由敏感度更高的HBsAg定量检测我们能发现什么？

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What can be revealed by extending the sensitivity of HBsAg detection to below the present limit?

今年7月份Journal of Hepatology上挺有意思的一篇文章，Journal of Hepatology Vol.49 (2008) 17–24，Via。采用自由基免疫测定HBsAg，检测下限达到0.025ng/ml。于是可以分别在一部分健康人群、非B非C型硬化患者、非B非C型肝癌患者、CHC患者、C型肝癌患者中检测到HBsAg。

结论是，目前的HBsAg定量检测敏感度太低了。这是日本人的文章。不知道国内的HBsAg定量敏感度和精确性如何。

PDF有300多K，权限不够，传不上来。

下面是Abstract：


Background/Aims
We investigated what can be revealed by extending the sensitivity of HBsAg detection to below the present limit.MethodsWeexamined the sensitivity of this immunoassay in comparison withreal-time PCR detection of HBV DNA using serially diluted sera from HBVcarriers. Low HBsAg was measured in 210 healthy volunteers and 368patients with non-B chronic liver diseases who were negative for HBsAgby a standard EIA method.

Results
The radicalimmunoassay was able to detect HBsAg at a concentration of 0.025 ng/ml.Low HBsAg was positive in 6 of 210 normal volunteers (2.86%), 5 of 65non-B, non-C cirrhosis patients (7.69%), 6 of 62 non-B, non-Chepatocellular carcinoma patients (9.68%: p = 0.04 vs. volunteers), 12 of 134 chronic hepatitis C patients (8.96%: p < 0.02 vs. volunteers), and 11 of 107 hepatocellular carcinoma patients complicated by chronic hepatitis C (10.28%: p < 0.008vs. volunteers). Although no HBV DNA was positive in healthyvolunteers, 9 patients with non-B chronic liver diseases were positivefor HBV DNA by real-time PCR analysis.

Conclusions
Increasingthe sensitivity of HBsAg detection to below the present limit hasrevealed that infection with HBV, including occult HBV, is far moreendemic than suspected previously.

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1. Introduction

About 350 million people worldwide are chronic carriers of the hepatitis B virus (HBV) [1]. The infection can cause acute and chronic liver diseases including fulminant hepatic failure, cirrhosis and hepatocellular carcinoma. Each year, acute and chronic HBV infection causes roughly one million deaths [1] and [2]. HBV infection occurs either vertically or horizontally. Therefore, surveillance of HBV infection in individuals is mandatory from a public health perspective, particularly in highly endemic areas. The diagnosis of chronic HBV infection is based on the persistent presence of viral envelope protein, hepatitis surface antigen (HBsAg), in the blood [3]. Recent advances in gene technology have prompted the concept of occult HBV, which is defined as HBV DNA detectable by sensitive polymerase chain reaction (PCR) among individuals negative for HBsAg [4] and [5] M. Torbenson and D.L. Thomas, Occult hepatitis B, Lancet Infect Dis 2 (2002), pp. 479–486. Article | PDF (240 K) | View Record in Scopus | Cited By in Scopus (114)[5]. Particularly in carriers with a low HBV load, it is essential to distinguish such carriers from donors in order to ensure safe blood transfusion [6]. Moreover, investigation of chronic infection with a low HBV load and its clinical significance is considered to make a significant contribution to prevention and treatment.

Recently, we have developed a radical immunoassay method that can detect HBsAg in serum with revolutionarily high sensitivity [7] and [8]. This radical immunoassay is based on measurement of peroxidase activity in peroxidase–antibody conjugates, in which a stable nitroxide radical is generated in the presence of H2O2, p-acetamidophenol (p-AP), and 1-hydroxy-2,2,5,5-tetramethyl-3-imidazoline-3-oxide (HTIO) [7], [8] and [9]. In this method, peroxidase activity is quantified by measuring the stable nitroxide radical by electron spin resonance (ESR) spectroscopy, which yields a markedly high degree of sensitivity. To expand the availability of this immunoassay, we further developed an automated ESR analyzer for measurement of HBsAg. We therefore expect that this new analytical method will make it possible to diagnose low HBV load carriers, including occult HBV carriers, with convenience, high sensitivity and low cost, and will become a powerful examination tool for worldwide use. In the present study, we investigated what would be revealed when the detection sensitivity for HBsAg was increased to below the present detection limit. In non-B chronic liver diseases including hepatocellular carcinoma (HCC), the positivity rate for low HBsAg was significantly high and individuals with occult HBV were not rare, suggesting the involvement of occult HBV in these diseases. Here we show that low-concentration HBs antigenemia exists in two forms, i.e. low HBsAg positive with low HBV DNA, and low HBsAg positive without HBV DNA.

