215007 Institute of Liver Diseases, Suzhou Fifth People’s Hospital, Suzhou, China (Tong Fuyi Wu Jianhong Cao Wengui Wu Xingfu Fei Xiaofeng Ding Longqi)
[Abstract] Objective: To investigate the relationship between hepatitis B virus markers (HBV-M) and HBV load (HBV-DNA), and to provide a scientific basis for proper clinical diagnosis and treatment. METHODS: HBV-M and HBV-DNA were detected in the sera of 670 patients by immunoluminescence and fluorescence quantitative polymerase chain reaction techniques, respectively. results: HBeAg-positive mode HBV-DNA > 5.0e4 cps/ml accounted for 96.3% (363/377), HBeAg-negative mode HBV-DNA > 5.0e4 cps/ml accounted for HBV-DNA load was significantly higher in the HBeAg-positive pattern than in the HBeAg-negative pattern (P < 0.05). HBV-DNA load was significantly higher in the 1,3,5 pattern than in the 1,4,5 pattern (P < 0.01). 18.8% (12/64),and among the anti-HBs positive patterns, HBV-DNA >5.0e4 cps/ml accounted for 16.7% (7/42). Conclusion: HBV-DNA load was significantly higher in HBeAg-positive pattern than in HBeAg-negative pattern. However, a few HBeAg-positive patterns had very low viral levels and HBeAg-negative patterns had high viral levels. There are also HBsAg-negative or/and anti-HBs-positive HBV infections. Therefore, only the combination of HBV-M and HBV load testing can correctly determine the disease and prognosis in clinical practice. Tong Fuyi, Department of Hepatology, Suzhou Fifth People’s Hospital, Suzhou, China
[Keywords] Hepatitis B virus; polymerase chain reaction; fluorescence quantification; enzyme immuno
Relationship between the models of serologic markers and the quantities of virus in patients infected with hepatitis B virus
Tong Fuyi,Shaowei,Cao Wengui,et al. Department of Infectious Diseases, Suzhou Fifth People’s Hospital, Suzhou 215007
[Abstract] Objective To understand the relationship between the models of serologic markers and the quantities of hepatitis B virus (HBV). Methods The serologic markers of HBV in 670 patients were detected by using the enzyme immunoassay and the quantities of HBV in those were measured by using Results The HBeAg-positive patients whose copies were more than 5.0e4 /ml accounted for 96.3%, and the The HBV copies of HBeAg-positive patients were significantly more than HBeAg-negative patients whose copies were more than 5.0e4 /ml accounted for 74.8%. The HBV copies of HBeAg-positive patients were significantly more than HBeAg-negative patients (P < 0.05). The HBV copies of anti-HBe-positive patients were significantly less than anti-HBe-negative patients (P < 0.01). The HBV-DNA was detected in some HBsAg -negative (anti-HBs-positive or not) patients.Conclusions The HBV copies of HBeAg-positive patients were significantly more than HBeAg-negative However, some HBeAg-positive patients' HBV copies are very low, and some HBeAg-negative patients' HBV copies are The HBV-DNA even could be detected in some HBsAg-negative (anti-HBs-positive or not) patients. It is helpful to judge accurate prognosis by testing the serologic marker and the quantities of virus.
It is helpful to judge accurate prognosis by testing the serologic marker and the quantities of virus. Quantitative PCR (Q-PCR) has become a reality due to the leap forward in polymerase chain reaction (PCR) technology in recent years. We used fluorescence quantitative polymerase chain reaction (FQ-PCR) method to detect HBV-DNA in 670 cases of HBV-infected patients to explore the relationship between HBV-M and HBV load.
1 Data and methods
1.1 Case collection: 670 cases were all inpatients of our hospital from January to October 2002. All cases had not used special antiviral drugs such as interferon, lamivudine, α1 thymidine, etc. in the last 3 months. Peripheral blood was collected, serum was isolated and tested on the same day or stored at -20°C.
1.2 Determination of HBV-M: AXSYMTM automated immunoluminescence diagnostic instrument from Abbott (USA), with reagents provided by Abbott as supporting reagents, operated by dedicated personnel. Positive reporting mode: HBsAg (S/N) ≥ 2.00; anti- HBs (Miu/ml) ≥ 10.00; HBeAg (S/CO) ≥ 1.00; anti- HBeAg (S/CO) ≤ 1.00; anti-HBc (S/CO) ≤ 1.00.
