Sir,

Hepatitis B virus (HBV) infection is a major health problem worldwide. The World Health Organization estimates that more than 400 million people are affected. Chronic hepatitis B frequently leads to end-stage liver disease and is the leading cause of liver cancer worldwide. Current HBV therapies are inadequate to eradicate chronic infection. However, anti-HBV drugs may ameliorate liver disease progression in the short term (with normalization of transaminases, reduction in serum HBV-DNA levels and/or HBeAg seroreversion) and in the long term (with prevention of liver cirrhosis and/or hepatocellular carcinoma). After many years of lamivudine being the only oral drug to treat chronic hepatitis B, marketing of adefovir was very welcome. Moreover, this nucleotide analogue was shown to be also effective in lamivudine-experienced patients, with a relatively low incidence of resistance, at least within the first 2 years of therapy but increasing to ∼30% at 5 years.1

Suboptimal response to adefovir, and subsequent selection of resistance mutations, has been associated with pharmacological issues, mainly derived from the low dose (10 mg daily) approved in an attempt to minimize renal toxicity. Adefovir resistance has been classically linked to a single mutation (rtN236T) within the D domain of the HBV polymerase gene.2 A number of other mutations, such as V84M and S85A (A domain), A181T/V (B domain), V207L, S213T, V214P and Q215S (C–D inter-domain) and 229W/V, P237H and N238T/D (D domain), have also been described in patients experiencing virological failure under adefovir.2

Between 8% and 15% of the patients starting adefovir show non-response initially, defined as a decay in serum HBV-DNA <1 log after 12 weeks of therapy. Prior lamivudine experience might influence adefovir activity, especially if an A181S/T/V mutation was selected when lamivudine failed. Besides, two polymorphic changes have been involved as a cause of ‘natural resistance’ to adefovir.3,4 Interestingly, the I233V mutation is particularly common among HBV genotype D. Controversy regarding the real impact of this change exists. The other change is L217R, which in the original report was present at baseline in HBV isolates from two patients who subsequently failed adefovir therapy.4

We investigated in a cross-sectional study the prevalence of L217R and I233V in chronic hepatitis B patients who had never been exposed to adefovir or tenofovir. Serum samples sent to our reference laboratory for HBV viral load, genotyping and drug resistance testing since 2004 were identified. DNA extraction from sera was carried out using the Qiagen DNA blood kit (Qiagen, Mannheim, Germany). The HBV polymerase from each isolate was amplified using a nested PCR, and then sequenced and analysed for mutational resistance patterns. The sequences from each isolate were typed using the Lasergene software (DNAStar Inc., Madison, USA).

A total of 172 specimens were initially selected, of which 14 were excluded subsequently due to prior adefovir experience. DNA amplification could not be made in another 32 samples due to low HBV-DNA levels. Thus, HBV virological characterization could be performed on samples collected from 126 patients (Table 1). All were Caucasians, with a mean age of 38 years; 67% were males. A total of 65 patients (52%) were co-infected with HIV, while the remainder were HBV-monoinfected individuals. Most subjects (62%) had been exposed to lamivudine.

Table 1

Main characteristics of the study population

 HIV-negative HIV-positive P 
No. of patients 61 65  
HBV genotype (A/D/E/F/G/mixed) 8/46/2/5/0/0 30/27/1/2/3/2 <0.01 
HBeAg+ 28 <0.001 
Risk group (IDU/MSM/others) 2/3/56 24/29/12 <0.001 
Prior lamivudine exposure 32 46 0.03 
 HIV-negative HIV-positive P 
No. of patients 61 65  
HBV genotype (A/D/E/F/G/mixed) 8/46/2/5/0/0 30/27/1/2/3/2 <0.01 
HBeAg+ 28 <0.001 
Risk group (IDU/MSM/others) 2/3/56 24/29/12 <0.001 
Prior lamivudine exposure 32 46 0.03 

The distribution of HBV genotypes was as follows: 73D (58%), 38A (31%), 7F (6%), 3E (2%), 3G (2%) and 2 mixed (1%). Interestingly, HBV genotype A was the most frequent in HIV-positive patients (46%), while genotype D was predominant in HIV-negative patients (75%; P = 0.01).

Sequence analyses showed that subgenotype A2 (31 patients, 82%) was significantly more frequent than A1 (7; 18%), without significant differences according to HIV status (P < 0.01). Interestingly, the 217R polymorphism was detected in all patients infected with subgenotype A2, while all the remaining patients, including those infected with subgenotype A1, harboured 217L. On the other hand, the I233V polymorphic change was detected in 2 of 73 patients infected with HBV genotype D. As expected, lamivudine-associated resistance mutations (M204V/I, with or without L180M) were observed in most patients (65 of 78; 83%) with prior exposure to the drug.

Preliminary findings have shown that initial non-response to adefovir in a subset of our study population was more frequently seen in HBV-A than in other genotypes (9 of 13 versus 2 of 41; P < 0.001) and was mainly driven by HBV-A subgenotype (8 of 9 A2 versus 1 of 4 A1; P = 0.05).5 The results also suggested that the presence of histidines at codons 122 and/or 126 in HBV subgenotype A2 could restore the susceptibility to adefovir in the presence of R217.

There are important implications derived from these findings. First, HBV subgenotype A2 is the most frequent genotype A variant in Europe in contrast with most Asian and African regions. In this regard, response to adefovir could be lower in European chronic hepatitis B patients. Second, introduction of HBV genotyping seems to be warranted before prescription of adefovir in treatment-naive subjects.

Funding

This work was supported in part by grants from Red de Investigación en SIDA (RIS G03/173), Fundación Investigación y Educación en SIDA (IES), NEAT and the VIRGIL European Network of Excellence on Antiviral Drug Resistance (LSHM-CT-2004-503359).

Transparency declarations

None to declare.

References

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