Division of Gastroenterology, University of Michigan Medical Center, Ann Arbor, Michigan,
1 Innogenetics NV, Ghent, Belgium
2
Received 12 March 2003/ Returned for modification 5 May 2003/ Accepted 18 May 2003
Hepatitis B virus (HBV) genotypes may influence HBeAg seroconversion
rates, mutational patterns in the precore (PC) and core promoter
(CP) regions, severity of liver disease, and response to antiviral
treatment. Development of rapid, simple, and standardized assays
to detect viral genotypes and common mutations in the PC and
CP regions can accelerate research on the clinical significance
of these variants. We aim to assess the accuracy of a line probe
assay in determining HBV genotypes and detecting HBV PC and
CP variants. HBV genotypes in 701 patients and PC and CP variants
in 600 patients with chronic HBV infection from China and the
United States were studied using the INNO-LiPA assay. All but
one (99.9%) sample were classified by the genotyping assay.
All eight genotypes, i.e., A to H, were found. The INNO-LiPA
genotyping assay results were completely concordant with those
of sequencing. Using the INNO-LiPA PC assay, 99.8 and 94.7%
samples were classifiable in the PC and CP regions, respectively.
The PC assay results were completely concordant with those of
sequencing in all samples that showed either wild-type or variant
sequence. The line probe assay was more sensitive in detecting
mixtures than was direct sequencing. By INNO-LiPA, only 50 and
27% of the samples, with mixed wild-type and variant sequence
in the PC and CP region, respectively, showed mixed sequence
by direct sequencing. INNO-LiPA is rapid, sensitive, and reliable—thus
enabling accurate determination of HBV genotypes and detection
of PC and CP variants in a large population of patients.
Hepatitis B virus (HBV) can be classified into seven genotypes—A
through G—based on an intergroup divergence of 8% or more
in the nucleotide (nt) sequence (
24,
27,
30). Recently, a new
genotype, H, has been described (
1) in patients from Nicaragua,
Mexico, and California. Studies to date have shown that genotype
A is most common in the United States and northwest Europe,
that genotypes B and C predominate in Asia, that genotype D
is most frequently found in the Mediterranean countries, and
that genotype F prevails in Central and South America (
9,
20,
25). The geographic distribution of genotypes E, G, and H is
less clear (
10). Besides differences in geographic distribution,
recent studies demonstrated that HBV genotypes are related to
hepatitis B e antigen (HBeAg) seroconversion, severity of liver
disease, and response to antiviral treatment (
11,
14,
28,
32).
As increasing data on the clinical significance of HBV genotypes
become available, HBV genotyping may evolve from a research
tool into being an essential clinical diagnostic test, very
much as HCV genotyping did. One hurdle in the introduction of
HBV genotyping to clinical practice is the lack of a simple,
rapid, and accurate test.
HBV replicates via reverse transcription of an RNA intermediate (33). Because of the lack of proofreading during reverse transcription, the HBV genome has a mutation rate of 1.4 x 10-5 to 3.2 x 10-5 substitutions per site per year (20). The most common naturally occurring HBV mutations are the G1896A mutation, which creates a premature stop codon in the precore (PC) region (4, 6), and the A1762T, G1764A dual mutation in the core promoter (CP) region, which down-regulates HBeAg production (5, 29). The PC mutation is most commonly found in association with HBV genotypes D and B and is therefore most prevalent in southern Europe and Asia (13, 14, 17). The dual CP mutations can be found with all four major genotypes (i.e., A through D) but are more commonly associated with HBV genotypes A and C (7, 14, 28). The PC and—to a lesser extent—the dual CP mutations are more often found in HBeAg-negative patients. Some studies suggest that these mutations may be associated with more active or severe liver disease (4, 6, 16), but both PC and CP variants have also been found in inactive hepatitis B surface antigen (HBsAg) carriers (8, 19, 23). Data on the relation between PC and CP variants and response to antiviral therapy are conflicting (3, 12, 15, 36). Despite more than a decade of research, the clinical significance of PC and CP variants remains unclear. This is in part related to the wide variety of mutations in the PC and CP regions besides the signature mutations mentioned above, the lack of standardization in nomenclature, and the heterogeneity of the tests used for the detection of these mutations.
