
 chronic hepatitis b virus ( hbv ) infection is a potentially life - threatening liver disease with serious complications such as cirrhosis and hepatocellular carcinoma .
the prevalence of hbv infection varies widely , with rates ranging from 0.1% to 20% worldwide .
iran is an area of intermediate endemicity and the prevalence of chronic hepatitis b infection is reported to be 1.7% in general population . 
 

it has been demonstrated that some hbs - ag negative individuals with positive anti - hbc continue to replicate hbv .
thus , the absence of hbs - ag in the blood of apparently healthy individuals may not be sufficient to ensure an hbv - free status .
anti - hbc can be detected throughout the course of both acute and chronic hbv infection .
it persists longlife after resolution , therefore the routine blood donor screening for anti - hbc has been implemented in some countries , resulting in a decrease in the risk of post - transfusion hbv infection . 
 
 in iran ,
the reported rates range from 5.1% in hamadan and 6.5% in shiraz to as high as 34% in sistan - balouchestan . nevertheless , the associated figures for isoalted anti - hbc prevalnce in other studies were as low as 0.56% in the united kingdom to as high as 76% in ghana however , most of previous studies reported isolated anti - hbc rates between 2 - 5% . despite this fact
, iranian health policy makers have not introduced anti - hbc screening among blood donors , partly because of the lack of empirical data on the benefit of introducing anti - hbc screening in a donor population with low prevalence of hbv . on the other hand ,
the indefinite deferral of donors with false - positive anti - hbc is a major disappointing side effect of anti - hbc screening in countries with low endemicity of hbv infection .
it is of utmost importance to differentiate whether isolated anti - hbc is due to false positive results or the prior exposure to hbv , because individuals with false - positive anti - hbc can benefit from vaccination and their blood can be safely transfused . to distinguish between the aforementioned conditions , evaluation of response to hb vaccination
has been proposed . 
 
 in the present study , we investigated the anti - hbs seroconversion in 2 groups of blood donors with isolated anti - hbc and their controls after hb vaccination .
we also attempted to find the predictors of non - response status among individuals with isolated anti - hbc . 

ninety individuals with isolated anti - hbc positive test and 100 healthy persons with negative serological markers of hepatitis b were recruited in the study as case and control groups , respectively . 
 

the exclusion criteria were : age > 64 years , previous hbv vaccination , organ transplantation , immunodeficiency disorders , hemodialysis , immunosuppressive therapy , contraindication for hbv vaccination ( i.e. prior history of anaphylactic reaction to vaccine ) , positive results for hbs - ag , anti - hbs , anti - hcv , or anti - hiv tests and aspartate aminotransferase ( ast ) or alanine aminotransferase ( alt ) levels above 3 times normal cutoff values . 
 
 also , none of the participants were positive for signs and symptoms of chronic hepatitis and cirrhosis .
prior to intervention , informed consents were obtained from all the participants , in accordance with the guidelines established by the ethics committee of zahedan university of medical sciences . 
 

all the participants were tested for hbv - dna by qualitative polymerase chain reaction ( pcr ) ( cinna gen inc . ,
tehran , iran ) that could detect as low as 100 copy / ml of viral genome .
the following reagents were added to each tube on ice : 1x pcr mixture 15 ul and taq - dna polymerase 0.4 ul . then the tubes were shaken and spun thoroughly . the pcr mixture contained primers amplifying 353 bp of the region of the core gene .
one drop ( 20 - 25 ul ) of mineral oil was also added to each tube . then , 10 ul dna ( with the use of specified pipette for sampling of dna ) was added to the mixture and spun on microfuge for 3 - 5 seconds .
the tubes were transferred to preheated thermocycler with the following program : 1 cycle of 60 seconds at 93c , 20 seconds at 61c and 40 seconds at 72c , followed by 35 cycles of 93c , 61c , and 72c for 20 , 20 , and 40 seconds , respectively .
finally , 10 iu / ml of amplified samples were directly analyzed in a 2% agarose gel without adding loading buffer .
afterwards , all subjects received three doses of 20 g of hepatitis b recombinant vaccine ( heberbiovac hbr , havana , cuba ) at 0 , 1 , and 6 months , if applicable ( see below ) .
the vaccine was injected intramuscularly into the deltoid muscle . 
 
 in order to quantify anti - hbs antibodies ,
individuals with high titer anti - hbs response ( anti - hbs > 50 miu / ml ) did not receive additional doses of the vaccine .
however , others completed the vaccination course , and another blood sample was collected 30 days after the third dose to measure anti - hbs level . 
 

anti - hbs titer 10 miu / ml 30 days after the first vaccination was considered as early primary response .
also , late primary response was defined as anti - hbs titer 10 miu / ml 30 days after the third dose of vaccines .
non - responders were individuals with < 10 miu / ml anti - hbs titers after receiving three doses of hb vaccine . 
 

data were analyzed using spss for windows software , version 11.5 ( spss inc . ,
odd s ratios ( ors ) were chosen to measure the association between dichotomous variables , and the results were adjusted for potential confounders by multivariate logistic modeling .
table 1 presents the demographic and important risk factors of the participants , comparing those with isolated anti - hbc to controls . 
 

