BACKGROUND
genotyping methods have been extensively applied to analyze the recent transmission dynamics of mycobacterium tuberculosis .
is6110-restriction fragment length polymorphism  is the reference technique for genotyping mtb  <cit>  because of its high discriminatory power.
however, the need for well-grown cultures and purified dna to obtain rflp data means that it takes a long time to produce results.
in addition, analysis of rflp band-patterns requires specific software, which makes it difficult to interpret and exchange data.
finally, rflp is limited when analyzing mtb strains with a low number of is <dig> copies.
different pcr-based genotyping approaches targeting the variable number of tandem repeats  have been developed to compensate for the limitations of rflp.
these include vntr analysis based on mycobacterial interspersed repetitive units   <cit> , which has been considered a good alternative to the reference method and has proven to be faster and easier to perform.
miru-vntr genotyping based on a 12-loci set  has been evaluated in several studies in different settings.
some authors  <cit>  have found it to show a discriminatory power equivalent to that of rflp and have considered it an alternative to is6110-rflp for epidemiological purposes  <cit> .
however, other authors have found limitations in its discriminatory power and incomplete correlation with the rflp analysis  <cit> , indicating that miru analysis should be combined with an additional genotyping method  <cit> .
recently, a new miru-vntr format has been developed to improve the discriminatory power of miru- <dig>  <cit> .
this new version targets  <dig> loci , although to date few studies have evaluated its efficiency  <cit> .
we compared the new 15-loci format of miru-vntr  with the miru- <dig> version and is6110-rflp in two independent samples in the context of molecular epidemiology studies.
these data could serve to clarify the final application of this new procedure in our setting.
RESULTS
i) miru- <dig> analysis of a convenience sample
a) clustering rates and discriminatory power
as an initial approach to comparing the efficiency of miru- <dig> with miru- <dig>  we studied the same convenience sample we had already used to test the efficiency of miru- <dig>  <cit> .
spoligotyping indicated that most of the isolates corresponded to the lam  and haarlem  lineages.
only  <dig> % of the isolates belonged to the beijing family.
rflp clustered 41% of the isolates in  <dig> clusters , whereas miru- <dig> clustered  <dig> % in  <dig> clusters .
miru- <dig> clustered  <dig> % of the isolates in  <dig> clusters .
therefore, the hunter-gaston discriminatory index  was higher for miru- <dig>  than for miru- <dig> .
the loci with the highest hgdis were qub <dig> , qub11b , and miru <dig> , two of which were not present in the miru- <dig> version .
if we consider the nine new loci that were not included in the miru- <dig> version, all of them showed hgdi values over  <dig> , whereas in miru- <dig> only three  showed equivalent values and the remainder showed hgdi values below  <dig>  .
b) correlation analysis
the correlation between rflp and miru- <dig> was higher than that observed with miru- <dig> in our previous study  <cit> .
miru- <dig> showed full or high correlation in 14/ <dig>  of the clusters defined by rflp, whereas miru- <dig> had shown an equivalent correlation in only 8/ <dig> clusters  .
miru- <dig> showed noncorrelation with the rflp data in only three clusters , whereas miru- <dig> had shown no correlation much more frequently.
the discrepancies shown by miru- <dig> corresponded to i) the addition of two isolates to an rflp cluster , although with low is <dig> band similarities , ii) the discrimination of an isolate with a double locus variation  , and iii) full splitting  of the largest cluster defined by rflp .
additionally, in one of these clusters with no correlation , miru- <dig> grouped with high correlation another isolate that showed 95% is <dig> band similarity .
miru- <dig> also defined a new cluster  between two isolates with low is <dig> band similarity  .
in our previous study  <cit> , miru- <dig> defined up to eight clusters not assessed by rflp.
when spoligotyping was applied as a second-line method in discrepant cases, all the isolates grouped by miru- <dig> that were unclustered by is6110-rflp  were split.
cluster r <dig>  which was subdivided by miru- <dig>  was split by spoligotyping .
ii) miru- <dig> analysis of a population-based sample
after observing that miru- <dig> improved the miru- <dig> data, we decided to re-evaluate miru- <dig> efficiency with an independent sample.
unlike the first convenience sample, the new one included all the mtb isolates in almería during the period 2003– <dig>  in total,  <dig> isolates were genotyped, corresponding to  <dig> autochthonous cases  and  <dig> immigrant cases .
