hepatitis b virus ( hbv ) infection is an important global health problem [ 1 , 2 ] especially in asia , africa , southern europe , and latin america .
about 2 billion people are infected with hbv worldwide [ 47 ] , among them , 400 million suffer chronic hbv infection . in brazil ,
the hbsag positivity ranges from 1.6% to 8.5% , corresponding to approximately 3 to 16 million infected individuals [ 813 ] . the residual hbv risk in screened blood transfusion ranges from 1  :  10,700   to 1  :  62,734   and is markedly higher compared to europe , north america [ 17 , 18 ] , australia , and japan .
further , the identification of blood donors with occult hbv infection ( obi ) individuals negative for hepatitis b antigen ( hbsag ) with detectable circulating hbv dna has created concern related to the safety of the blood supply [ 21 , 22 ] .
hepatitis b virus continues to offer the greatest risk of transfusion - transmission infection despite hbsag screening of blood donations .
residual risk of hbv transfusion - transmission results from occult hbv infection , window period donations , and possible escape variants .
there is a need to develop cost - effective algorithms using available serological and nat methods to increase blood safety without compromising the availability of blood .
false - positive results in anti - hbc testing and false - negative results in nat methods can hamper the identification of occult hbv infection .
in anti - hbc testing , false - positive result to anti - hbc are attributed to nonspecific reactions associated with competitive anti - hbc enzyme immunoassay , possiblly due to iga or igm - related molecules produced from nonspecifically activated hbv - specific b lymphocytes or cross - reactivity with other serum components . in donor testing using nat pooling methods , the low viral load found in most obis may affect blood safety and screening algorithms in which confirmation depends upon dna detection .
the reasons for low hbv dna levels in absence of detectable hbsag in obi remain undefined , but it is conjectured that both host and viral factors are important in suppressing viral replication and maintaining control of the infection [ 5 , 6 , 23 ] .
t - cell mediated responses contribute to the control of viral replication , protect against reinfection , and may lead to spontaneous resolution of infection in obi .
patients who successfully clear the virus present a t - cell response to hbsag , core and polymerase proteins [ 25 , 26 ] . additionally , increased hbcag- and hbeag - specific t - helper cell responses are also seen in patients with viral clearance .
the t - cell response in individuals with an anti - hbc only profile showed a typical protective memory when compared to anti - hbc - negative blood donors that have not been exposed to hbv .
cellular and humoral immune response pressure against hbv envelope proteins are the principal mechanisms originating obi . in this context , the aim of this study was to develop algorithms / methods to confirm anti - hbc test results .
a new method is proposed : detection of hbcag - specific t - cell responses .
the blood donor population from colsan - associao beneficente de coleta de sangue ( so paulo , brazil ) was tested between january 2010 and october 2011 .
we selected 2,126 donors found positive only for anti - hbc from a total of 125,068 donations . during the process , obi donors were identified , and their hbcag - specific t - cell response was analyzed and compared to donors with chronic ( hbsag positive ) and recovered ( anti - hbc only ) infection . as control subjects , we enrolled 100 healthy blood donors that had no reactivity for hbv including hbv pcr , and who were nonreactive for hepatitis c virus ( hcv ) , human immunodeficiency virus ( hiv ) , human t - lymphotropic virus ( htlv ) , syphilis , and chagas diseases .
all subjects gave their informed consent , and the study was approved by the unifesp ethical committee .
blood units collected at colsan - associao beneficente de coleta de sangue ( so paulo , brazil ) were screened for the presence of serum anti - hbc and hbsag using a commercial enzyme linked immune - assay ( elisa ) ( biomrieux , france ) .
hbe , anti - hbe , anti - hbs , and core igm testing was performed using the elfa system ( biomreiux , france ) .
all the samples were also screened by elisa for possible hcv , htlv , hiv infections , syphilis , and chagas diseases ( elisa ) ( biomrieux , france ) .