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2. Methods

2.1. Chemical

p-AP and sodium azide were purchased from Wako Pure Chemical Industries, Osaka, Japan. HTIO was from Aldrich (Milwaukee, USA). 3-Morpholinopropanesulfonic acid (MOPS) was from Dojindo Laboratories (Kumamoto, Japan). Dimethyl sulfoxide (DMSO) was from Kanto Chemical (Tokyo, Japan). All other chemicals and reagents were of analytical grade.

2.2. Principle of HBsAg detection by the radical immunoassay method

Fig. 1 shows the principle of the radical immunoassay. Phenoxy radicals are produced from p-AP by the action of horseradish peroxidase in the presence of H2O2. One of the hydroxylamine compounds, HTIO, is converted to stable nitroxide radicals by oxidation of phenoxy radicals. Peroxidase activities are quantified by measuring these stable nitroxide radicals with the automated ESR analyzer. The radical immunoassay method can amplify peroxidase activity by 106 times (Fig. 1) [9].



Fig. 1. Principle of the radical immunoassay method. [This figure appears in colour on the web.]

Beads coated with HBs-antibody, a peroxidase-labeled monoclonal antibody against HBsAg (anti-HBs peroxidase conjugate) and HBsAg-negative controls (human sera non-reactive for both HBsAg and anti-HBsAg) attached to AUSZYME II (Abbott Laboratories, N. Chicago, IL) were used. An aliquot of 240 μl serum was incubated with both 60 μl of anti-HBs peroxidase conjugate and a bead at 37 °C for 30 min. After washing the beads with 0.01% W1A (Sigma Chemical Company, St. Louis, MO, USA) solved in distilled water 11 times, they were again incubated with 200 μl of reagent (2 mM p-AP, 0.017 mM HTIO, DMSO and 33  mM MOPS, pH 6.5) and 100 μl of 0.001% H2O2 for 30 min. Then, 50 μl of 100 mM NaN3 was added to the reaction mixture to stop the enzyme reaction (Fig. 1). All the reagents were prepared with milli-Q water.

HBsAg levels in the sera were determined by the radical immunoassay method, using an automated electron spin resonance (ESR) analyzer (Tohoku Seiki Industry, Yamagata, Japan), equipped with a pipettor, an incubator, a washer, and a reader station. The ESR spectroscopic settings for measurement were done automatically. The signal intensity of the middle-field component of the triplet nitroxide radical was measured. The result was expressed as the signal to noise (S/N) ratio, calculated by dividing the signal intensity of the sample (signal) by that of a paired HBsAg-negative serum sample (noise) (Fig. 1).

2.3. Confirmation of the specificity of HBsAg detection

To confirm the specificity of HBsAg detection by the radical immunoassay method, we performed an absorption test using HBsAb-coated ferrite particles (Fujirebio Diagnostics Inc., Tokyo, Japan). HBsAg panel serum (adr, genotype C, Institute of Immunology Co., Ltd., Tokyo, Japan) at a concentration of 0.1 ng/ml was incubated with the HBsAb-coated ferrite particles for 30 min at room temperature. Then, the ferrite particles were removed using a magnet, and HBsAg in the absorbed panel serum was measured by radical immunoassay. Our preliminary study showed that a concentration of 0.1 ng/ml detected by the present HBsAg panel serum was nearly equal to 0.2 IU/ml by the WHO International Standard.

2.4. Establishment of the cut-off value

The radical immunoassay was conducted in 146 healthy volunteers whose ALT level was normal and who had exhibited negative results for HBsAg (HBsAg II EIA Cobas Core, Roche Diagnostics Corp., Indianapolis, IN) and anti-HBc (Enzygnost Anti-HBc monoclonal, Boehringer Diagnostic GmbH, Germany) according to the standard ELISA. The cut-off value was established based on the distribution of the S/N ratio obtained from healthy volunteers [7].

