Journal of Clinical Virology 67 (2015) 31–35

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Case report

Occult infection with HBV intergenotypic A2/G recombinant following acute hepatitis B caused by an HBV/A2 isolate夽 José Júnior Franc¸a de Barros a , Luciana Rego Peres b , Paulo Sérgio Fonseca de Sousa b , Francisco Campello do Amaral Mello a , Natalia Motta de Araujo a , Selma de Andrade Gomes a , Christian Niel a,∗ , Lia Laura Lewis-Ximenez b a b

Laboratório de Virologia Molecular, Instituto Oswaldo Cruz, Fiocruz, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brazil Laboratório de Referência Nacional para Hepatites Virais, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brazil

a r t i c l e

i n f o

Article history: Received 18 November 2014 Received in revised form 20 March 2015 Accepted 22 March 2015 Keywords: Brazil Genotype A Genotype G HBsAg loss Occult hepatitis B Recombinant virus

a b s t r a c t Viral and host factors leading to occult hepatitis B virus (HBV) infection (OBI) are not fully understood. Whether HBV genotype may influence the occurrence and course of OBIs is unknown. Here, we describe the case of a patient infected with HBV genotype A2 who developed symptomatic acute hepatitis and did not seroconvert after loss of HBsAg and HBeAg. The acute phase of hepatitis B was followed by a period of more than 2 years during which the DNA of an intergenotypic HBV/A2/G recombinant was intermittently detected in serum. © 2015 Elsevier B.V. All rights reserved.

1. Why this case is important? Anti-hepatitis B core (anti-HBc) IgM antibodies and hepatitis B surface antigen (HBsAg) are the main serological markers of the acute phase of hepatitis B virus (HBV) infection. The presence of hepatitis B ‘e’ antigen (HBeAg) and HBV DNA in serum indicates active viral replication. Resolution of infection is accompanied by the normalization of serum alanine aminotransferase (ALT) levels and seroconversion to anti-HBs and anti-HBe. However, HBV may persist in liver and/or serum of patients after recovery from self-limited acute viral hepatitis [1–3]. The long-lasting persistence of HBV genome in the liver (with or without detectable HBV DNA in the serum) of HBsAg negative individuals is termed occult HBV infection (OBI).

Abbreviations: HBV, hepatitis B virus; OBI, occult hepatitis B infection; HBsAg, hepatitis B surface antigen; HBeAg, hepatitis B ‘e’ antigen; Anti-HBc, anti-hepatitis B core; HCV, hepatitis C virus; HDV, hepatitis delta virus; HIV, human immunodeficiency virus; ALT, alanine aminotransferase; PCR, polymerase chain reaction. 夽 Nucleotide sequences: Nucleotide sequences described in this work have been deposited to Genbank with following accession numbers: KP033193 to KP033197 ∗ Corresponding author at: Laboratory of Molecular Virology, Oswaldo Cruz Institute, Fiocruz, Av. Brasil, 4365, 21040-360 Rio de Janeiro, Brazil. Tel.: +55 21 2562 1895; fax: +55 21 2562 1825. E-mail address: niel@ioc.fiocruz.br (C. Niel). http://dx.doi.org/10.1016/j.jcv.2015.03.019 1386-6532/© 2015 Elsevier B.V. All rights reserved.

HBV genotype G (HBV/G) was first identified in 1990 in a persistently infected homosexual man [4] and recognized as a new, separate genotype 10 years later [5]. HBV/G infection occurs mostly in the context of co-infection or recombination with HBV/A [6], although monoinfection has also been described [7]. Few studies have described the natural history of persistent HBV infection in patients who do not seroconvert after HBsAg clearance. Here we report the case of a patient who was infected with an HBV/A2 isolate during the acute phase of hepatitis B. In the subsequent, occult phase of infection, DNAs of both an HBV/G isolate and a recombinant A2/G virus were detected in the serum, in the absence of the original HBV/A2 isolate. 2. Case description A Caucasian 32-year-old man was referred to our outpatient unit (Fiocruz, Rio de Janeiro, Brazil) in October 2008 showing a clinical picture typical of acute hepatitis. He tested positive for HBsAg, HBeAg and IgM anti-HBc, with jaundice and ALT levels reaching 555 IU/mL. He had no history of surgery, organ transplant or blood transfusion, tested negative for HCV, HDV, HIV and syphilis, denied using illicit drugs and declared having a stable heterosexual relationship. ALT levels returned to normal after 5 weeks. IgM anti-HBc antibodies, HBsAg and HBeAg were detected for 17 weeks, and on the 21st were negative except for HBeAg which was

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Table 1 Serological, biochemical and molecular characteristics of the patient during follow-up. Weeksa Vaccination

1st dose 2nd dose

Total anti-HBc

HBsAg

Anti-HBs

HBeAg

Anti-HBe

ALT (IU/mL)