serum samples were stored at 20c and thawed immediately before use . for detection of hbv dna , dna was extracted and subsequently subjected to a commercial test for hbv - dna ( cobas ampliprep / cobas taqman hbv test , v2.0 ) with a range from as low as 20  iu / ml to as high as 1.7  e  +  08  iu / ml . peripheral blood mononuclear cells ( pbmcs )
were freshly isolated from heparinized blood by ficoll - hypaque density gradient centrifugation with lymphoprep ( axis - shield , oslo , norway ) as previously described .
subsequently , cells were resuspended in complete rpmi-1640 medium ( gibco , invitrogen , beijing , china ) , which contained 10% heat - inactivated fetal bovine serum ( gibco , invitrogen , australia ) , 2  mm l - glutamine , 100  u / ml penicillin , and 100  g / ml streptomycin .
t - cells were cultured at 10 cells per ml in rpmi-1640 ( gibco , invitrogen , beijing , china ) supplemented with 10% heat - inactivated human ab serum , 2  mmol / l l - glutamine , 100  u / ml penicillin , and 100  g / ml streptomycin and incubated overnight at 37c and 5 percent co2 .
recombinant hbcag ( rhbcag ) expressed in escherichia coli are based on cloned hbv dna of hbv genotypes a , b , c , d , and f was purchased from diasorin , saluggia , italy .
seventeen overlapping 20-mer peptides covering the entire hbv core sequence ( amino acids 1 to 183 ) were synthesized with a multiple - peptide synthesizer using standard 9-fluorenylmethoxy carbonyl chemistry ( syro , multisyntech , bochum , germany ) .
recombinant - hbcag ( 10  g / ml ) was added , and cells were further incubated for 5 days .
control cultures included 2  g / ml phytohemagglutinin ( pha ) , medium alone , and recombinant hcv ns5 protein , ( american research products , belmont , ma ) .
results were expressed as counts per minute ( cpm ) for pha - stimulated cultures and as stimulation index ( si ) , for example , the ratio between median cpm in the hbcag - containing cultures and median cpm of control hcv ns5-containing cultures .
the 99th percentile of hbcag - specific si in 10 healthy blood donors was 2.01 . for practical purposes ,
the antigen used for the human ifn- elispot assays was the same used in hbcag t - cell response ( rhbcag ) .
multiscreen - ip 96-well plates ( millipore , billerica , ma ) were coated overnight at 4c with 10  g / ml anti - human ifn- monoclonal antibody ( 1-dik ; mabtech , sweden ) .
plates were washed seven times with dpbs and blocked with rpmi 1640 supplemented with 10% fetal bovine serum for 2 hours at room temperature . freshly isolated or frozen - thawed
after washing , 50  l of 1  g / ml biotinylated monoclonal antibody to inf- ( 7-b6 - 1 ; mabtech , sweden ) was added to each well and incubated for 2 hours at room temperature ; plates were subsequently washed seven times and 50  l per well of 1  g / ml streptavidin - alkaline phosphatase ( mabtech , sweden ) was added , and plates were further incubated for 1 hour at room temperature .
plates were washed seven times , and 100  l per well of bcip / nbt ( diluted by distilled water ; zymed bcip / nbt substrate kit , invitrogen , camarillo , ca ) was added .
after 10 minutes , the colorimetric reaction was stopped by distilled water and washed three times with distilled water .
plates were air dried , and the immunospot s4 macro analyzer ( cellular technology ltd , usa ) was used for spot counting .
results were expressed as numbers of spot - forming cells ( sfc ) per 10 pbmcs .
the number of specific ifn--secreting cells was calculated by subtracting the value of the unstimulated control from the value of the stimulated sample .
the positive control consisted of pbmcs stimulated with 10  g / ml of pha , and the criteria for a positive response for the ex vivo elispot assays were more than 5  sfc per well and more than twice the number of sfc than the unstipulated control well .
therefore , in this study , more than 20  sfc per 10 pbmcs would be a positive response .
comparison of paired data from patients at different times of followup was performed by wilcoxon 's signed - rank test .