2.5. Comparison of HBsAg detection by various assay systems

To confirm the sensitivity of the radical immunoassay, we conducted the following experiments: HBsAg panel serum (adr, genotype C, Institute of Immunology Co., Ltd.) was serially diluted and then measured by EIA (HBsAg II EIA Cobas Core, Roche Diagnostics Corp.), the chemiluminescence method (Architect HBsAg, Dainabot Co., Ltd., Tokyo, Japan) and the radical immunoassay method.

2.6. Hepatitis B surface antigen and HBV DNA detection by serial dilution of sera obtained from HBV carriers

To compare the sensitivity of HBsAg detection by radical immunoassay with that of HBV DNA by real-time PCR, serial dilution tests were conducted with sera from HBV carriers. HBV DNA was measured using a real-time direct test for HBV (HBV-Direct Mag, JSR, Tokyo, Japan), which combines the use of a DNA extraction system based on magnetic beads coated with polyclonal anti-HBsAg and the real-time detection method [10] and [11]. The PCR primers and probe used were designed using Primer Express software (Applied Biosystems, CA, USA) and were available for eight HBV genotypes (A–H) on the basis of alignment with their sequences [10]. The detection limit of the test is 1.0 log10 copies per ml.

2.7. Measurement of HBsAg in sera from normal volunteers and patients with non-B chronic liver diseases

We analyzed sera from an additional 210 healthy volunteers, being different from the volunteers who participated in the establishment of the cut-off value, 65 patients with cirrhosis of unknown cause (non-B, non-C cirrhosis), 62 patients with hepatocellular carcinoma of unknown cause (non-B, non-C hepatocellular carcinoma), 134 patients with HCV chronic hepatitis or cirrhosis, and 107 patients with hepatocellular carcinoma complicated by HCV chronic hepatitis or cirrhosis. All the examined cases were negative for HBsAg by ELISA (HBsAg II EIA, Cobas). The sera of patients with non-B chronic liver diseases positive for HBsAg by the radical immunoassay method were further subjected to measurement of anti-HBs (Lumipulse II HBsAb, Fujirebio Diagnostics Inc.), anti-HBc (Lumipulse II HBcAb, Fujirebio Diagnostics Inc.), and HBV DNA. All patients gave their informed consent.

2.8. Statistical analysis

Comparisons of values between two groups were performed using the Mann–Whitney U test. Statistical analysis of HBsAg positivity rate among the groups was conducted by Fisher’s exact test. Differences at p < 0.05 were considered to be statistically significant. The analysis software used was the Statistical Package for the Social Sciences (SPSS Inc., Chicago, IL) version 12.

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3. Results

3.1. Establishment of the cut-off value

The minimum and maximum S/N ratios of the healthy volunteers were 0.63 and 2.15, respectively (Fig. 2A). The data from four volunteers were excluded from this analysis by the Smirnov test because of their abnormal distribution. Calculations of the logarithm of S/N ratios showed that in the healthy volunteers the minimum and maximum values were −0.1973 and 0.2389, respectively (Fig. 2B). The distribution of S/N ratios in the healthy volunteers was considered to be a normal distribution with a skewedness of 0.008 and a kurtosis of 3.249 (Fig. 2B). From these data, S/N ratios of <means + 2SD (S/N = 2.208) were considered to be negative (−), greater-or-equal, slantedmeans + 4SD (S/N = 3.249) to be positive (+), and greater-or-equal, slantedmeans + 2SD and <means + 4SD to be undeterminable (±).

Fig. 2. Establishment of the cut-off values. (A) The minimum and maximum S/N ratio for 146 normal volunteers. (B) Logarithm of the S/N ratio of 146 normal volunteers. The distribution of S/N ratios in the negative control group was considered to be a normal distribution.

3.2. Specificity and sensitivity of HBsAg detection by radical immunoassay

The absorption of HBsAg by the HBsAb-coated ferrite particles revealed a marked decrease in the ESR signal, as shown in Fig. 3. The radical immunoassay was able to detect 0.025 ng/ml HBsAg, with an undeterminable range of 0.01 ng/ml, while the lowest determinable level of EIA was 1.2 ng/ml and that of the chemiluminescence immunoassay 0.2 ng/ml (Table 1).

Fig. 3. Confirmation of specificity by radical immunoassay. HBsAg panel serum at a concentration of 0.1 ng/ml was incubated with HBsAb-coated ferrite particles and HBsAg was absorbed. A drastic decrease of the ESR signal was observed after absorption of HBsAg by the HBsAb-coated ferrite particles.

