Eur J Clin Microbiol Infect Dis DOI 10.1007/s10096-015-2522-7

REVIEW

Acute hepatitis B virus infection or acute exacerbation of chronic hepatitis B infection: the differential serological diagnosis R. A. A. Pondé 1,2,3,4

Received: 6 October 2015 / Accepted: 2 November 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract Acute exacerbations of chronic hepatitis B are common, and may even be the first presentation of hepatitis B virus (HBV) infection. Sometimes, patients involved in these scenarios may have mistaken diagnosis of acute hepatitis B. The reason for the confusion is that the two forms of infection manifestation resemble remarkably in clinical, biochemical, and serological features, such as apparent rapid onset of severe disease, advanced grades of encephalopathy, high aminotransferases and prolonged international normalized ratios (INRs), as well as positivity for HBsAg and for IgM anti-HBc antibodies and DNA detection. Therefore, these two entities cannot be distinguished easily without historical information of HBV-associated chronic infection or recent HBV exposure, information that is often inaccurate. Considering the different prognoses, treatment strategies, and the epidemiological impact in the public health context, the correct diagnosis is extremely important. Despite the lack of effective and reliable tests to differentiate between acute infection and acute exacerbation of chronic HBV infection, the expression and kinetic evaluation of viral markers present in the circulation of individuals infected, the observation of physical-chemical

* R. A. A. Pondé [email protected] 1

Laboratory of Human Virology, Institute of Tropical Pathology and Public Health, Federal University of Goiás, Goiânia, Goiás, Brazil

2

Central Goiana de Sorologia, Imuno-hematologia e Biologia Molecular, Goiânia, Goiás, Brazil

3

SUVISA—Superintendência de Vigilância em Saúde, Secretaria Estadual de Saúde, Coordenação Estadual de Controle das Hepatites Virais (CECHV), Goiânia, Goiás, Brazil

4

Rua 7A Edifício RIOL, Nº 158, 1º andar, sala 101, setor aeroporto, Goiânia, Goiás 74-075-030, Brazil

properties of specific antibodies, and the combination of these findings represent some strategies in serology that could assist in differentiating between the two entities, or at least in the guidance for the correct diagnosis.

Introduction Acute exacerbations of chronic hepatitis B virus (HBV) infection are common in regions with intermediate and high prevalence rates, and may be the first clinical manifestation of HBV infection. Although it is not yet a consensus, this clinical entity is characterized by the sudden re-emergence or increase of HBV/DNA in serum, resulting from viral reactivation in a patient with resolved or chronic/inactive HBV infection, usually being associated with immune imbalance [1]. The clinical expression of cases of acute exacerbation of chronic infection resembles the clinical manifestations in acute infection after recent exposure to the virus. In both conditions, patients may suffer from flu-like prodromic symptoms, together with jaundice, abdominal discomfort, and pruritus, in addition hepatosplenomegaly that is also common in both situations. Jaundice is more common in acute infection and splenomegaly is more common in acute exacerbation. However, clinical examination may not differentiate between the two clinical entities. In the laboratory context, there are no significant differences in regards to the biochemical assays, such as peak bilirubin serum level, prothrombin time prolongation, and serum albumin level, except for a tendency for higher levels of serum transaminases in acute infection cases. However, none of the biochemical parameters can differentiate between acute infection and acute exacerbation of chronic infection [2, 3]. Regarding the serological findings, the difficulties in the differential diagnosis are similar. Although the serological pattern of viral markers that define acute and