correlation between hbv dna and hbc - specific si was assessed by spearman 's  test .
a total of 2,126 of 125,068 donations , ( 1.7% ) were positive for anti - hbc , and none of them had detectable hbsag in the serum .
reactivity only hbsag was found in 125 ( 0.1% ) donations , and hbsag plus anti - hbc was observed in 563 ( 0.45% ) donations .
both hbsag and anti - hbc were absent in 122,254 ( 97.75% ) . among 2,126
( hbsag/anti - hbc+ ) only 21 samples had detectable hbv dna . among the 125 samples with primary infection ( hbsag+/anti - hbc ) , 27 samples had detectable hbv dna . among 563 samples with chronic infection ( hbsag+/anti - hbc+ ) , 438 had detectable hbv dna ( figure 1 ) . between january 2010 and july 2010 ,
when hbv dna screening was performed using minipools of 6 samples , we did not observe positive samples in a total of 12.000 screened samples from blood donors with hbsag and anti - hbc negative .
in the 21-month study period , 21 blood donors with anti - hbc alone profile were identified as obi ( 1 in each 5955 donors ) . in the same period ,
the number of blood donors with reactivity for hbsag and anti - hbc was 113 ( 1 in each 1106 blood donors ) . among the group of 2,126 samples with anti - hbc only , test reactivity ( represented as co / s ) ranged between 0.008 and 0,975 .
the samples were divided in two groups based on a threshold of co / s = 0.1 and further tested for hbv dna .
21 samples had hbv dna detectable and co / s  0.1 and then was classified as obi , and 2,105 anti - hbc only segregating into 1,293 anti - hbc co / s  0.1 and 812 co / s < 0.1 ( figure 1 ) .
the specificity of the anti - hbc response in these two groups of anti - hbc only samples was investigated further by analyzing the hbcag t - cell response through t lymphocyte proliferation and inf- response to specific and nonspecific stimuli .
the proliferative response of peripheral blood lymphocytes to nonspecific ( pha ) and specific stimuli ( hbv core antigen ; hcv ns5 antigen ) was analyzed in hbv carriers , obi , healthy donors , and two groups based on a threshold of co / s  0.1 and co / s  0.1 .
the relevant finding was the observation that anti - hbc only subjects with co / s  0.1 ( n = 1.293 ) did not have either hbcag - specific t - cells or detectable hbv dna in any analyzed samples .
false - positives  for anti - hbc , corroborated by the absence of reactivity to anti - hbc in the subsequent donations . the median signal value obtained in the  false - positive  group was equal to healthy blood donors ( si < 3,0 ) .
all anti - hbc only with co / s  0.1 ( n = 833 ) presented hbcag - specific t - cell responses ; however , 21 out of 833 presented detectable hbv dna ( obi ) .
the median si obtained in hbv carriers and the obi group was 13,6 and 12,2 , respectively .
the subjects ( n = 812 ) with co / s  0.1 and hbcag t - cell response and undetectable hbv dna are classified as spontaneous hbv resolvers .
spontaneous hbv resolvers showed a large peripheral blood mononuclear cell ( pbmc ) response ( si = 36,2 ) to hbcag when compared to hbv carriers and obi ( si = 12,2 ) ( p < 0.001 ) ( figure 2 ) . in the subset of 21 obi subjects , 9 collected samples viral load
was quantified in all donations and fluctuated between < 10 and 118  iu / ml , whereas in three samples hbv dna was not detected , but hbcag t - cell response remained positive in all the study period ( figure 3 ) .
elispot - inf- assays were positive in hbc only with co / s  0.1 , obi , and positive control .
the magnitude of t - cell responses to hbcag in hbc only with co / s  0.1 ranged from 1,020 to 1,628  iscs/10 pbmcs , whereas among obi ranged from 132 to 548  iscs/10 pbmcs , among positive controls from 160 to 582  iscs/10 pbmcs , and among hbc only co / s > 0.1 and healthy blood donors from 8 to 15  iscs/10 pbmcs .
