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Serological Markers
Diagnosis of HBV infection is based on the detection of three pairs of antigens and their related antibodies: (a) hepatitis B surface antigen/hepatitis B surface antibody (HBsAg/HBsAb), (b) hepatitis B e antigen/hepatitis B e antibody (HBeAg/HBeAb) and (c) hepatitis B core IgG/hepatitis B core antibody (HBcAg/HBcAb).[3] To note, HBcAg is undetectable in serum and can only be shown in hepatocytes by specific immunohistochemistry methods.
HBsAg was the first developed serological marker of HBV infection; it appears within 4–10 weeks after virus exposure and several weeks before symptoms manifestation and transaminase increase. HBsAg becomes undetectable in patients who recover from acute hepatitis B 4–6 months after the onset.[4] In general, the persistence of HBsAg for more than 6 months suggests the development of a chronic disease. The disappearance of HBsAg is followed by the occurrence of anti-HBs antibody in plasma after several weeks. In most cases, anti-HBs will persist indefinitely, thus developing some immunity to future HBV infection.[5] In some situations, anti-HBs may decrease to undetectable levels. This does not imply a greater susceptibility to HBV recurrence or a loss of immunological protection. There is some evidence that, despite the absence of anti-HBs, the mere re-exposure to a new viral inoculum induces an immunological response, as a result of which anti-HBs levels become detectable in plasma. On the other hand, a long window period may elapse as the disappearance of HBsAg (a few weeks to several months) until the time at which anti-HBs grows detectable. During this period, detection of IgM anticore (IgM anti-HBc) may become the only serological marker to diagnose HBV infection.[6]
Although HBsAg frequently co-exist with anti-HBs, the former has been reported in almost 25% of patients with HBV infection. The detection of these antibodies usually reveals either very low or no neutralizing ability. Furthermore, these antibodies represent no risk for the patient and are unrelated to any specific change in the course of the disease. This serological profile is usually detected during an active HBV replication.[7]
Anti-HBc consists of two molecular fractions (total IgG and IgM) that are detectable in serum during the course of either the acute or the chronic disease. IgM antibodies against hepatitis B core antigen (anti-HBc IgM) occur during the course of acute viral infection and typically decline to undetectable levels within 6 months after the onset. However, there is evidence that this marker may become positive at a low titre during episodes of chronic hepatitis B reactivation.[8] On the contrary, IgG anti-HBc, in the absence of HBsAg, persists indefinitely and may be the only serological marker remaining after recovery from an acute hepatitis B infection (Table 1). The presence of IgG anti-HBc in the absence of HBsAg usually indicates a past HBV infection. The incidence of this event varies between blood banks in USA (0.6%) and Argentina (2–10%). The event itself could be a contra-indication for organ transplantation as it harbours a potential risk of HBV transmission (occult HBV infection).[9] To note, the frequency of occurrence of people who become infected with HBV and remain asymptomatic is as high as 65%. This situation explains the accidental detection of anticore antibody in blood banks, as well as the patients' ignorance about their carrier condition for this memory antibody.
There are some unusual profiles or combinations of some markers that remain uncommon laboratory findings. The report of such events tends to maximize the accuracy of results of the submitted tests[10] (Table 2).
Isolated detection of anti-HBc may occur in four different instances: (a) during the window period of acute hepatitis B, that is when the anti-HBc profile comprises predominantly the IgM class; (b) several years after recovery from acute hepatitis B, when anti-HBs levels have decreased down to undetectable levels; (c) whenever a false-positive test results or (d) after many years of HBv infection, when HBsAg levels become undetectable.[11]
On the other hand, evidence shows that the isolated anticore may be associated with HBV DNA as a detectable event in serum in up to 1/3 of patients (0–30%). The presence of viraemia in the absence of HBsAg is usually associated with a occult hepatitis B infection and poses some potential implications regarding virus transmission.[12] This topic must be taken into account because of its high impact on HBV transmission from anti-HBc-positive donors to liver transplant recipients (50–70%). Likewise, low levels of HBV replication confirmed by PCR (polymerase chain reaction) assays were observed in serum and tissue from HBsAg-negative patients with cirrhosis and hepatocellular carcinoma (HCC).[13,14] The same serological pattern was found in non-A, non-E fulminant hepatitis patients.
HBeAg is a viral soluble protein usually found in serum during HBV acute infection. It is a substitute marker of replication and as such it is frequently associated with high concentrations of HBV DNA in plasma. A good example of the high infecting power of these patients is offered by chronic HBV carrier mothers who have high levels of HbeAg. Such mothers have a high chance to transmit HBV to newborns during delivery (90%), compared with that of negative-HBeAg carriers (10–20%).[15] The presence of HBeAg for more than 10 weeks in acute hepatitis B carriers highly suggests the development of a chronic HBsAg carrier condition.
