model: The developer designs the system, in a platform independent manner that focuses upon the application. Timing and resource constraints are expressed in the model.

• RAM -Viewpoint of the analysis model: The resulting RCM design is formal and lends itself to static analysis that is mapped to the actual run-time platform. The analysis includes type checking, execution order, real-time requirements such as response times and worst-case execution times. This analysis helps in reducing late, costly and time-consuming testing efforts of, e.g., temporal errors. Furthermore, mathematical models and supporting tools provide formal evidence of fulfilling requirements. • RRM -Viewpoint of the run-time platform model: The RCM design together with the RAM analysis is utilized to synthesize the code for the actual run-time platform. This automated synthesis prevents error prone and costly integration errors. The run-time platform may be the Rubus Kernel or some other Real Time Operating System.

These concepts have proven to be effective in providing scalability from small to large real-time applications implemented by various organizations.

Rubus Products

The main products; namely Rubus Tool Suite and Rubus RTOS that Arcticus delivers to its customers are portrayed in Figure 3. It is important to note that the real-time application developed using Rubus Tool Suite can be executed on a variety of real-time platforms, that is, various hardware and various real-time operating systems.

Figure 3: The Rubus Product Suite.

The Rubus Tool Suite Product

The Rubus Tool Suite (Integrated Component Model Development Environment) that is utilized by customers in developing, simulating and implementing time critical and non-time critical applications.

Fig. 2. The Rubus tool chain.

• Raises the level of abstraction thus addressing the increasing complexity problem for embedded software. • Formal and early reasoning. The system architecture can be described early in the system life cycle as high-level models and alternative designs can be rapidly developed and analyzed to try out different solutions. Note that the design can be analyzed without writing any source code. Furthermore, system model documentation facilitates maintenance and further development activities. • Code synthesis. There is a separation between the runtime model and the program code. The user works on a platform independent model, then selects the specific target platform, and Rubus-ICE generates the framework code. Productivity is increased since the auto-generation automates code generation that is often error prone. • Traceability. The system architecture documentation is kept up to date with the implementation resulting in traceability from design to implementation and vice versa. The Rubus Designer is used to interactively describe and analyse the application developed using the Rubus Component Model (RCM) [START_REF] Hänninen | The Rubus Component Model for Resource Constrained Real-Time Systems[END_REF]. The structure of the application is developed by means of Software Circuits (SWCs), lowest-level hierarchical components, and their interconnections. The user defines the input and output ports of SWCs and connects them in a manner similar to hardware diagrams. The Rubus Inspector supports platform-independent formal and semi-formal "Model-in-the-Loop testing environment. It provides for unit testing (SWCs and assemblies, i.e., containers of SWCs) as well as for sub-system testing (node and network) or complete system testing (distributed system with multiple nodes and networks). Test inputs are provided by the user however, it is also possible to generate tests that utilize LabView/Simulink and Matlab environments. The Rubus Analyser provides for the user-friendly presentation of off-line and on-line information about the execution behavior of the system. It enables connection with the development host system to download information such as trace data and run-time information in real-time. Postrun-time analysis of host data for formal analysis can be compared with the original model data to verify the realtime properties initially set by the designer. Another powerful characteristic of the Rubus Analyser is its support for pre-runtime timing analysis including response-time analysis of tasks with offsets [START_REF] Mäki-Turja | Tighter response-times for tasks with offsets[END_REF], shared-stack analysis [START_REF] Bohlin | Bounding shared-stack usage in systems with offsets and precedences[END_REF], response-time analysis of Controller Area Network (CAN) and its higherlevel protocols [START_REF] Mubeen | Integrating Mixed Transmission and Practical Limitations with the Worst-Case Response-Time Analysis for Controller Area Network[END_REF], distributed end-to-end delay analysis [START_REF] Feiertag | A Compositional Framework for End-to-End Path Delay Calculation of Automotive Systems under Different Path Semantics[END_REF], [START_REF] Mubeen | Support for end-to-end response-time and delay analysis in the industrial tool suite: Issues, experiences and a case study[END_REF], as well as early timing analysis of black box nodes (whose internal software architectures are not available) [START_REF] Mubeen | End-to-end Timing Analysis of Black-box Models in Legacy Vehicular Distributed Embedded Systems[END_REF]. Rubus RTOS. The Rubus tool chain is complemented by Rubus RTOS that is certified in ISO 26262:2011 safety standard. Rubus Kernel provides support for RCM in achieving an optimal real-time software system. Its main features include support for both time-and event-triggered execution of threads, inter-thread communication, static allocation of resources, scalability and portability. The combination of a dynamic and static scheduling supported by the Rubus Kernel enables the design of optimal real-time software systems. The kernel can be ported to various targets and development environments including Freescale MPC-processors, Texas DSP, Infineon's xc167-processors and various C-compiler environments such as Green Hills, WindRiver, Tasking, Microsoft VS and GCC. Rubus Simulator provides for testing and verifying the composite of the Rubus Kernel and application software. Rubus Kernel provides support for the Rubus Component Model (RCM) in achieving an optimal real-time software system. The main features of the Rubus Kernel are:

