{'fraction_non_alphanumeric': 0.03919716646989374, 'fraction_numerical': 0.032192050373868554, 'mean_word_length': 4.605779836752703, 'pattern_counts': {'":': 0, '<': 27, '<?xml version=': 0, '>': 7, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 1, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Clinical features of HBsAg seroclearance in hepatitis B virus carriers in South Korea: A retrospective longitudinal study

AIM To investigate the characteristic features of hepatitis B surface antigen (HBsAg) seroclearance among Korean hepatitis B virus (HBV) carriers. METHODS Carriers with HBsAg seroclearance were selected by analyzing longitudinal data collected from 2003 to 2015. The period of time from enrollment to the negative conversion of HBsAg (HBsAg-NC) was compared by stratifying various factors, including age, sex, hepatitis B e antigen (HBeAg), HBV DNA, sequential changes in the signal-to-cutoff ratio of HBsAg (HBsAg-SCR), as measured by qualitative HBsAg assay, and chronic liver disease on ultrasonography (US-CLD). Quantification of HBV DNA and HBsAg (HBsAg-QNT) in the serum was performed by commercial assay. RESULTS Among the 1919 carriers, 90 (4.7%) exhibited HBsAg-NC at 6.2 ± 3.6 years after registration, with no differences observed among the different age groups. Among these carriers, the percentages of those with asymptomatic liver cirrhosis (LC) and hepatocellular carcinoma (HCC) at registration were 31% and 7.8%, respectively. The frequency of HBsAg-NC significantly differed according to the HBV DNA titer and US-CLD. HBeAg influenced HBsAg-NC in the 40-50 and 50-60 year age groups. HBsAg-SCR < 1000 was correlated with an HBsAg-QNT < 200 IU/mL. A gradual decrease in the HBsAg-SCR to < 1000 predicted HBsAg-NC. Six patients developed HCC after registration, including two before and four after HBsAg-NC. The rate at which the patients developed new HCC after HBsAg seroclearance was 4.8%. LC with excessive drinking and vertical infection were found to be risk factors for HCC in the HBsAg-NC group. CONCLUSION HCC surveillance should be continued after HBsAg seroclearance. An HBsAg-SCR < 1000 and its decrease in sequential testing are worth noting as predictive markers of HBsAg loss.


INTRODUCTION
Hepatitis B virus (HBV) is the most important cause of liver cirrhosis (LC) and hepatocellular carcinoma (HCC) in endemic areas worldwide [1,2] . The natural course of HBV infection is associated with immuno logical changes that occur in three phases: tolerance, eradication, and recovery [3] . These phases are classified based on the serum aminotransferase level, hepatitis B e antigen (HBeAg) and HBV DNA titers, which represent hepatitis and viral replication, respectively [4,5] . Recovery is defined as ceasing of the self-replicating activity of the HBV genome and its transition to a nonreplicating state. In general, a serum HBV DNA level of below 2000 IU/mL is considered to indicate an inactive hepatitis B surface antigen (HBsAg) carrier state [3,57] . Once the HBV genome is inactivated, it remains inert throughout life, HBeAg becomes negative, and HBsAg is cleared in approximately 40% of patients after 25 years of follow up [3] . On the other hand, a significant proportion of carriers with HBeAg loss harbor the G1896A mutation, the socalled eminus mutation [2] . In Korea, where most HBV carriers harbor genotype C2 [810] , most carriers over the age of 40 are infected with HBV with basal core promoter (BCP) double mutations (A1762G and A1764T), and more than half of these individuals have the G1896A mutation [9,10] . These mutations are associated with HBeAgnegative chronic hepatitis that is frequently reactivated [2,11,12] , and HBeAg serocon version is associated with the development of LC and HCC in twothirds of carriers [2,7,13] . Because the turning point of seroconversion generally occurs near the age of 40 [11] , the recovery phase and timing of mutations usually overlap with the development of LC and HCC at this time [2,14] . However, HCC may also develop after HBsAg seroclearance [2,14] . These results highlight the difficulty of determining when and how the negative conversion of HBsAg (HBsAgNC) in the serum takes the risk out of HCC. Thus, Korean HBV carriers represent a good model to study the clinical significance of HBsAg seroclearance in individuals with genotype C. This study investigated the long term process of HBsAg seroclearance to elucidate the outcomes and predictive factors.

