BACKGROUND
in spite of the wide availability of cost effective interventions for its control, tuberculosis  is still a major global health problem and the leading cause of death from a curable infectious disease  <cit> .
an increasing amount of evidence indicates that mycobacterium tuberculosis' ability to spread varies from strain to strain and that different strains have different geographical and/or host specificities  <cit> .
since the discovery of dna polymorphisms in m. tuberculosis, molecular typing of strains has become invaluable for the study of epidemiology of tb  <cit> .
restriction fragment length polymorphism typing using the insertion element is <dig> as a probe has been applied since the early 1990s and still is the most reliable method for m. tuberculosis strain differentiation  <cit> .
the use of additional m. tuberculosis genotyping approaches further allows investigators not only to document outbreaks and track epidemics locally, but also to gain an insight into the global migration and expansion of strains  <cit> .
several polymerase chain reaction -based techniques have been proposed for m. tuberculosis strain typing.
the most widely used is spoligotyping, which detects presence or absence of  <dig> short variable spacer sequences interspersed with direct repeats in the direct repeat  region of the chromosome  <cit> .
a more recent approach consists of the analysis of polymorphisms in  <dig> to  <dig> loci containing variable number of tandem repeats of mycobacterial interspersed repetitive units   <cit> .
these two pcr-based methods have been adapted for high-throughput genotyping and combined provide the basis for research on evolutionary genetics of m. tuberculosis  <cit> .
the information on m. tuberculosis population diversity gathered so far in global databases provides a robust platform for research on phylogeny and virulence  <cit> .
ultimately, this knowledge is contributing to the design of rational measures for the control of tb.
paraguay is an inland south american country with  <dig>  million inhabitants and an area of  <dig>  km2; it shares borders with bolivia, brazil and argentina  <cit> .
politically, the country consists of  <dig> departments, in addition to asuncion, the capital city.
the paraguay river runs from north to south dividing the territory in two distinct geographical regions with a remarkable difference in population density.
the fertile oriental region has  <dig>  km <dig> and as many as  <dig>  inhabitants per km <dig>  to the northwest is the chaco, an arid region with a surface of  <dig>  km <dig> and only one inhabitant per km2; its people are mainly settled in sparse aboriginal communities.
a total of  <dig>  tb cases were reported countrywide in  <dig>  yielding a case notification rate of  <dig>  cases per  <dig>  inhabitants.
in absolute numbers, most cases occur in asuncion and the central department, its contiguous densely populated area; both are located in the oriental region.
in the oriental region the incidence mirrors the national rate whereas in the chaco the rate is  <dig> to  <dig> times higher.
there is considerable under-reporting of tb in paraguay; the world health organization estimates the true incidence in the country as 50– <dig> cases per  <dig>   <cit> .
in  <dig> the national tb program implemented a project on anti-tb drug resistance surveillance sponsored by who/usaid.
at that time, mycobacterial culture was seldom performed in the country and case finding was mainly based on acid fast bacilli smear examination of symptomatic patients.
the availability of isolates from this project permitted this first-ever strain typing study in paraguay.
this investigation was undertaken to provide both a nationwide view of the m. tuberculosis population structure and a preliminary assessment of the feasibility and usefulness of genotyping for epidemiological purposes in this limited-resource and endemic tb setting.
RESULTS
population characteristics
of the  <dig> strains included in the study,  <dig>  were isolated from male patients.
one hundred forty-six patients  were  <dig> to  <dig> years old .
two hundred and sixteen patients  were native-born; one patient was an immigrant from south korea; the place of birth was not registered for the remaining three patients, but their demographic data suggested brazilian origin.
sixty-seven strains  were isolated from patients diagnosed in either asuncion or the central department ;  <dig>  originated from other areas in the oriental region and  <dig>  were obtained from the sparsely populated chaco.
one hundred and eighty-four strains  were isolated from newly diagnosed patients and the remaining  <dig>  from patients previously treated for tb.
thirty-three strains were drug resistant, six of which were multidrug resistant .
lineage assignment according to spoligotyping
the  <dig> strains were classified into  <dig> different spoligopatterns that could be assorted in three groups.
the first group included  <dig> strains matching  <dig> shared international types  already present in an updated version of the spoldb <dig> database  <cit> .
for these previously described sits, table  <dig> presents the frequencies found in our study as compared to frequencies in the international database at the time of this analysis, as well as their clade denomination.