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chronic HBV infection is fully recognized, in cases of acute exacerbation of chronic infection, IgM anti-HBc antibodies can be detected, in addition to the viral markers HBsAg and HBeAg. The latter two are due to their persistence in the bloodstream, or as a result of the seroreversion, which comprises an underlying phenomenon when the respective antibodies are present. This serological profile may suggest acute infection. The similarities between acute exacerbation of chronic infection and acute infection in the clinical and laboratory context make it difficult to distinguish between the two clinical entities. Given this situation, the diagnosis of acute HBV infection can be established mistakenly, unless the past clinical history of the patient it is known, as well as their epidemiological history. Even considering the natural difficulties to establish the diagnosis, it is important to differentiate between acute exacerbation and acute HBV infection, since the two clinical conditions have different prognoses and might require different therapeutic strategies. Most patients with acute infection recover spontaneously and the treatment may be required only in a small number of individuals, i.e., in those that evolve to fulminant hepatitis. On the other hand, patients presenting acute exacerbation of chronic infection generally need therapy, since the hepatocellular dysfunction can lead to hepatic decompensation [4]. Despite the lack of effective and reliable tests for differentiation between acute exacerbation of a chronic infection and HBV-related acute infection, the analysis not only qualitative of the viral markers but also their kinetic evaluation, the observation of some physical-chemical properties of specific antibodies, and the correlation of the results observed, includes strategies in serology that could assist in differentiating between the two entities and, consequently, in the guidance for the correct diagnosis. These features and strategies are summarized in this review.

Expression and evaluation of the levels of IgM anti-HBc antibodies It has been reported that IgM anti-HBc antibodies are present in approximately 10–15 % of patients with chronic hepatitis B, especially in those during acute exacerbation episodes of chronic hepatitis B [5]. Accordingly, other studies have shown that, during these episodes, IgM anti-HBc antibodies can be detected in almost three-quarters of chronically infected patients, even in low titers [3, 6, 7]. The IgM production during episodes of acute exacerbations of chronic infection may be the result of inflammation and hepatocellular lysis during flare-ups of the disease, with consequent release of high concentrations of the nucleocapsid protein, leading to the activation of B cells. In addition, other mechanisms related to the conformational structure of the epitope of core antigen has

been proposed [8]. Since the presence of IgM anti-HBc may suggest persistent viral replication, its detection could be used as an indicator for acute exacerbation in chronic patients. It is possible that the IgM antibodies detection during episodes of acute exacerbation is the laboratory finding of greatest potential to induce to the misdiagnosis of acute HBV infection. This occurs obviously by the fact that antiHBc IgM is recognized as a classic marker of acute infection, due to their production and detection in high levels during the primary immune response, after HBcAg interaction with cells of the immune response, in particular, after HBcAg interaction direct with B lymphocytes [9]. A phenomenon similar to what occurs in cases of acute exacerbation arises, however, with greater vigor. Additionally, the mistake in diagnosis also occurs due to the currently standardized tests’ sensitivity, which attain an analytical sensitivity as low as 7 Paul-Ehrlich-Institut (PEI) units [10], and, therefore, they are capable of detecting low levels of IgM antibodies, which are produced during episodes of acute relapse of chronic infection. The same performance could not be observed from the old enzyme immunoassays, which were standardized at a threshold value corresponding to 600–700 PEI units, and, therefore, were able to give a positive result only in the presence of the high IgM levels that are usually produced during acute HBV infection [10–12], being, however, unable to detect the low levels produced during acute exacerbations. Consequently, with the introduction of assays with high sensitivity, the low levels of IgM anti-HBc produced during episodes of acute exacerbation of chronic infection could be detected in the circulation of chronically infected individuals, leading to the misconception in the diagnosis of acute infection. In view of this situation, and to minimize the possibility of misdiagnosis, it has been suggested that the quantitation of IgM anti-HBc could allow distinction between acute infection and acute exacerbation of chronic infection, since high levels may be suggestive of acute infection, whereas low levels may indicate acute exacerbation of chronic infection. From this premise, some authors have suggested different parameters for evaluation. For example, Kumar et al., using the microparticle enzyme immunoassay (MEIA; Abbott), found that titers higher than 1:1000 could be seen in 80 % of acutely infected patients and that in 70 % of chronically infected patients, the IgM titers were below 1:1000 or were negative [7]. Despite this important observation, these limits do not seem to demonstrate the sensitivity and specificity required to differentiate between the two clinical conditions. On the other hand, Han et al. demonstrated that IgM anti-HBc titers >1:10,000, detected by enzyme immunoassay (EIA), had a sensitivity and specificity of 96.2 and 93.1 %, respectively, for the diagnosis of acute infection [13], whereas Rodella et al., using a quantitative chemiluminescent immunoassay (CLIA; Abbott Architect), found that an average sample/cutoff (S/CO) value of >10 for IgM identified acute infection. In acute exacerbation,