1 ) abnormal level was defined as levels > 1.5 times upper than normal limit 
 
 2 ) ns : not significant 
 
 3 ) linear by linear association ( chi - squared for linear trend ) ; exact method 
 
 participants of the case group were significantly older , and mostly married .
the case group had also higher male / female ratio , and more history of transfusion and tattoo ( p<0.05 ) . however , history of infection in spouse , familial history of hbv infection , and serum ast and alt levels was not significantly different between cases and controls . 
 

totally , 19 ( 21.1% ) cases and 3 ( 3% ) controls did not seroconvert ( non - responder ) after three doses of hb vaccine ( p<0.0001 ) .
however , primary response was observed in 43 ( 47.8% ) isolated anti - hbc positive cases and 92 ( 92% ) controls ( p<0.0001 ) , of whom 15 cases and two controls seroconverted after the first dose of hb vaccine ( early responder ) .
furthermore , anti - hbs titer  50 miu / ml 30 days after the first dose of vaccine was significantly higher among cases ( 31.1% vs. 5% , p<0.0001 ) .
two ( 2.2% ) cases with positive isolated anti - hbc had detectable hbv dna and were therefore , excluded from the analysis .
both of these two hbv dna positive cases did not seroconvert after receiving 3 doses of vaccination .
none of the cases and controls showed adverse effects after receiving recombinant hb vaccine . 
 

table 2 compares non - responders ( excluding 2 hbv dna positive cases ) with primary responders ( including early and late responders ) . according to univariate analysis , serum anti -
hbc positivity , age , and marital status were significantly different between the two groups ; however , in multivariable analysis , only anti - hbc positivity and age remained independently associated with non - responding status .
indeed , anti - hbc positive subjects were 12.2 times ( 95% ci : 3.2 - 46.4 , p<0.0001 ) more likely to fail seroconversion . similarly , age  50 years was 3.6 times more likely to lead in non - responsive state ( 95% ci : 1.0 - 12.3 , p<0.04 ) . also , anti -
hbc positive participants were more likely to develop anti - hbs titer  50 miu / ml 30 days after receiving the first dose of vaccine ( table 3 ) . 
 

1 ) abnormal level was defined as levels > 1.5 times upper than normal limit 
 
 2 ) ns : not significant 
 
 3 ) linear by linear association ( chi - squared for linear trend ) ; exact method . 
 
 * participants with anti - hbs titer  50 miu / ml 30 days after the first dose of vaccine and those with positive hbv dna were excluded 
 
 as noted earlier , early response ( anti - hbs titer 10 miu / ml 30 days after the first vaccination ) was found in 17 subjects .
we compared early responders to late responders ( the table is not included ) and found a trend toward a higher seroprotective response rate after the first vaccination in the subjects with abnormal baseline alt levels and those aged>50 years ( ors : 6.9 and 8 , p<0.02 and p<0.001 , respectively ) . 


 data were analyzed using spss for windows software , version 11.5 ( spss inc . ,
odd s ratios ( ors ) were chosen to measure the association between dichotomous variables , and the results were adjusted for potential confounders by multivariate logistic modeling .
table 1 presents the demographic and important risk factors of the participants , comparing those with isolated anti - hbc to controls . 
 

1 ) abnormal level was defined as levels > 1.5 times upper than normal limit 
 
 2 ) ns : not significant 
 
 3 ) linear by linear association ( chi - squared for linear trend ) ; exact method 
 
 participants of the case group were significantly older , and mostly married .
the case group had also higher male / female ratio , and more history of transfusion and tattoo ( p<0.05 ) . however , history of infection in spouse , familial history of hbv infection , and serum ast and alt levels was not significantly different between cases and controls . 
 
 totally , 19 ( 21.1% ) cases and 3 ( 3% ) controls did not seroconvert ( non - responder ) after three doses of hb vaccine ( p<0.0001 ) .
however , primary response was observed in 43 ( 47.8% ) isolated anti - hbc positive cases and 92 ( 92% ) controls ( p<0.0001 ) , of whom 15 cases and two controls seroconverted after the first dose of hb vaccine ( early responder ) .
furthermore , anti - hbs titer  50 miu / ml 30 days after the first dose of vaccine was significantly higher among cases ( 31.1% vs. 5% , p<0.0001 ) .
two ( 2.2% ) cases with positive isolated anti - hbc had detectable hbv dna and were therefore , excluded from the analysis .
both of these two hbv dna positive cases did not seroconvert after receiving 3 doses of vaccination . none of the cases and controls showed adverse effects after receiving recombinant hb vaccine . 
 

