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The diagnosis of infections due to hepatitis B virus (HBV) is usually made on the basis of surface antigen (HBsAg) in the patient’s serum. The numerous viral markers available enable the evolution of the infection to be followed over time and a precise definition to be made of the different stages up to the chronic state, which occurs in a small percentage of cases but can lead to the development of cirrhosis or hepatocarcinoma. Modern molecular biology studies have revealed that, even when surface antigen is not detectable, the viral genome can persist indefinitely in infected host cells, most often within the liver, but sometimes also detectable in the serum. The persistence of HBV DNA in the absence of HBsAg is defined as an occult hepatitis B virus infection1–4.
This particular form of infection has been known for some time, but only in recent years have ever increasingly careful studies been carried out in this field, thanks to the availability of molecular biology techniques. Despite the fact that the occult infection can be associated with serological markers of previous HBV infection (anti-HBc and/or anti-HBs), a relatively high percentage of subjects are negative for all the viral markers5. In this context, the new molecular biology methods are of fundamental importance for a specific diagnosis of the occult infection and for identifying the HBV DNA in the liver and peripheral blood.
One of the most important aspects of occult hepatitis B virus infection is its clinical relevance. It has now been well documented that these occult infections can be transmitted by both blood transfusions6 and organ transplants7–9, while the role that this type of infection plays as a risk factor for the development of hepatocarcinoma has not yet been clarified definitely and is still under investigation10,11.
Occult hepatitis B virus infection seems to be associated in a small proportion of cases with viruses that are genetically mutated and undetectable using the commonly employed methods for the determination of HBsAg. More often the occult state of the infection appears to be related to a marked reduction in viral replication and gene expression, responsible for both the negativity for HBsAg and the fact that HBV DNA levels are very low or undetectable in the serum, but present in hepatic tissue.
Here we present the cases of two periodic blood donors referred to the Service of Immunohaematology and Transfusion (SIT) of Parma University Hospital in the period between July 1, 2005 and July 31, 2006. One of these donors was referred by the provincial AVIS blood donation centre in Parma and the other by the SIT of Piacenza. Both donors were negative for HBsAg and positive for HBV DNA.
In the period between July 1, 2005 and July 31, 2006, virological and serological screening of 32,608 donations from periodic donors in the city of Parma and the surrounding province were carried out in the SIT of Parma. Screening for HBsAg was carried out using the Hepanostika HBsAg Ultra test (Ortho Clinical Diagnostics)12 while the presence of HBV DNA was determined using the Cobas AmpliScreen (Roche) amplification test. This molecular biology test was performed by analysing samples in pools of 20, and had a sensitivity for HBV of 120 IU/mL. During the period of the study, no samples were positive for HBsAg while two (out of 32,608) units were positive for HBV DNA (case I and case II). Quantitative assays of the HBV DNA (PCR COBAS TaqMan HBV test, Roche) and nested ‘in-house’ PCR of the pre-core region and a fragment of the S region of HBV were carried out on the positive units and subsequently collected samples and the related products of amplification were sequenced.
The viral genotype of HBV was determined by nested amplification of a second fragment of the S region and subsequent restriction fragment length polymorphism (RFLP) analysis13.
DNA was extracted using a QIAmp MinElute Virus Spin QIAGEN kit (Qiagen GMBH, Hilden, Germany) according to the manufacturer’s instructions. A Perkin Elmer DNA 480 thermal cycler was used for the amplification of the DNA. Part of the protein S was then amplified, using the primers listed in Table I, in order to determine the genotype of the infecting virus. The HBMR2 primer used in the first round of amplification was also used in the second round of amplification. The primers listed in Tables II and III were used to investigate whether there were any mutations in the S region or pre-core region, respectively.
The amplification products were purified using a Seq-Prep GD400 kit (GeneDia) to eliminate the primers, dNTP and dimers and were then subjected to capillary electrophoresis on a DNA Analysis System CEQ 2000 (Beckman Coulter). Sequencing was carried out with a CEQ Dye Terminator Cycle Sequencing kit (Beckman), using the same primers as those employed for the amplification reaction.
The first case was a 55-year old, Italian, male periodic donor (U.A.), who had already made 50 donations. The donor had not been vaccinated against hepatitis B and he had no history of known risk factors for hepatitis B. A unit of blood collected on September 4, 2005 was negative for HBsAg but positive for HBV DNA (Table IV); the value of alanine aminotransferase (ALT) was normal. The unit of blood donated by this subject in September 2005 was eliminated and the donor was definitively suspended from the SIT.
All the donations made in the past had been negative for HBsAg, while this was the first donation tested for HBV DNA which was found to be positive. In the light of these findings, the donor was recalled in order to take further samples for investigations. As shown in Table IV, further tests confirmed the presence of a low titre of HBV DNA, together with serological evidence of anti-HBc, anti-HBs and anti-HBe. Furthermore, the absence of HBsAg, in the presence of a normal value of ALT, was confirmed.