Patients
Among chronic HBV carriers who visited the Hepatology Center of Bundang Jesaeng General Hospital between March 2003 and September 2015, all patients with HBsAg seroclearance were recruited. The clinical and laboratory data were retrospectively recorded at baseline and during followup. Chronic carriers were defined as those with HBsAg positivity on two successive tests performed at least six months apart. Registration or entry was defined as the initial visit to the center. The baseline data included the data collected at the first visit. HBsAg seroclearance was defined as two consecutive negative HBsAg tests performed at least six months apart.

Clinical data at baseline and during follow-up
The patients underwent tests for biochemical liver function, hepatitis B virological markers, and alpha fetoprotein, as well as liver ultrasonography (US). The virological markers assessed included HBsAg, anti HBs, HBeAg, antiHBe, and HBV DNA. The antiHBs or antiHBe test was performed as clinically indicated. The antiHCV test was performed at baseline and was repeated if the alanine aminotransferase level was elevated to more than twice the upper limit of normal.
The signaltocutoff ratio of the HBsAg (HBsAgSCR) level was measured for each patient by qualitative assay.

Methods for testing virological markers
The HBsAg, antiHBs, HBeAg and antiHBe levels were determined by ARCHITECT qualitative assay, which is a chemiluminescent microparticle immunoassay (CMIA) that measures the resulting chemilumines cent reaction as relative light units (RLUs) (Abbott Laboratories, North Chicago, IL, United States). There is a direct relationship between the amount of HBsAg in a sample and the RLUs detected by ARCHITECT assay (Abbott Laboratories). The antiHCV levels were determined using a thirdgeneration enzyme immunoassay (Abbott Laboratories). HBV DNA was tested using a commercially available realtime poly merase chain reaction (RTPCR; Roche Molecular Systems, Pleasanton, CA, United States), according to the manufacturer's instructions. The lower limit of detection for the Roche assays is 20 IU/mL. The viral load data obtained with the Roche assay were converted to international reference units for analysis using approximations for 1 pg (283000 copies) and 1 IU/mL (5.8 copies/mL).

Quantitative HBsAg assay
The data on the HBsAg levels in 177 patients were separately collected to perform correlation analysis with the HBsAgSCRs obtained with CMIA. The HBsAg quantification (HBsAgQNT) was measured by electrochemiluminescence immunoassay using an Elecsys HBsAg II Quant Assay (Roche Diagnostics, Indianapolis, IN, United States), according to the manufacturer's instructions. This approach quantifies HBsAg against an internal World Health Organization reference standard in IU/mL.

Statistical analysis
To compare the characteristics between groups, either the χ 2 test or Fisher's exact test was used to analyze categorical variables, and Student's ttest or Wilcoxon nonparametric test was used to analyze continuous variables. Statistical data were expressed as the mean ± SD. The correlation between the HBsAgSCR and HBsAgQNT was analyzed. The statistical procedures were performed with Rproject version 3.2 [15] . P values of < 0.05 were considered significant.

HBsAg seroclearance rate
Negative seroconversion of HBsAg was observed in 90 of 1919 carriers (4.7%), with an estimated annual rate was 0.76%. The rate of HBsAg loss was higher in the carriers over the age of 40 than in the younger carriers (5.7% vs 2.5%, P = 0.001). Among the carriers with HBsAg seroclearance, male predominance was more significant in the 4050 and 5060 year age groups than in the 3040 and > 60 year age groups (P < 0.0001). The period of time until HBsAg clearance after registration was 6.48 ± 3.76 years. This value did not significantly differ among the age groups (P = 0.148) ( Table 1).