the second group consisted of  <dig> strains distributed within  <dig> newly created sits, as shown in figure  <dig>  five of these new sits,  contained strains only identified in this study.
each of the remaining eight newly created sits involved a single strain in this study matching with a previously orphan strain in spoldb <dig>  the third group included the remaining  <dig> strains that did not match with any other spoligopattern in this study nor in spoldb <dig> .
the frequencies of major m. tuberculosis spoligotype families as defined in the spoldb <dig> database  <cit>  ranked in the following order: latin-american & mediterranean   <dig> % , haarlem  <dig> % , s clade  <dig> % , an ill-defined t superfamily   <dig> % , x clade  <dig> %  and beijing clade  <dig> % .
the sole beijing strain in the study was pansusceptible and had been obtained from a  <dig> year-old female south korean immigrant living in asuncion.
twenty-two  strains showed patterns that did not fit within any of the major clades described in spoldb <dig>
is <dig> rflp analysis and epidemiological clustering
patterns of the  <dig> isolates analyzed by is <dig> rflp contained between four and  <dig> bands .
the sole strain showing an rflp pattern with four bands belonged to the x lineage .
one hundred and nineteen distinct rflp patterns were observed.
when only is <dig> rflp was considered for clustering analysis,  <dig> isolates were grouped in  <dig> clusters of  <dig>   <dig>   <dig>   <dig> and  <dig> strains.
when both rflp and spoligotype were analyzed together, four rflp clusters were further split off and five strains were classified as orphan .
* rflp clusters marked with an asterisk contain one or more strains with different spoligopattern and numbers in brackets indicate cluster size after subtraction of strains with different spoligopattern.
concordant rflp and spoligotype clustering involved 15/ <dig> , 20/ <dig>  and 25/ <dig>  strains from the chaco, the metropolitan area and the oriental region, respectively.
no association was found between clustering and gender , age , treatment history  or drug resistance .
demographic evidence was consistent with transmission in particular settings such as the tacumbu jail in asuncion, indigenous communities in the chaco, and a single household in the metropolitan area.
strains harboring the sit <dig> of the s clade grouped in the rflp cluster c together with two other strains harboring rare spoligotypes: an orphan strain  and a strain of the new sit  <dig> .
these two novel spoligopatterns could be visually considered to be derived from the sit  <dig> associated to this rflp cluster .
similarly, identical or closely-related rflp patterns were exhibited by strains classified within each of five new shared spoligotypes identified exclusively in paraguay.
these are sits  <dig> ,  <dig> ,  <dig> ,  <dig> and  <dig> .
in cluster a, is <dig> rflp grouped strains of the sit <dig> together with one strain harboring an orphan spoligotype different from but closely related to sit  <dig>  most strains in this cluster  had been isolated from inmates in the tacumbu jail.
apart from these two orphan strains grouped by rflp in clusters a and c, the remaining  <dig> strains with orphan spoligotypes did not match with any of approximately  <dig> rflp patterns contained in the database at the malbran institute, which gathers patterns from strains isolated in argentina and other south american countries.
miru-vntr analysis of new spoligopatterns
results of miru analysis of  <dig> selected strains with either new sits or orphan spoligotypes are summarized in table  <dig> and figure  <dig>  miru typing confirmed the clonal nature of two of the five newly described sits restricted so far to paraguay: sit 2650-mit  <dig> of the lam lineage and sit 2643-mit  <dig> of the haarlem lineage.
this latter clone is hereafter named "tacumbu" after the men's correctional facility in asuncion where it was most frequently found; it included another strain in the same rflp cluster with an orphan but related spoligopattern  isolated from another inmate in the same jail .
discussion
we present herein a first insight into the biodiversity of the m. tuberculosis epidemic in paraguay.
at the same time, we provide evidence of the suitability of is <dig> rflp as a genotyping tool for epidemiological studies in the country, either as a stand-alone tool or, still better, in combination with spoligotyping.
even though the characteristics of the study sample favored phylogenetic rather than epidemiological analysis, rflp analysis by itself produced a concise picture of tb transmission patterns in paraguay.
in particular, it served to identify transmission in specific settings such as indigenous communities in the chaco and the tacumbu jail in asuncion.
the percentage of clustering in different regions was congruent with the respective regional incidence rates, i.e.
