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1st Study of Occult Hepatitis B prevalence in Community Setting
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"Occult hepatitis B virus infection in a North American community-based population"
Journal of Hepatology
April 2005
Gerald Y. Minuka*email address, Dong-feng Suna, Julia Uhanovaa, Manna Zhanga, Shauna Caouettea, Lindsay E. Nicollea, Adam Gutkina, Karen Doucettea, Bruce Martinb, Antonio Giulivic
a Section of Hepatology, Department of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada
b Department of Community Health Sciences, University of Manitoba, Winnipeg, Manitoba, Canada
c Health Canada, Ottawa, Ontario, Canada
"...The results of this study, if representative of the entire community, indicate that 18% of inhabitants with serologic-evidence of recovery from previous HBV infection and 8.1% of HBV seronegative individuals have occult HBV infections..
... Whether occult HBV infection is clinically relevant remains unclear. Studies documenting acute hepatitis developing in recipients of HBsAg negative, anti-HBc and HBV-DNA positive blood and the finding of HBV-DNA in the sera and livers of patients with cryptogenic hepatitis and hepatocellular carcinoma are reasons for concern [23-27]. On the other hand, lack of transmission of HBsAg negative/HBV-DNA positive blood to chimpanzees in some (but not all) reports [28,29] and failure of other groups to identify associations between occult HBV and significant chronic liver disease argue against such concerns..
... Unfortunately, neither age, gender, nor liver biochemistry findings appear to assist in the identification of occult HBV carriers from those without such infections. Thus, if occult HBV is subsequently demonstrated to result in an adverse outcome or is found to be transmissible to others, HBV-DNA testing in all individuals would be the only means at the present time of identifying individuals at risk for these outcomes.."
See Editorial
Introduction
Abstract
Author Discussion
Results
Study methods & patients
References
INTRODUCTION
Occult hepatitis B virus (HBV) infection can be defined as the presence of HBV-DNA in blood or liver in the absence of detectable serum hepatitis B surface antigen (HBsAg) 1. Two common findings and possible explanations for occult HBV are low levels of viral replicative activity and/or mutations to the 'a' epitope of the S gene that encodes amino acid residues 100-160 of HBsAg (so-called 'S mutants or variants'). Although occult HBV infection is more common in individuals with serologic evidence of recovery from previous HBV exposure (antibody to hepatitis B core and surface antigen; anti-HBc and anti-HBs, respectively, positive) it has also been described in those who are HBV seronegative (anti-HBc and anti-HBs negative) 2.
The prevalence of occult HBV is variable, and depends to a large extent on the prevalence of HBV in the general population and the methods used to detect HBV-DNA 2. Thus, occult HBV infection is most common in regions of the world, where HBV is endemic, less common in regions with intermediate HBV prevalence rates and least common in areas, where HBV is relatively uncommon. As the majority of individuals with occult HBV have viral loads less than 105 viral copies/ml (typically 101-103copies/ml), studies employing non-polymerase chain reaction (PCR) based assays (sensitivity >105copies/ml) tend to report lower prevalence rates than those employing PCR based assays 3.
The clinical sequelae (if any) of occult HBV infection have yet to be determined. Associations with fulminant hepatic-failure, unexplained chronic hepatitis and hepatocellular carcinoma raise concerns that the natural history of this condition may not be benign in all cases [4-7].
To date, documentation of occult HBV prevalence rates has been limited to blood or organ donors and selected patient populations [1,2]. To our knowledge, there have been no published reports of occult HBV in community-based populations. The principal purpose of the present study was to document the prevalences of occult HBV infection and the most common S-variant (nt 587 or G145R), in the inhabitants of an isolated North American community.
ABSTRACT
Background/Aims:
Occult hepatitis B virus (HBV) infection [HBV-DNA detection in hepatitis B surface antigen (HBsAg)-negative individuals] may cause acute and/or chronic liver disease. The objective of this study was to document the prevalence of occult HBV in an isolated, North American Inuit community.
Methods:
Four hundred and eighty seven HBsAg negative sera (61% of the community population) were available for HBV-DNA testing by real time PCR. Of these, 80 (Group 1) had serologic evidence of resolved HBV infection and 407 (Group 2) were HBV-seronegative.