table 2 compares non - responders ( excluding 2 hbv dna positive cases ) with primary responders ( including early and late responders ) . according to univariate analysis , serum anti -
hbc positivity , age , and marital status were significantly different between the two groups ; however , in multivariable analysis , only anti - hbc positivity and age remained independently associated with non - responding status .
indeed , anti - hbc positive subjects were 12.2 times ( 95% ci : 3.2 - 46.4 , p<0.0001 ) more likely to fail seroconversion .
similarly , age  50 years was 3.6 times more likely to lead in non - responsive state ( 95% ci : 1.0 - 12.3 , p<0.04 ) .
also , anti - hbc positive participants were more likely to develop anti - hbs titer  50 miu / ml 30 days after receiving the first dose of vaccine ( table 3 ) . 
 

1 ) abnormal level was defined as levels > 1.5 times upper than normal limit 
 
 2 ) ns : not significant 
 
 3 ) linear by linear association ( chi - squared for linear trend ) ; exact method . 
 

* participants with anti - hbs titer  50 miu / ml 30 days after the first dose of vaccine and those with positive hbv dna were excluded 
 
 as noted earlier , early response ( anti - hbs titer 10 miu / ml 30 days after the first vaccination ) was found in 17 subjects .
we compared early responders to late responders ( the table is not included ) and found a trend toward a higher seroprotective response rate after the first vaccination in the subjects with abnormal baseline alt levels and those aged>50 years ( ors : 6.9 and 8 , p<0.02 and p<0.001 , respectively ) . 


 in our setting , we vaccinated isolated anti - hbc as well as healthy blood donors and monitored their response to hbv vaccination .
a total of 19 ( 21.1% ) cases and 3 ( 3% ) controls did not seroconvert after receiving 3 doses of hb vaccine .
prior investigators reported a wide range of non - responding state , sunbul 9.1% , coz yatacho 9.7% .
therefore , at least 20% of isolated anti - hbc positive cases may suffer from occult hepatitis b infection , for whom a diminished ability to mount an anti - hbs response is proposed . 
 

furthermore , in our study , anti - hbs titer  50 miu / ml 30 days after the first dose of vaccine was developed in 28 ( 31.1% ) cases and 5 ( 5% ) controls .
scanty studies have addressed anamnestic rates ; however , in a study reported by sunbul and colleagues , out of 33 ( 42.4% ) subjects with isolated anti - hbc showed rapid high seroconversion , while in a report by ural and co - workers , this rate was 41.7% ( 20 out of 48 ) .
our lower rate could in part be explained by lower prevalence of hbv in our region . in such patients
, further vaccination may not be necessary . finally , 43 ( 47.8% ) subjects with isolated positive anti - hbc and 92 ( 92% ) controls showed primary response after the vaccination protocol .
nevertheless , most of previous researchers assigned their subjects in two groups of responders ( either primary or anamnestic ) and non - responders .
accordingly , our cumulative responder rate would be 78.9% among subjects with isolated positive anti - hbc , which is in agreement with reports by koh ( 70.6% ) , kabir ( 79.8% ) , and silva ( 80% ) , but lower than what reported by ural ( 89.6% ) , pereira ( 90% ) , yatacho ( 90.3% ) and sunbul ( 90.9% ) .
, discrepancies in response rate to hb vaccination among isolated anti - hbc subjects can be explained by serologic kit sensitivity , defining cut off points , and inclusion criteria for borderline results .
therefore , in our region , nearly 80% of isolated anti - hbc subjects can be safely included in the donor pool , simply based on a vaccination protocol .
notably , the isolated anti - hbc subjects with false positive results ( 47.8% in our setting ) are susceptible to hepatitis b infection and should receive a complete course of hb vaccine .
however , they may lose hb vaccination because of misinterpretation of anti - hbc results . 
 

on the other hand , our results revealed that individuals with isolated positive anti - hbc were significantly older , mostly married , had higher male / female ratio , history of transfusion , tattoo and abnormal serum ast and alt levels when compared with controls .
the higher rate of anti - hbc positivity among older subjects mirrors a cohort effect of hbv infections acquired decades ago when hbv was moderately endemic in iran and hbv national vaccination program had not been commenced .
furthermore , only two ( 2.2% ) isolated anti - hbc positive cases had detectable hbv dna .
recent studies suggest that 1 - 2% of anti - hbc reactive units contain low levels of hbv dna .
however , in studies conducted on 4930 healthy blood donor volunteers in fars and hamadan provinces , 6.5% and 5.1% were isolated anti - hbc positive cases , among whom 16 ( 12.3% ) and 3 ( 1.2% ) were hbv dna positive , respectively . 
 