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the S/CO values were 10 demonstrated 100 % sensitivity and a specificity of 99 % for acute infection diagnosis, being, therefore, of practical importance. Finally, and in accordance with the findings of Rodella et al. [14], Dao et al., showed that the cutoff index value for IgM anti-HBc of 5.0, obtained by an indirect IgM capture immunoassay, could be enough to differentiate acute HBV infection from acute exacerbation of chronic HBV with a positive predictive value of 86 % and a negative predictive value of 89 %. In addition, their studies suggested that determining the IgM anti-HBc levels with a quantitative assay or at least a higher cutoff value than 1.0 is a readily available single test with excellent predictive capacity [15]. Table 1 demonstrates some studies on IgM anti-HBc quantitation conducted from different assays, suggesting a threshold value and its sensitivity/specificity for the differentiation of acute hepatitis from acute exacerbation of chronic infection. In light of these studies, the evaluation of IgM antibody levels presents itself as interesting strategy to be used in the differentiation of the two manifestations of HBV infection. By its applicability, some authors have proposed that the index of IgM antibodies that define acute infection cases should be reconsidered [15, 17]. However, it is remarkable the diversity of reference values suggested from various studies, a fact that would make it difficult to establish a standard reference value for the analysis of cases. In addition, there is no general agreement in the literature and among the producers of test kits on whether a clear distinction is possible and which titer could serve as the cutoff level, which could be associated partly to the lack of a generally accepted reference sample. Consequently, the lack of standardization of commercialized assays and the absence of any valid clinical threshold make the use of this marker quite unreliable [11]. In summary, in practice, the presence of IgM anti-HBc positivity is associated with acute infection and is necessary but not sufficient to diagnose acute hepatitis B. However,

Table 1

knowing that higher IgM anti-HBc titers have been suggested to be associated with a highly active host immune response, quantitative IgM anti-HBc testing comprises a good strategy to more accurately distinguish between acute and non-acute cases. A second feature, still associated with the detection of IgM antibodies, that could be used to aid in distinguishing between acute infection and acute exacerbation is the evaluation of the activity of IgM anti-HBc antibodies in the molecular weight fractions 19S and 7-8S. The 19S and 7-8S immunoglobulin fractions can be prepared by rate-zonal centrifugation of sera from patients with hepatitis B (acute or chronic active hepatitis) and, so, tested for IgM anti-HBc [18]. Some studies have shown that serum from patients with acute infection tend to demonstrate a high activity of 19S IgM anti-HBc, whereas the 7S IgM anti-HBc activity has proved to be higher than 19S IgM anti-HBc activity in the sera from patients with chronic hepatitis, which could identify cases of acute exacerbations [19, 20]. This feature can be particularly important when the titers of IgM antibodies are not high enough to guide the correct diagnosis, but merely only detectable. However, it must be highlighted that, during reactivation of chronic infection, the IgM-specific anti-HBc can re-emerge as a 19S IgM fraction, although the ratios are usually low when compared to those observed in acute disease (1:1000 >1:10,000 >10

96.2 % 70.0 % 96.2 % 100 %

89.7 % 77.6 % 93.1 % 99 %

CLIA MEIA EIAa CLIA

[16] [7] [13] [14]

Dao et al. (2012) Huang et al.(2006)

5.0 2.4–2.5

86%b 90.0 %

89%c 90.0 %

IgM capture immunoassayd AxSYMe CORE-M

[15] [17]

a

Shanghai Kehua Bio-engineering Co., Ltd.

b

PPV positive predictive value

c

NPV negative predictive value

d

ADVIA Centaur IgM anti-HBc assay (Siemens Diagnostics)

e

AxSYM CORE-M (Abbott)