Results:
HBV-DNA was detected in 14/80 (18%) and S-variants in 12/14 (86%) samples from Group 1. In Group 2, HBV-DNA was detected in 33/407 (8.1%) and S-variants in 17/33 (52%). In all cases (Groups 1 and 2) viral loads were low (<105 viral copies/ml) and clinical or biochemical features did not distinguish HBV-DNA positive from negative individuals. However, S-variants were more common (P<0.0001) in older age groups.
Conclusions:
The results of this study indicate that in this community-based population; (1) the prevalence of occult HBV infection is 18% in those with serologic evidence of previous HBV infection and 8.1% in HBV seronegative individuals, (2) age, gender and liver biochemistry findings do not identify those with occult HBV and (3) S-variants are present in the majority of individuals with occult HBV.
AUTHOR DISCUSSION
The results of this study, if representative of the entire community, indicate that 18% of inhabitants with serologic-evidence of recovery from previous HBV infection and 8.1% of HBV seronegative individuals have occult HBV infections. Moreover, age, gender and liver biochemistry findings do not help distinguish these individuals from those without occult HBV infection. Finally, the results also indicate the majority of occult infections, particularly amongst the elderly, are associated with the presence of S-variants.
Because this study provides the first data on the prevalence of occult HBV infection in a community-based population, no similar reports are available for comparative purposes. However, studies describing the prevalence of occult HBV in seropositive (anti-HBc+anti-HBs); blood donors (0-17%), patients with 'cryptogenic' chronic hepatitis with or without HCC (30-35%), and individuals followed for chronic HBV that spontaneously seroconverted from HBsAg positive to negative (55%), suggest that prevalence rates are lower in unselected community-based populations with the same serologic profile, than in these groups (summarized in Ref. 2). The reason for this finding has yet to be determined. A likely contributing factor is the selection bias inherent in non-general population based studies, particularly the non-blood donor populations. Also to be considered is the stringent definition we employed for occult HBV. Specifically, each sample had to test positive on two separate occasions by two rather than one set of HBV primers. Indeed, had samples been considered positive by only one set of primers, prevalence rates would have been approximately 2-3 times higher in both study groups (data not shown). Also to be considered are differences in the sensitivities of the assays employed 3. However, real time PCR (the assay employed in the present study) is at least as sensitive and in many cases, more sensitive (10 viral copies/ml) than many commercially available HBV-DNA detection systems and thus, false negative results would be expected to be less common in the present study.
Regarding HBV-DNA detection in HBV seronegative individuals, to date, rates of 0, 1.7, 5.9 and 10% have been reported in the medical literature [10-14]. Thus, the 8.1% described in the present study is within but towards the higher range of these reports. Perhaps the increased sensitivity of real time PCR contributed to this finding. Of note, the size of the study populations in the earlier reports (5, 20, 34, 58 and 119 individuals, respectively) are considerably smaller than the 407 individuals that constituted Group 2 (HBV seronegative) in the present study.
A somewhat unexpected finding in this study was the high prevalence (86% in Group 1 and 52% in Group 2) of S-variants in those who were HBV-DNA positive. Previous studies have suggested that such variants are more common in patients with long-standing or more advanced HBV infections, but others have not confirmed these findings [15-19]. That S-variants were significantly more common in the older age groups of Groups 1 and 2 subjects would be in keeping with the former findings. Our study subjects had not been provided with either passive or active HBV immunoprophylaxis, interventions most commonly associated with S-mutant development [20-22].
Unfortunately, neither age, gender, nor liver biochemistry findings appear to assist in the identification of occult HBV carriers from those without such infections. Thus, if occult HBV is subsequently demonstrated to result in an adverse outcome or is found to be transmissible to others, HBV-DNA testing in all individuals would be the only means at the present time of identifying individuals at risk for these outcomes.