• Support the execution of Time-Triggered Red threads.

• Support the execution of Interrupt-Triggered Green threads.

• Support execution of Event-Triggered Blue threads.

• Support communication between different types of threads.

• Support statically allocated resources.

• Support scalability and portability.

• Support the instrumentation of runtime analysis.

The combination of a dynamic and static scheduling supported by the Rubus Kernel enables the design of optimal real-time software systems.

The Rubus Kernel can be ported to various targets and development environments on customer request and includes, amongst others, Freescale MPC-processors, Texas DSP, Infineons xc167-processors and various C-compiler environments such as Green Hills, WindRiver, Tasking, Microsoft VS and, GCC.

Contributions from Mälardalens University

Since the conception of the first Rubus products, cooperation with Mälardalen University (and other academic institutions) has been of great importance to drive the development of the Rubus tool-suite Vice versa, experience and feedback from industrial use of Rubus have inspired many research projects over the years. A graphic history of different component-models that have been developed in academia and by Arcticus is shown in Figure 7. Rubus was initially developed from the joint academic and industrial project VIA, project BASEMENT and its conceptual component model [START_REF] Hansson | Basement: an architecture and methodology for distributed automotive real-time systems[END_REF]. Later, an SSF-funded project, SAVE, coordinated by Mälardalen University with partners from KTH, Linköping University, and Uppsala University based the SaveComp component model on the BASEMENTconcept and the experiences from early Rubus models. In a technology-transfer project MULTEX, funded by KKS, Mälardalen University worked with Arcticus Systems to define the next version of RCM using concepts from SaveComp. This resulted in the first version of Rubus where components could be used in time-, event-and interrupt-triggered threads. In 2006, a KKS-funded project at Mälardalen University, called PROGRESS, provided the ProCom component model [START_REF] Sentilles | A Component Model for Control-Intensive Distributed Embedded Systems[END_REF] that inspired further development of RCM. A new technologytransfer project, EEMDEF, funded by KKS resulted in the extension of RCM and Rubus tool chain to support the modeling and development of distributed embedded systems [START_REF] Mubeen | Communications-Oriented Development of Component-Based Vehicular Distributed Real-Time Embedded Systems[END_REF].

Another KKS-funded project FEMMVA resulted in the extension of timing analysis framework in Rubus tool chain to support the specification, analysis and validation of end-to-end path delay constraints namely age and reaction [START_REF] Feiertag | A Compositional Framework for End-to-End Path Delay Calculation of Automotive Systems under Different Path Semantics[END_REF], [START_REF] Mubeen | Support for end-to-end response-time and delay analysis in the industrial tool suite: Issues, experiences and a case study[END_REF], [START_REF]AUTOSAR Techincal Overview[END_REF]. Further, the results of the project provided foundations for the translation of timing constraints and design-level models from EAST-ADL to RCM. Since 2012, and the finalization for Rubus Component Model Version 4.0, Arcticus has further intensified the academic cooperation with Mälardalen University and with participation in several larger European projects. Many projects are a result of the continued cooperation with Mälardalen University. Finally, a Worst Case Execution Time (WCET) analysis project done in cooperation with Mälardalen University provided an important step toward adding WCET analysis of the C-source code as a complement to measurement on target processors.

