subjects - the sample comprised 32 brazilian healthy volunteers 
 ( donors ) selected from students and workers from the advanced laboratory of public 
 health , oswaldo cruz foundation ( fiocruz ) , salvador , state of bahia , brazil , and the 
 immunology laboratory , heart institute , university of so paulo ( usp ) , so paulo , state 
 of so paulo , brazil . they were divided into 
 two groups according to the tst response : latent mtb infection ( 16 subjects , 
 tst - positive ) and uninfected controls ( 16 subjects , tst - negative ) , with a mean age of 35 
  11 years ( tst - positive ) or 30  7 years ( tst - negative ) . the tst was performed 
 according to the mantoux method with ppd , following the manufacturer s instructions 
 ( statens serum institute , denmark ) . tst was considered positive when the induration was 
  10 mm after 48 h of ppd application . all of the individuals gave their written 
 informed consent to participate in this study , which was approved by the institutional 
 review boards of the school of medicine , usp ( protocol 250/04 ) and fiocruz ( protocol 
 51/2004 ) . peptide selection - sequences of three important antigens expressed by 
 mtb were used : groel2 protein , accession p9wpe6.1 ( fleischmann et al . 2002 ) , pbp-1 , accession p9wgu0.1 ( andersen & hansen 1989 ) and 19 kda antigen , accession s02753 
 ( ashbridge et al . 1989 ) . these sequences were 
 screened by tepitope and we predicted seven epitopes that were used in the elispot 
 assay . the seven peptides were predicted to bind to at least 12 out of the 25 hla - dr 
 molecules in the tepitope matrix , as previously described ( bian et al . they correspond to an inner nonamer core selected 
 as the hla - binding motif with flanking amino acids added when possible ( e.g. , conserved 
 flanking sequences ) at either or both the n and c - terminal ends , to increase the 
 efficiency of in vitro peptide presentation to cd4 t cells ( bian et al . peptide synthesis - peptides of the mtb proteins were synthesised by 
 solid phase technology using 9-fluorenylmethoxycarbonyl strategy , with the c - terminal 
 carboxyl group in amide form . peptide purity and quality were assessed by reverse - phase 
 high performance liquid chromatography and mass spectrometry , as described elsewhere 
 ( atherton & sheppard 1989 ) . the peptides 
 were dissolved in dimethyl sulfoxide ( dmso ) for stock solutions of 25 mg / ml . 
 pbmc separation - pbmc suspensions were prepared from 50 ml heparinised 
 venous blood by density gradient centrifugation using ficoll - hypaque , 1.077 the cells were washed twice with phosphate - buffered saline ( pbs ) 
 ( gibco , uk ) and were kept frozen at -196c in liquid nitrogen in foetal calf serum ( fcs ) 
 containing 10% dmso ( merck , germany ) until testing . after thawing , the pbmcs were 
 suspended in rpmi-1640 ( gibco ) , supplemented with 200 mm glutamine , 100 u / ml penicillin , 
 100 u / ml streptomycin and 10% fcs , 100 mm non - essential amino acids and 100 mm 
 pyruvate . ifn- elispot assays - the ifn- elispot assay was performed as 
 described previously ( sun et al . briefly , cryopreserved pbmcs ( 10 cells / well ) were incubated with 5 m of 
 each peptide on a 96-well multiscreen elispot plate ( multiscreen maips4510 ; millipore , 
 usa ) previously coated with 50 l / well of capture anti - human ifn- monoclonal antibody 
 ( mab ) diluted 1/1,000 ( igg1/b - b1 ; diaclone , france ) in pbs ( 1 l / ml of mab ) at 4c 
 overnight . the remaining peptide binding 
 sites were blocked with 100 l rpmi/10% fcs for 2 h at 37c . the medium was removed just 
 before the addition of cell suspensions ( 100 l ) and stimulating agents ( 100 l ) . the 
 mtb peptides ( 5 m ) were added to each well of the plates . pbmcs incubated with 0.5 g / ml phytohemagglutinin 
 ( sigma , france ) or rpmi/10% fcs served as the positive and negative controls , 
 respectively . at the end of the incubation , the cells were removed from the elispot 
