Review

The case report by Huysman and colleagues raises a number of provocative questions and conclusions regarding hepatitis B.¹ First, can hepatitis B virus (HBV) be transmitted via bodily fluids such as semen and saliva from a hepatitis B surface antigen (HBsAg) carrier whose serum HBV DNA levels are repeatedly verified as negative? Second, what is the likelihood of acquiring lamivudine (Epivir, GlaxoSmithKline)-resistant HBV among treatment-naive patients? Finally, Huysman and coworkers demonstrate that, in the treatment of hepatitis B, prevention is superior to therapy.

Transmission of Hepatitis B Virus in the Absence of Detectable Hepatitis B Virus DNA

If undetectable levels of HBV DNA in the serum can be associated with transmission of the infection through bodily fluids, especially during sexual contact, significant public health implications may result. In order to establish the validity of this possible route of HBV transmission, one must have concrete scientific evidence based on well-designed clinical studies. As Huysman and colleagues cited in their discussion, most of the original studies addressing this issue were published in the 1980s.^2–4 The limitations of these studies include lack of accuracy and of standardization of HBV DNA quantification. Over the past decades, significant advances have been made in the diagnosis and treatment of hepatitis B. We, therefore, must critically examine prior studies and determine whether their results still hold true with the availability of more sensitive HBV DNA quantification methods.

Fagan and coworkers reported in 1986 that a patient with hepatitis B achieved undetectable HBV DNA levels in serum but had HBV DNA levels that could still be detected in other bodily fluids such as urine, semen, and saliva.² However, it is important to emphasize that the assays utilized for detecting HBV DNA levels in serum and other bodily fluids were heterogeneous. HBV DNA levels in sera, for instance, were measured by spot hybridization assay, whereas southern blots were applied to detect HBV DNA in urine, saliva, and semen. As the threshold for HBV DNA detection in these tests was different, definitive conclusions about the transmission of HBV via bodily fluids in the absence of HBV DNA in serum cannot be drawn from this study. The results of the study conducted by Jenison and associates actually indirectly refuted the hypothesis that HBV can be transmitted in the setting of undetectable HBV DNA in serum.³ All 3 subjects with detectable HBV DNA levels in semen had at least 100-fold higher levels of HBV DNA in their corresponding serum samples.

In the case report by Huysman and colleagues, the partner of their patient could theoretically transmit HBV during unprotected sex, but the causal relationship could not be established, as the partner's semen sample was not tested for HBV DNA. Furthermore, sequencing results were unavailable to determine whether both the patient and his partner had the same strain of HBV. Carefully conducted studies utilizing sensitive HBV DNA assays on different bodily fluids are essential to draw a definitive conclusion to this question.

There are many commercially available assays to quantify HBV DNA. Until recently, HBV DNA titers had been reported in different units of measurement such as copies/mL, genome equivalents/mL, or mega-equivalents/mL. Since the World Health Organization developed an international standard for HBV DNA nucleic acid amplification techniques, serum HBV DNA titers can and should be uniformly expressed in IU/mL to ensure comparability among different assays.⁵ The ideal HBV DNA quantification assay should be sensitive and reproducible and have broad dynamic range of at least 5 logs. Real-time polymerase chain reaction (PCR) quantification assays possess these properties and, therefore, are recommended for HBV DNA baseline determination and monitoring during therapy.^6–8

The Prevalence of Lamivudine-Resistant Hepatitis B Virus Among Treatment-Naive Patients

It is interesting that the authors chose to treat the patient with 1 mg of entecavir (Baraclude, Bristol-Myers Squibb) versus the recommended 0.5 mg dose for treatment-naive HBV patients. Entecavir 1 mg daily is the US Food and Drug Administration-approved dose for patients with known lamivudine-resistance or history of being refractory to lamivudine therapy.^9–10 Lamivudine is a nucleoside analog that directly inhibits HBV DNA polymerase.¹¹ Lamivudine resistance has been attributed mainly to a substitution of valine or isoleucine for methionine in the tyrosine-methionine-aspartate-aspartate (YMDD) motif in the catalytic site of the HBV polymerase gene rtM204V/I.^12–13 In some patients, this is accompanied by a second mutation substituting methionine for leucine in an upstream region (rtL180M).^12–13 Entecavir, a cyclo-pentyl guanosine analogue, is a potent inhibitor of HBV DNA polymerase, inhibiting both the priming and elongation steps of viral DNA replication.¹⁴ Cross-resistance between entecavir and lamivudine have been described in in-vitro and clinical observations.^15–16 Entecavir resistance occurs in the setting of preexisting lamivudine-resistance plus additional substitutions at reverse transcriptase positions T184, S202, and/or M250.¹⁷ Thus, entecavir is less potent and is associated with a significantly higher rate of resistance when used in patients with lamivudine-resis-tance compared with treatment-naive subjects, despite a higher dose of medication.^16,18,19 The reported entecavir resistance rate is less than 1% in 4 years for treatment-naive patients compared with 39% among those with a history of being refractory or resistant to lamivudine.²⁰