IV. PERSPECTIVE OF THE END USER

We consider two end-users of the Rubus tool chain as part of the selected collaboration.

A. BAE Systems Hägglunds

BAE Systems is a renowned global manufacturer of advanced defense, combat and security systems that can be used in the air, land and sea. BAE Systems Hägglunds excels at manufacturing state-of-the-art combat vehicles. According to BAE Systems Hägglunds, the main reasons for selecting RCM and Rubus tool chain for the development of embedded software in their products are as follows.

• Requirement for the time-and event-triggered execution: They require time-triggered architectural approach that is needed to execute the software threads that compose majority of the repetitive functions in their products. The support for event-triggered execution of threads is needed to service various internal and external events in the system. Both the time-and event-triggered modeling and execution of software circuits are fully supported by RCM, Rubus-ICE and Rubus RTOS. • Requirement to support background threads and services: They require the support to implement background threads that are appropriate for services of diverse character such as diagnostics as well as vital functions for the implementation of communication protocols such as TCP/IP. This requirement is fully supported by the Rubus tool chain. • Requirement of pre-runtime timing analysis: They require pre-runtime guarantees on the predictability of both time-and event-triggered threads. Using RCM and the Rubus tool chain, they provide pre-runtime guarantees for time-triggered software circuits by using the correctby-construction offline schedule that is generated by the Rubus scheduler. Whereas in the case of event-triggered software circuits, response-time analysis and end-to-end delay analysis in Rubus-ICE provides pre-run-time guarantees on the timing behavior of the software circuits. • Requirement of coupling the software components with Simulink: The behavior of control functions are developed using Simulink. There is a requirement to couple each software component in the software architecture with Simulink. The coupling to Simulink is achieved at a suitable level via a modified code generator such that the generated code becomes an entry point for a software circuit. The software circuit is then allocated to a timetriggered chain with an appropriate periodicity. After their adoption of the Rubus tool chain, they have provided the industrial needs, requirements and use cases during the evolution of the Rubus tool chain. Moreover, they have provided evaluation of the methods, techniques, results as well as corresponding extensions in the Rubus tool chain in a realistic industrial environment.

B. Volvo Construction Equipment (VCE)

VCE is the oldest and one of the world-leading manufacturers of heavy construction-equipment vehicles, e.g., wheel loaders, articulated haulers and excavators. The company has been successfully applying model-based software development, using the Rubus tool chain, to provide computer-controlled functionality in the vehicles since 1997. VCE has used Rubus in almost all of their products, most notably, for controlling the gear shift and various other ECUs. According to VCE, Rubus has proven to work efficiently from small applications with only a few objects up to very large applications with thousands of objects. Some of their experiences are listed below.

• Development and testing times: Small development time is of essence for VCE. The component concept in Rubus offers advantages such as divide-and-conquer strategies, i.e., breaking down of an application into smaller components that can be distributed to a large number of developers. This enables development of vehicle functionality in parallel, hence resulting in shortening the development time. The support for model-in-the-loop testing and creation of unit tests for each component in the Inspector, one of the tools in Rubus-ICE, has resulted in reduction of the amount of bugs that end up during the integration of a system. As a result, time to test has been shortened after using the Rubus tool chain. V. SUMMARY In this section we summarize our experiences by a set of quotes from key stake-holders in our collaboration.

• "In order to be competitive as a tool provider in the embedded systems domain, we strive to be active, agile and fast in identifying the needs and challenges of our customers and provide them solutions based on the state-of-the-art techniques and research results" (Arcticus Systems).

• "To remain academically excellent, and scientifically relevant, we find the long-term cooperation within the team highly invigorating. Over the years the collaboration have had a profound impact on our whole research environment at MDH" (Prof. Mikael Sjödin, Mälardalen University).

• "Based upon my long-term cooperation with Arcticus Systems, I am pleased with their work towards implementing a very useful real-time kernel and supporting tool suite. It has been personally satisfying since Arcticus Systems has made usage of several of the concepts that I previously developed" (Harold "Bud" Lawson).