HBV DNA

Mutations in the ‘a’ determinantb

Lamivudine resistance

Genotyping

rt180

rt204

Direct sequencing

Sequencing of clones

Pos Pos Pos Pos Pos Pos Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg

Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos

Pos Pos Pos Pos Pos Pos Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg

Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg

Pos Pos Pos Pos Pos Pos Ind Neg Neg Neg Neg Neg Neg Neg Neg Neg

Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg

555 250 42 42 34 42 17 15 10 36 20 20 31 25 25 40

Pos Pos Pos Pos Pos Pos Pos Posc Pos Neg Neg Neg Neg Neg Neg Neg

No No No No No No No – No – – – – – – –

WT WT WT WT WT WT WT – Mut – – – – – – –

WTd WT WT WT WT WT WT – Mute – – – – – – –

A2 A2 A2 A2 A2 A2 A2 A2 A2/G – – – – – – –

A2 nd nd nd nd nd nd – G + A2/G – – – – – – –

Neg Neg

Pos Pos

Neg Neg

Neg Pos

Neg Neg

Neg Neg

29 21

Pos Neg

No –

Mut –

Mute –

A2/G –

nd –

Neg Neg Neg

Pos Pos Pos

Neg Neg Neg

Pos Pos Pos

Neg Neg Neg

Neg Neg Neg

25 10 19

Neg Neg Neg

– – –

– – –

– – –

– – –

– – –

3rd dose

Ind – indeterminate; nd – not done. a Weeks after onset of jaundice. b Amino acids P120, T123, C124, T126, Q129, N131, M133, F134, K141, D144 and G145. c PCR positive for pre-core/core region only. d Lamivudine resistance, due to YIDD motif, was detected in 12% of the molecules by pyrosequencing. e Lamivudine resistance, due to YVDD motif, was detected in 100% of the molecules.

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2 4 7 9 13 17 21 25 30 39 48 60 73 88 96 114 119 127 140 144 147 156 165

IgM anti-HBc

J.J.F. de Barros et al. / Journal of Clinical Virology 67 (2015) 31–35

indeterminate. After loss of HBsAg and HBeAg, the patient was accompanied for an additional 3 years, because of the absence of seroconversion. Anti-HBe antibody test remained negative until the end of follow-up. Furthermore, anti-HBs antibodies did not appear spontaneously, but after administration of a second dose of hepatitis B vaccine (Table 1). The patient had no signs of liver dysfunction or lesion during follow-up and was not submitted to any antiviral treatment. HBV DNA was detected by nested PCR of genomic regions preS/S, precore-core or both, in all nine samples collected 2–30 weeks after the onset of jaundice. Although PCR assay results were negative for seven samples obtained between the 39th and the 114th week after the peak of serum ALT level, HBV DNA was successfully amplified at the 127th week (2.5 years). Meantime, vaccination of the patient had been initiated at the 114th week, and HBV DNA remained undetectable after completion of the vaccination schedule (Table 1). By direct nucleotide sequencing of PCR products and phylogenetic analysis, it was found that the HBV isolate present

A

33

in the serum during the acute, symptomatic phase of infection (2–25 weeks), belonged to genotype A, subgenotype A2. Its serological subtype was adw2, according to the deduced amino acid sequence of the S region. However, the sequences derived from the samples collected at the 30th and 127th week appeared to be related to genotype G on the phylogenetic tree (not shown). BLAST analysis showed a very high [98%] percent identity between those sequences and that of a Canadian A/G recombinant strain (GenBank accession number EU833889) [5]. The hypothesis of an intergenotypic recombination was confirmed by using two different recombinant analysis tools, namely SimPlot [8] and jpHMM [9], and the recombination breakpoint between genotypes G and A2 was located between nucleotides 447 and 480 (Fig. 1). In an attempt to detect an eventual, preexisting coinfection during the acute phase of the disease, 12 clones derived from the first blood sample (2 week) were sequenced, but all belonged to genotype A2, without recombination. However, sequencing of 10 clones derived from the blood sample collected at the 30th week after the onset of

Genotype G

Genotype A2

100

Percent of permuted trees

90 80 70 60 50 40 30 20 10 0 200

300

400

500

600

700

800

Position on HBV genome

B

1

Window, 200 bp; Step, 20 bp; GapStrip, On; Reps, 100; Kimura (2-parameter); T/t, 2,0, Neighbor-Joining