the frequency of positive ifn- elispot responses in spontaneous hbv resolvers indicated a higher chance to have demonstrable hbv - specific t - cell responses than among positive controls ( p < 0.001 ) and among obi ( p < 0.001 ) ( figure 4 ) .
serological markers of hbv exposure or vaccination ( anti - hbs ) were observed in 9.1% ( 3/40 ) from primary infection ( hbv carriers ) ; 21% ( 21/100 ) of healthy donors ; 23,5% ( 303/1293 ) of hbc only with co / s  0.1 ; 32.4% ( 263/812 ) of hbc only with co / s  0.1 ; and 10,2% in obi .
reactivity to igm anti - core was observed in 2.5% ( 1/40 ) and 5.0% ( 2/21 ) of hbv carriers and obi , respectively .
hbe antigen was observed in all blood donors with obi and in hbv carriers but not in healthy donors and hbc only .
however , 10.5% ( 2/21 ) of obi and 12.5% of ( 5/40 ) hbv carriers showed anti - hbe - positive . in healthy donors and
hbc only , no reactivity to anti - hbe and core igm was observed ( figure 5 ) .
hepatitis b virus presents a higher residual risk of transmission by transfusion than hepatitis c virus ( hcv ) or human immunodeficiency virus ( hiv ) , especially in countries of south america , like brazil , where the prevalence of hbsag in the general population ranges from 2 to 20% . in this study we found hbv dna detectable in 21.6% of blood donors with primary infection ( hbsag ( + ) anti - hbc (  ) and 77.8% of chronic blood donors ( hbsag ( + ) anti - hbc ( + ) ) .
these data are consistent with the observation by several authors about hbv persistence in chronic infection [ 29 , 30 ] .
the persistence of hbv dna was observed like obi in 0.98% of the 1.7% of blood donors which were anti - hbc positive , corroborating results of previous studies   that observed 3.3% obi subjects into of 5.6% of blood donors with anti - hbc alone profile .
the same assays for detection of hbsag and anti - hbc were used in both studies , and the detection limit in hbv dna pcr was similar . in the general blood donor population ,
the prevalence of obi in this study was 1 in 5,955 ( 0.017% ) , and , in the same period , hbv carrier rate was 1 in 1,106 blood donors .
the obi among blood donors varies from country to country in different parts of the world .
for example , in the european countries such as poland , italy , spain , and germany , the obi prevalence rates of 0.006% , 0.22% , 0.05% , and 0.0006% , respectively , have been reported by candotti et al . .
in addition , it seems that the prevalence of obi in blood donors has a regional variance , not only internationally but also nationally .
this national discrepancy can be observed in brazil , where hbv prevalence differs regionally , varying from 27% in the north east to 275.6% in north west . in this context ,
testing for anti - hbc is important , principally in the detection of cases where hbsag is negative and hbv dna detectable ( obi ) .
however , a high frequency of reactivity to anti - hbc is observed in brazil ranging from 1.0% to 5.0% in northeast , midwest , southeast , and south of brazil but can reach alarming levels of 75.6% in the amazonia region . in this study , we observed a 1.7% of frequency of reactivity to anti - hbc in the absence of hbsag , according with frequency found in southeast of brazil .
however , the presence of hbv dna observed was low representing only 0.98% of the 1.7% of anti - hbc only blood donors .
these data raise the question  what is the meaning of anti - hbc reactivity in absence of detectable hbv dna in blood donors ?  in the serological routine in blood bank , it is not uncommon to find isolated reactivity for anti - hbc and hbv dna undetectable in first donation followed by nonreactive anti - hbc results in the next donation , suggesting that the initial result was false - positive .
the determination of the frequency of true anti - hbc only in blood donors is very important to blood safety strategy and to indentify recovered infection , escape mutants , and obi .
algorithms including multiple successive assays are necessary but not sufficient to identify true reactivity to anti - hbc . to overcome this difficulty ,
japanese blood banks have set up an anti - hbc cutoff agglutination title of 32 and , below this level , donations are considered noninfectious ( presumably false - positive or with undetectable anti - hbs ) .