It is well documented that long-term lamivudine monotherapy leads to the emergence of lamivudine-resis-tant virus in patients with chronic hepatitis B. Approximately 25% of patients developed resistance within 1 year of treatment, and more than 40% after 2 years, which increases to 53% and 67% after 3 and 4 years, respec-tively.^21–24 More recently, naturally occurring rtM204V/I and rtL180M mutant viruses were reported in HBV carriers who have never received lamivudine.^25–31 Feeney and colleagues examined 108 samples from treatment-naive HBV patients from diverse ethnic backgrounds and HBV genotypes.²⁵ Among them, 34% were born in Ireland and the remainder were from Africa (22%), Asia (17.4%), Eastern Europe (10.1%) and other regions (16.5%). Genotypes were available in 98 samples. Lamivudine-resistant mutations were present in 16 (14.8%) of the patients tested. However, it is unclear whether these patients actually developed de novo lamivudine-resistant mutations or were infected with lamivudine-resistant viruses that persisted. The actual transmission rate of the lamivudine-resistant viruses is unknown. Thibault and associates documented a case of primary infection with lamivudine-resistant HBV via direct sequencing that was acquired presumably via homosexual activity. The patient was receiving a lamivudine-containing highly active antiretroviral treatment regimen when he developed acute hepatitis B. His HBsAg was negative 3 months prior to the onset of acute hepatitis, providing clues that lamivudine-resistant HBV can be transmitted from one individual to another. It would be more conclusive and convincing, however, if the source patient had been identified and tested.³²

In the case study by Huysman and coworkers, the patient's genotype profile did not reveal polymerase inhibitor-induced resistant mutants. However, the sensitivity of the different genotypic assays for the detection of drug resistance varies significantly in their ability to identify minor strains of viruses (Table 1).³³ Direct sequencing, for example, has relatively low sensitivity and can only detect mutant viruses if they exceed 15–50% of the total viral population. The advantage of direct sequencing is its ability to identify new mutations. Line probe and matrix assisted laser desorption/ionization time-of-flight assays, in contrast, can identify very low levels (5%) of the mutant viruses. It is, therefore, important to know which resistance assay was applied. This is of clinical relevance to the case study patient of Huysman and associates, as it would influence the treatment choice if subpopulations of polymerase inhibitor-induced resistant mutants coexisted with the wild-type virus. Standardization of the genotypic resistance assays is necessary to determine the incidence and prevalence of the transmission of these nucleos(t)ide-induced resistant HBV mutations.

Table 1.

Comparison of Different Genotypic Assays for Detection of Drug Resistance

Method	Detection Threshold	Information details	Commercially Available	Complexity of interpretation
Direct sequencing	15–50%	High	Yes	High
RFLP	5–10%	Low	No	Intermediate
RT-PCR	5–10%	Low	No	Intermediate
LiPA	5%	Low	Yes	Low
Flourescence	Not determined	Intermediate	No	Intermediate
MALDI-TOF	<5%	Intermediate	No	High

Adapted from Sablon and Shapiro.³³

RFLP

restriction fragment length polymorphism

RT-PCR

real-time polymerase chain reaction

LiPA

line probe assay

MALDI-TOF

matrix assisted laser desorption/ionization time-of-flight assay.

Prevention is Superior to Therapy

In this clinical case study, the patient recalled being vaccinated against hepatitis B in 2000. It is not known if he received all 3 of the recommended doses of the vaccine nor whether he had postvaccination testing. In November 2005, it was noted that he had an anti-HBs titer less than 3 mIU/mL, indicating either failed immunity or lack of immunity.

Among healthy adults receiving immunization with HBV vaccine at baseline, 1 month, and 6 months, approximately 90% develop protective antibodies. Response rates range from 20% to 30% after the first injection, approximately 75–80% after the second injection, and approximately 90–95% following the third injection.^34–35 Response rates to the HBV vaccine tend to be lower in populations with chronic conditions such as hepatitis C, alcoholic liver disease, renal failure on dialysis, HIV, and liver transplantations.^36–40 Other factors that are associated with reduced response include male gender, smoking cigarettes, obesity, and older age (>40 years).^41–43 In order to identify true nonresponders, the Center for Disease Control (CDC) recommends evaluating the anti-HBs titer at 1–6 months following the last dose of the vaccine when utilizing the recommended vaccination schedule. Individuals who achieved a titer greater than 10 mIU/mL are considered to be immune.⁴⁴ From the very first publication of the Advisory Committee on Immunization Practices in 1982, men having sex with men were targeted as adults at high risk for infection and vaccination was recommended. Despite subsequent guidelines to reinforce the importance of vaccination in this high-risk group, the incidence of new HBV cases increased from 7% to 18% among this population from 1990 to 2004.⁴⁵

The CDC does not recommend postimmuniza-tion testing for the general population because of the high response rate.⁴⁶ However, the CDC does recommend postvaccination testing for high-risk groups such as healthcare workers, public safety workers, chronic hemodialysis patients, HIV-infected persons, and sex or needle-sharing partners of HBsAg-positive persons. The patient in Huysman and colleagues' case study had a sex partner with chronic hepatitis B. Accordingly, he should have had postvaccine testing after receiving a full course of HBV vaccine. Testing would have identified failed immunity or lack of immunity in the patient's case and would have allowed the opportunity for further intervention to prevent the acquisition of hepatitis B.