 plates by washing nine times : three times with pbs , three times with pbs containing 
 0.05% tween-20 ( sigma ) and three times again with pbs . spots were detected after the 
 addition of the second biotinylated anti - human ifn- mab ( b - g1 ; diaclone ) ( 100 l / well , 
 4 h , 37c in 5% co2 ) , diluted 1/500 in pbs - bovine serum albumin ( pbs - bsa ) 
 ( gibco ) ( 1 l/500 l of biotinylated antibody ) . the plates were washed three times with 
 pbs , followed by streptavidin - alkaline phosphatase conjugate ( amersham , france ) ( 100 
 l / well , 1 h , 37c in 5% co2 ) diluted 1/1000 in pbs - bsa ( 1 l / ml of 
 streptavidin - alkaline phosphatase conjugate ) , washed three times with pbs and incubated 
 with 50 l / well of chromogen substrates 5-bromo-4-chloro-3-indolyl - phosphate toluidine 
 and 4-nitroblue tetrazolium ( sigma ) , which were incubated at room temperature ( 25c ) 
 until the appearance of violet spots ( ~ 30 min ) . antigen - specific t cells , expressed as spot forming cells 
 ( sfc)/10 pbmcs frequencies , were measured using an automated 
 stereomicroscope ( ks elispot ; carls zeiss , germany ) . the sfc number was calculated after 
 subtracting the negative control values ( wells with cells in the absence of peptide ) 
 from the same subject . the positivity cut - off was  30 ifn- sfc/10 pbmcs , 
 which was above the mean  3 standard deviation of the highest response found among all 
 uninfected control subjects to any peptide ( data not shown ) . the magnitude of response 
 to each given peptide or the sum of the sfc of the total peptides was calculated as the 
 sfc/10pbmcs of peptides with responses above the positivity cut - off . comparisons of the number of ifn- sfc/10 
 pbmcs between the clinical groups were performed with a non - parametric one - way anova , 
 kruskal - wallis and dunn s test . the mann - whitney u test was used to 
 calculate the differences between the groups . three mtb antigenic proteins , groel2 , pbp-1 and 19 kda , were scanned with the tepitope 
 algorithm at a 3% threshold . seven peptide sequences predicted to bind to multiple 
 hla - dr molecules were synthesised ( table i ) . table isequences of peptides derived from mycobacterium tuberculosis 
 ( mtb ) sequences selected by tepitope algorithmpeptidehla - dr molecules 
 predicted to bind to peptide ( % ) sequencegroel2 ( 215 - 229)76dpyillvsskvstvkgroel2 ( 463 - 477)60aekvrnlpaghglnapbp-1 ( 59 - 75)84tllyplfnlwgpafherpbp-1 ( 118 - 133)88kglmnialaisaqqvnpbp-1 ( 351 - 365)64qvhfqplppavvklspbp-1 ( 352 - 371)84vhfqplppavvklsdaliat19 kda ( 127 - 141)48gshykitgtatgvdm 
 a : 3% threshold ; hla : human leukocyte antigen . next , we tested if the ifn- response to the seven peptides could discriminate between 
 tst - positive and tst - negative healthy donors . the mean size (  standard deviation ) of 
 ppd induration detected for tst - negative individuals was 0.71  2.02 mm and for 
 tst - positive individuals was 11.14  4.07 mm . pbmcs from the 16 tst - positive and 16 
 tst - negative individuals were tested in the presence of each peptide for an ifn- 
 elispot assay . the frequency of peptide recognition by the tst - positive group was 
 significantly higher than the tst - negative group . eighty - eight percent ( 14 out of 16 
 donors ) of tst - positive donors recognised at least one peptide and 31% ( 5 out of 16 
 donors ) had responses to three or more peptides , while in the tst - negative group , 31% ( 5 
 out of 16 donors ) responded to any peptides and only 12% ( 2 out of 16 donors ) responded 
 to three or more peptides ( p = 0.002 ) ( fig . . 
 table iirecognition of human leukocyte antigen ( hla)-dr promiscuous epitopes from 
 mycobacterium tuberculosis antigens by peripheral blood 
 mononuclear cells ( pbmc ) from tuberculin skin test ( tst)-positive and 
 tst - negative subjects using the interferon ( ifn)- elispot assaypeptidesifn- sfc/106 
 pbmc ( n ) 
 tst - positivetst - negative 