actually japanese blood bank defers all donors with any reactive result for anti - hbc . in this study we had relevant findings about the correlation between threshold ( co / s ) values in anti - hbc competition assay and hbv dna detection . among anti
- hbc only subjects with detectable hbv dna , all had co / s  0,1 . however , among blood donors with co / s > 0.1 , none had detectable hbv dna .
distinguishing in this group between hbv exposure and false - positive to anti - hbc appears to be defined by the hbcag t - cell response .
we found that , among the anti - hbc only group with co / s < 0.1 , and in the presence of hbv dna , all donors had a response for hbcag t - cell in all subjects that we classified as occult hbv infection . in anti - hbc
only co / s > 0.1 and in the absence of hbv dna when there was an hbcag t - cell response , we classified these individuals as having spontaneous hbv clearance , and in absence of hbcag t - cell response we classified them as false - positive to anti - hbc .
these findings are corroborated by the difference in magnitude of hbcag t - cell response between the groups ( hbv carriers and healthy groups ) , obi subjects with serial collected samples and elispot - inf- data .
we found a large hbcag t - cell response for spontaneous hbv clearance , positive controls , and obi , all subjects with previous hbv exposure when compared healthy group .
we observed hbv dna fluctuation in 9 cases of obi with serial collected samples ; however , the presence of hbcag t - cell response was observed in all serially collected samples . in the follow - up of cases of obi , we observed fluctuation of viral load but the hbcag t - cell response was always positive .
this is very important to the identification of obi because it eliminates several problems that are due to false results in hbv dna detection , like viral load , assay sensitivity , fluctuation of viral load , repeat of the hbv dna detection , and other factors ( mutant , variants , mosaic , and quasispecies ) . in the other 11 remaining obis , we observed linear hbv dna detection in two subsequent donations .
these blood donors will be followed for more two donations , and the results will be analyzed . in transfusion medicine , assays to detect anti - hbc
hbcag specific t - cell response showed high sensibility in distinguishing  true positive  for  false - positive  to anti - hbc .
the results observed in hbcag- t - cell response corroborate with elispot - inf- data . we observed that spontaneous hbv resolvers presented higher frequency of positivity in this assay when compared to hbv carrier , healthy donors , anti - hbc false - positive results , and obi ( p > 0.001 ) .
in addition , the magnitude of t - cell responses to hbcag in hbv spontaneous resolvers was clearly higher when compared to hbv carriers , healthy donors , anti - hbc false - positive results , and obi ( p > 0.001 ) .
these data suggest that antibody titers indeed correlated with hbcag- t - cell response and elispot - inf-. the presence of immune response was found in elispot - inf- in spontaneous hbv resolvers , and obi and hbv carriers are according to findings of zerbini et al . who reported that the presence of memory t - cell response in anti - hbc positive individuals with obi could be important for maintaining viremia under control but persistent .
despite all the evidence pointing to efficiency of hbcag t - cell response to confirm anti - hbc result in the competition assay , the discrepancy of hbcag t - cell response in the obi and positive groups when compared to findings of zerbini and cols in 2008 and bes and cols in 2012 was an important point to elucidate .
we suggest that the discrepancy between our results and these researchers could be principally due to process of selection of study groups , because if we introduce the group classified for us as false - positive to anti - hbc , the magnitude of hbcag t - cell response is equal between obi and positive control , if it is considered to be the median of stimulation index found in true positive and false - positive to anti - hbc , agreeing with zerbini and bes studies .
hbcag t - cell response proved to be an important test to eliminate false - positive results for anti - hbc and that could be used in the research and in blood bank routine . in brazil
, the cost of discarding anti - hbc false - positive donation is high , and the cost to use hbcag t - cell response in the laboratory that had previous laminar flow cabinet and co2 incubator is low , five times smaller than the cost of false - positive donation .