Table 1.
Comparison of the sensitivity between EIA, CLIA, and radical immunoassay using HBsAg panel serum
HBsAg (ng/ml)        EIA        CLIA        Radical immunoassay (S/N ratio)
1.2        +        +        +(27.95)
0.8        −        +        +(25.64)
0.2        −        +        +(13.42)
0.1        −        −        +(9.31)
0.05        −        −        +(6.27)
0.025        −        −        +(3.70)
0.010        −        −        ±(2.33)

HBsAg panel serum: adr, genotype C CLIA, chemiluminescent immunoassay.

3.3. Comparison of radical immunoassay and HBV RTD-direct test

The levels of HBV DNA and HBsAg in the diluted sera from 9 HBV carrier patients were measured simultaneously by HBV RTD-direct test and the radical immunoassay. In case those original sera were diluted to an HBV DNA concentration of 1.2–2.4 LOG IU/ml, the sera were positive for HBsAg by the radical immunoassay. When the sera were further diluted to a level of less than 1.0 LOG IU/ml HBV DNA, the diluted sera were still positive for HBsAg by the radical immunoassay (Table 2).

Table 2.

Comparison of sensitivity between HBV RTD-direct test and radical immunoassay
Case (genotype)        Diluted sera from HBV carriers        Further dilution of the diluted sera from HBV carriers
        HBV RTD-direct (PCR: LOG IU/ml)        Radical immunoassay (S/N)        HBV RTD-direct (PCR: LOG IU/ml)        Radical immunoassay (S/N)
1 (B)        1.8        +(35.4)        10 times Negative (less than 1)        +(5.0)
2 (C)        1.7        +(339)        10 times Negative (less than 1)        +(31.3)
3 (B)        1.3        +(12.4)        4 times Negative (less than 1)        +(4.2)
4 (B)        1.2        +(23.2)        5 times Negative (less than 1)        +(6.3)
5 (B)        1.3        +(13.3)        4 times Negative (less than 1)        +(4.3)
6 (C)        1.4        +(82.9)        30 times Negative (less than 1)        +(6.8)
7 (C)        2.4        +(461)        100 times Negative (less than 1)        +(16.4)
8 (C)        1.2        +(10.7)        3 times Negative (less than 1)        +(3.7)
9 (B)        1.2        +(9.6)        3 times Negative (less than 1)        +(3.9)
Full-size table

3.4. Examination of HBsAg by radical immunoassay in healthy volunteers and patients with non-B chronic liver diseases

Serum HBsAg was examined by the radical immunoassay method using the automatic ESR analyzer in healthy volunteers and patients with non-B, non-C liver cirrhosis and non-B, non-C hepatocellular carcinoma. As shown in Fig. 4, HBsAg was positive in 6 of the 210 volunteers, 5 of the 65 non-B, non-C liver cirrhosis patients, and 6 of the 62 non-B, non-C hepatocellular carcinoma patients (P = 0.04 vs. volunteers). We also examined the low level of HBsAg in the sera from HCV-positive patients. Even though HBsAg was negative by routine EIA, the radical immunoassay showed positivity for HBsAg in 12 of the 134 patients with chronic hepatitis C (p < 0.02 vs. volunteers) and 11 of the 107 patients with HCV-positive HCC (p < 0.008 vs. volunteers). The positivity rate of HBsAg in patients with non-B chronic liver diseases (34 out of 368 patients) was significantly higher than that in normal volunteers (p < 0.005).

Fig. 4. Examination of HBsAg by the radical immunoassay method in normal volunteers and patients with chronic liver diseases.

3.5. Details of low HBsAg-positive patients with chronic non-B liver diseases

HBV DNA detection was performed in sera from 6 volunteers and 34 patients with chronic liver diseases who were positive for HBsAg by radical immunoassay. No HBV DNA-positive case was found among 6 volunteers who showed low HBsAg positivity by radical immunoassay. One patient with non-B, non-C liver cirrhosis with a S/N ratio of 7.13, two patients with non-B, non-C HCC with S/N ratios of 12.2 and 9.34, three patients with chronic hepatitis C with S/N ratios of 11.6, 5.46 and 4.53, and three patients with HCC complicated by HCV with S/N ratios of 10.8, 8.86 and 5.57 were positive for HBV DNA. The S/N ratios for HBV DNA-positive cases tended to be higher than those for HBV DNA-negative cases (Table 3).

Table 3.