• "From the end-user's perspective some remarkable features of Rubus include user-friendly tools, simplicity to understand and use by the engineers, light run-time foot print and automation" (VCE). • "The good thing about Rubus is that it supports both timeand event-triggered execution. With the time-triggered execution, majority of our functions are supported well due to their repetitive nature. The communication and diagnostic services in our systems are well supported by utilizing the event-triggered execution in Rubus. Interoperability between Rubus and Simulink eases our effort for the development of control logic" (BAE Systems). "Automated interoperability to support information exchange among tools and models during the development of cyber-physical systems is going to be the next big thing in the vehicle industry (industrial members of the collaboration)."

VI. ONGOING WORK

In modern vehicles, contemporary functions and advanced features require new levels of computational power and more complex coordination among subsystems. Multi-core platforms offer an efficient support for running such computationintensive vehicle functions by executing various activities in parallel. However, multi-core platforms are more prone to unpredictable behavior as compared to the single-core platforms.

How to provide predictable execution of vehicle software in parallel on a contemporary multi-core ECU with shared caches and memory banks? It is still an unsolved problem. This problem is expected to increase in the future with the introduction of more advanced architectures with cluster sets of cores having more advanced memory interconnects. A seamless tool chain for model-and component-based development of vehicle software its predictable execution on multicore platforms is missing in the state of the practice. Currently, the selected collaboration is working to achieve these goals in the ongoing projects4 such as EMC 2 , DPAC, ASSUME and PreView. Another ongoing work includes certification of parts of the tool chain according to the ISO 26262 safety standard.

  III. ACADEMIC PERSPECTIVESince the conception of the first Rubus products, cooperation with Mälardalen University (and other academic institutions) has been of great importance to drive the development of the Rubus tool chain. Vice versa, experience and feedback from industrial use of Rubus have inspired many research projects over the years. A graphic history of different component models that have been developed in academia and by Arcticus Systems is shown in Fig.3.Confidential/proprietary information of Arcticus Systems AB is contained herein and may not be disclosed, displayed, used reproduced or copied without prior written consent. Failure to comply with this notice may result in liability for costs, damages or losses. © 2015, Arcticus Systems AB. All rights reserved. Rubus® is a registered trademark of Arcticus Systems.

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 8

Figure 7 :

 7 Figure 7: Contributions to the Rubus Component Model. As mentioned above, Rubus was initially developed from the joint academic and industrial project VIA project BASEMENT and its conceptual component-model. In development of the first Rubus tools, MDH

Fig. 3 .

 3 Fig. 3. Contributions to the Rubus Component Model and tool chain.

  Pre-runtime timing analysis: A lot of functionality in the vehicles, produced by VCE, have real-time require-ments. VCE is required to verify such requirements before the functionality is deployed in the vehicles. The preruntime timing analysis support in Rubus-ICE, including response-time analysis and distributed end-to-end delay analysis, has proven to yield good results in respect to early verification, feasibility validation and timing checks.• Certification: VCE needs to deliver certified vehicle functionality according to safety standards. Since the Rubus RTOS is certified according to ISO-26262:2011 ASIL-D, it serves the purpose for VCE. The Rubus tool suite is currently undergoing the certification process. VCE has contributed to the evolution of the Rubus tool chain by not only supplying industrial needs, requirements and use cases but also providing the efficacy of the new methods, techniques, results and corresponding extensions.

	• Support for interoperability: Interoperability of infor-
	mation and models during the development of vehicle
	software is very important for VCE. Such a support
	is provided by the Rubus tool chain since it allows
	integration with other tools such as Simulink/Matlab.
	Using Rubus, there are various means of either using the
	application programming interfaces to read/write data or
	directly access the model information using scripts such
	as Python.
	• Information management: Rubus tool suit uses XML-
	files as an exchange format. Such a format enables the
	use of existing change management systems such as SVN,
	ClearCase and GIT. Merging is also facilitated since the
	information is human readable but still possible for a
	machine to process. It also means that creating model
	information from other tools or means is relatively easy.
	Hence, information management becomes easier.

•

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