Roles of HBeAg, the HBV DNA titer and BCP mutations in HBsAg seroclearance
Among the 90 carriers with HBsAg seroclearance, 82.2% were HBeAgnegative at registration. Among these HBeAgnegative carriers, 95.3% had no history of antiviral treatment and were considered to be in the inactive phase. The time interval to HBsAg clearance did not differ between the HBeAgpositive and negative carriers (P = 0.982). Among the carriers with HBsAg seroclearance who had a treatment history, the time interval to HBsAg clearance did not differ between the HBeAgpositive and negative carriers (8.13 ± 3.12 vs 7.95 ± 3.45 years, P = 0.4442). However, this time interval was longer for the carriers who had received treatment than for those who had not received treatment (7.95 ± 3.34 vs 5.52 ± 3.48 years, P = 0.017), suggesting that antiviral therapy does not lead to HBsAg seroclearance [16,17] . On the other hand, the time interval from entry to HBsAg seroclearance was significantly longer for the HBeAgpositive carriers than for the HBeAgnegative carriers in the 40s and 50s age groups (9.24 ± 2.49 years vs The overall rate of HBsAg seroclearance was 4.7%, and the estimated annual rate of HBsAg seroclearance was 0.76%. The rate of HBsAg seroclearance was significantly higher in the 40-50 and 50-60 year age groups than in the 20-30 and 30-40 year age groups (P = 0.001). Male predominance was observed in the 40s and 50s age groups but was not clear in the > 59 year age group. The time interval from entry to HBsAg loss was longer in the 40-50 and 50-60 year age groups than in the 20-30 and 30-40 year age groups.
0.0057). Further, the mean age in the < 1000 group tended to be higher than that in the ≥ 1000 group (46.2 ± 11 years vs 50 ± 11.1 years old, P = 0.0593). However, there was no difference in the sex ratio or the prevalence of HCC or LC between the two groups.
6.18 ± 3.63 years, P = 0.013) ( Table 2). Likewise, the time interval was significantly longer for the carriers with an HBV DNA level of > 2000 IU/mL than for those with a level of < 20 IU/mL (8.31 ± 3.26 years vs 4.92 ± 3.36 years; P = 0.0005) ( Table 3). Data on the BCP double mutations, A1762G and A1764T, and the eminus mutation, G1896A, were available for 18 patients. Women were predominant in the wildtype BCP group, whereas men were predominant in the mutant group (male/female = 1/6 vs 9/2, respectively; P = 0.0014). The BCP mutation carriers tended to be older than the wildtype carriers (41.9 ± 10.8 years vs 48.5 ± 7.6 years old, P = 0.0725). However, the time interval from entry to HBsAg seroclearance did not differ between the groups.

Utility of the signal-to-cutoff ratio of the qualitative HBsAg level
A loglinearshaped correlation was detected between the HBsAgSCR and HBsAgQNT ( Figure 1A), and a good correlation with a linear drift curve was observed between an HBsAgSCR < 1000 and an HBsAgQNT < 100 IU/mL (y = 0.0674x + 0.9902, R² = 0.916) ( Figure  1B). No correlation was observed between these two values in the patients with HBsAgQNT levels of over 200 IU/mL ( Figure 1C). An inverse correlation was observed in the patients with very high HBsAg titers of over 10000 IU/mL ( Figure 1D), indicating a prozone effect on the HBsAgSCR caused by excess antigen. Based on the HBsAgSCR value of 1000, we arbitrarily divided the 90 carriers with HBsAg seroclearance into two groups. The period of time from entry to HBsAg seroclearance was significantly shorter in the < 1000 group than in the ≥ 1000 group (5.07 ± 3.74 years vs 7.47 ± 3.05 years, P = 0.0008). Moreover, a sequential decrease in the HBsAgSCR was predictive of HBsAgNC in every case ( Figure 2). The HBeAg positive rate was significantly higher in the ≥ 1,000 group than in the < 1000 group (25% vs 11.9%, P = Overall, the time interval of HBsAg seroclearance did not differ between the two groups (P = 0.982). However, when limited to the 40s and 50s age groups, the time interval to HBsAg seroclearance was significantly longer in the HBeAg-positive group than in the HBeAg-negative group (P = 0.013), but it did not significantly differ in the other age groups. In the 60s age group, the time interval to HBsAg seroclearance did not differ between the HBeAg-positive and negative groups (P = 0.1208).
Park YM et al . Clinical features of HBsAg seroclearance Figure 1 Signal-to-cutoff ratio of hepatitis B surface antigen was measured by qualitative assay and partially reflects the quantity of hepatitis B surface antigen in the serum. A: There was a log-linear correlation between the HBsAg-SCR and HBsAg-QNT; B: There was a good correlation with a linear drift curve between an HBsAg-SCR < 1000 and an HBsAg-QNT < 100 IU/mL (y = 0.0674x + 0.9902, R² = 0.916); C: HBsAg-QNT levels of greater than 200 IU/mL were not correlated with the HBsAg-SCR; D: Very high HBsAg titers of more than 10000 IU/mL were inversely correlated with the HBsAg-SCR, which was caused by a prozone effect. HBsAg-SCR: Signalto-cutoff ratio of HBsAg; HBsAg-QNT: Quantity of HBsAg; HBsAg: Hepatitis B surface antigen.