we can not forget the stigma in the donor to receive a result with positive serology and indirect cost for the public health service to conduct additional tests in order to confirm the positive anti - hbc result .
the presence of hbcag t - cell response in the absence of hbv dna in cases of obi suggests that hbcag t - cell response could be a complementary or in specific cases an alternative to nat .
even with detecting other serological markers of hepatitis b like core igm , hbeag , anti - hbe and anti - hbs , the identification of obi in anti - hbc only is difficult .
firstly , we investigated the concentration of anti - hbs in obi , spontaneous hbv resolvers , and hbv carrier , and we observed that frequency was 10.2% , 32,4% , and 9.1% , respectively .
several studies showed that some individuals with obi or hbv , who have recovered from hbv infection , produce neutralizing anti - hbs . in these same individuals
we may observe continuous hbv dna replication at low levels that are detectable for years in the liver , peripheral blood mononuclear cells or serum [ 32 , 33 , 3639 ] .
the frequency of anti - hbs found in spontaneous hbv resolvers was higher than hbv carriers and obi probably due to important immunity role of anti - hbs in protection against hbv infection .
the presence of anti - hbs in obi has been reported by many authors with frequency varying from 0.5% to 15% still tested positive for serum hbv dna , through at a very low titer which is in accordance according with our findings in this study .
another serological marker studied was anti - hbe , an important marker of hbv chronic carriage .
we observed in obi ( 10.5% ) and hbv carrier ( 12.9% ) the presence of anti - hbe in lower concentration when compared to levels of anti - hbc .
this data could be explained by the variable proportion of samples with hbc alone profile , in which anti - hbe is detected .
although , with lower titers of anti - hbe compared to anti - hbc , there comes a point when the former is no longer detectable , leaving the latter as the only serological marker of infection .
hbeag was observed in all blood donors with viremia , including obi and hbv carrier .
igm anti - hbc is the first antibody detectable in acute hbv infection , which is usually detected within 1 month after appearance of hbsag .
the presence of igm anti - hbc with high index value usually indicates a recent hbv infection , and this antibody usually disappears within 6 months .
however , the index value of igm anti - hbc may increase to levels usually detectable in acute infection in 1020% of chronic hepatitis b patients with acute exacerbation or hepatitis flare that often leads to a misdiagnosis of acute hepatitis b . in our study
we observed that the frequency of high index value of igm anti - hbc was 5.0% in obi and 2.5% in hbv carrier , suggesting a recent hbv infection .
our data suggest that hbcag - specific t - cell response could be used to confirm anti - hbc serological status distinguish previous exposure to hepatitis b virus and anti - hbc false - positive .
 
 background . during routine donor screening in the blood bank , it is not uncommon to find isolated reactivity for anti - hbc in the absence of detectable hbv dna in a first donation but absence of reactivity to anti - hbc in subsequent donations , suggesting a false - positive result for anti - hbc . study design and methods . 
 the blood donor population was screened between january 2010 and october 2011 . 
 we selected 2,126 donations positive only for anti - hbc from a total of 125,068 donations . during the process 
 , obi donors were identified , and their hbcag - specific t - cell response was analyzed and compared to donors with chronic ( hbsag positive ) and recovered ( anti - hbc only ) infection . 
 we analyzed correlations between signal levels ( co / s ) in the competitive assay for anti - hbc and hbv dna detection . 
 results . in the 21-month study period , 
 21 blood donors with anti - hbc alone were identified as obi ( 1 in each 5955 donors ) . 
 the relevant finding was the observation that anti - hbc only subjects with co / s  0.1 did not have either hbcag - specific t - cells or detectable hbv dna and obi subjects presented with co / s  0.1 and hbcag t - cell response . in the subset of 21 obi subjects , 9 donors remained positive for hbcag t - cell response after four collections . in all 9 samples , 
 we observed hbv dna fluctuation . conclusion . 
 our data suggest that hbcag - specific t - cell response could be used to confirm anti - hbc serological status , distinguishing previous exposure to hepatitis b virus from anti - hbc false - positive results . 