Currently, HBV genotypes and mutations in the PC and CP region can be determined by several methods, including direct sequencing (26, 30), restriction fragment length polymorphism analysis (15, 18, 34), line-probe assay (14), colorimetric point mutation assay (2), ligase chain reaction assay (22), and enzyme-linked immunosorbent assay for genotype-specific epitopes (35). Direct sequencing is the most accurate and permits detection of the common as well as uncommon mutations but is also the most expensive and tedious. The other tests are simpler, but they have not been well validated and can only detect specific known genotypes or PC and CP mutations. Development of rapid, simple, and standardized assays that can detect all known genotypes and small amounts of the most common known mutations in the PC and CP regions can accelerate progress in research on the clinical significance of HBV genotypes and PC and CP variants.
The aim of this study is to assess the accuracy of a line probe assay in the determination of HBV genotypes (i.e., INNO-LiPA HBV genotyping) and the detection of HBV PC and CP variants (INNO-LiPA HBV PC).
Stored sera from chronic HBV-infected patients enrolled in two
studies on HBV genotypes and variants were studied. The first
study involved Chinese patients seen in the Hepatitis Clinic
at Queen Mary Hospital in Hong Kong from 1984 to 1992 (
11).
Residual sera previously collected for HBV testing were used
for this study. The second study involved consecutive chronic
HBV-infected patients seen in 17 liver centers across the United
States who participated in a study of molecular epidemiology
of HBV infection between March 2001 and March 2002 (
10). The
use of human sera was approved by the institutional review boards
of each of the participating centers, and written consent was
obtained from all patients. Sera were stored at -80°C prior
to testing for HBV genotype and PC and CP variants.
HBV genotyping. Sera from 335 patients in the Hong Kong study and 366 patients from the U.S. study were used to determine HBV genotypes using a line probe assay (INNO-LiPA HBV genotyping assay; Innogenetics NV, Ghent, Belgium). HBV DNA was extracted as described previously (7), and amplification was carried out by PCR assay using primers in the HBV S gene region as described by Stuyver et al. (31). Samples with detectable HBV DNA after nested PCR were tested by the INNO-LiPA HBV genotyping assay according to the manufacturer's instructions. Briefly, 10 µl of amplified products was denatured at room temperature for 5 min and hybridized for 1 h at 50°C to specific oligonucleotide probes coated on reaction strips. Probes were designed based on genotype-specific sequence motifs in the amplified region. Three genotype-specific motifs were selected for HBV genotype A, two genotype-specific motifs were selected each for HBV genotypes B through F, and one genotype-specific motif was selected for HBV genotype G. After hybridization, conjugate solution (i.e., streptavidin labeled with alkaline phosphatase) was added and allowed to bind to biotinylated hybrid for 30 min at room temperature. Color development involved incubation with substrate (5-bromo-4-chloro-3-indolylphosphate -nitroblue tetrazolium chromogen) for 30 min at room temperature. The reaction strips were then aligned against a plastic reading card and interpreted based on the manufacturer's instructions. Classifying a sample as belonging to a specific genotype depended on the hybridization of the amplified DNA to all of its genotype-specific lines. When lines corresponding to more than one genotype were detected, a mixed genotype was reported unless there was only a single positive line for the second genotype, in which case the result was reported as a single genotype. Genotype G was an exception because there was only one genotype-specific line. Motifs specific for genotype H were not included in the strips, but sequence analysis showed that reaction to a combination of lines 11 and 15 is specific for genotype H. Figure 1 shows a few examples of genotyping results obtained.
fig.ommitted
|
FIG. 1. Examples of results obtained by INNO-LiPA genotyping assay. A sample is classified as a specific genotype based on hybridization of the amplified DNA to all of its genotype-specific lines. Samples that react to a combination of lines 11 and 15 are classified as genotype H. Based on the manufacturer's manual, samples 4 and 5 were classified as genotypes A and C, respectively, and sample 6 as coinfection of genotypes A and G.
| |
To validate the results of INNO-LiPA HBV genotyping assay, all
samples with mixed genotypes, all samples with genotypes E,
F, G, and H, and about 10% of randomly selected samples from
genotypes A through D were sequenced at the DNA sequencing core
facility, University of Michigan Medical Center, by using the
standard protocol for the Applied Biosystems DNA sequencer 377
(Perkin Elmer Corp., Foster City, Calif.). Primer sequences
used in the second round of PCR were used for bidirectional
sequencing. Sequencing results were compared with published
HBV S-gene sequences to ascertain HBV genotypes.
