Detection of Highly Prevalent Hepatitis B Virus Coinfection among HIV-Seropositive Persons in Ghana

Study population.Paired serum and plasma samples were collected from 838 consecutive, unselected HIV-infected patients with unknown HBsAg status attending for routine care at the Komfo Anokye Teaching Hospital (KATH) in Kumasi, Ghana. The samples were stored at −20°C prior to shipping on dry ice to the Royal Free Hospital in London, where they were kept at −70°C before testing, avoiding multiple freeze-thaw cycles. Stored serum samples from a further 1,300 HIV-infected patients were tested for HBsAg at the KATH serology laboratory using the rapid Determine assay. Of these, 178 samples tested HBsAg positive and were retrieved for repeat testing in London. The study was approved by the Committee on Human Research Publications and Ethics at the Kwame Nkrumah University of Science and Technology of Kumasi, Ghana.

Control panels.The World Health Organization (WHO) 2nd International Standard (IS) for HBsAg (genotype A, subtype adw2) was used to evaluate the sensitivity of the assays (National Institute for Biological Standards and Control, Potters Bar, United Kingdom; code no. 03/262). The panel included the positive controls A to D (encompassing 4-fold dilutions) and the negative control E, each reconstituted in 1 ml of distilled water. In addition, a seroconversion panel was tested comprising samples collected prospectively from a donor with acute hepatitis B infection, at days 0 (first sample), 36, and 43 (Boston Biomedica, Inc. [BBI], West Bridgewater, MA).

Detection of HBsAg mutants was investigated using the Coleman panel of recombinant HBsAg mutant samples, which was provided by Abbott (Abbott Laboratories, Abbott Park, IL) (10). The panel comprised five single mutants (E129H, M133L, D144A, G145R, and T123A), two double mutants (P142L G145R and P142S G145R), two insertion mutants (122NTins and 122RAins), and a wild-type positive control. A second panel of recombinant HBsAg mutants (genotype D) comprised 22 site-directed single mutants spanning codons 120 to 147 and a wild-type positive control. The expression vector, constructed using pCH under the cytomegalovirus immediate-early promoter-enhancer 078 pCHsAg An b-glob, was kindly donated by Ulrike Protzer (Technische Universität München, Munich, Germany). The wild-type and mutated HBsAg were tested by transfection of human hepatoma HuH7 cells with pCHsAg plasmids using FuGene6 (Roche Diagnostics GmbH, Mannheim, Germany). After 72 h, supernatants were collected for analysis. The same supernatant was used for all tests.

Serology assays.Serum samples and control panels were tested for the presence of HBsAg, using five assays. These comprised three reference assays, the automated chemilumiscent immunoassays Architect HBsAg (Abbott Diagnostics, Maidenhead, United Kingdom) and Liaison HBsAg Ultra (Diasorin, Bracknell, United Kingdom), and the manual Murex version 3 plate enzyme immunoassay (Abbott Diagnostics). In addition, the samples and control panels were tested by two rapid immunochromatography (lateral flow) assays, the Determine HBsAg assay (Inverness Medical, Stockport, United Kingdom) and the Vikia HBsAg assay (bioMérieux, Basingstoke, United Kingdom). The manufacturers' reported respective sensitivity and specificity are >99% and >99% for the three reference assays and >95% and >99% for the Determine test and >98% and >99% for the Vikia test. Serum samples were centrifuged at 10,000 relative centrifugal force (RCF) for 15 min prior to testing to remove debris. All assays were performed according to the manufacturers' instructions. In the Architect assay, results were reported as IU/ml and specimens with concentration values of ≥0.05 IU/ml were considered reactive. In the Liaison assay, results were reported as index values, and specimens with values of ≥0.9 were considered reactive. In the Murex assay, the mean absorbance of the replicates of the negative control (NC) was calculated and the reactivity was identified by the formula NC + 0.05; results were reported as sample absorbance/cutoff (s/co) values and regarded as reactive if the s/co value was ≥1. Reactivity was confirmed in neutralization assays by pretreatment of samples with anti-HBs antibodies, using the appropriate Architect, Liaison, or Murex HBsAg confirmatory assay according to the manufacturer's instructions; specimens showing neutralization values of >60% were considered positive. The Determine and Vikia rapid tests were read 15 min after addition of 50 μl and 75 μl of sample, respectively. A positive result was indicated by the presence of a clear antigen band and a clear control band on the strip; invalid results, defined by the lack of a clear control band, underwent repeat testing. The HBV e antigen (HBeAg) and anti-HBe antibody were measured by the Architect assay (Abbott Diagnostics).