Characteristics of 34 cases
Patient        Age/sex        Diagnosis        Radical immunoassay (S/N ratio)        HBV DNA (LOG IU/ml)        Anti-HBs        Anti-HBc
1        58/M        Non-B, non-C liver cirrhosis        7.13        1.2        –        +
2        69/M        Non-B, non-C liver cirrhosis        4.86        –        –        –
3        57/F        Non-B, non-C liver cirrhosis        4.28        –        +        –
4        54/M        Non-B, non-C liver cirrhosis        3.72        –        –        –
5        68/F        Non-B, non-C liver cirrhosis        3.31        –        –        +
6        72/M        Non-B, non-C HCC        12.20        2.3        –        +
7        75/F        Non-B, non-C HCC        9.34        1.3        –        –
8        73/M        Non-B, non-C HCC        4.12        –        +        +
9        68/M        Non-B, non-C HCC        4.10        –        –        –
10        66/F        Non-B, non-C HCC        3.68        –        –        +
11        69/M        Non-B, non-C HCC        3.29        –        –        –
12        64/M        Chronic hepatitis C        11.60        1.6        –        –
13        69/M        Chronic hepatitis C        8.64        –        +        +
14        53/F        Chronic hepatitis C        7.42        –        –        –
15        47/F        Chronic hepatitis C        7.15        –        –        +
16        36/M        Chronic hepatitis C        5.92        –        +        –
17        53/F        Chronic hepatitis C        5.46        2.0        –        –
18        54/F        Chronic hepatitis C        5.23        –        –        +
19        64/M        Chronic hepatitis C        4.53        1.8        +        +
20        50/M        Chronic hepatitis C        4.25        –        –        –
21        59/M        Chronic hepatitis C        4.07        –        –        +
22        60/M        Chronic hepatitis C        3.81        –        +        –
23        36/F        Chronic hepatitis C        3.37        –        –        –
24        69/M        HCC with HCV        10.8        1.2        –        –
25        70/F        HCC with HCV        8.86        1.3        +        +
26        61/M        HCC with HCV        6.25        –        –        –
27        65/F        HCC with HCV        5.57        1.0        –        –
28        64/M        HCC with HCV        5.12        –        –        +
29        62/M        HCC with HCV        4.41        –        –        +
30        73/F        HCC with HCV        4.23        –        –        –
31        59/F        HCC with HCV        3.74        –        –        +
32        54/M        HCC with HCV        3.46        –        +        –
33        71/F        HCC with HCV        3.35        –        –        –
34        73/F        HCC with HCV        3.54        –        –        –

Anti-HBs positivity (+): greater-or-equal, slanted5 mIU/ml Anti-HBc positivity (+): greater-or-equal, slanted50% inhibition.

Two of 9 HBV DNA-positive cases were positive for anti-HBs and 6 of 25 HBV DNA-negative cases were positive for anti-HBs. Four of 9 HBV DNA-positive cases were positive for anti-HBc and 10 of 25 HBV DNA-negative cases were positive for anti-HBc. As shown in Table 3, there was no clear relationship between HBV DNA positivity, anti-HBs positivity, and anti-HBc positivity.

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4. Discussion

Fig. 5 summarizes the status of low HBs antigenemia clarified by highly sensitive HBsAg measurement. In combination with radical immunoassay and real-time PCR, we found two groups of patients with low HBs antigenemia: low HBs antigenemia with HBV DNA and low HBs antigenemia without HBV DNA. The former group is considered to represent occult HBV carriers. In the latter group, it is uncertain whether there is actual infection with a low HBV load, and may simply imply a false positive. Our present study demonstrated that our radical immunoassay method is a promising new tool for screening occult HBV carriers from healthy subjects and patients with various liver diseases.

Fig. 5. Summary of low HBsAg and HBV DNA measurement in patients with non-B chronic hepatitis. We found two groups: low HBs antigenemia with HBV DNA, which was often observed in patients with non-B chronic liver diseases, and low HBs antigenemia without HBV DNA, which was predominant in healthy volunteers.