PC and CP variants. Sera from 322 patients in the Hong Kong study and from 278 patients in the U.S. study were tested for HBV PC and CP variants by line probe assay (INNO-LiPA HBV Precore Research Version; Innogenetics NV). Two hundred forty-eight (41%) patients were HBeAg positive, 327 (55%) were HBeAg negative, and the remaining 25 (4%) were not tested for HBeAg.
Except for the primers (14) and reaction strips, the procedure was similar to that for HBV genotyping. The probes were designed to determine the nucleotide sequences at position 1896 in the PC region (G versus A) and positions 1762 (A versus T) and 1764 (G versus A and G versus T) in the CP region. Multiple probes were applied on the strip for each motif, taking into account the extensive variability surrounding the specific nucleotide positions assessed by the assay.
To validate the results of INNO-LiPA HBV PC assay, all samples with indeterminate results and 10% randomly selected samples were sequenced. Sequencing results were compared with a prototype sequence obtained by aligning published HBV PC and X-gene sequences of the respective HBV genotype. All indeterminate results were later retested using a newer version—the INNO-LiPA HBV PC commercial assay—which included six additional probes covering polymorphisms in the CP region. Figure 2 shows examples of different patterns of PC and CP sequences obtained.
| fig.ommitted |
FIG. 2. Examples of results obtained by INNO-LiPA PC assay. WT, wild type.
| |
HBV genotyping. Of the 701 samples tested for HBV genotype, 700 (99.9%) could
be classified. All eight HBV genotypes, A to H, were found (Table
1). Of the 75 samples sequenced, the results of sequencing were
completely concordant with those of the INNO-LiPA HBV genotyping
in 72 samples (Table
2). Three (0.4%) samples hybridized to
multiple genotype-specific lines. Based on the manufacturer's
instructions, samples 4 and 5 were interpreted as a single genotype—A
and C, respectively—and sample 6 was interpreted as true
coinfection of genotypes A and G (Fig.
1). Direct sequencing
of 300 nucleotides of the S gene (codons 120 to 220) revealed
that all three samples had sequences that correspond to two
genotypes, with samples 4 and 5 more closely resembling genotype
C and sample 6 being most similar to genotype G (Fig.
3).
| fig.ommitted |
TABLE 1. Prevalence of HBV genotypes as determined by the INNO-LiPA genotyping assay
| |
| fig.ommitted |
TABLE 2. Comparison of HBV genotyping results of the INNO-LiPA genotyping assay and sequencing
| |
| fig.ommitted |
FIG. 3. S gene nucleotide sequences of three samples that hybridized to probes corresponding to multiple genotypes in the INNO-LiPA genotyping assay. The sequence of each sample is compared to prototype sequences of two genotypes based on the results of the INNO-LiPA genotyping assay. Dashes (-) represent sequences identical to those of the prototypes of both genotypes. Sequences that match with the first genotype but not the second are shown in regular font; those that match with the second genotype but not the first are shown in boldface; and those that match with neither genotype are shown in boldface italic. *, sample 4 showed an additional band corresponding to genotype C on INNO-LiPA. ", sample 5 showed an additional band corresponding to genotype E on INNO-LiPA.
| |
PC stop codon (G1896A) and CP (A1762T, G1764A) variants. A total of 600 samples were tested for PC stop codon and CP
variants by INNO-LiPA HBV PC research version assay.