 having compared non - responders to primary responders ( table 2 ) , anti - hbc positivity , age , and marital status showed significant differences in univariable analysis .
however , in multivariable analysis , only anti - hbc positivity and age ( > 50 years ) remained independently associated with non - responding status . on the other hand , when we compared primary responders with those who developed anti - hbs titer  50 miu / ml 30 days after the first dose of vaccine ( table 3 ) , positivity for anti - hbc and age > 50 years were the most relevant factors in developing higher anti - hbs titers to hbv vaccine .
therefore , isolated anti - hbc subjects aged > 50 years would more likely either develop anti - hbs titers  50 miu / ml 30 days after receiving the first dose of vaccine or never respond to hb vaccine . as a result , one month after the first dose of hb vaccine in isolated anti - hbc subjects aged > 50 years , we can make a decision regarding whether to advise a strict follow - up ( occult hbv infection ) or assure the patient that he / she has a resolved hbv infection .
interestingly , when we compared the early and late responders ( the table is not included ) , anti - hbc positivity , abnormal alt level , and age > 50 years were associated with a higher probability of early response .
therefore , anti - hbc positive subjects aged > 50 years with abnormal alt levels should be offered the first dose of hb vaccine and evaluated one month later in order to decide for their future follow - up . 
 

various molecular modalities have been developed to determine if isolated anti - hbc subjects could transmit hbv infection .
however , they will further increase the testing costs and require trained personnel and equipment , which may not be possible in many blood banks around the country .
thus , studying the cost effectiveness of  monitoring the response to hb vaccination could be worth of considering as a diagnostic tool .
we also suggest future studies focusing on evaluating the short - course vaccination protocols such as double - dose / double - time vaccination in order to make sooner decision . 
 
 in conclusion ; using vaccination , we found that half of the subjects with isolated positive anti - hbc were falsely positive for the test and more than 75% of subjects with isolated positive anti - hbc could benefit from vaccination and could be included in donor pool .
moreover , one - fifth of subjects were non - responders and may have occult hbv infection . because at least 5% of the donor population in iran is anti -
hbc positive , blindly rejecting anti - hbc positive donors would cause the exclusion of a consistent number of donors , most of whom could safely be included in the donor pool .
therefore , hb vaccination may be used as a diagnostic tool for clarifying the situation of the subjects with isolated anti - hbc and can help in reducing the blood supply problems . 


 this study was financially supported by zahedan university of medical sciences and iranian blood transfusion organization research center ( zahedan branch ) . 

 background 
 it is important to differentiate whether isolated anti - hbc is due to false 
 positive results or the prior exposure to hepatitis b virus , because individuals 
 with false - positive anti - hbc can benefit from vaccination and their blood can 
 be safely transfused . to distinguish between these two conditions , we evaluated 
 the serologic response to hepatitis b vaccine . 

 
 methods 

 ninety subjects with isolated anti - hbc ( cases ) and 100 subjects with totally 
 negative hepatitis b serologic markers ( controls ) were recruited to receive 
 three doses of hepatitis - b ( hb ) vaccine . 
 thirty days after the first dose of the 
 vaccine , anti - hbs titers were checked and individuals with anti - hbs titer > 50 
 miu / ml did not receive additional doses of the vaccine . 
 however , others completed 
 the vaccination course , and another blood sample was collected 30 days 
 after the third dose to measure anti - hbs level . 
 results 
 nineteen ( 21.1% ) cases and three ( 3% ) controls had no sero - conversion 
 ( anti - hbs titers < 10 miu / ml ) 30 days after the third dose ( p<0.0001 ) . 
 primary 
 response , defined as the development of anti - hbs antibody titers 10 
 miu / ml 30 days after the third dose , was observed in 43 ( 47.8% ) cases and 92 
 ( 92% ) controls ( p<0.0001 ) . 
 also , 31.1% of cases developed anti - hbs titers  
 50 miu / ml 30 days after the first dose of vaccine , but the rate was significantly 
 lower ( 5% ) in the control group ( p<0.0001 ) . 
 furthermore , half of the individuals 
 with positive isolated anti - hbc developed protective levels of anti - hbs 
 after three doses of hb vaccination . 

 
 conclusion 

 more than 75% of individuals with positive isolated anti - hbc can benefit 
 from vaccination and can be included in donor pool . 
 also , one fifth seemed to 
 have occult hbv infection . 
 so hb vaccination may be used as a diagnostic 
 tool for clarifying the situation of the subjects with isolated anti - hbc . 
 