highest in the chaco, intermediate in the densely populated metropolitan area, and lowest in the rest of the oriental region.
we did not find correlation of clustering with previous tb treatment or drug resistance, suggesting that in paraguay most patients develop drug resistant tb individually through selective pressure imposed by poorly constructed or inadequately supervised treatment regimens rather than through transmission of resistant strains.
the age and gender composition of patients in our sample reflects the predominance of tb among young male people in paraguay.
this disease distribution is common to many low income settings worldwide and could be attributed to socioeconomic and cultural barriers in the access to health care  <cit> .
in the present study, however, we failed to find association of clustering with gender or age, probably due to the small size of the sample and the short sampling period.
in this sense, we are aware of the fact that genotype clustering analysis underestimates transmission and that this bias is inversely proportional to length of time and size of the sample  <cit> .
the relative frequencies of major m. tuberculosis spoligotype families were roughly in range with the overall frequencies described for countries in the south american region  <cit> .
the largely predominant lam lineage, identified in more than half of the strains in our study, was ubiquitous in the country.
both its high degree of dissemination and its preponderance among the new  patterns are manifestations of the current adaptative evolution of the lam lineage in paraguay.
second in frequency, the haarlem clade was found mainly in the metropolitan area.
third in frequency, the s clade was found predominantly in the oriental region.
the strains classified within the ill-defined t family were widely distributed within the country.
the is <dig> low-banding pattern x genotype family was rather unusual and the beijing genotype was absent in the native population of our study.
the sole beijing strain identified in the study was isolated from an east asian immigrant who most probably acquired her infection prior to arriving in paraguay.
an example of congruence between phylogenenetic and epidemiological findings is the fact that the four most prevalent sits in our study accounted for two thirds of the rflp/spoligotyping clustered cases, including cases with epidemiological links .
these were sit <dig> of the lam <dig> clade, sit <dig> of the lam <dig> clade, sit <dig> of the s clade and the newly-created sit <dig> of the haarlem clade.
as shown by their active ongoing transmission, these sits could be regarded as highly successful m. tuberculosis genotypes in paraguay.
interestingly, two of these m. tuberculosis sub-lineages seem to have a restricted geographical distribution: sit <dig> has been so far found only in brazil  and paraguay  and the newly created sit <dig>  is restricted to paraguay alone.
is <dig> rflp and miru typing confirmed the clonality of this latter phylogeographically specific genotype, which is hereby designated as "tacumbu" genotype.
indeed, this clone is completely new as it created not only a new sit  but also matched a rare mit  in the database within the haarlem clade.
although different mutation mechanisms may converge into identical spoligopatterns, the main force driving variation in the dr region appears to be deletion of single or contiguous direct variable repeat sequences  <cit> .
our data supports this kind of evolution for some actively trasmitting clones, which may represent emerging genotypes in paraguay.
for example, the main chain of transmission in tacumbu jail included an orphan strain that shared identical mit and rflp with other strains in the cluster and harbored a spoligotype that could well have evolved from the same tacumbu sit through deletion of two contiguous spacers.
likewise, sit <dig>  endemic in paraguay , has probably evolved to the new sit <dig>  by the loss of one spacer.
similar deletions in the dr region could have also occurred in two rare strains harboring mit <dig> and fitting within the largest rflp cluster together with  <dig> strains of the s clade.
in addition to rapid evolution of some predominant strains, spoligotyping revealed the geographical specificity of a number of m. tuberculosis strains in our study.
similar to findings reported in venezuela, brazil and suriname  <cit>  a number of spoligopatterns in our study were unique among more than  <dig>  strains recorded in the spoldb <dig>  most of these strains proved further their singularity when tested by rflp and/or mirus.
moreover, almost one in ten strains did not fit into any of the major m. tuberculosis phylogenetic clades described in the updated database and one in five spoligopatterns in the study were rarely or never described outside paraguay.
CONCLUSIONS
paraguay's current tb epidemic seems to consist of a wide diversity of sublineages among which a few lam, haarlem and s genotypes prevail and are evolving actively.
the lineages of tubercle bacilli currently thriving in this rather secluded south american niche may reflect the local host-pathogen adaptation of strains introduced into the country during past migrations from european countries  <cit> .