As shown in our data comparing the HBV RTD-direct test and radical immunoassay using diluted sera from HBV carriers, the radical immunoassay still showed positivity for HBsAg when the HBV RTD-direct test indicated negativity for HBV DNA (less than 1.0 LOG IU/ml). In low-HBV load carriers, the proportion of HBsAg and HBV DNA concentrations in sera may differ among individuals. However, approximately 26.5% of low-HBs antigenemia patients with non-B chronic liver diseases proved to be positive for low HBV DNA, and were considered to be occult HBV carriers. In comparison with HBV DNA, which exists in an episomal or integrated pattern, HBV-associated proteins such as HBsAg fluctuate to a lesser degree [12]. Therefore, radical immunoassay for measurement of low HBsAg seems to be advantageous to HBV DNA measurement by PCR.

Occult HBV is defined as the presence of HBV DNA detectable by sensitive PCR among individuals testing negative for HBsAg [5], [13], [14] and [15]. There are two main reasons for occult HBV: mutations in the S gene of HBV DNA or a low level of HBV viremia [16] and [17]. Bréchot et al. have provided very strong evidence that most cases of occult HBV are related to very low levels of HBV (low HBV load) rather than to HBV mutants that do not express or produce aberrant HBV surface protein [17]. Full-length genome analysis has shown that multiple alterations in the HBV genome may have a synergistic effect in down-regulation of HBsAg production, making it difficult to establish a specific mutation in a particular gene [18]. Therefore, we expected that our radical immunoassay system would be able to screen the majority of occult HBV carriers conveniently because of its high sensitivity. Testing of whole samples by real-time PCR of HBV DNA may be of interest regarding the proportion of HBV DNA positivity in HBsAg-negative samples and will reveal the proportion of HBV DNA positivity in HBsAg-positive samples by the radical immunoassay method, thus validating its rationality.

In the present study, an important issue was whether individuals with low HBsAg positivity without HBV DNA were HBV carriers. We confirmed the specificity of HBsAg detection by an absorption test using diluted HBsAg panel serum and anti-HBsAb. Although we did not show the data, we also performed the absorption test on several low HBsAg-positive cases and confirmed its specificity. Our analytical results therefore appear to be reliable. Natural clearance of HBsAg has been reported to occur in chronic HBV carriers [19] and [20]. Among 34 cases with low HBsAg positivity and HBV DNA negativity, 23.5% were positive for anti-HBs and 41.2% were positive for anti-HBc. We speculate that some cases showing low HBsAg but negativity for HBV DNA might reflect the process of natural clearance of HBsAg. In the present study, we were unable to obtain data indicating that patients with low HBsAg positivity with HBV DNA negativity were HBV carriers. Further study will be required to clarify this issue.

In areas where HBV infection is highly endemic, vertical and horizontal transmissions are now a social health problem [21]. Although the prevalence of occult HBV infection is speculated to be high in these endemic areas, the PCR method cannot be used routinely for screening large numbers of samples because of the complexity of sample preparation, the expense of reagents including Taq polymerase, and the time taken for sample processing. Moreover, the sensitivity of PCR analysis depends on DNA concentration, selection of PCR primers, and assay conditions [22]. Thus, PCR analysis of occult HBV is not suitable as a universal screening method. Occult HBV carries a risk of transmission [23] and [24]. It is anticipated that the radical immunoassay method will become a powerful tool for worldwide prevention of vertical and horizontal transmission of occult HBV, including cases with a low virus load.

In conclusion, the radical immunoassay method has high sensitivity for HBsAg detection. This method demonstrated that a low concentration of HBsAg was present in healthy volunteers and a higher percentage of patients with non-B chronic liver diseases. The present findings clearly demonstrate that infection with HBV, including occult HBV, is far more prevalent than previously thought, as a result of increasing the sensitivity of HBsAg detection to below the present limit.

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Acknowledgements

Thisstudy was supported by a Grant-in-Aid from the Ministry of Health,Labor and Welfare, Japan. The authors also thank the editors andreviewers for their helpful comments and suggestions.


References

[1] J.E. Maynard, Hepatitis B: global importance and need for control, Vaccine 8 (1990), pp. S18–S20. Abstract |  View Record in Scopus | Cited By in Scopus (157)

[2] M. Kane, Global programme for control of hepatitis B infection, Vaccine 13 (1995), pp. S47–S49. Abstract | PDF (380 K) |  View Record in Scopus | Cited By in Scopus (226)

[3] W.M. Lee, Hepatitis B virus infection, N Engl J Med 337 (1997), pp. 1733–1745.  Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (1009)

[4] C. Brechot, F. Degos, C. Lugassy, V. Thiers, S. Zafrani and D. Franco et al., Hepatitis B virus DNA in patients with chronic liver disease and negative tests for hepatitis B surface antigen, N Engl J Med 312 (1985), pp. 270–276.  View Record in Scopus | Cited By in Scopus (131)