the most widely represented countries of origin in the study population were morocco , romania , and mali .
correlation analysis
when the rflp-clustered cases from almería were analyzed by miru- <dig>  full or high correlation was obtained in 83% of the clusters .
miru- <dig> showed no correlation in the remaining five rflp clusters , and discriminated 12/ <dig> isolates grouped in them .
the discrepancies were as follows: i) one isolate clustered by rflp  showed a dlv ; ii) splitting of two rflp clusters  with isolates showing differences in more than four loci: and iii) three and four isolates clustered by rflp  showed differences in two to eight miru- <dig> loci.
additionally, miru- <dig> subdivided rflp cluster  <dig> into three different miru types.
in order to understand the significance of the concordances and discrepancies between the clusters defined by rflp and miru- <dig>  we evaluated the epidemiological evidence found for these clusters.
therefore, we labeled the clusters according to a gradient of evidence of epidemiological links between their cases .
for the rflp clusters with full or high correlation with miru data, we obtained epidemiological data in 22/ <dig> clusters.
of these, epidemiological evidence  was detected in 16/ <dig> clusters .
for the  <dig> patients included in these  <dig> rflp clusters, we detected epidemiological links for 43/ <dig> cases with available epidemiological data .
for the remaining  <dig> cases no epidemiological evidence was found.
epidemiological data were available for four of the five clusters that were split by miru- <dig>  and no links were found for all the representatives which were discriminated by miru- <dig> .
a review of these cases reveals the following: a) in cluster  <dig> , the only isolate without epidemiological links was clearly split by miru- <dig> .
this corresponded to an unrelated ghanian patient in a cluster of spanish cases; b) in a further two clusters  involving two cases each , miru- <dig> showed discrepancies in five and six loci respectively, and once again, no epidemiological connections were found; c) no epidemiological links could be found for the remaining cases involved in cluster  <dig> ; d) cluster  <dig> , involving different nationalities, was also split by miru- <dig>  according to the geographic origin/socio-cultural background of the cases .
no links were found between the multinational patients grouped in rflp cluster  <dig>  but links were found between the cases in at least one of the split subgroups .
discussion
during the last few years, the search for an alternative to rflp, has led to the development of a pcr-based technique, miru-vntr  <cit> .
this technique has proven to be fast and easy to perform, and it allows the direct exchange of data between laboratories.
different combinations of miru and other vntr loci have been published  <cit> , but most of the studies have focused on a set of  <dig> miru loci  <cit> .
this format offers a higher discriminatory power  than other pcr-based genotyping techniques  <cit> .
other studies  <cit> , however, have revealed lower discriminatory power and a low correlation with rflp data, which improved if complemented with spoligotyping.
in a previous study  <cit> , we focused on isolates with high-copy-number fingerprints, to try to evaluate miru- <dig> in a more challenging situation than that of studies performed in circumstances that could favor miru, e.g.
high proportion of low-copy-number rflp fingerprints, low variability of circulating strains due to the prevalence of specific genetic families, etc.
we found that, compared with rflp, miru- <dig> overestimated candidates for recent transmission, by grouping a higher number of isolates and defining a higher number of clusters.
furthermore, 53% of miru- <dig> clusters showed a low or no correlation with rflp data.
these data urge caution when considering substituting rflp with miru- <dig>  and suggest that incongruent study conclusions could be due to geographic differences in the genotypic composition of circulating strains.
supply et al  <cit>  have recently published a proposal for a new refined set of  <dig> miru-vntr loci.
when the advanced set was tested on  <dig> isolates including representatives of the main mtb lineages, a subset of  <dig> loci  was considered to have the highest efficiency because it contained 96% of the resolution obtained with the whole 24-loci set.
however, the miru- <dig> design has received little attention  <cit> ; therefore, additional studies in a variety of socio-epidemiological backgrounds are necessary to fully evaluate the usefulness of this new strategy.
this was the aim of our analysis.
we began by evaluating the new 15-loci miru version with the same convenience sample that was used to evaluate miru- <dig>  the genetic lineages which were over-represented in this sample were lam and haarlem, and only three beijing strains were found, thus ruling out enrichment in strains which have been found to be poorly discriminated by miru- <dig>  <cit> .
miru- <dig> showed higher discriminatory efficiency by grouping  <dig> % fewer isolates than miru- <dig> and showing a higher hgdi value .