LC and HBsAg seroclearance
We classified the patients with HBsAg seroclearance into the following three groups according to their US findings: normal, noncirrhotic chronic liver disease (CLD) and cirrhotic. The period of time from study entry to HBsAg loss was significantly shorter in the normal US group than in the CLD and cirrhotic US groups (5.05 ± 3.92, 6.86 ± 3.51 years and 6.8 ± 2.98 years, respectively; normal US vs CLD, P = 0.0259; normal vs cirrhotic US, P = 0.0366) (

Hepatocellular carcinoma and HBsAg seroclearance
Among the 90 patients with HBsAg seroclearance, thirteen were diagnosed with HCC, including seven (7.8%) at registration (HCC group1) and six (6.7%) during followup (HCC group2). Among the patients in HCC group2, two developed HCC prior to HBsAg loss, and four developed HCC at 0.66 ± 0.63 years after HBsAg loss, corresponding to a rate of 4.8% among the 83 carriers without HCC at entry. There was no difference in the time interval from entry to HBsAg loss between HCC group1 and group2 (6.78 ± 2.77 years and 5.77 ± 3.18 years, respectively). In addition to LC, the risk factors for HCC included excessive drinking in three men and vertical infection in one woman.    [19] , 1%1.9% in Caucasian carriers [5,17] , 2.5% in the Goto Islands of Japan [20] , and 3.08% in China [21] . Compared with data from Taiwan [18] , HBsAg seroclearance in Korea was significantly reduced in all age groups as follows: 0.55% vs 0.77% in the 2030 year, 0.45% vs 1.07% in the 3040 year, 0.92% vs 1.65% in the 4050 year, and 0.89% vs 1.83% in the 50 and over age groups. This variability may be due to various factors, such as geographic differences in genotypes, age, gender, viral loading and fibrosis at enrollment. For instance, almost all Korean carriers are known to be infected with genotype C, whereas 60% and 34% of Taiwanese carriers are infected with genotypes B and C, respectively [6,22] . Carriers with an initial HBeAgpositive result show the gradual negative conversion of HBeAg and HBV DNA before the loss of HBsAg [14] . In carriers with an initial HBeAgnegative result, HBV DNA is cleared from the serum before HBsAgNC, although low HBV DNA titers are persistently detected in some patients [23] . These results are interrelated in that the HBsAg loss is usually preceded by a long period of inactive disease [17] . The time period from entry to HBsAg loss in our study is similar to that reported in a previous sixyear study conducted in China [21] . Notably, no significant differences in this time interval were observed accord ing to age, the HBeAg status or the HBV DNA titer at the time of enrollment. Two opposite conclusions have been made regarding the role of HBV DNA in HBsAg seroclearance. One is that HBV DNA is not a dependent factor for HBsAgNC [6,19] , while the other suggests that lower viral loads are predictive of HBsAg seroclearance [24] . In our study, the HBV DNA titer was associated with the relative period of time from detection of viral activity to HBsAg seroclearance, and this period was at least 1.5 times longer in the carriers with HBV DNA levels ≥ 2000 IU/mL than in those with levels < 20 IU/mL 40s. However 50s, HBeAg exhibited this effect only in the 40s and 50s age groups, indicating its agedependent role in predicting HBsAg loss, in contrast with the HBV DNA titer. The baseline HBsAg level is known to be a better predictor of HBsAg seroclearance than other fac tors [6,16,19,21,2426] . This statement applies to inactive carrier, in whom a lower HBsAg level is more predictive of HBsAg seroclearance. Reportedly, the optimum cut off baseline HBsAg level has varied among studies, for example, studies have reported levels of < 10 IU/mL [6,21] , < 100 IU/mL [19,24] , < 200 IU/mL [25,26] , and < 751 IU/mL [16] . In addition, it has been reported that the predictive capacity of the HBsAg level can be improved by considering it in combination with other factors, such as a normal platelet count [21] , old age [19] , an undetectable HBV DNA level [24] , the HBV DNA level at 12 mo after HBeAg seroconversion [27] , and a yearly ≥ 0.5 log IU/mL reduction [25,26] .