Molecular assays.To quantify the HBV DNA load, HBsAg-positive samples underwent nucleic acid extraction by the m2000sp automated system (Abbott Laboratories), followed by quantitative real-time PCR using an assay that was calibrated against the 2nd World Health Organization (WHO) International Standard for HBV DNA and showing a lower limit of quantification of 14 IU/ml. Samples with detectable HBV DNA underwent population sequencing of the polymerase gene reverse transcriptase (RT) domain (amino acids 1 to 344) and the surface (S) gene (amino acids 1 to 226) as previously described (5). Briefly, plasma DNA was extracted using the NucliSens EasyMag platform (bioMerieux). A 1.4-kb amplicon comprising RT and surface gene was amplified by nested PCR and sequenced using a 3730 DNA analyzer (Applied Biosystems, Warrington, United Kingdom). Sequence analysis was performed using SeqScape software V 2.6 (Applied Biosystem).

Analysis.Confirmed positive results were defined by reactivity in all three reference assays or reactivity in one or two reference assays followed by confirmation in the respective neutralization assay. Invalid results in the two rapid tests were analyzed with the negative results. Sensitivity for each assay was defined by the number of correctly identified positive (true-positive) results divided by the total number of confirmed positive results (true positive + false negative). Specificity was defined by the number of correctly identified negative (true-negative) results, divided by the total number of confirmed negative results (true negative + false positive). The 95% confidence intervals (CI) for specificity and sensitivity were calculated. The positive predictive value (PPV) of the assays was calculated by the formula true positives/(true positives + false positives), whereas the negative predictive value (NPV) was defined by the formula true negatives/(true negatives + false negatives). To facilitate the comparative analysis, the strength of the signal expressed as fold reactivity above the assay interpretative cutoff was categorized as follows: Architect and Liaison signals 1- to 10-fold above cutoff, +; >10- to 40-fold above cutoff, ++; >40- to 100 fold above cutoff, +++; and >100-fold above cutoff, ++++; Murex s/co value of 1 to 3, +; s/co value of >3 to 10, ++; s/co value of >10 to 20, +++; and s/co value of >20, ++++. Discrepant results were defined as a marked reduced reactivity in one or two of the three reference assays relative to the third assay. The latter was defined as a signal strength of + relative to a strength of +++ or ++++ in one other test or a signal strength of ++ relative to a strength of ++++ in one other test. Among coinfected patients who tested either negative or positive with the rapid tests, proportions with HBV DNA of >14 and >2,000 IU/ml were compared by the z-test, whereas the median HBV DNA load values were compared using the Kruskall-Wallis test.

HBsAg seroprevalence in HIV-1-infected patients with unknown HBsAg status.Among 838 samples from HIV-1-infected patients with unknown HBsAg status, 140 showed confirmed HBsAg positivity, including 134 specimens that were HBsAg positive in all three reference assays and 6 specimens that were HBsAg positive in one (n = 2) or two (n = 4) reference assays. This yielded an HBsAg seroprevalence of 16.7% (95% CI, 14.2 to 19.2%).

Characteristics of HBsAg-positive patients.Among HBsAg-positive patients, 37/140 (26.4%) were HBeAg positive and 67/140 (47.9%) were anti-HBe antibody positive; the remainder lacked HBV e antigen markers. The majority of HBsAg-positive patients (118/140; 84.3%) showed a detectable (>14 IU/ml) HBV DNA load, and the median HBV DNA loads were 5,346 IU/ml overall and 8,279 IU/ml (range, 23 to 506,000,000 IU/ml) among viremic patients. HBV DNA sequencing was attempted with all samples showing a detectable HBV DNA load. However, the success rate was low for samples with HBV DNA levels of <200 IU/ml despite a nested PCR, due to the small volume of sample available for testing. Among 86/118 (72.9%) viremic patients with an HBV DNA sequence, the distribution of HBV genotypes was E in 82/86 (95.3%) and A in 4/86 (4.7%). Antiviral treatment status was not known for individual coinfected patients, but in the whole cohort, one-third of patients were receiving lamivudine-based antiretroviral therapy. The overall prevalence of major drug resistance mutations in RT was 4/86 (4.6%), comprising the lamivudine resistance mutations V173L, L180M, and M204V in three patients and the unusual combination of V173L plus the tenofovir resistance mutation A194T in the fourth patient.