this increase in resolution was due to the nine new loci whose hgdi values were higher than  <dig> , whereas only three in the previous set achieved equivalent values, which justified the selection of these loci in the new advanced design.
with regard to the correlation with rflp, our previous study revealed that only  <dig> out of  <dig> rflp clusters fitted well with miru- <dig> data, and it was necessary to include spoligotyping to improve these results.
miru- <dig> alone increased the number of clusters with good correlation with rflp data to as many as 14/ <dig>
in this study, we considered full or high values as indicative of a good correlation between miru- <dig> and rflp.
correlation was considered to be high when miru-grouped isolates sharing rflp patterns showed high is <dig> band similarity , or isolates sharing rflp types showed subtle differences  in the miru analysis.
this decision was supported by studies that found epidemiological links for clusters including isolates with subtle differences in rflp isolates  <cit>  and by reports that some miru loci have faster molecular clocks  <cit> , which could even lead to slvs between isolates linked to an ongoing transmission event.
although good correlation was generally found in our convenience sample, no correlation with rflp was observed for three rflp clusters.
miru- <dig> added two isolates in the r <dig> rflp cluster, which showed only low is <dig> band similarity percentages with the other clustered isolates.
the spoligotypes for these two isolates were also different from the r <dig> representative pattern, although they belonged to the same genetic lineage .
this could be due to the fact that miru- <dig> misassigned these isolates and we were probably observing a genotypic convergence phenomenon.
on the other hand, the rate of miru changes is relatively lower than that of is6110-rflp and spoligotyping, so it could also be possible that the miru type in this case is shared by different representatives of the t <dig> lineage.
in the remaining two noncorrelated clusters, we observed that miru- <dig> split them to a different extent, and we detected differences  among isolates sharing an rflp type.
in addition, the largest rflp cluster was fully split by miru- <dig>  with differences in 2– <dig> loci for all the isolates.
splitting of rflp clusters by miru- <dig> has been found in clusters involving strains with low-copy-band fingerprints   <cit> , which is somehow expected, although in this case the strain had seven bands.
splitting of rflp clusters, even those with high-copy-band strains has been described elsewhere  <cit> , and these findings urge caution in assuming certainty for all defined clusters.
in order to fully understand the meaning of the concordances and discordances between rflp and miru- <dig> data, we used an unselected population-based sample with available epidemiological data to interpret potential discrepancies.
we chose the mtb isolates cultured in the province of almería during a  <dig> -year period to ensure quality in the clustering assignation and to increase the observation time of other studies  <cit> .
we decided not to consider the orphan cases  and to focus exclusively on the analysis of the rflp clusters detected in this population sample because they have epidemiological value, as they are used as markers for recent transmission.
the ability of miru- <dig> to classify as orphan those cases unclustered by rflp is being evaluated in an ongoing prospective population-based study.
as with our findings in the convenience sample, the correlation between miru- <dig> and rflp genotypes was good; full or high correlation with the rflp data was detected for  <dig> of the  <dig> clusters genotyped by miru- <dig>  we found epidemiological links in  <dig> % of the clusters and in  <dig> % of the cases with available epidemiological data.
these percentages of "epidemiologically proved" clusters agree with those of other studies, and it is well known that higher values are only obtained if highly refined epidemiological surveys are followed up  <cit> .
it is noteworthy that almost all the clusters  without epidemiological links involved two cases, and it is generally assumed that the detection of links in transmission chains involving a reduced number of cases produces a lower yield.
it is interesting that, of the four rflp clusters with epidemiological information available which were split by miru- <dig>  no links were found for all the representatives which were discriminated by miru- <dig>  this suggests that miru- <dig> was able to detect some cases that were falsely clustered by rflp.
moreover, in at least one case, miru- <dig> redefined epidemiologically-consistent subclusters within a common rflp cluster which was not epidemiologically supported.
CONCLUSIONS
the data obtained by miru- <dig> in this study and elsewhere  <cit>  suggest that the new design is very efficient at assigning clusters confirmed by epidemiological data.
if we add this to the speed with which it provides results, miru- <dig> could be considered a suitable tool for real-time genotyping.
this could be essential in study populations such as ours, which is undergoing an epidemiological transformation due to the marked increase in tuberculosis among immigrants.
the complexity of this situation reduces the efficiency of standard epidemiological approaches and demands new strategies such as miru- <dig> to allow rapid identification of clusters.