 in spite of the wide availability of cost effective interventions for its control, tuberculosis is still a major global health problem and the leading cause of death from a curable infectious disease. an increasing amount of evidence indicates that mycobacterium tuberculosis' ability to spread varies from strain to strain and that different strains have different geographical and/or host specificities. since the discovery of dna polymorphisms in m. tuberculosis, molecular typing of strains has become invaluable for the study of epidemiology of tb. restriction fragment length polymorphism typing using the insertion element is as a probe has been applied since the early 1990s and still is the most reliable method for m. tuberculosis strain differentiation. the use of additional m. tuberculosis genotyping approaches further allows investigators not only to document outbreaks and track epidemics locally, but also to gain an insight into the global migration and expansion of strains. several polymerase chain reaction -based techniques have been proposed for m. tuberculosis strain typing. the most widely used is spoligotyping, which detects presence or absence of short variable spacer sequences interspersed with direct repeats in the direct repeat region of the chromosome. a more recent approach consists of the analysis of polymorphisms in to loci containing variable number of tandem repeats of mycobacterial interspersed repetitive units. these two pcr-based methods have been adapted for high-throughput genotyping and combined provide the basis for research on evolutionary genetics of m. tuberculosis. the information on m. tuberculosis population diversity gathered so far in global databases provides a robust platform for research on phylogeny and virulence. ultimately, this knowledge is contributing to the design of rational measures for the control of tb. paraguay is an inland south american country with million inhabitants and an area of km2; it shares borders with bolivia, brazil and argentina. politically, the country consists of departments, in addition to asuncion, the capital city. the paraguay river runs from north to south dividing the territory in two distinct geographical regions with a remarkable difference in population density. the fertile oriental region has km and as many as inhabitants per km to the northwest is the chaco, an arid region with a surface of km and only one inhabitant per km2; its people are mainly settled in sparse aboriginal communities. a total of tb cases were reported countrywide in yielding a case notification rate of cases per inhabitants. in absolute numbers, most cases occur in asuncion and the central department, its contiguous densely populated area; both are located in the oriental region. in the oriental region the incidence mirrors the national rate whereas in the chaco the rate is to times higher. there is considerable under-reporting of tb in paraguay; the world health organization estimates the true incidence in the country as 50– cases per. in the national tb program implemented a project on anti-tb drug resistance surveillance sponsored by who/usaid. at that time, mycobacterial culture was seldom performed in the country and case finding was mainly based on acid fast bacilli smear examination of symptomatic patients. the availability of isolates from this project permitted this first-ever strain typing study in paraguay. this investigation was undertaken to provide both a nationwide view of the m. tuberculosis population structure and a preliminary assessment of the feasibility and usefulness of genotyping for epidemiological purposes in this limited-resource and endemic tb setting.  population characteristics of the strains included in the study, were isolated from male patients. one hundred forty-six patients were to years old. two hundred and sixteen patients were native-born; one patient was an immigrant from south korea; the place of birth was not registered for the remaining three patients, but their demographic data suggested brazilian origin. sixty-seven strains were isolated from patients diagnosed in either asuncion or the central department; originated from other areas in the oriental region and were obtained from the sparsely populated chaco. one hundred and eighty-four strains were isolated from newly diagnosed patients and the remaining from patients previously treated for tb. thirty-three strains were drug resistant, six of which were multidrug resistant. lineage assignment according to spoligotyping the strains were classified into different spoligopatterns that could be assorted in three groups. the first group included strains matching shared international types already present in an updated version of the spoldb database. for these previously described sits, table presents the frequencies found in our study as compared to frequencies in the international database at the time of this analysis, as well as their clade denomination. the second group consisted of strains distributed within newly created sits, as shown in figure five of these new sits, contained strains only identified in this study. each of the remaining eight newly created sits involved a single strain in this study matching with a previously orphan strain in spoldb the third group included the remaining strains that did not match with any other spoligopattern in this study nor in spoldb. the frequencies of major m. tuberculosis spoligotype families as defined in the spoldb database ranked in the following order: latin-american & mediterranean %, haarlem %, s clade %, an ill-defined t superfamily %, x clade % and beijing clade %. the sole beijing strain in the study was pansusceptible and had been obtained from a year-old female south korean