PC variants. In the PC region, 358 (60%) samples had wild-type (G) sequence at nucleotide 1896, 59 (10%) had the G1896A mutation, and 182 (30%) had a mixture of wild-type and variant sequences (Table 3). A PC variant was more commonly found in HBeAg-negative than in HBeAg-positive patients—54 versus 22% (P < 0.001), respectively. A PC variant alone was found in 16 and 2% (P < 0.001), a mixture of PC and wild type sequence in 37% and 20% (P < 0.001), and wild-type sequence alone in 47 and 78% (P < 0.001) of HBeAg-negative and HBeAg-positive patients, respectively. Forty-seven samples with interpretable results in the INNO-LiPA PC assay were sequenced. The results of sequencing were completely concordant with those of the INNO-LiPA PC assay in all 37 samples that showed either wild-type or variant sequence. In the remaining 10 samples, INNO-LiPA PC assay showed a mixture of wild-type and variant sequence, while direct sequencing revealed mixed sequences in 5 samples, variant sequence alone in 4 samples, and wild-type sequence alone in 1 sample (Table 4).
| fig.ommitted |
TABLE 3. Prevalence of HBV PC and CP variants as determined by the INNO-LiPA PC assay
| |
| fig.ommitted |
TABLE 4. HBV PC results obtained by using the INNO-LiPA PC assay and sequencinga
| |
Only one sample (0.2%) had an indeterminate sequence. Direct
sequencing of this sample showed the following point mutations:
A
1846T, C
1856T, T
1858C, and T
1893C. The T
1893C mutation likely
accounted for the indeterminate result in the INNO-LiPA HBV
PC assay as it occurred in the region that should hybridize
to the probes used for the detection of the G
1896A mutation.
A single mismatch is sufficient to abolish hybridization to
a probe under the conditions of this assay.
CP variants. In the CP region, INNO-LiPA HBV PC research version assay revealed that 216 (36%) samples had wild-type sequence (A at nucleotide 1762 and G at nucleotide 1764), 148 (24.7%) had the classical dual mutation (A1762T, G1764A), 186 (31%) had a mixture of wild type and classical dual mutation, 2 (0.3%) had a single mutation at nucleotide 1764, 14 (2.4%) had a mixture of wild type and single mutation at position 1762 or 1764, 2 (0.3%) had mixtures of single and dual mutations, and 32 (5.3%) had indeterminate sequences (Table 3). CP variants were more common in HBeAg-negative patients, with the dual CP variant alone found in 35% of HBeAg-negative and 25% of HBeAg-positive (P = 0.013) patients.
Forty-six samples with interpretable results in the INNO-LiPA PC assay were sequenced. The results of sequencing were completely concordant with those of the INNO-LiPA assay in all 31 samples that showed either wild-type or variant sequence. In the remaining 15 samples, the INNO-LiPA assay showed a mixture of wild-type and variant sequence, while direct sequencing revealed mixed sequences in 4 samples, variant sequence alone in 7 samples, and wild-type sequence alone in 4 samples (Table 5).
| fig.ommitted |
TABLE 5. HBV CP results using the INNO-LiPA PC assay and sequencing
| |
Thirty-two (5.3%) samples had indeterminate sequence in the
CP region. Of these, 8 (25%), 9 (28%), and 15 (49%) were from
patients with HBV genotypes A, B, and C, respectively. Indeterminate
results were obtained in 2.5 and 8.6% of samples from patients
in the Hong Kong and U.S. studies, respectively (
P = 0.0016).
Direct sequencing showed deletions in and around nucleotides
1762 to 1764 in 10 samples and point mutations involving another
nucleotide(s) in the remaining 22 samples. Figure
4 shows a
few examples of direct sequencing results for indeterminate
samples.
| fig.ommitted |
FIG. 4. CP nucleotide sequences of samples that were indeterminate in the INNO-LiPA PC assay. All the samples shown and the prototype sequence used were genotype C. Dashes (-) represent sequences identical to those of the prototype sequence. ///, deletions; nt, nucleotide.
| |
All the indeterminate samples were retested using the INNO-LiPA
HBV PC commercial version assay. Under the conditions of the
new assay, one sample then showed a wild-type sequence and 7
showed the classical dual mutations, whereas 24 samples remained
indeterminate.
Among the 24 samples that remained indeterminate by this latest version, 10 had deletions that spanned nucleotides 1762 to 1764 and 10 samples had other mutations at nucleotides 1762 and 1764 or at both nucleotide positions. Of the remaining four samples, two had wild-type sequence and two had the classical dual mutation; all four had an additional mutation at nucleotide 1760 that likely prevented hybridization to the probe.