Whether occult HBV infection is clinically relevant remains unclear. Studies documenting acute hepatitis developing in recipients of HBsAg negative, anti-HBc and HBV-DNA positive blood and the finding of HBV-DNA in the sera and livers of patients with cryptogenic hepatitis and hepatocellular carcinoma are reasons for concern [23-27]. On the other hand, lack of transmission of HBsAg negative/HBV-DNA positive blood to chimpanzees in some (but not all) reports [28,29] and failure of other groups to identify associations between occult HBV and significant chronic liver disease argue against such concerns [1,30,31].
One of the important limitations of this study was the absence of liver tissue. Thus, we cannot comment on whether prevalence rates of occult HBV would have been higher had tissue also been analyzed or whether histologic evidence of chronic liver disease existed in these individuals. Both the remoteness of the study population and paucity of biochemical evidence of active liver disease precluded performing liver biopsies in these individuals.
In summary, occult HBV infection was documented in 18% of community residents with serologic evidence of resolved HBV infection and 8% of those with no serologic markers for HBV infection. Age, gender and liver biochemistry findings did not help to distinguish individuals with occult HBV from other members of the community. Finally, the majority of occult infections were associated with S-variants which were most common in older age groups.
RESULTS
Study population
The demographics of the 720 community inhabitants who volunteered to participate in the original study are provided in Table 2. From these individuals, a total of 516 sera representing 61% of the community's population were available in sufficient amounts for HBV-DNA analyses. Of these, 29 had tested positive for HBsAg, 80 negative for HBsAg but positive for anti-HBc and anti-HBs (Group 1) and 407 negative for all three HBV serologic markers (Group 2). The ages and gender distributions for Groups 1 and 2 are provided in Table 3.
3.2. HBV-DNA results
3.2.1. Group 1
The results of HBV-DNA testing are provided in Table 4. Fourteen of the 80 (18%) Group 1 individuals were HBV-DNA positive. Their mean age (45.0+15.5 years) and gender distribution (36% male) were similar to those who were HBV-DNA negative (44.3+15.0 years and 42% male, respectively). HBV-DNA levels were low (mean; 3.38 log10 viral copies/ml, range: 2.87-3.9 log10 viral copies/ml).
Twelve of the 14 HBV-DNA positive Group 1 individuals (86%) were S-variant positive. In six of the 12, the S-variant was the only form of HBV detected (wild type negative) while in the remaining six, mixtures of S-variant plus wild type were present. The mean age of the S-variant positive subjects was 47.7+17.3 years (range: 27-79 years). Nine (75%) were female and 3/12 (25%) male.
3.2.2. Group 2
As shown in Table 4, compared to Group 1, fewer subjects in Group 2 were HBV-DNA positive (33/407, 8.1% versus 14/80 (18%), respectively, P<0.01). The mean age of those in Group 2 who were HBV-DNA positive (19.6+11.3 years) was younger than those in Group 1 (45.0+15.5 years, P<0.0001), but older than the remainder of Group 2 who were HBV-DNA negative (16.5+10.7 years). However, the latter difference did not research statistical significance (P=0.06). Fifteen of 33 (45%) HBV-DNA positive individuals in Group 2 were male, not significantly different from the 36% in Group 1 (P<0.09) and similar to the remaining 203/374 (54%) of Group 2 who were HBV-DNA negative (P=0.37). The mean HBV-DNA level in Group 2 was 3.40 log10 viral copies/ml (range: 2.40-4.73 log10 viral copies/ml).
Seventeen of 33 (52%) HBV-DNA positive samples from Group 2 were S-variant positive. Of these, seven were S-variant positive alone while 10 represented co-infections with wild type HBV. The mean age of the S-variant positive individuals was 17.0+11.6 years and five (29%) were male.
3.3. Overall
Table 5 provides the results of age specific prevalence rates for HBV-DNA, wild type, S-variant and mixed wild type/S-variant infections for the entire HBV-DNA positive cohort. Although the prevalence of HBV-DNA tended to increase with each age group, the trend was not statistically significant (P=0.22). This was not the case with S-variants in that prevalence rates increased significantly with age (<11, 11-20, 21-30, 31-40, 41-50 and >50 years; 4.2, 4.8, 5.6, 6.8, 5.9 and 18.8%, respectively, P<0.00001).