[5] M. Torbenson and D.L. Thomas, Occult hepatitis B, Lancet Infect Dis 2 (2002), pp. 479–486. Article | PDF (240 K) |  View Record in Scopus | Cited By in Scopus (114)

[6]C. Matsumoto, K. Tadokoro, K. Fujimura, S. Hirakawa, S. Mitsunaga andT. Juji, Analysis of HBV infection after blood transfusion in Japanthrough investigation of a comprehensive donor specimen repository, Transfusion 41 (2001), pp. 878–884.  Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (26)

[7] T. Matsuo, H. Shinzawa, H. Togashi, M. Aoki, K. Sugahara and K. Saito et al., Highly sensitive hepatitis B surface antigen detection by measuring stable nitroxide radical formation with ESR spectroscopy, Free Radic Biol Med 25 (1998), pp. 929–935. Article | |   PDF (85 K)View Record in Scopus | Cited By in Scopus (10)

[8] M. Aoki, T. Saito, H. Watanabe, T. Matsuo, K. Saito and H. Togashi et al.,Clinical significance of a highly sensitive enzyme immunoassay ofhepatitis B surface antigen using a novel electron spin resonancetechnique, J Med Virol 66 (2002), pp. 166–170.  Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (5)

[9] M. Aoyama, M. Shiga, H. Ohya and H. Kamada, A novel ESR method for horseradish peroxidase activity using a combination of p-acetamdophenol and hydroxylamine, and its application to enzyme immunoassays, Anal Sci 14 (1998), pp. 1107–1113.  Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (4)

[10] M. Mukaide, Y. Tanaka, S. Katayose, H. Tano, M. Murata and M. Hikata et al.,Development of real-time detection direct test for hepatitis B virusand comparison with two commercial tests using the WHO internationalstandard, J Gastroenterol Hepatol 18 (2003), pp. 1264–1271.  Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (9)

[11] H. Sakugawa, K. Kobashigawa, T. Nakayoshi, T. Yamashiro, T. Maeshiro and K. Tomimori et al., Monitoring low level hepatitis B virus by a newly developed sensitive test, Hepatol Res 26 (2003), pp. 281–286. Abstract |  View Record in Scopus | Cited By in Scopus (5)

[12]T. Tsurimoto, A. Fujiyama and K. Matsubara, Stable expression andreplication of hepatitis B virus genome in an integrated state in ahuman hepatoma cell line transfected with the cloned viral DNA, Proc Natl Acad Sci USA 84 (1987), pp. 444–448.  Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (57)

[13] T. Uchida, M. Shimojima, K. Gotoh, T. Shikata, E. Tanaka and K. Kiyosawa et al., “Silent” hepatitis B virus mutants are responsible for non-A, non-B, non-C, non-D, non-E hepatitis, Microbiol Immunol 38 (1994), pp. 281–285.  View Record in Scopus | Cited By in Scopus (47)

[14] J.P. Allain, Occult hepatitis B virus infection, Transfus Clin Biol 11 (2004), pp. 18–25. Article | PDF (216 K) |  View Record in Scopus | Cited By in Scopus (28)

[15] G. Raimondo, T. Pollicino, I. Cacciola and G. Squadrito, Occult hepatitis B virus infection, J Hepatol 46 (2007), pp. 160–170. Article | PDF (765 K) |  View Record in Scopus | Cited By in Scopus (0)

[16]S. Grethe, M. Monazahian, I. Bohme and R. Thomssen, Characterization ofunusual escape variants of hepatitis B virus isolated from a hepatitisB surface antigen-negative subject, J Virol 72 (1998), pp. 7692–7696.  View Record in Scopus | Cited By in Scopus (51)

[17]C. Brechot, V. Thiers, D. Kremsdorf, B. Nalpas, S. Pol and P.Paterlini-Brechot, Persistent hepatitis B virus infection in subjectswithout hepatitis B surface antigen: clinically significant or purelyoccult?, Hepatology 34 (2001), pp. 194–203. Abstract |  Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (211)

[18]V. Chaudhuri, R. Tayal, B. Nayak, S.K. Acharya and S.K. Panda, Occulthepatitis B virus infection in chronic liver disease: full-lengthgenome and analysis of mutant surface promoter, Gastroenterology 127 (2004), pp. 1356–1371. Abstract |  View Record in Scopus | Cited By in Scopus (24)

