High levels of hepatitis B virus DNA in body fluids from chronic carriers

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Summary

Chronic infection with hepatitis B virus (HBV) is a major global health problem. Transmission is mainly blood-borne, although the route of infection during horizontal transmission in childhood is unclear. Nosocomial outbreaks of HBV have been widely reported, but have mainly focused on blood-borne transmission. There is limited knowledge of the viral load levels in other body fluids. In the present study, chronic HBV carriers were tested for the presence of HBV DNA in serum, saliva, nasopharyngeal fluid, urine and tears by means of qualitative and quantitative polymerase chain reaction (PCR) methods. Twenty-five patients who were positive for HBV DNA with both PCRs were included. Low titres in real-time PCR corresponded with weak bands in the qualitative assay. HBV DNA was found in two urine samples, 10 saliva samples, five nasopharyngeal swabs and in tear fluid from four patients. One highly viraemic HBeAg-positive carrier with serum HBV DNA levels of 7 × 109 genome copies had high copy numbers detected in both saliva and nasopharyngeal fluid. These results demonstrate that highly viraemic HBV carriers may have high titres of HBV DNA in other body fluids. This has particular importance for infection control programmes and regulations, underlining the importance of aiming towards regular HBV DNA testing and thus infectivity assessment of chronic carriers in order to prevent transmission.

Introduction

Infection with hepatitis B virus (HBV) may lead to chronic carriage, with a risk of developing progressive liver disease and hepatocellular carcinoma. The clinical course varies depending upon the age of the patient at the time of infection and also upon the patient's immune response. The vast majority of adults resolve the disease, whereas around 90% of those infected in infancy proceed to become chronic carriers.1 Globally, the most common route of transmission for HBV is vertical, from mother to child. Although efficient vaccines have been commercially available for more than 20 years, the number of chronic carriers does not appear to have changed appreciably worldwide and totals more than 350 million.

Although hepatocytes are the primary target cells for HBV, both HBV proteins and HBV DNA can be found in the systemic circulation. There is a discrepancy between the number of complete virions (Dane particles) and the number of empty viral surface particles (HBsAg) found in the circulation of infected patients. It has been postulated that the vast excess of ‘empty shells’ serves to absorb neutralizing antibodies to surface proteins during infection.2

HBV is a member of the family Hepadnaviridae and has several unique characteristics. Its partially double-stranded genome has a compact organization, with no non-coding regions and overlapping open reading frames. It replicates through an intermediate reverse transcription step. The host range is very narrow (only humans, chimpanzees and a few other primates can be infected), which has impeded a large number of animal studies of HBV. HBV does not infect standard laboratory cell lines, and the infectivity of patient samples has thus had to be tested indirectly by antigen or DNA detection.2 Earlier studies have shown the presence of HBsAg in several body fluids such as saliva, semen, breast milk, urine, and pancreatic and biliary secretions.3, 4, 5, 6, 7 A number of very early reports supported or discarded the infectivity of body fluids other than blood. However, HBV DNA was not tested for in these studies, and with the excess of circulating surface antigen, antigen testing is not ideal for judging infectivity in HBV patients. Instead, HBV DNA detection by polymerase chain reaction (PCR) has replaced HBsAg and especially HBeAg/anti-HBe testing in many clinical settings. The only study comparing in vivo infectivity of an HBV inoculum with in vitro testing by PCR was performed by Ulrich et al. by inoculation of chimpanzees.8 These authors suggested that specimens in which no HBV DNA can be amplified by PCR are unlikely to contain infectious HBV.

A previous study showed that HBV DNA can often be amplified from the urine of chronic carriers, usually with a 100- to 1000-fold lower titre than in serum.7 Most infection control recommendations only include warnings about blood-borne and sexual transmission of HBV, although Milne et al. showed horizontal transmission among children in New Zealand.9

The aim of the present study was to assess the potential infectivity of other body fluids from HBV-carrying patients, correlating detection of HBV DNA by PCR with the infecting HBV genotype, the patient's alanine aminotransferase (ALT) level and their HBeAg/anti-HBe status.

Section snippets

Patients and samples

Chronic HBV carriers attending the (outpatient) Department of Infectious Diseases at the University Hospital of Lund, who had tested HBV DNA positive in serum by PCR on previous occasions, were tested for the presence of HBV DNA in serum and other body fluids. Twenty-five patients were included in the study (12 females and 13 males) and the mean age was 30 years. Six patients originated from South-east Asia, seven patients from Central Asia or the Middle East, seven patients from South-east

Results

The qualitative HBV DNA PCR yielded positive results in the serum samples from 25 patients; five were HBeAg positive and 20 were anti-HBe positive. All the serum samples with a positive qualitative PCR also had a positive quantitative real-time PCR. The results of the two PCRs corresponded well, with lower HBV DNA titres in the quantitative assay being mirrored by weak bands visualized by ultraviolet light in ethidium-bromide-stained agarose gels in the qualitative assay. There was minimal

Discussion

Transmission of HBV occurs most commonly through blood, with transmission from mother to child at birth, not in utero, being the most common route in some regions of the world. Horizontal transmission in early childhood accounts for a large proportion of infections in both Africa14 and New Zealand.9 Intrafamilial spread is particularly important in these cases, as is transmission between preschool and school children through sores and open wounds.15 In Europe and North America, most infections

Acknowledgements

The authors wish to thank Ann Åkesson and Maria Sandberg. This work was supported by a grant (ALF) from the Faculty of Medicine, Lund University, Sweden.

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