1.3 Quantitative determination of HBV-DNA: Real-time fluorescence quantitative PCR method was used. AcuGen System TMAG 9900 QRM Guantitation RoboMaster fully automated quantitative PCR instrument was used, and the reagents were AcuGen System supporting reagents, provided by Shenzhen Baiyaktai (Biotronics) Company. The detection sensitivity was 5.0e4cps/ml.
1.4 Statistical treatment: χ2 test was used for the comparison of rates.
2 Results
2.1 Case data: a total of 670 patients, 515 males and 155 females. The male:female ratio was 3.3:1, and the age ranged from 7 to 72 years, of which 89.6% were aged 15-60 years (600/670).
2.2 HBV-M patterns: 14 HBV-M patterns were found. 8 HBsAg-positive patterns totaled 591 cases, accounting for 88.2% of the total number of cases (591/670), of which 1,3,5 patterns accounted for 50.3% (297/591) and 1,4,5 patterns accounted for 30.6% (181/591). 6 HBsAg-negative patterns totaled 64 cases, accounting for The total number of HBsAg-negative patterns was 9.6% (64/670) of the total number of cases, including 34.4% (22/64) for the 2,4,5 pattern, 23.4% (15/64) for the 4,5 pattern, and 21.9% (14/64) for the 2,5 pattern. 15 cases were completely negative for HBV-M.
Table The relationship between HBV-M and HBV-DNA load
Table 1 The relationship between the marker of HBV and the quantities of HBV
Hepatitis B virus marker Number of cases HBV-DNA load the quantities of HBV (cps/ml)
the marker of HBV n ＜5.0e4(%) 5.0e4~4.9 e6(%) 5.0 e6~4.9 e8(%) ＞5.0 e8(%)
(-) (+) (++) (+++)
1,3,5 297 8(2.7) 69(23.2) 124(41.8)△ 96(32.3)▲
1,4,5 181 46(25.4) 67(37.0) 55(30.4)△ 13(7.2)▲
1,3,4,5 67 4(6.0) 20(29.9) 35(52.2) 8(11.9)
1,5 23 6(26.1) 10(43.5) 4(17.4) 3(13.0)
1,2,3,5 11 1(9.1) 3(27.3) 4(36.4) 3(27.3)
1,2,4,5 9 1(11.1) 4(44.4) 3(33.3) 1(11.1)
1,2,3,4,5 2 1(50.0) 1(50.0)
1 1 1(100.0)
2,4,5 22 18(81.8) 3(13.6) 1(4.5)
2,5 14 12(85.7) 2(14.3)
2 5 4(80.0) 1(20.0)
2,4 1 1(100.0)
4,5 15 12(80.0) 1(6.7) 2(13.3)
5 7 5(71.4) 2(28.6)
Negative 15 14(93.3) 1(6.7)
Total 670 132 186 226 126
* 1: HBsAg, 2: anti-HBs, 3: HBeAg, 4: anti-HBe, 5: anti-HBc. △: P < 0.05; ▲: P < 0.01
2.3 HBV load: among the HBsAg-positive patterns: HBeAg-positive pattern HBV-DNA > 5.0e4cps/ml accounted for 96.3% (363/377),analysis of HBeAg-negative pattern HBV-DNA > 5.0e4cps/ml accounted for 74.8% (160/214). HBV-DNA load was significantly higher in HBeAg-positive pattern than in HBeAg-negative pattern (χ2=6.07,P<0.05). For patterns 1,3,5, HBV-DNA <5.0e4cps/ml accounted for only 2.7%, while HBV-DNA >5.0e4cps/ml accounted for 97.3% (289/297), of which HBV-DNA >5.0e6cps/ml accounted for 41.8% (124/297) and HBV-DNA >5.0e8cps/ml accounted for 32.3% (96/297).1,4,5 Among the patterns, HBV-DNA <5.0e4cps/ml accounted for 25.4%, while HBV-DNA >5.0e4cps/ml accounted for 74.6% (135/181), of which HBV-DNA >5.0 e6 cps/ml accounted for 30.4% and HBV-DNA >5.0e8 cps/ml accounted for only 7.2%.1 HBV-DNA load was significantly higher in the 1,3,5 patterns than in the 1,4,5 patterns (P < 0.01). e8 cps/ml; HBV-DNA <5.0e4 cps/ml accounted for 83.3% (35/42) of the anti-HBs positive pattern. In 2 cases, 5 markers of HBV-M were present simultaneously. (see table)
3 Discussion
HBV-M can manifest itself in many forms after HBV infection. The results of this study revealed 14 antigenic antibody patterns. the most common pattern of HBsAg positivity was 1,3,5 pattern (43.0%). Among the HBsAg-negative patterns, 1,5 pattern (29.1%) and 2,4,5 pattern (27.8%) were the most common, followed by 4,5 pattern (19.0%) and 2,5 pattern (17.7%). And 2 cases were found to be positive for HBV-M.