Finally, the index value used to report some determinations should not be understood as a quantitative measure, though, indirectly, it usually provides information regarding quantification. The index value indicates the way our sample behaves in reference to the cut-off value for the assay. Both the cut-off value and the index value vary according to each commercial assay. Index values close to the cut-off value may be associated with false-positive results so, in such situations, it is recommended to repeat the analysis on a new sample and/or use an alternative technique. All these situations must be discussed considering the patient's clinical context, including the remaining serological and virological markers.[16]
When preparing the report, it is also important to inform the technique used, the results collected, the index value of the sample and the potential index value range.
The difficulties for diagnosis in HBsAg detection in the presence of HBsAg mutants depend on the assay design, especially when polyclonal antibodies, monoclonal antibodies or a combination of both are used.
HBeAg behaviour reflects different clinical settings. It has been shown that persistent presence of HBeAg in serum for many years is associated with an increased risk of cirrhosis or HCC. Progression to cirrhosis is quite frequent in patients older than 40 years with this profile.[17]
Likewise, HBeAg seroconversion usually implies an abrupt reduction in HBV DNA levels as well as the clinical and histological remission of the disease. Early HBeAg seroconversion, as well as a therapy-induced HBe seroconversion, are associated with an improved prognosis and a longer, complication-free survival.[18,19]
One of the most important therapeutic goals is to achieve a seroconversion from HBe to anti-HBe. This is a desirable fact, not only because it halts the evolution of the disease but also because it increases the likelihood of HBsAg loss. HBsAg loss associated with the appearance of anti-HBs is much more frequent in patients who experience a HbeAg loss. Recent investigations show that low titres of HBeAg for the first 3 months of treatment with peg-INF are associated with a higher rate of HBe-anti-HBe seroconversion.[20]
Hepatitis B virus DNA is the most sensitive and specific marker of viral replication. It may be found in serum, liver tissue and bone marrow, as well as in peripheral blood mononuclear cells. HBV DNA can be measured both qualitatively and quantitatively (HBV viral load).[21] Qualitative commercial assays are exclusively applied for testing nucleic acids in blood and haemoderivatives, thus they do not have any clinical application. In-house qualitative methods are not recommended as no standardized limit of detection expressed in IU/ml has been established. The most frequently used quantitative methods are shown in Table 3.[12,22]
It is underscoring that the high sensitivity of quantitative assays allows its use whenever it is necessary to detect HBV DNA. On the other hand, the comparison of results derived from the various methods usually presents some inconsistencies. For this reason, use of the same kind of assay to monitor the response to a specific drug during treatment is recommended. To prevent mistakes in viral load interpretation, the physician ordering the test should clearly indicate the methodology to be used and the reason why such study is ordered (pre- or intratreatment quantification).[16]
Results above the upper limit of detection are not valid; in these situations, specimens should be diluted until the correct value is reached. These dilutions must be done in accordance with the sample matrix. On the other hand, when HBV DNA levels are below the lower limit of detection, it does not necessarily mean absence of viral replication.
The used method, the active range of quantification, the value in IU/ml, copies/ml and logs must always be included in the report. Values below the lower limit of quantification should not be informed.