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IgG avidity is defined as the strength with which IgG binds to antigenic epitopes expressed by a given protein, which increases as the IgG matures. The antibody matures gradually during the 6 months following primary infection. This means that IgG produced within the first 3–5 months following a primary infection exhibits low avidity (or bind weakly with the antigen), whereas IgG produced several months or years after a primary infection exhibit high avidity (or bind strongly with the antigen). Therefore, low IgG anti-HBc avidity is an accurate indicator of primary infection within the preceding 3 to 4 months. On the other hand, high avidity excludes primary infection within the preceding 3 months. The avidity index is calculated using the ratio of the readings obtained from samples prepared with a dissociating agent (urea, guanidine) under conditions whereby hydrogen bonds between antigens and anti-HBc antibodies would be disrupted [21, 22]. The relative amount of anti-HBc antibody is, thus, assessed using the ratio between the signals obtained for each sample over the assay cutoff and a composite avidity index can be calculated taking into account both the dissociation index and the relative anti-HBc level [22]. The avidity index has been defined arbitrarily from each study, according to the studied populations, serological history, and, also, according to the clinical and laboratory data of the patients involved, parameters to be considered in order to minimize the chances of mistakenly classifying a sample in each case [14, 22]. For example, an arbitrary cutoff for the avidity index set at 0.70 appeared as the best option in the study of Rodella et al. in order to discriminate the two phases of acute hepatitis B, i.e., one that was followed by a recent exposure (or prior to a probable chronic infection) from an acute flare of an already established chronic HBV infection [14]. To calculate this index, Rodella et al. adopted the same experimental procedure employed by Suligoi et al. [21] for assessing the avidity of anti-HIV antibodies. For each sample, two aliquots of 0.1 mL each were subjected to a pretest 1:10 dilution, respectively with the AxSYM working buffer (B) and with 1 M guanidine chlorhydrate (G). Both aliquots were vortexed and incubated for 30 min, and then assayed for antiHBc by the automated AxSYM Core assay (Abbott GmbH). Since this is a competitive assay in which reactive samples are lower than the cutoff (S/CO < 1), the avidity index has been calculated by dividing the reciprocal (1 divided by S/CO) of the S/CO value obtained on the G aliquot by the reciprocal of the S/CO value of the B aliquot. On the other hand, the avidity index marker validated on commercial and in-house HBV seroconversion panels by Terkmani et al., and defined as a reference, it was 4.5. In this case, anti-HBc measurement was performed using Monolisa Anti-HBc PLUS (Bio-Rad, Marnes-la-Coquette, France), and a dissociation index was calculated using the ratio of the readings obtained with/ without 6 M urea as the dissociating agent. The avidity index, thus, was calculated considering both the dissociation index

and the relative anti-HBc level. From such a reference, an avidity index below or equal to 4.5 was identified as being highly predictive for recent hepatitis B (related to the apparition of HBsAg), typically within the previous 3 months [22]. Despite IgG avidity testing being increasingly recognized as a valuable tool, given its importance and the clinical significance, IgG avidity testing should not be used alone and without an understanding of the limitations of the technique. Serology remains an important tool for the diagnosis and management of infectious disease [23]. The avidity index has been a test used to identify acute infection caused by several other viral agents, such as cytomegalovirus, rubella, and West Nile virus, in addition acute infection caused by hepatitis C virus (HCV) and hepatitis A virus (HAV) [24–27].

Expression and kinetics evaluation of surface antigen of HBV (HBsAg) HBsAg is used typically as a qualitative serological marker for diagnosing an ongoing HBV infection, and this restricted use has neglected, in a way, the importance of this marker as a diagnostic tool (and prognosis), since with the development of assays for HBsAg quantification, it is possible to assess and monitor its levels in the bloodstream, which allows to predict the possibility of its clearance, depending on the outcome observed. Based on this observation, the quantitative determination of HBsAg in the bloodstream may present as an additional resource used in the differentiation between acute infection and chronic infection. Accordingly, some studies have shown that, in cases of acute hepatitis, the HBsAg levels in the circulation are lower than in cases of chronic infection [13, 15], which has been associated with the greater robustness of immune response and greater immune control of infection. Although important, this statement should be treated with caution because there has not been defined a cutoff limit that could establish the differentiation between the two entities. Besides, depending on the infection phase, acutely infected individuals may have high levels of HBsAg, in the same way that chronically infected individuals can also express lower levels in the bloodstream [28]. Thus, the definition of a cutoff could not be safe and reliable. On the other hand, it has been shown that the HBsAg kinetics in the bloodstream are different in the two clinical situations. In the acute infection context, after infection with wild-type HBV, HBsAg may be undetectable for several weeks, but always with a progressive increase in concentration until reaching final concentrations of 10 000–100 000 ng/ mL with 2–4 days of doubling time [29]. If acute HBV is resolved, HBsAg decreases with an initial half-life of 8 days