Assay performance with serum samples.The sensitivity and specificity of the five HBSAg assays are summarized in Table 1. The two rapid assays showed a sensitivity of 69.3% for Determine and 70.7% for Vikia, whereas specificity and PPV were 100% for both assays. The NPVs were 94.2% and 94.5% for Determine and Vikia, respectively. Among 140 HBsAg-positive samples, invalid results (no control band) requiring repeat testing were obtained in 5 (3.6%) samples by Determine and 20 (14.3%) samples by Vikia. After repeat testing, one sample showed a persistently invalid result by Determine, and three samples showed a persistently invalid result by Vikia. The lowest HBsAg concentration (as measured by the Architect assay) that showed reactivity in the Determine and Vikia assays was 1.5 IU/ml. The median HBsAg concentrations of samples that tested falsely negative in the rapid assays (excluding invalid results) were 0.16 IU/ml (range, 0.05 to 3.4 IU/ml) for Determine and 0.15 IU/ml (range, 0.05 to 1.0 IU/ml) for Vikia. The proportion of patients with a detectable HBV DNA load was 34/43 (79.1%) among those who tested falsely negative by Determine (including one invalid result) and 84/97 (86.6%) among those who tested positive (P = 0.38); the median HBV DNA loads were 598 IU/ml (range, <14 to 950,405 IU/ml; interquartile range [IQR], 30 to 4,908 IU/ml) and 10,905 IU/ml (range, <14 to 506,000,000 IU/ml; IQR, 252 to 1,750,000 IU/ml) in the two groups, respectively (P = 0.0005); 15/43 (34.9%) and 60/97 (61.9%) patients, respectively, showed an HBV DNA level of >2,000 IU/ml (P = 0.006). Similarly, the proportions of patients with a detectable HBV DNA load were 31/41 (75.6%) among those who tested falsely negative by Vikia (including three invalid results) and 86/99 (86.9%) among those who tested positive (P = 0.17); the median HBV DNA loads were 587 IU/ml (range, <14 to 950,405 IU/ml; IQR, 30 to 3,848 IU/ml) and 10,905 IU/ml (range, <14 to 506,000,000 IU/ml; IQR, 252 to 1,750,000 IU/ml) in the two groups, respectively (P = 0.0003); 13/41 (31.7%) and 61/99 (61.6%) patients, respectively, showed an HBV DNA level of >2,000 IU/ml (P = 0.002).

The 178 samples that had tested HBsAg positive by the Determine assay in Ghana were tested by the Architect and Liaison assays, and all were confirmed as HBsAg positive, yielding 100% specificity for the test when performed on site.

Assay performance with reference reagents.With the 2nd International HBsAg standard, the Architect and Liaison assays showed identical levels of performance, with reactivity that declined from 187- and 181-fold above the cutoff at a 1:4 dilution to 46- and 44-fold, 12- and 12-fold, and 3- and 3-fold above the cutoff at 1:16, 1:64, and 1:256 dilutions, respectively (Table 2). The Murex assay also detected all dilutions, whereas the two rapid assays were positive with the 1:4 dilution but negative with the 1:16 dilution, corresponding to an HBsAg concentration of 2.3 IU/ml. With the seroconversion panel, the levels of performance of the three reference assays were similar overall, whereas the rapid tests remained negative up to day 43 after the first sampling (Table 3).

Assay performance with mutant recombinant HBsAg.With the Coleman HBsAg mutant panel, all mutants were detected by the three reference assays, with the highest relative strength of reaction seen with the Murex assay (Table 4). Low signal strength with Architect relative to Murex was seen with T123A and the 122NT and 122RA insertion mutants. The rapid assays detected some but not all of the mutants, although it should be noted that overall HBsAg concentrations were generally low relative to the expected assay sensitivities of >1.5 to 3.4 IU/ml for Determine and >1.5 to 2.3 for Vikia. With the Cologne HBsAg mutant panel, all mutants were detected by the Architect assay and HBsAg concentrations were generally higher than those seen with the Coleman panel. Of the 22 mutants, 16 (72.7%) were detected by Determine and 18 (81.8%) by Vikia. The samples that escaped detection by Determine despite an HBsAg concentration of >3.4 IU/ml comprised the P120G, C137W, and C147S mutants. The Vikia assay detected both the P120G and C137W mutants but missed the C147S mutant.

Analysis of false-negative and discrepant samples.We analyzed the HBV surface gene sequence of samples that gave false-negative or discrepant results and had detectable HBV DNA (Table 5). Among six samples that showed a false-negative result in one (n = 4) or two (n = 2) reference assays, the HBV DNA loads were <14 IU/ml in two samples and 23, 24, 199, and 6,479 IU/ml in the other four samples, respectively. A sequence was obtained from the latter sample, which gave a false-negative result in the Architect assay; this showed the mutation T123A (Table 5). There were 12 samples that showed discrepant results in one or two of the reference assays relative to the third reference assay, as indicated by marked differences in the strength of the signal. These included 10 samples with reduced reactivity by Architect, 6 samples with reduced reactivity by Liaison, and 1 sample with reduced reactivity by Murex. The HBV DNA load was <14 in four samples and ranged between 82 and 135,772 IU/ml in the remaining eight samples, of which six yielded a sequence (Table 5). The T123A mutation again appeared to have a significant impact on detection by the Architect assay. Major HBsAg immune escape mutants were detected in two samples, including E164D in the sample with the RT mutations V173L and 194T. In addition, three samples with an HBsAg concentration of >1.5 IU/ml escaped detection by one or both rapid assays, as confirmed by repeat testing. The HBV DNA loads were 23, 59, and 4,596 IU/ml in the three samples, respectively; a sequence was obtained from the latter sample, but no mutations were identified in the surface gene.