This large study of 701 chronic hepatitis B patients from China
and the United States showed that the HBV genotype as well as
PC and CP variants can be determined for 99.9, 99.8, and 94.7%
of samples, respectively, by using a rapid and simple assay.
The entire assay from DNA extraction to development of color
reaction can be completed in 12 h, and as many as 30 samples
can be run simultaneously. The ease of the line probe assay
enables it to be applied in the clinical diagnostic laboratory
or in research laboratories that conduct epidemiology research
for which a large number of samples need to be tested, as was
the case in this study. We validated the line probe assay results
by direct sequencing of 10% of interpretable samples in the
genotyping and PC assays, and we found that the INNO-LiPA assay
results were completely concordant with those of sequencing.
The INNO-LiPA assay was able to detect all HBV genotypes, i.e., A to H. Almost all the patients from Hong Kong had genotypes B or C, whereas all eight genotypes were found in the U.S. patients, with A and C being most common. Although genotype H-specific probes were not included in the INNO-LiPA HBV genotyping assay, comparison of the published sequence of genotype H and the positions of the probes on the reaction strips suggest that genotype H samples yield a unique pattern with reaction to a combination of lines 11 and 15. In this study, we found three U.S. patients but none of the Hong Kong patients exhibiting this pattern. Direct sequencing of amino acids 128 to 227 of the HBV polymerase gene revealed a histidine at position 134 in all three samples, a finding which is in agreement with the unique amino acids previously reported for genotype H (1).
In accordance with previous reports, the prevalence of PC variant in our study was significantly higher in HBeAg-negative than in HBeAg-positive patients. The prevalence of the dual CP variant alone was also higher in HBeAg-negative than in HBeAg-positive patients. As had been reported with the INNO-LiPA DR assay for the detection of lamivudine-resistant mutations (21), the line probe assay is more sensitive for the detection of mixtures than is direct sequencing. In this study, of the 10 samples with mixed wild-type and variant PC sequence, only 5 (50%) were found to have mixed sequence by direct sequencing. Similarly, of 15 samples with mixed wild-type and variant CP sequence, only 4 (27%) had mixed sequence by direct sequencing. Thus, the line probe assay may enable earlier detection of PC and CP mutations as they become selected during the course of chronic HBV infection. Differences in sensitivities of HBV variant detection techniques and result reporting (reporting of predominant sequence versus reporting of mixed sequences) may account for some of the discordant results in the published literature regarding prevalence of HBV variants and correlation between these variants and severity of liver disease and response to antiviral treatment.
The line probe assays have limitations. The most important limitation is that they are only capable of detecting the presence of the commonly occurring known mutations and the established HBV genotypes. Thus, the current version of the genotyping assay did not include probes that specifically detect genotype H. As additional HBV genotypes are identified, new probes have to be designed, and the assays must consequently be upgraded. Similarly, the PC assay is designed to detect only the classical stop codon mutation (i.e., G1896A). Other mutations, such as start codon mutation and amino acid substitutions in codon 29, cannot be detected. Another limitation is that polymorphisms within the region of the probes can prevent annealing and thus yield indeterminate results. This is a major reason for indeterminate results in the CP region. Despite the addition of new probes to account for polymorphisms in this region, 75% of the samples with indeterminate results in the latest research version PC assay still had indeterminate results in the improved commercial version assay. Likewise, the line probe assay is unable to characterize samples with deletions in the region of the probes.
In conclusion, we demonstrated that the line probe assay, which is simple and rapid, can be used for large-scale testing for HBV genotypes as well as for PC and CP variants. These assays can be applied to samples from patients in different parts of the world with a >99% level of accuracy for the determination of HBV genotypes and the PC (G1896A) variant. The accuracy for the detection of CP variants is slightly lower, 95%, because of frequent polymorphisms and deletions in this region. We also found that the line probe assay is more sensitive than direct sequencing for the detection of mixed sequences and hence permits earlier detection of newly selected mutations.
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作者：
Munira Hussain Chi-Jen Chu Erwin Sablon and Ann 2007-5-10