DISCUSSION
Because the quantitative HBsAg test is expensive, we analyzed the utility of the HBsAgSCR in predicting HBsAg seroclearance. A good linear correlation was observed between an HBsAgSCR < 1000 and HBsAg QNT < 200 IU/mL. In addition, a prozone effect on the HBsAgSCR caused by excess antigen was observed among the carriers with excessive HBsAg titers of > 10000 IU/mL, most of whom had a very high serum HBV DNA level of > 7 log10 IU/mL. With the exception of those carriers with highly replicative infections, the HBsAgSCR was significantly reduced in the HBeAg negative carriers compared with the HBeAgpositive carriers. Moreover, the period of time from a given viral state to HBsAg loss tended to be shorter, as shown in Figure 2. A gradual decrease in the repeated tests during followup usually indicated HBsAg loss within five years. These results suggest that sequential HBsAgSCR data are very useful for predicting HBsAg seroclearance.
The rate of LC at entry of 31.1% for the carriers with HBsAg seroclearance is similar to that reported  previously [14] . These results indicate that LC is present in an appreciably high rate of carriers with HBsAg seroclearance. On the other hand, significant fibrosis has been reported to be more prevalent in patients with HBsAg seroclearance who are > 50 years of age compared with those who are < 50 years of age [19,28] . In our study, the prevalence of LC was significantly increased among the males who were approximately forty years of age. In addition, the carriers in whom LC/CLD was detected on US exhibited a longer time interval from entry to HBsAg seroclearance than those with normal US results. These results suggest that the correlation between age and HBsAg loss is meaningful when it is considered together with gender and LC. In addition, the frequency of HBsAg loss was higher in the carriers over the age of 40 than in the younger carriers, and this trend is similar to trends reported in Taiwan [18] , Japan [20] , and New Zealand [29] . These results might be associated with a high rate of HBsAg seroclearance during longterm followup, as reported in Chu and Liaw's study [18] . Inactive carriers generally have a good prognosis. Indeed, none of the patients developed HCC following HBsAg loss (median followup of 72 mo) in a community study conducted in Kawerau, New Zealand [19] , or in a study of Caucasians [5] . In contrast, in studies performed in Hong Kong and the United States, 1.4%2.4% of the patients developed HCC after HBsAg seroclea rance [7,14,28] . In our study, 4.82% of the carriers with HBsAg seroclearance developed HCC after HBsAg loss, indicating that HBsAg seroclearance does not guarantee patient safety out of HCC.
In one study, HBsAg seroclearance in individuals < 50 years of age has been shown to be associated with a lower risk of the development of HCC [28] . In another study, a low baseline level of albumin and family histories of HBsAg positivity and HCC have been demonstrated to be associated with a high risk of development of HCC, even in individuals who are < 50 years of age at the time of HBsAg clearance [14] . In our study, HCC developed in one patient over 50 years of age and in three patients over 60 years of age after HBsAg seroclearance. All patients had asymptomatic LC at registration and no evidence of deterioration of liver function during followup. In addition, none of these patients had a family history of HCC. Three men were excessive drinkers, and one woman had a vertical HBV infection.
In conclusion, HBsAg seroclearance does not indicate safety out of HCC, particularly in patients with LC. Spontaneous HBsAg loss might occur in a large proportion of cryptogenic LC and HCC cases in Korea, and surveillance should be continued after HBsAg loss in the same manner as for HBsAgpositive patients. Sequential HBsAgSCR data measured with the conventional test are very useful for the longterm management of carriers, similar to HBsAg levels.