[19]H. Adachi, S. Kaneko, E. Matsushita, Y. Inagaki, M. Unoura and K.Kobayashi, Clearance of HBsAg in seven patients with chronic hepatitisB, Hepatology 16 (1992), pp. 1334–1337.  Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (43)

[20] R. de Franchis, G. Meucci, M. Vecchi, M. Tatarella, M. Colombo and E. Del Ninno et al., The natural history of asymptomatic hepatitis B surface antigen carriers, Ann Int Med 118(1993), pp. 191–194.  View Record in Scopus | Cited By in Scopus (128)

[21] J.H. Kao and D.S. Chen, Global control of hepatitis B virus infection, Lancet Infect Dis 2 (2002), pp. 395–403. Article | |   PDF (228 K)View Record in Scopus | Cited By in Scopus (217)

[22]M.J. Gandhi, G.G. Yang, B.J. McMahon and G.N. Vyas, Hepatitis B virionsisolated with antibodies to the pre-S1 domain reveal occult viremia byPCR in Alaska Native HBV carriers who have seroconverted, Transfusion 40 (2000), pp. 910–916.  Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (13)

[23] V. Thiers, E. Nakajima, D. Kremsdorf, D. Mack, H. Schellekens and F. Driss et al., Transmission of hepatitis B from hepatitis-B-seronegative subjects, Lancet 2 (1988), pp. 1273–1276. Abstract |  View Record in Scopus | Cited By in Scopus (120)

[24]M. Torbenson, R. Kannangai, J. Astemborski, S.A. Strathdee, D. Vlahovand D.L. Thomas, High prevalence of occult hepatitis B in Baltimoreinjection drug users, Hepatology 39 (2004), pp. 51–57.  Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (34)


Theauthors who have taken part in the research of this paper declared thatthey do not have a relationship with the manufacturers of the materialsinvolved either in the past or present and they did not receive fundingfrom the manufacturers to carry out their research. Dr. Kainuma is anemployee of Tohoku Seiki Industries, Ltd., Yamagata, Japan.

^.^

What can be revealed by extending the sensitivity of HBsAg detection to below the present limit?

Hitoshi Togashi1, , , Chika Hashimoto1, Junji Yokozawa1, Akihiko Suzuki1, Kazuhiko Sugahara1, Takafumi Saito1, Ichiro Yamaguchi2, Hala Badawi3, Norikazu Kainuma4, Masaaki Aoyama5, Hiroaki Ohya5, Takao Akatsuka5, Yasuhito Tanaka6, Masashi Mizokami6 and Sumio Kawata1

1 Departmentof Gastroenterology, Course of Internal Medicine and Therapeutics,Yamagata University Faculty of Medicine, Yamagata University HealthAdministration Center, 1-4-12 Kojirakawa-machi, Yamagata 990-8560,Japan
2 Murayama Public Health Center, Yamagata Prefecture, Japan  
3 Medical Microbiology, Theodor Bilharz Research Institute, Giza, Egypt  
4 Tohoku Seiki Industries, Ltd., Yamagata, Japan  
5 Institute for Life Support Technology, Yamagata Public Corporation for Development of Industry, Yamagata, Japan  
6 Departmentof Clinical Molecular Informative Medicine, Nagoya City UniversityGraduate School of Medical Sciences, Nagoya, Japan

Received 10 August 2007;  
revised 6 March 2008;  
accepted 21 March 2008.  
Associate Editor: R.P. Perrillo.  
Available online 22 April 2008.

FORRESTFU

目标组不太理想。特别是健康组，没有提到表面抗体情况。
146 healthy volunteers whose ALT level was normal and who had exhibited negative results for HBsAg (HBsAg II EIA Cobas Core, Roche Diagnostics Corp., Indianapolis, IN) and anti-HBc (Enzygnost Anti-HBc monoclonal, Boehringer Diagnostic GmbH, Germany) according to the standard ELISA. The cut-off value was established based on the distribution of the S/N ratio obtained from healthy volunteers [7].

至于健康组之外的其他组，测出表面抗原是天经地义的。

良药尽欢颜

这篇文章很重要，大家要重视，
这就是为什么有人长期小三，阴性，最后还硬化的原因，因此一定要提高定量检测水平。

无悔这一生男人

看不懂哦，很想看，怎么办?