immigrant living in asuncion. twenty-two strains showed patterns that did not fit within any of the major clades described in spoldb is rflp analysis and epidemiological clustering patterns of the isolates analyzed by is rflp contained between four and bands. the sole strain showing an rflp pattern with four bands belonged to the x lineage. one hundred and nineteen distinct rflp patterns were observed. when only is rflp was considered for clustering analysis, isolates were grouped in clusters of and strains. when both rflp and spoligotype were analyzed together, four rflp clusters were further split off and five strains were classified as orphan. * rflp clusters marked with an asterisk contain one or more strains with different spoligopattern and numbers in brackets indicate cluster size after subtraction of strains with different spoligopattern. concordant rflp and spoligotype clustering involved 15/, 20/ and 25/ strains from the chaco, the metropolitan area and the oriental region, respectively. no association was found between clustering and gender, age, treatment history or drug resistance. demographic evidence was consistent with transmission in particular settings such as the tacumbu jail in asuncion, indigenous communities in the chaco, and a single household in the metropolitan area. strains harboring the sit of the s clade grouped in the rflp cluster c together with two other strains harboring rare spoligotypes: an orphan strain and a strain of the new sit. these two novel spoligopatterns could be visually considered to be derived from the sit associated to this rflp cluster. similarly, identical or closely-related rflp patterns were exhibited by strains classified within each of five new shared spoligotypes identified exclusively in paraguay. these are sits,,, and. in cluster a, is rflp grouped strains of the sit together with one strain harboring an orphan spoligotype different from but closely related to sit most strains in this cluster had been isolated from inmates in the tacumbu jail. apart from these two orphan strains grouped by rflp in clusters a and c, the remaining strains with orphan spoligotypes did not match with any of approximately rflp patterns contained in the database at the malbran institute, which gathers patterns from strains isolated in argentina and other south american countries. miru-vntr analysis of new spoligopatterns results of miru analysis of selected strains with either new sits or orphan spoligotypes are summarized in table and figure miru typing confirmed the clonal nature of two of the five newly described sits restricted so far to paraguay: sit 2650-mit of the lam lineage and sit 2643-mit of the haarlem lineage. this latter clone is hereafter named "tacumbu" after the men's correctional facility in asuncion where it was most frequently found; it included another strain in the same rflp cluster with an orphan but related spoligopattern isolated from another inmate in the same jail. discussion we present herein a first insight into the biodiversity of the m. tuberculosis epidemic in paraguay. at the same time, we provide evidence of the suitability of is rflp as a genotyping tool for epidemiological studies in the country, either as a stand-alone tool or, still better, in combination with spoligotyping. even though the characteristics of the study sample favored phylogenetic rather than epidemiological analysis, rflp analysis by itself produced a concise picture of tb transmission patterns in paraguay. in particular, it served to identify transmission in specific settings such as indigenous communities in the chaco and the tacumbu jail in asuncion. the percentage of clustering in different regions was congruent with the respective regional incidence rates, i.e. highest in the chaco, intermediate in the densely populated metropolitan area, and lowest in the rest of the oriental region. we did not find correlation of clustering with previous tb treatment or drug resistance, suggesting that in paraguay most patients develop drug resistant tb individually through selective pressure imposed by poorly constructed or inadequately supervised treatment regimens rather than through transmission of resistant strains. the age and gender composition of patients in our sample reflects the predominance of tb among young male people in paraguay. this disease distribution is common to many low income settings worldwide and could be attributed to socioeconomic and cultural barriers in the access to health care. in the present study, however, we failed to find association of clustering with gender or age, probably due to the small size of the sample and the short sampling period. in this sense, we are aware of the fact that genotype clustering analysis underestimates transmission and that this bias is inversely proportional to length of time and size of the sample. the relative frequencies of major m. tuberculosis spoligotype families were roughly in range with the overall frequencies described for countries in the south american region. the largely predominant lam lineage, identified in more than half of the strains in our study, was ubiquitous in the country. both its high degree of dissemination and its preponderance among the new patterns are manifestations of the current adaptative evolution of the lam lineage in paraguay. second in frequency, the haarlem clade was found mainly in the metropolitan area. third in frequency, the s clade was found predominantly in the oriental region. the strains classified within the ill-defined t family were widely distributed within the country. the is low-banding pattern x genotype family was rather unusual and the beijing genotype was absent in the native population of our study. the sole beijing strain