 genotyping methods have been extensively applied to analyze the recent transmission dynamics of mycobacterium tuberculosis. is6110-restriction fragment length polymorphism is the reference technique for genotyping mtb because of its high discriminatory power. however, the need for well-grown cultures and purified dna to obtain rflp data means that it takes a long time to produce results. in addition, analysis of rflp band-patterns requires specific software, which makes it difficult to interpret and exchange data. finally, rflp is limited when analyzing mtb strains with a low number of is copies. different pcr-based genotyping approaches targeting the variable number of tandem repeats have been developed to compensate for the limitations of rflp. these include vntr analysis based on mycobacterial interspersed repetitive units, which has been considered a good alternative to the reference method and has proven to be faster and easier to perform. miru-vntr genotyping based on a 12-loci set has been evaluated in several studies in different settings. some authors have found it to show a discriminatory power equivalent to that of rflp and have considered it an alternative to is6110-rflp for epidemiological purposes. however, other authors have found limitations in its discriminatory power and incomplete correlation with the rflp analysis, indicating that miru analysis should be combined with an additional genotyping method. recently, a new miru-vntr format has been developed to improve the discriminatory power of miru-. this new version targets loci, although to date few studies have evaluated its efficiency. we compared the new 15-loci format of miru-vntr with the miru- version and is6110-rflp in two independent samples in the context of molecular epidemiology studies. these data could serve to clarify the final application of this new procedure in our setting.  i) miru- analysis of a convenience sample a) clustering rates and discriminatory power as an initial approach to comparing the efficiency of miru- with miru- we studied the same convenience sample we had already used to test the efficiency of miru-. spoligotyping indicated that most of the isolates corresponded to the lam and haarlem lineages. only % of the isolates belonged to the beijing family. rflp clustered 41% of the isolates in clusters, whereas miru- clustered % in clusters. miru- clustered % of the isolates in clusters. therefore, the hunter-gaston discriminatory index was higher for miru- than for miru-. the loci with the highest hgdis were qub, qub11b, and miru, two of which were not present in the miru- version. if we consider the nine new loci that were not included in the miru- version, all of them showed hgdi values over, whereas in miru- only three showed equivalent values and the remainder showed hgdi values below. b) correlation analysis the correlation between rflp and miru- was higher than that observed with miru- in our previous study. miru- showed full or high correlation in 14/ of the clusters defined by rflp, whereas miru- had shown an equivalent correlation in only 8/ clusters. miru- showed noncorrelation with the rflp data in only three clusters, whereas miru- had shown no correlation much more frequently. the discrepancies shown by miru- corresponded to i) the addition of two isolates to an rflp cluster, although with low is band similarities, ii) the discrimination of an isolate with a double locus variation, and iii) full splitting of the largest cluster defined by rflp. additionally, in one of these clusters with no correlation, miru- grouped with high correlation another isolate that showed 95% is band similarity. miru- also defined a new cluster between two isolates with low is band similarity. in our previous study, miru- defined up to eight clusters not assessed by rflp. when spoligotyping was applied as a second-line method in discrepant cases, all the isolates grouped by miru- that were unclustered by is6110-rflp were split. cluster r which was subdivided by miru- was split by spoligotyping. ii) miru- analysis of a population-based sample after observing that miru- improved the miru- data, we decided to re-evaluate miru- efficiency with an independent sample. unlike the first convenience sample, the new one included all the mtb isolates in almería during the period 2003– in total, isolates were genotyped, corresponding to autochthonous cases and immigrant cases. the most widely represented countries of origin in the study population were morocco, romania, and mali. correlation analysis when the rflp-clustered cases from almería were analyzed by miru- full or high correlation was obtained in 83% of the clusters. miru- showed no correlation in the remaining five rflp clusters, and discriminated 12/ isolates grouped in them. the discrepancies were as follows: i) one isolate clustered by rflp showed a dlv; ii) splitting of two rflp clusters with isolates showing differences in more than four loci: and iii) three and four isolates clustered by rflp showed differences in two to eight miru- loci. additionally, miru- subdivided rflp cluster into three different miru types. in order to understand the significance of the concordances and discrepancies between the clusters defined by rflp and miru- we evaluated the epidemiological evidence found for these clusters. therefore, we labeled the clusters according to a gradient of evidence of epidemiological links between their cases. for the rflp clusters with full or high correlation with miru data, we obtained epidemiological data in 22/ clusters. of these, epidemiological evidence was detected in 16/ clusters. for the patients included in these rflp clusters, we detected epidemiological links for 43/ cases with available epidemiological data. for the remaining cases no