Background
Chronic hepatitis B virus (HBV) infection follows a unique natural course, consisting of immune tolerance, eradication, and a recovery phase. Intricate changes in the balance between host and viral factors stimulate the phase transition. Prognosis of chronic carriers is determined by a complicated process with consideration of phase transition. Seroclearance of hepatitis B surface antigen (HBsAg) is the final step of the recovery phase. However, the clinical features of HBsAg seroclearance have not yet been completely elucidated.

Research frontiers
The recovery phase usually overlaps with the occurrence of escape mutations in the HBV genome and the development of liver cirrhosis (LC) and hepatocellular carcinoma (HCC). Thus, it is difficult to determine when and how the negative conversion of HBsAg (HBsAg-NC) has occurred to a sufficient extent to have preventive effects for HCC. The research hotspot is clarification of the clinical features associated with spontaneous HBsAg seroclearance, including the outcome and predictive markers, to facilitate the development of a patient management strategy both during and after the recovery phase.

Innovations and breakthroughs
The results of the present study have suggested that the phase transition to recovery occurs spontaneously in most patients and that asymptomatic LC is prevalent among these patients (31%). Thus, a significant proportion of chronic carriers are at risk of HCC at the time of recovery. In fact, the data showed that HCC developed among the patients with LC during the recovery phase both pre-and post-HBsAg seroclearance. On the other hand, it is known that the HBsAg quantity, in combination with the HBV DNA titer, is a predictive marker for HBsAg seroclearance. The authors have demonstrated for the first time that a signal-to-cutoff ratio of the qualitative HBsAg level (HBsAg-SCR) of < 1000 and its sequential decrease are notable predictive markers of HBsAg loss. Our data support the value of sequential HBsAg-SCR data.

Applications
The results of our study may facilitate the design of a management strategy for HBV carriers both pre-and post-HBsAg seroclearance. In particular, patients with evidence of chronic liver disease or LC should be carefully monitored for the development of HCC, even after the spontaneous loss of HBsAg.

Terminology
Serum HBsAg positivity lasting for six months is a serological indicator for chronic HBV infection. HBsAg seroclearance refers to the spontaneous HBsAg-NC in the serum. The HBsAg-SCR is the signal-to-cutoff ratio of HBsAg, as measured by qualitative assay, and the quantification of HBV DNA and HBsAg (HBsAg-QNT) is the quantity of HBsAg in the serum, as measured by quantitative assay. The HBsAg-SCR is determined according to the signal intensity of an antigen-antibody reaction. The authors found that carriers with a very high HBV DNA titer of > 7 logs IU/mL exhibited a prozone effect, in which the HBsAg-SCR was paradoxically reduced by excess antigen. In contrast, in the HBV DNA-negative patients, an HBsAg-SCR < 1000 was strongly correlated with an HBsAg-QNT < 200 IU/mL. Otherwise, the HBsAg-SCR was not correlated with the HBsAg-QNT.

Peer-review
This invited manuscript written by Park et al investigated the clinical features of HBsAg serological clearance among almost 2000 Korean HBV carriers. This is a valuable study that included large-scale clinical sample analyses; the significant conclusion of this study is that liver cancer surveillance should be continued after HBsAg seroclearance, particularly in patients with cirrhosis. The title describes the contents of the paper. The abstract is informative and completely self-explanatory; it briefly presents the topic, states the scope of the experiments, provides the significant data, and notes the major findings and conclusions. The purpose or purported significance of the article is explicitly stated. The research study methods are complete enough to enable the experiments to be reproduced. All figures and tables are necessary and appropriate. The discussion interprets the findings in view of the results