identified in the study was isolated from an east asian immigrant who most probably acquired her infection prior to arriving in paraguay. an example of congruence between phylogenenetic and epidemiological findings is the fact that the four most prevalent sits in our study accounted for two thirds of the rflp/spoligotyping clustered cases, including cases with epidemiological links. these were sit of the lam clade, sit of the lam clade, sit of the s clade and the newly-created sit of the haarlem clade. as shown by their active ongoing transmission, these sits could be regarded as highly successful m. tuberculosis genotypes in paraguay. interestingly, two of these m. tuberculosis sub-lineages seem to have a restricted geographical distribution: sit has been so far found only in brazil and paraguay and the newly created sit is restricted to paraguay alone. is rflp and miru typing confirmed the clonality of this latter phylogeographically specific genotype, which is hereby designated as "tacumbu" genotype. indeed, this clone is completely new as it created not only a new sit but also matched a rare mit in the database within the haarlem clade. although different mutation mechanisms may converge into identical spoligopatterns, the main force driving variation in the dr region appears to be deletion of single or contiguous direct variable repeat sequences. our data supports this kind of evolution for some actively trasmitting clones, which may represent emerging genotypes in paraguay. for example, the main chain of transmission in tacumbu jail included an orphan strain that shared identical mit and rflp with other strains in the cluster and harbored a spoligotype that could well have evolved from the same tacumbu sit through deletion of two contiguous spacers. likewise, sit endemic in paraguay, has probably evolved to the new sit by the loss of one spacer. similar deletions in the dr region could have also occurred in two rare strains harboring mit and fitting within the largest rflp cluster together with strains of the s clade. in addition to rapid evolution of some predominant strains, spoligotyping revealed the geographical specificity of a number of m. tuberculosis strains in our study. similar to findings reported in venezuela, brazil and suriname a number of spoligopatterns in our study were unique among more than strains recorded in the spoldb most of these strains proved further their singularity when tested by rflp and/or mirus. moreover, almost one in ten strains did not fit into any of the major m. tuberculosis phylogenetic clades described in the updated database and one in five spoligopatterns in the study were rarely or never described outside paraguay.  paraguay's current tb epidemic seems to consist of a wide diversity of sublineages among which a few lam, haarlem and s genotypes prevail and are evolving actively. the lineages of tubercle bacilli currently thriving in this rather secluded south american niche may reflect the local host-pathogen adaptation of strains introduced into the country during past migrations from european countries. 
lam genotypes were ubiquitous and predominated among both rflp clusters and new patterns, suggesting ongoing transmission and adaptative evolution in paraguay. we present a picture of the biodiversity of mycobacterium tuberculosis in paraguay, an inland south american country harboring million inhabitants with a tuberculosis notification rate of 38/. lineages currently thriving in paraguay may reflect local host-pathogen adaptation of strains introduced during past migrations from europe. concordant clustering by is restriction fragment length polymorphism and spoligotyping identified transmission in specific settings such as the tacumbu jail in asuncion and aboriginal communities in the chaco. our study contributed to the database new sits and orphan spoligopatterns. spoligopatterns of strains matched shared international types already present in an updated version of spoldb the global spoligotype database at pasteur institute, guadeloupe.
 we present a picture of the biodiversity of mycobacterium tuberculosis in paraguay, an inland south american country harboring million inhabitants with a tuberculosis notification rate of 38/.  a total of strains collected throughout the country in were classified by spoligotyping into different patterns. spoligopatterns of strains matched shared international types already present in an updated version of spoldb the global spoligotype database at pasteur institute, guadeloupe. our study contributed to the database new sits and orphan spoligopatterns. frequencies of major m. tuberculosis spoligotype lineages in our sample were as follows: latin-american & mediterranean %, haarlem %, s clade %, t superfamily %, x clade % and beijing clade %. concordant clustering by is restriction fragment length polymorphism and spoligotyping identified transmission in specific settings such as the tacumbu jail in asuncion and aboriginal communities in the chaco. lam genotypes were ubiquitous and predominated among both rflp clusters and new patterns, suggesting ongoing transmission and adaptative evolution in paraguay. we describe a new and successfully evolving clone of the haarlem sub-lineage, sit which is thus far restricted to paraguay. we confirmed its clonality by rflp and mycobacterial interspersed repetitive unit typing; we named it "tacumbu" after the jail where it was found to be spreading. one-fifth of the spoligopatterns in our study are rarely or never seen outside paraguay and one-tenth do not fit within any of the major phylogenetic clades in spoldb  lineages currently thriving in paraguay may reflect local host-pathogen adaptation of strains introduced during past migrations from europe. 