RFLP analysis, carried out on the 485 bp amplification product of the S region of the HBV DNA, showed the presence of a D genotype, subtype ayw. Analysis of the nucleotide sequence of the 440 bp of the S region of the HBV DNA extracted from the donor’s plasma, aligned with a “consensus” reference sequence (BioEdit Sequence Alignment programme, version 7.0) for genotype D, showed mutations I110K, T116N, S117I, P120S, A128V, and Y134P (Figure 1).
These mutations are included between the first and third MHR antigenic region. Sequencing of the 256 bp fragment of the pre-core region of the viral DNA extracted from the donor showed that this region had a wild-type sequence.
The second case was a 60-year old, Italian, male donor (C.E.) who had not been vaccinated against hepatitis B and had no declared risk factors for HBV infection; this donor had already made 58 donations up to November 2005, all of which had been negative for HBsAg. At the time of the donation on in May 25, 2006, the man was negative for HBsAg and positive for HBV DNA (Table V). The unit of blood donated by this donor in May was eliminated and the donor was definitively suspended from the SIT.
The negativity for HBsAg found in the first sample was confirmed in subsequent samples taken on June 7, 2006 and July 26, 2006. Testing for serological markers of HBV, carried out on the same samples, showed negativity for HBeAg and IgM anti-HBc and anti-HBe antibodies, while the samples were positive for anti-HBs and anti-HBc antibodies. The viral load was low (57–62 UI/mL) and the viral genotype was type A, subtype ayw. Amino acid sequencing of the S region did not reveal any mutations. In contrast, analysis of the nucleotide sequence of the amplified fragment of the pre-core region revealed the substitution of an adenine for a guanine at nucleotide 1896 (G1896A) (Figure 2). As known 14, this mutation leads to the substitution of codon 28 (TGG), which codes for the amino acid tryptophan, by a stop codon (TAG) responsible for blocking the transcription of HBeAg.
The data presented here show how the introduction of molecular biology tests for the detection of HBV DNA enabled the identification of two cases of occult HBV infection. In fact, according to recent definitions, the persistence of HBV DNA in concomitance with the lack of HBsAg is indicative of an occult HBV infection1–4. Numerous mechanisms have been proposed to explain how the HBV genome persists in the host’s body in the absence of viral markers that reveal its existence, or of important clinical signs. The hypotheses concerning the pathogenesis of occult HBV infection include the contemporaneous presence of other viruses that can interfere with the replication of HBV14,15, control of the replication by the immune system16,17 and inefficient viral replication as a result of mutations of the virus18.
In consideration of the hypothesis that the lack of detection of HBsAg in the serum of the subjects tested in our centre could have been due to mutations in the HBV genome, we sequenced a part of the S region. There are numerous studies indicating a relation between point mutations in the genome of the virus and the lack of synthesis of a HBV proteins that can be recognised by the standard diagnostic test19–29 In both the subjects analysed in our study, sequencing of the S and pre-core regions of the HBV genomes considered revealed the presence of point mutations.
In the first case, the mutations were found between the first and third antigenic MHR regions which represents the fundamental part of the transcription of the S antigen, known to be exposed on the cell surface and involved in antibody binding. In agreement with published data22.23 indicating that most of the MHR variants emerge as a consequence of selective pressure and can facilitate the replication of resistant viral strains capable of eluding diagnostic tests, in case I, six mutations were found between positions 110 and 134 of the S protein. The viral mutations found in donor U.A. have not been described in the literature and it is impossible to be sure that they are involved in the altered antigenicity of the S protein; however, the fact that they are found in a region highly exposed to immunological pressure makes it legitimate to consider that this mechanism is involved in the lack of ability to detect the HBsAg in the serum.
Furthermore, in donor U.A., the presence of a low viral load in the circulation together with anti-HBs and anti-HBc antibodies and the lack of HBsAg, raises the possibility that the occult infection is the consequence of a series of concomitant events, including the presence of mutations in the determinant regions associated with a strong reduction in viral replication and gene expression.
The finding of the HBV variant in the pre-C/C gene in donor C.E. is also of particular clinical and epidemiological interest. This mutation, which consists of the substitution of an adenine in the place of a guanine at nucleotide 1896, leads to the replacement of codon 28 (TGG), which in the wild-type virus codes for tryptophan, by a translation stop codon (TAG), which blocks the translation of HBeAg. This is, in fact, the most common mutation of the pre-core region19–21. The wild-type phenotype (G1896) is associated with a favourable outcome of HBV infection, while the pre-core mutation (A1896) can be associated with severe liver disease in patients with chronic HBeAg positivity who have lost circulating antigen in the serum29.
On the basis of knowledge acquired so far and the results of this study it can be concluded that, in rare cases, blood donors can be unconscious carries of HBV infection, due to viral strains that, as a result of their structural and immunological characteristics, elude the screening for HBsAg in the serum/plasma, thus compromising the safety of transfusion of blood or blood components.
This study shows that more sensitive molecular tests are needed in order to guarantee greater efficacy in the recognition of occult HBV infections and the highest possible level of safety of blood and blood components destined for transfusion.
From the point of view of the laboratory diagnosis of HBV infection, it is clear that tests to detect viral DNA are of critical diagnostic importance also in cases of apparently resolved infection (HBsAg-negative and anti-HBV-positive).