To ensure quality control from laboratories using these molecular techniques, it is recommended to establish an integrated quality management system and to subscribe to an accreditation program ensuring assay reproducibility, sensitivity and specificity.[16]
The availability of more sensitive methods such as real-time PCR has improved the investigation of the relationship between HBV replication levels and the natural history of the disease.[23] A good example of this is the REVEAL-HBV study, which mainly included Asian HBeAg-negative patients and demonstrated a close relationship between elevated HBV DNA levels and more cases of cirrhosis and HCC.[24] On the other hand, HBV DNA quantification is currently becoming a key tool for selecting patients eligible for treatment, for guiding response to antiviral therapy and also for the differentiation of settings related to liver disease progression.[25–29]
Although HBV DNA determination describes the viral status at the moment of detection, it does not represent the balance between development of host immunity and viral replication. It is important to point out that HBV DNA levels can fluctuate in negative-HBeAg patients and become temporarily undetectable; this situation makes physicians consider a differential diagnosis associated with the inactive carrier state.[30]
Although changes observed in viral load levels in patients with low HBV DNA titres do not represent a flaw in method sensitivity, they have contributed to the development of complementary methods with a high clinical impact, such as HBe and HBsAg quantification[31]
血清学标志物
HBV感染的诊断是基于三对抗原及相关抗体的检测：（1）乙肝表面抗原/ B型肝炎表面抗体（乙肝表面抗原/乙肝表面抗体），（B）B型肝炎e抗原/乙肝e抗体（ e抗原/抗 - HBe）和（c）乙型肝炎核心抗体/乙型肝炎核心抗体（HBcAg的/ HBcAb阳性）[3]注意到，核心抗原是不可检测的血清中，并且可以仅在肝细胞中通过特定的免疫组化方法显示。
乙肝表面抗原是乙肝病毒感染的第一个发达的血清学标志物;它病毒暴露和几个星期前的症状表现和转氨酶升高后，将出现4-9个星期内。乙肝表面抗原检测不到就在谁从一开始4-6个月后，急性乙肝患者康复[4]一般情况下，乙肝表面抗原为6个月以上的持久性提出一种慢性疾病的发展。 HBsAg的消失之后是抗HBs抗体在血浆中的数周后发生。在大多数情况下，抗HBs将无限期持续，因而发展了一些免疫将来HBV感染[5]在一些情况下，抗HBs可能会降低到检测不到的水平。这并不意味着更容易感染乙肝复发或免疫保护的损失。有一些证据表明，尽管没有抗HBs的，仅仅再暴露到一个新的病毒接种物诱导的免疫应答，其结果是抗HBs抗体水平成为可检测的血浆中。在另一方面，长的窗口期可能经过的乙肝表面抗原的消失（几周到几个月），直到其抗HBs检测的生长时间。在此期间，检测的IgM anticore（IgM抗-HBc抗体）可能成为唯一的血清学标记物来诊断HBV感染[6]。
尽管HBsAg的经常并存抗HBs，前者已报道在患有HBV感染几乎25％。这些抗体的检测通常揭示要么非常低或没有中和能力。此外，这些抗体表现为患者无风险，是无关的，在疾病过程中的任何特定的变化。此血清学配置文件是一个积极的HBV复制过程中通常会检测到。[7]
抗HBc由两个分子级分（总IgG和IgM）可检测血清中的任一的急性或慢性疾病过程中的。 IgM抗体抗乙肝核心抗原（抗HBc IgM抗体）发生急性病毒感染过程中，通常在发病后下降到检测不到的水平6个月内。然而，有证据表明，该标记物可在慢性乙型肝炎再激活事件成为以低滴度阳性[8]与此相反，IgG抗-HBc抗体，在不存在的HBsAg，持续下去，可能是唯一的血清学标记物从急性乙型肝炎感染恢复后剩余的（表1）。 IgG抗-HBc阳性在没有乙肝表面抗原的存在通常表明过去的HBV感染。在美国（0.6％）和阿根廷（2-10％），血库之间的这种事件的发生变化。事件本身可能是一个禁忌症的器官移植，因为它藏着乙肝病毒传播（隐匿性HBV感染）的潜在风险。[9]要注意，出现的人谁感染上乙肝病毒和保持无症状的频率是一样高为65％。这种情况说明了偶然发现在血库anticore抗体，以及病人的无知有关此存储器抗体的载体的条件。