447 - 480

S

Genotype G Genotype A2

800

Fig. 1. HBV genome recombination analyses using two programs – SimPlot and jpHMM. (A) Bootscan analysis performed with SimPlot (version 3.5.1.) showing recombination between genotypes G and A2 in the small S open reading frame. (B) Schematic diagram of jpHMM results showing part of the circular HBV genome with alternate genotype G (purple) and genotype A2 (red) regions. The region of recombination, between nucleotides 447 to 480 (HBV genome numbering beginning at the unique EcoRI site), is shown. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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jaundice allowed to detect a mixture of two virus subpopulations, one of which was from genotype G and the other consisting of the recombinant A2/G (Table 1). Lamivudine resistance of the HBV isolates was evaluated by analyzing the sequence of the reverse transcriptase (rt) domain of the viral polymerase. In the first sample (2 week), none of the 12 sequenced clones showed lamivudine resistance. Still, pyrosequencing of PCR products revealed that about 12% of the molecules showed the rtM204I mutation leading to the lamivudine resistance YIDD motif (ATT coding for isoleucine) while 88% were wild-type. In contrast, lamivudine resistance was detected at a rate of 100% by pyrosequencing of PCR products obtained at the 30th and 127th weeks and by sequencing of all 10 clones derived from the sample collected at the 30th week. Interestingly, lamivudine resistance was not due to YIDD-, but to YVDD motif (rtM204 V mutation). Results obtained from pyrosequencing showed valine residue encoded by GTG and GTT triplets in 94–98% and 2–6% of the molecules, respectively. In addition, the compensatory mutation rtL180M was present in the HBV/G isolate as well as in the A2/G recombinant, but not in the HBV/A2 strain (Table 1). With respect to occult infection, no typical mutation (at the amino acids residues P120, T123, C124, T126, Q129, N131, M133, F134, K141, D144 or G145) was observed in the ‘a’ antigenic determinant, which would be able to make HBsAg undetectable from the 21st week.

all Brazilian HBV/G isolates described until now [14–16] showed the rtL180 M substitution, associated or not with the rtM204 V mutation. As HBV/G is more frequent among HBV–HIV coinfected patients and a number of them are under lamivudine treatment, a possible explanation is that this would have facilitated the dissemination of lamivudine resistant HBV/G isolates, even among the treatment naïve subjects, like the patient under study. Whether these lamivudine resistance mutations play a role in the ability to develop anti-HBs antibodies is unknown. In conclusion, this study shows the importance of following the patients who do not seroconvert after acute hepatitis B because of the eventual occurrence of OBI. The timely vaccination of people who do not seroconvert may result in viral clearance [17] and protect against reinfection and reactivation. The importance of the HBV genotype in OBI cases, as well as the influence of intergenotypic recombination events on the natural history of HBV infection, are not well understood and deserve further investigation.

3. Other similar and contrasting cases in the literature

Ethical approval

An HBV/G monoinfection has been described in an HBsAg negative German blood donor who tested repeatedly positive for HBV DNA [7]. Furthermore, a recent study has reported the case of a 42-year-old male Dutch blood donor with OBI for whom the absence of HBsAg detection was attributed to the genotype (G) of the HBV isolate. The individual was initially tested anti-HBs negative, but seroconversion occurred spontaneously during follow-up [10]. To our knowledge, however, no HBV intergenotypic recombinant involved in a case of OBI has been described.

Ethical approval was given by the Oswaldo Cruz Foundation Ethics Committee for Medical Research (registration number 575/10).

Funding This work was supported by CNPq. Competing interests The authors have declared that no competing interests exist.

Acknowledgement The DNA Sequencing Platform PDTIS/FIOCRUZ is acknowledged for performing nucleotide sequencing. References

4. Discussion OBI is characterized by periods of transient HBV viremia alternating with periods during which the viral DNA remains undetectable in serum [11]. For this reason, the importance of the detection of anti-HBc and anti-HBs antibodies, in addition to HBsAg marker, has been recently reaffirmed [12]. In the case described here, HBV subpopulations were followed up during and after acute hepatitis B in an anti-HBc positive patient who did not seroconvert to either anti-HBs or anti-HBe after negativation of HBsAg and HBeAg. The substitution of the original HBV/A2 strain by both an HBV/G isolate and an intergenotypic A2/G recombinant was observed. Infection with HBV/G, although very rare in some parts of the world, as the far East [13], is relatively frequent in the Western hemisphere where it has been described in Canada, the United States, Mexico, Argentina, Colombia and Brazil, mostly in men who have sex with men. Noteworthy, a delay in detecting HBsAg during HBV/G infections has been observed which might be explained by either decreased genotype G-specific synthesis of incomplete viral forms or the lower sensitivity to genotype G of the current HBsAg assays [7]. In the present case, the genome region coding for the ‘a’ determinant was from genotype G (in both HBV/G isolate and the A/G recombinant), and this may have contributed to the seroclearance of HBsAg without seroconversion. The HBV/G isolate showed both the primary rtM204 V lamivudine resistance and compensatory L180 M mutations. Interestingly,

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A2 isolate.

Viral and host factors leading to occult hepatitis B virus (HBV) infection (OBI) are not fully understood. Whether HBV genotype may influence the occu...
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