epidemiological evidence was found. epidemiological data were available for four of the five clusters that were split by miru- and no links were found for all the representatives which were discriminated by miru-. a review of these cases reveals the following: a) in cluster, the only isolate without epidemiological links was clearly split by miru-. this corresponded to an unrelated ghanian patient in a cluster of spanish cases; b) in a further two clusters involving two cases each, miru- showed discrepancies in five and six loci respectively, and once again, no epidemiological connections were found; c) no epidemiological links could be found for the remaining cases involved in cluster; d) cluster, involving different nationalities, was also split by miru- according to the geographic origin/socio-cultural background of the cases. no links were found between the multinational patients grouped in rflp cluster but links were found between the cases in at least one of the split subgroups. discussion during the last few years, the search for an alternative to rflp, has led to the development of a pcr-based technique, miru-vntr. this technique has proven to be fast and easy to perform, and it allows the direct exchange of data between laboratories. different combinations of miru and other vntr loci have been published, but most of the studies have focused on a set of miru loci. this format offers a higher discriminatory power than other pcr-based genotyping techniques. other studies, however, have revealed lower discriminatory power and a low correlation with rflp data, which improved if complemented with spoligotyping. in a previous study, we focused on isolates with high-copy-number fingerprints, to try to evaluate miru- in a more challenging situation than that of studies performed in circumstances that could favor miru, e.g. high proportion of low-copy-number rflp fingerprints, low variability of circulating strains due to the prevalence of specific genetic families, etc. we found that, compared with rflp, miru- overestimated candidates for recent transmission, by grouping a higher number of isolates and defining a higher number of clusters. furthermore, 53% of miru- clusters showed a low or no correlation with rflp data. these data urge caution when considering substituting rflp with miru- and suggest that incongruent study conclusions could be due to geographic differences in the genotypic composition of circulating strains. supply et al have recently published a proposal for a new refined set of miru-vntr loci. when the advanced set was tested on isolates including representatives of the main mtb lineages, a subset of loci was considered to have the highest efficiency because it contained 96% of the resolution obtained with the whole 24-loci set. however, the miru- design has received little attention; therefore, additional studies in a variety of socio-epidemiological backgrounds are necessary to fully evaluate the usefulness of this new strategy. this was the aim of our analysis. we began by evaluating the new 15-loci miru version with the same convenience sample that was used to evaluate miru- the genetic lineages which were over-represented in this sample were lam and haarlem, and only three beijing strains were found, thus ruling out enrichment in strains which have been found to be poorly discriminated by miru-. miru- showed higher discriminatory efficiency by grouping % fewer isolates than miru- and showing a higher hgdi value. this increase in resolution was due to the nine new loci whose hgdi values were higher than, whereas only three in the previous set achieved equivalent values, which justified the selection of these loci in the new advanced design. with regard to the correlation with rflp, our previous study revealed that only out of rflp clusters fitted well with miru- data, and it was necessary to include spoligotyping to improve these results. miru- alone increased the number of clusters with good correlation with rflp data to as many as 14/ in this study, we considered full or high values as indicative of a good correlation between miru- and rflp. correlation was considered to be high when miru-grouped isolates sharing rflp patterns showed high is band similarity, or isolates sharing rflp types showed subtle differences in the miru analysis. this decision was supported by studies that found epidemiological links for clusters including isolates with subtle differences in rflp isolates and by reports that some miru loci have faster molecular clocks, which could even lead to slvs between isolates linked to an ongoing transmission event. although good correlation was generally found in our convenience sample, no correlation with rflp was observed for three rflp clusters. miru- added two isolates in the r rflp cluster, which showed only low is band similarity percentages with the other clustered isolates. the spoligotypes for these two isolates were also different from the r representative pattern, although they belonged to the same genetic lineage. this could be due to the fact that miru- misassigned these isolates and we were probably observing a genotypic convergence phenomenon. on the other hand, the rate of miru changes is relatively lower than that of is6110-rflp and spoligotyping, so it could also be possible that the miru type in this case is shared by different representatives of the t lineage. in the remaining two noncorrelated clusters, we observed that miru- split them to a different extent, and we detected differences among isolates sharing an rflp type. in addition, the largest rflp cluster was fully split by miru- with differences in 2– loci for all the isolates. splitting of rflp clusters by miru- has been found in clusters involving strains with low-copy-band