The extent and frequency of liver biochemistry abnormalities in HBV-DNA positive individuals from Groups 1 and 2 were similar to age and gender matched HBV-DNA negative controls.
Two subjects with occult HBV (both belonging to Group 2) were positive for 1762/1764 basal core promoter mutations.
Materials and methods
Baker Lake is an Inuit (Eskimo) community located approximately 200miles inland from the Northwest shore of Hudson Bay, Canada. In 1980, the year serum samples were obtained, the community's population was approximately 850. In response to advertisements on local radio and posters placed in public meeting areas, 720 (85% of the community) agreed to participate in a study designed to document the prevalence of viral hepatitis in the community. The results of the 1980 study revealed 4.0% of the population were HBsAg positive and 27% had serologic evidence of previous HBV infection 8. Consent was obtained at the time of the study to store sera for future testing related to viral hepatitis when more sensitive and specific assays became available or new hepatotropic agents were discovered. Sera were stored at -70¡C until thawed for testing.
HBV-DNA testing was performed on two separate occasions using two independent sets of primers 9. Briefly, DNA was extracted from sera using a high pure viral nuclei acid isolation kit (Roche Diagnostics, Laval, Quebec, Canada). The sequences of the primers, anchor and sensor probes used in this study are summarized in Table 1. Wild type HBV adw1 subtype was purchased from ATCC (Manassas, VA, USA). The HBV fragment was cut from a pBR322 plasmid at the BamH I site, separated on 1% agarose gel, and reamplified. The purified DNA concentration was determined with a spectrophotometer at 260nm, and the corresponding copy number of HBV virus calculated. Serial dilutions in HBV-DNA dilution buffer (Nuclisens Elution Buffer: Biomerieux bv/biolab-Merieux S.A.) ranging from 1 to 1A-105 copy per 1ul volume of dilutant were prepared. Quantitative PCR of serum HBV copy number based on six dilutions (external standards: wild type HBV, adv 1 in PAM 6 plasmid (1A-1010copies/ml) purchased from ATCC), was performed in the presence of 2ul of genomic DNA of patient isolate. The lowest detection limit of this assay (10 viral copies/ml) was analyzed in parallel with standards and calculated by genome copies/ml.
PCR reactions were performed in a total volume of 20ul containing 2ul of DNA template, 2ul of LightCycler DNA Master Hybridization Mixture (Taq DNA polymerase, reaction buffer, dNTP mixture and 10mmol/l MgCl2), 1.6ul of 25mmol/l MgCl2, 0.2umol/l each of the probes, 0.5umol/l each of the primers. Samples were loaded into composite plastic/glass disposable capillaries, centrifuged, and placed in the LightCycler sample carousel (Roche Diagnostics, Laval, Quebec, Canada).
After amplification was complete, a melting curve was generated. The melting curves were converted to melting peaks (Tm) by plotting the negative derivative of the fluorescence with respect to temperature (-dF/dT) against temperature. Melting curves and quantitative analysis of the data were performed using LightCycler analysis software 3.5.
S-variant detection was based on melting peaks derived from sensor probes designed to contain the wild-type sequence. A point mismatch at nt 587 resulted in a lower Tm of 53.75¡C relative to wild-type; 60.89¡C 9. The positive mutant control for the assay was purchased from ATCC.
To determine the sensitivity of the detection system for mutant HBV-DNA among wild type HBV-DNA, mutant and wild type DNA were mixed at mutant: wild type ratios of: 100/0; 50:50; 25:75; 10:90; 5:95; 2:98; and 0/100 prior to processing for melting analysis.
In order to prevent carryover contamination during PCR, each step of the procedure was performed in a separate room with dedicated equipment and directional flow from the beginning of the procedure to the end. Negative controls containing serum or water were also included in each extraction run, and an extra negative control containing water was included at each PCR.
Student's t-tests were performed for parametric and Mann-Whitney tests for non-parametric data. A Chi-square test of association (F-test when warranted) was used to examine differences in proportions and an Armitage test for trends in proportions. Unless otherwise specified, the results provided represent the mean+SD. P values less than 0.05 were considered significant. All statistical analyses were performed using the Number Cruncher Statistical Systems 2001 (Kaysville, Utah).
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