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until it has disappeared completely from serum after several weeks to months. In patients in whom a decrease in HBsAg concentrations by more than 50 % within the first 4 weeks is observed, it is expected that there is resolution of acute hepatitis in 95 % of cases [30–32]. In about 20 % of cases of acute resolving hepatitis B, HBsAg disappears much faster, so that samples taken in the late acute phase may be HBsAg-negative. In this last condition, the acute infection diagnosis should be established only by the IgM antibodies presence and HBV/ DNA detection. A detailed and recent study demonstrated that HBsAg levels decline more or less slowly depending on the length of the period of its persistence in the circulation. For individuals in whom the HBsAg persisted for less than 3 months, the levels of this marker declined faster than in those individuals with persistent HBsAg over 3– 6 months. In individuals with persistent HBsAg for >6– 12 months, the HBsAg levels declined more slowly than in subjects with persistent HBsAg for 3–6 months. In patients who evolved to chronic infection, it was observed re-elevation of HBsAg at 12 weeks after the acute infection onset [33]. In this study, it was also shown that the persistence time of HBsAg in the bloodstream may vary depending on genotype. In chronic infection, contrary to what occurs in acute infection, the HBsAg concentration may fluctuate over time and, under certain circumstances, may not be detectable, as seen in low-replication phases, when infection resolves spontaneously or after successful antiviral therapy [34, 35]. Accordingly, several recent studies have highlighted the differences in HBsAg titers throughout the natural history of chronic HBV infection [36, 37], and demonstrated the occurrence of variations in HBsAg levels at different stages of chronic hepatitis B (immune-tolerant, immune-active, and inactive) [38], as well as changes in HBsAg levels during the natural progression of disease in untreated patients [39]. As HBsAg quantitation assays make it possible to demonstrate that exchanges in quantitative measurement of this marker occur during the infection development, the HBsAg quantitative evaluation, as well as the subsequent monitoring of its levels, may present as an additional resource on the characterization of acute infection (obviously, if the HBsAg kinetics expression is compatible with acute infection), and, therefore, in the exclusion of acute exacerbation cases [31, 40]. These latter cases would be characterized by the development of persistent surface antigenemia, suggesting chronic liver damage. However, it should be considered the low sensitivity that this strategy provides. In turn, a quantitative analysis of the HBsAg concentration demonstrating persistently stable or increased values comprises an excellent prognostic strategy, indicating progression to chronicity [2, 33].

Evaluation of the hepatitis B e antigen (HBeAg) /anti-HBe antibodies profile HBeAg has been found more frequently in patients with acute infection compared with those with chronic infection, but the difference is not statistically significant. The differentiation between the two clinical conditions based on the presence of HBeAg has been hampered because, although this marker is more prevalently detected in acute cases, in chronic patients who are HBeAg-positive, the HBeAg seroconversion may be delayed for years, or even not develop in some patients [41]. In cases of acute exacerbation, the difficulty remains since, in these clinical situations, the occurrence of seroreversion is common, which would make this marker detectable in the circulation [7, 42]. However, similar to what is observed with respect to HBsAg (and DNA levels, see below), lower titers of HBeAg are more commonly seen in patients with acute infection, after recent exposure. Thus, the quantification of this marker may have some benefit, and the study of Han et al. showed that levels of HBeAg with an S/CO value of

Acute hepatitis B virus infection or acute exacerbation of chronic hepatitis B infection: the differential serological diagnosis.

Acute exacerbations of chronic hepatitis B are common, and may even be the first presentation of hepatitis B virus (HBV) infection. Sometimes, patient...
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