 p1p2p3p4p5p6p7p8p9p10p11p12p13p14p15p16n1n2n3n4n5n6n7n8n9n10n11n12n13n14n15n16pbp-1 ( 59 - 75)0 
 40 

 55 

 60 
 0 
 55 
 2500723200 
 65 
 0 
 100 
 00000010310153 
 30 
 270720pbp-1 ( 118 - 133 ) 
 30 
 10 
 155 
 0000 
 30 
 013101767250 
 60 
 00005010301037 
 87 

 43 
 2710pbp-1 ( 351 - 365 ) 
 60 

 30 

 140 

 230 
 002500230712750 
 40 
 15 
 30 
 200005735200 
 83 
 23 
 50 
 27pbp-1 ( 352 - 371)2010 
 135 

 60 
 200000 
 37 
 7 
 30 

 183 
 52030 
 95 
 5100510701072020 
 40 

 50 
 7groel2 ( 215 - 229)40 
 30 

 35 

 80 
 50000 
 30 

 50 

 33 

 30 
 0 
 40 
 05 
 30 
 50005170527032307groel2 ( 463 - 477 ) 
 40 

 60 

 110 

 130 
 250050132317 
 37 
 025 
 30 
 0050055720101000017019 kda ( 127 - 141 ) 
 55 
 10 
 95 
 102010 
 40 
 051023 
 13 123 
 05 
 37 
 025000001317151301723 
 37 
 0peptides recognised54750111021261150300000000012220sum of spots225160725560055403006750635676540267015500000000030170831370sfc/10pbmc were calculated according to the following equation : 
 ( average spots in duplicate peptide wells - average spots in duplicate 
 control wells ) x 5 . positive responses (  30 sfc/10pbmc ) are 
 highlighted by a gray background . sfc/10pbmc were calculated according to the following equation : 
 ( average spots in duplicate peptide wells - average spots in duplicate 
 control wells ) x 5 . positive responses (  30 sfc/10pbmc ) are 
 highlighted by a gray background . fig . 1 : number of mycobacterium tuberculosis ( mtb ) peptides 
 recognised by tuberculin skin test ( tst)-positive ( tst+ ) and tst - negative 
 ( tst- ) donors . peripheral blood mononuclear cells ( pbmcs ) from 16 tst - positive 
 and 16 tst - negative healthy donors were assayed with seven human leukocyte 
 antigen - dr promiscuous peptides derived from mtb protein sequences . each 
 peptide was tested individually at concentration of 5 m and the number of spot 
 forming cells ( sfc ) interferon-/106 pbmc were calculated according to the 
 following equation : ( average spots in duplicate peptide wells - average spots 
 in duplicate control wells ) x 5 . the 
 columns represent the number of tst - positive and tst - negative donors tested 
 that were negative or able to show positive responses in the elispot assay in 
 response to different numbers of peptides ( p = 0.01 , mann - whitney 
 u test ) . each of the selected peptides induced ifn- production by pbmcs from at least 31% of the 
 tst - positive donors . the frequency of responders to individual peptides ranged from 31% 
 to pbp-1 ( 118 - 133 ) , pbp-1 ( 352 - 371 ) and 19 kda ( 127 - 141 ) to 56% to groel2 ( 215 - 229 ) 
 ( fig . 2 ) . in contrast , among tst - negative 
 donors , there was no response to groel2 ( 463 - 477 ) peptide and the most frequently 
 recognised peptides were pbp-1 ( 351 - 365 ) and pbp-1 ( 352 - 371 ) , with 19% of 
 responders . 
 fig . 2 : proportion of tuberculin skin test ( tst)-positive ( tst+ ) and tst - negative 
 ( tst- ) donors that recognised selected mycobacterium 
 tuberculosis ( mtb ) peptides . peripheral blood mononuclear cells 
 ( pbmcs ) from 16 tst - positive and 16 tst - negative healthy donors were tested 
 with seven mtb derived peptides and the spot forming cells interferon-/106 
 pbmc were calculated . the columns represent the proportion of tst - positive and 
 tst - negative individuals that recognised at list one peptide ( p = 0.001 , 
 mann - whitney u test ) . the magnitude of responses to the peptides ( sum of ifn- sfc of all peptides with 
 positive responses ) was higher in the tst - positive group than in the tst - negative group 
 ( fig . the total number of ifn- sfc in 
 response to mtb peptides was 182  230 sfc/10pbmcs among the tst - positive 
 donors , compared to 36  62 sfc/10pbmcs in the tst - negative donors ( p = 
 0.007 ) . the magnitude of the responses per individual ranged from 0 - 725 sfc/106 
 pbmcs among the tst - positive individuals and 0 - 170 sfcs/10 pbmcs among 
 the tst - negative individuals ( table ii ) . the bars represent the magnitude of response of each peptide 
 ( overall sum of spots of all individuals with positive responses ) . positive 
 responses > 30 spot forming cells ( sfc ) interferon ( ifn)-/106 peripheral 
 blood mononuclear cells ( pbmcs ) ( p = 0.007 , mann - whitney u 
 test ) . the pbp-1 ( 351 - 365 ) mtb peptide induced the highest magnitude of response , 627 
 sfc/10pbmcs , followed by pbp-1 ( 352 - 371 ) , with 446 sfc/106 
 pbmcs ( fig . the highest magnitude of response was to pbp-1 ( 352 - 371 ) , with 185 sfc/106 
 pbmcs . in addition , a response to groel2 ( 463 - 477 ) was observed only in the 
 tst - positive group ( p = 0.02 ) . taking into consideration that all 32 healthy donors were subjected to bcg vaccination , 
 the combination of tst and positivity at the ifn- elispot test raised the overall 
 positivity value from 50% ( 16/32 ) to 59% ( 19/32 ) for recognition of mtb 
 antigens . our results suggest that the peptides predicted by tepitope are able to identify a 