fingerprints, which is somehow expected, although in this case the strain had seven bands. splitting of rflp clusters, even those with high-copy-band strains has been described elsewhere, and these findings urge caution in assuming certainty for all defined clusters. in order to fully understand the meaning of the concordances and discordances between rflp and miru- data, we used an unselected population-based sample with available epidemiological data to interpret potential discrepancies. we chose the mtb isolates cultured in the province of almería during a -year period to ensure quality in the clustering assignation and to increase the observation time of other studies. we decided not to consider the orphan cases and to focus exclusively on the analysis of the rflp clusters detected in this population sample because they have epidemiological value, as they are used as markers for recent transmission. the ability of miru- to classify as orphan those cases unclustered by rflp is being evaluated in an ongoing prospective population-based study. as with our findings in the convenience sample, the correlation between miru- and rflp genotypes was good; full or high correlation with the rflp data was detected for of the clusters genotyped by miru- we found epidemiological links in % of the clusters and in % of the cases with available epidemiological data. these percentages of "epidemiologically proved" clusters agree with those of other studies, and it is well known that higher values are only obtained if highly refined epidemiological surveys are followed up. it is noteworthy that almost all the clusters without epidemiological links involved two cases, and it is generally assumed that the detection of links in transmission chains involving a reduced number of cases produces a lower yield. it is interesting that, of the four rflp clusters with epidemiological information available which were split by miru- no links were found for all the representatives which were discriminated by miru- this suggests that miru- was able to detect some cases that were falsely clustered by rflp. moreover, in at least one case, miru- redefined epidemiologically-consistent subclusters within a common rflp cluster which was not epidemiologically supported.  the data obtained by miru- in this study and elsewhere suggest that the new design is very efficient at assigning clusters confirmed by epidemiological data. if we add this to the speed with which it provides results, miru- could be considered a suitable tool for real-time genotyping. this could be essential in study populations such as ours, which is undergoing an epidemiological transformation due to the marked increase in tuberculosis among immigrants. the complexity of this situation reduces the efficiency of standard epidemiological approaches and demands new strategies such as miru- to allow rapid identification of clusters. 
during the last few years, pcr-based methods have been developed to simplify and reduce the time required for genotyping mycobacterium tuberculosis by standard approaches based on is6110-restriction fragment length polymorphism. first, we analyzed the same convenience sample that had been used to evaluate miru- in a previous study, and the new 15-loci version offered higher discriminatory power and better correlation. if we add this to the speed with which it provides results, miru- could be considered a suitable tool for real-time genotyping. to understand the meaning of the discrepancies still found between miru- and rflp, we analyzed the epidemiological data for the clustered patients. the data obtained by miru- suggest that the new design is very efficient at assigning clusters confirmed by epidemiological data. in most cases, splitting of rflp-clustered patients by miru- occurred for those without epidemiological links, and rflp-clustered patients with epidemiological links were also clustered by miru- suggesting a good epidemiological background for clustering defined by miru- second, we evaluated miru- on a population-based sample and, once again, good correlation with the rflp clustering data was observed. of these, miru-12-vntr has been considered a good alternative.
 during the last few years, pcr-based methods have been developed to simplify and reduce the time required for genotyping mycobacterium tuberculosis by standard approaches based on is6110-restriction fragment length polymorphism. of these, miru-12-vntr has been considered a good alternative. nevertheless, some limitations and discrepancies with rflp, which are minimized if the technique is complemented with spoligotyping, have been found. recently, a new version of miru-vntr targeting loci has been proposed to improve the miru- format.  we evaluated the new miru- tool in two different samples. first, we analyzed the same convenience sample that had been used to evaluate miru- in a previous study, and the new 15-loci version offered higher discriminatory power and better correlation. second, we evaluated miru- on a population-based sample and, once again, good correlation with the rflp clustering data was observed. to understand the meaning of the discrepancies still found between miru- and rflp, we analyzed the epidemiological data for the clustered patients. in most cases, splitting of rflp-clustered patients by miru- occurred for those without epidemiological links, and rflp-clustered patients with epidemiological links were also clustered by miru- suggesting a good epidemiological background for clustering defined by miru-  the data obtained by miru- suggest that the new design is very efficient at assigning clusters confirmed by epidemiological data. if we add this to the speed with which it provides results, miru- could be considered a suitable tool for real-time genotyping. 