有一些标记，仍然鲜见实验室发现了一些不同寻常的个人资料或组合。此类事件的报告倾向于最大化提交的测试[10]（表2）的结果的准确性。
分离检测抗HBc的，可能会出现在四个不同的实例：（a）在急性乙型肝炎的窗口期，即当抗HBc信息主要包含IgM类; （b）由急性乙型肝炎，当抗HBs水平已降低到检测不到的水平恢复后数年; （c）在经过多年的乙肝病毒感染时，HBsAg水平成为不可检测的假阳性检测结果或（d）。[11]
另一方面，有证据表明，该隔离anticore可以与HBV DNA的相关联的可检测事件在血清中的患者（0-30％）至三分之一。病毒血症在没有乙肝表面抗原的存在通常与隐匿性乙肝病毒感染相关联，并提出了有关病毒传播的一些潜在影响。[12]这个话题，必须采取因从乙肝病毒传播的高影响到反HBc-积极捐助肝移植（50-70％）。同样地，HBV的复制，通过PCR（聚合酶链式反应）检测证实低浓度均在血清和组织从HBsAg阴性患者的肝硬化和肝细胞癌（HCC）的观察。[13,14]相同的血清型被发现在非A，非E暴发性肝炎患者。
大三阳通常是在乙肝急性感染中发现血清中病毒的可溶性蛋白。这是复制的替代标记物，因此它经常与血浆高浓度的HBV DNA的关联。这些患者感染的高功率的一个很好的例子就是谁拥有高水平的HBeAg阳性慢性HBV携带者母亲提供。这样的母亲有较高的机会传递（90％）期间，乙肝病毒传播给新生儿，与阴性，e抗原携带者（10-20％）相比，[15]大三阳的超过10周的急性乙肝携带者的存在强烈建议对慢性HBsAg携带者病情的发展。
最后，用于报告一些测定的指标值不应被理解为是一种定量测量，但是，间接地，它通常提供关于量化的信息。该指数值表明我们的样品表现在参照该分析的截止值的方式。根据各商业测定两者的截止值和索引值而有所不同。接近的截止值的索引值可与假阳性结果，因此相关联的，在这种情况下，建议重复上一个新的样品的分析和/或使用其它的技术。所有这些情况下，必须将讨论考虑患者的临床情况下，包括余下的血清学和病毒学标志物。[16]
当制备的报告，它也是重要的通知中使用的技术，其结果收集的样本的索引值和所述电势的索引值的范围。
对于诊断中的HBsAg检测HBsAg的突变的存在的困难，取决于测定的设计，特别是当多克隆抗体，单克隆抗体，或两者的组合来使用。
大三阳的行为反映了不同的临床环境。它已经显示，e抗原的血清中持久存在多年的肝硬化或肝癌的风险增加相关联。进展为肝硬化是年龄超过40岁，此配置文件的病人相当频繁。[17]
同样，HBeAg血清转换通常意味着突然降低HBV DNA水平以及疾病的临床和组织学缓解。早期的HBeAg血清学转换，以及一个治疗引起的HBeAg血清转换，都具有改善预后，延长，无并发症生存率[18,19]
其中最重要的治疗目标是实现从HBe抗体Â血清转化成抗HBe。这是一个理想的事实，不仅是因为它停止了疾病的发展，但也因为它增加了HBsAg消失的可能性。与抗HBs的出现相关联的HBsAg消失是很多患者谁遇到HBeAg转阴更加频繁。最近的研究表明，HBeAg阳性滴度较低的前3个月的治疗与PEG-INF文件都具有较高的HBE-抗HBe血清学转换有关。[20]
乙型肝炎病毒DNA是病毒复制的最敏感和特异的标记物。它可能的血清，肝组织和骨髓，以及在外周血单核细胞中被发现。 HBV-DNA可以定性和定量（HBV病毒载量）。测定[21]定性商业测定法是只适用于检测在血液和血液衍生物的核酸，因此它们不具有任何临床应用。内部的定性方法，不建议作为检测没有标准化的限制以IU表示/毫升已经建立。最经常使用的量化方法被示于表3[12,22]。
它强调指出的定量分析的高灵敏度允许其使用时，它是必要的，以检测HBV DNA。另一方面，从各种方法得出的结果进行比较常表现为一些不一致。出于这个原因，使用相同种类的检测，以监测治疗过程中对特定药物的响应的建议。为了防止病毒载量的解释错误时，医生下令测试应清楚地注明所使用的方法以及为什么这样的研究是有序的原因（前或intratreatment定量）。[16]
结果高于检测上限是无效的;在这种情况下，样品应稀释至正确的值为止。这些稀释液必须根据样品基质进行。另一方面，当HBV DNA水平低于检测下限，这并不一定意味着不存在病毒复制。
所使用的方法中，量化的活性范围，以IU/值毫升，拷贝/ ml和日志必须总是包含在报告中。低于定量下限值应不被告知。
为了确保使用这些分子技术实验室的质量控制，建议建立一个完整的质量管理体系和订阅的认证计划，确保实验的重复性，灵敏度和特异性。[16]
更敏感的方法，如实时PCR的可用性提高了HBV复制水平与疾病的自然史之间的关系进行调查。[23]这方面的一个很好的例子就是REVEAL-HBV的研究，主要包括亚洲HBeAg阴性患者表现出升高的HBV DNA水平和肝硬化和肝癌的病例多有密切的关系。[24]另外，乙肝病毒DNA定量正在成为选择的患者适合接受治疗，指导应对的关键工具抗病毒治疗，也为相关的肝脏疾病进展设置的分化[25-29]。
尽管HBV DNA的测定描述在检测时刻的病毒状态，它不表示宿主的免疫力及病毒复制的发展之间的平衡。它指出，HBV DNA水平可以在负HBeAg的患者发生波动，变得暂时不能检测到是很重要的;这种情况使得医生考虑与非活动性携带状态相关的鉴别诊断。[30]
虽然在病毒载量水平观察患者低HBV DNA滴度的变化并不代表方法的灵敏度的一个缺陷，他们已经促成了互补的方法具有较高的临床影响，如HBe抗体和乙肝表面抗原定量的发展[31]
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发表于 2014-10-7 17:10