HBeAg is the mRNA expression of the pre-C/C region of the HBV genome and is closely related to HBV-DNA, so usually HBeAg-positive individuals have active viral replication and are highly infectious, while HBeAg-negative individuals have low viral replication levels and are less infectious. The results of our study also confirmed that the HBV-DNA level in HBeAg-positive patients was significantly higher than that in HBeAg-negative patients (P < 0.05). This is consistent with most reports [1]. However, in the HBeAg-positive pattern, there were still 3.7% of patients with HBV-DNA <5.0e4 cps/ml, which may be related to the suppression of HBV replication by strong immune pressure of the organism or due to the integration of HBV-DNA in the blood into the hepatocytes. In the HBeAg negative pattern, there are still 74.8% of patients with HBV-DNA > 5.0e4 cps/ml and even 7.2% of patients with HBV-DNA > 5.0e8 cps/ml, which may be related to HBV gene mutations, especially when the pre-C gene or C gene promoter is mutated, e antigen is difficult to be expressed and viral replication is still present [2,3].
The disappearance of HBsAg and the production of anti-HBs mostly implies the recovery of HBV infection or the protective effect of hepatitis B vaccination of the organism. The results of this study showed that in HBsAg negative, anti-HBs positive pattern, there are still 16.7% of patients with HBV-DNA > 5.0e4 cps/ml, only the viral level is mostly not high, which may be related to the low level of viral replication.HBsAg negative HBV infection is a hot spot of clinical concern for several years. The reasons for this are: (i) the amount of antigen is too low to reach detectable levels; (ii) it exists as an antigen-antibody complex, and when antibodies are dominant the antigen is masked and the kit is not sensitive and cannot be detected; (iii) HBV-S gene variants, in which some variants in the main hydrophilic region do not produce HBsAg [4]. It may be due to mutations in the S gene encoding HBsAg: the “a” determinant cluster of the S gene is the major antigenic determinant cluster, and a single conserved amino acid substitution in this region can alter the antigenicity of HBsAg. A variation in the “a” determinant cluster affects the overall antibody response. HBsAg is detected by conventional immunodiagnostic reagents, and the anti-HBs produced by the body cannot neutralize the mutated HBV, and there are different subtypes of HBV with geographical distribution and polymorphism in the sequence of the “a” determinant cluster [5]. In our study, we found that HBsAg negative or/and anti-HBs positive patients had HBV infection in a small number of sera, and HBsAg and HBeAg titers were correlated with HBV-DNA levels [6]. levels were correlated [6]. In addition, anti-HBs test results may be related not only to the source of reagents but also to differences in the source of the hepatitis B vaccine received [7].
The viral replication process is also accompanied by the expression of packaging proteins, and these heterologous proteins (antigens) stimulate the body’s immune system to produce the corresponding antibodies. Thus, antigenic antibodies reflect the level of viral expression and, indirectly, the level of viral replication. Serological methods can also be used for the analysis of the etiology of hepatitis B (etiologic diagnosis), infectivity, disease progression and prognosis. However, the expression of HBV and the strength of the body’s immune response are influenced by several factors, and it is possible that immunological indicators do not exactly match clinical phenomena and viral load [8]. Due to the widespread use of HBV reverse transcriptase inhibitors and the widespread presence of viral variants, many clinical phenomena can no longer be fully explained by immunological indicators alone. Serologic testing is not a complete substitute for HBV-DNA testing. Viral load is a more quantitative indicator than serology, and may be more accurate and sensitive than serological indicators in reflecting clinical phenomena and their changing trends. However, the two cannot completely replace each other. Therefore, clinical testing of HBV-M must be accompanied by testing of HBV-DNA levels, and the combination of the two can correctly determine the disease and prognosis, if only one-sided emphasis on one side is biased.
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