 significant number of individuals with latent mtb infection . moreover , in our study , the 
 use of these peptides allowed us to identify individuals with latent mtb infection not 
 tracked by the tst test . the tepitope is an algorithm that uses a matrix that predicts the binding of 25 hla - dr 
 molecules . this allows the identification of epitopes with high binding affinity and the 
 ability to generate a strong t cell response . the use of this algorithm can lead to the 
 identification of a greater number of epitopes , which would display a higher sensitivity 
 and specificity for detecting mtb , as reported elsewhere ( zhang et al . the two methods used in this study to assess latent mtb infection , the tst and the ifn- 
 elispot , showed similar results in 88% of the evaluated individuals who were 
 tst - positive . these findings are similar to the published data , in which there is a 
 higher correlation of igra tests and tb when compared to tst and tb ( bartalesi et al . furthermore , it was possible to identify tst - negative 
 individuals ( 31% ) who responded to peptides predicted by tepitope . in this respect , 
 ewer et al . ( 2003 ) reported that elispot 
 offers a more accurate approach than tst for the identification of individuals who have 
 latent tb infection and could improve tb control by more precise targeting of preventive 
 treatment . however , possible antigenic cross - reactions or non - specific responses can not 
 be excluded and should be addressed . the advantage of using the igra with seven 
 promiscuous peptides is that would ensure larger number of responder individuals 
 independent of hla class ii and also a greater range or a greater number of recognised 
 peptides . to date , no fully effective methodology is available that can unequivocally 
 differentiate individuals who actually have latent mtb infection from those who were bcg 
 vaccinated or even those that have active mtb infection . the most widely and currently 
 used test for the screening of latent mtb infection remains the tst because it is 
 considered a low cost point - of - care - test ( ewer et al . therefore , it remains the 
 method of choice for screening for latent mtb infection , primarily due to a better 
 cost / benefit ratio when compared to tests based on igra ( lewinsohn et al . ( 2009 ) report that 
 immunocompromised persons infected with mtb have a high occurrence of false - negative 
 results in the tst and , in such cases , they recommend to tailor the application of igra 
 assays for latent mtb infection in different high - risk groups and advise caution in 
 their use in immunosuppressed patients . the results of the tst may show false positive results in individuals previously 
 immunised with bcg or because the population of the study has high rates of exposure to 
 mtb ( soborg et al . another relevant aspect 
 is that 70 - 80% of individuals with pulmonary tb have a diameter of the induration 
 greater than 10 mm by tst and this value is reduced in individuals co - infected with 
 hiv-1 ( idh et al . 2010 ) . this combination of 
 novel mtb cd4 t - cell epitopes should be tested in a larger cohort of 
 individuals with latent tb to confirm its potential to discriminate latent mtb infection 
 from no tb infection and may be included in elispot - based ifn- response assays to 
 identify individuals with such conditions .
the interferon ( ifn)- response to peptides can be a useful diagnostic marker of 
 mycobacterium tuberculosis ( mtb ) latent infection . 
 we identified promiscuous and 
 potentially protective cd4 + t - cell epitopes from the most conserved 
 regions of mtb antigenic proteins by scanning the mtb antigenic proteins groel2 , 
 phosphate - binding protein 1 precursor and 19 kda antigen with the tepitope algorithm . 
 seven peptide sequences predicted to bind to multiple human leukocyte antigen 
 ( hla)-dr molecules were synthesised and tested with ifn- enzyme - linked immunospot 
 ( elispot ) assays using peripheral blood mononuclear cells ( pbmcs ) from 16 mantoux 
 tuberculin skin test ( tst)-positive and 16 tst - negative healthy donors . 
 eighty - eight 
 percent of tst - positive donors responded to at least one of the peptides , compared to 
 25% of tst - negative donors . 
 each individual peptide induced ifn- production by pbmcs 
 from at least 31% of the tst - positive donors . 
 the magnitude of the response against 
 all peptides was 182  230 x 106 ifn- spot forming cells ( sfc ) among 
 tst - positive donors and 36  62 x 106 sfc among tst - negative donors ( p = 
 0.007 ) . 
 the response to groel2 ( 463 - 477 ) was only observed in the tst - positive group . 
 this combination of novel mtb cd4 t - cell epitopes should be tested in a larger cohort 
 of individuals with latent tuberculosis ( tb ) to evaluate its potential to diagnose 
 latent tb and it may be included in elispot - based ifn- assays to identify 
 individuals with this condition .