Brief Report
March 2011

Overestimation of Incidence of Hepatitis B Virus Mixed-Genotype Infections by Use of the New Line Probe INNO-LiPA Genotyping Assay

ABSTRACT

The new version of the INNO-LiPA HBV genotyping assay (Innogenetics) developed to identify all hepatitis B virus (HBV) genotypes, A to H, has been evaluated in comparison with sequencing of PCR-amplified HBV DNA from 200 samples before or after cloning. The genotyping data obtained with INNO-LiPA were in agreement with those from direct sequencing in the 179 samples characterized by the two methods. INNO-LiPA revealed 28 mixed infections. However, sequencing after molecular cloning confirmed only 15 of them and did not identify any that were of genotype H (n = 9). Our study demonstrates that INNO-LiPA overestimates mixed infections as a result of erroneous genotype H detection.
The most accurate method for hepatitis B virus (HBV) genotyping (3, 6) is based on phylogenetic analysis after DNA sequencing of the entire viral genome (11, 13), often restricted to the gene S (12). However, in case of multiple-genotype infection, only the most abundant genotype is identified. Such mixed infections have been identified using a commercial assay based on reverse hybridization on strips carrying genotype-specific probes, the INNO-LiPA HBV genotyping assay (Innogenetics, Ghent, Belgium) (4, 5, 14). The aim of the study was to evaluate the reliability of a new version of INNO-LiPA HBV genotyping assay in the detection of mixed-genotype infections by comparison with sequencing of PCR-amplified HBV DNA, before and after molecular cloning, in a population of 200 HBV surface antigen (HBsAg)- and anti-HBc-HBV-DNA-positive French blood donors who were selected in accordance with the HBV genotype distribution previously described in this population (15). The INNO-LiPA was used according to the manufacturer's instructions using biotinylated primers specific for gene S. The hybridization patterns were interpreted according to the chart provided: (i) a single genotype when at least one genotype-specific probe was reactive; (ii) a mixed-genotype infection when multiple genotype-specific lines were reactive for more than one genotype; (iii) an indeterminate profile when single lines were observed for more than one genotype, except for the combination of two probes (one D probe and one F probe), which was classified as genotype H (this genotype has no specific probes); and (iv) a single genotype (the one with a complete hybridization profile) when an isolated reactivity for a second genotype was observed.
First, the ability of the INNO-LiPA to detect the minor HBV genotype in a dual mixture was evaluated by testing mixtures of two native samples of genotypes A and D, respectively. Five ratios, from 50/50 to 90/10, of genotypes A and D, respectively, were constituted and adjusted to a final viral load (VL) of 1.70 × 105 IU/ml (COBAS TaqMan HBV assay; Roche Diagnostics, Meylan, France). The native samples were added as controls at concentrations of 1.70 × 105 IU/ml and 1.70 × 104 IU/ml for genotypes A and D, respectively. All samples were analyzed by INNO-LiPA, direct sequencing, and molecular cloning as described below.
The INNO-LiPA results obtained in the 200 included samples were compared to those previously obtained by the direct sequencing of the S-PCR product (nt 108 to 552 of the S gene) (15). In the case of mixed infections or indeterminate or single-genotype profiles associated with an isolated reactivity with INNO-LiPA, sequencing after molecular cloning of PCR DNA products was performed in the TOPO-TA vector (Invitrogen, Cergy-Pontoise, France) according to the manufacturer's instructions. For each sample included, cloning was initially performed with 10 recombinant HBV S-gene-specific clones generated from S-PCR DNA products and sequenced with M13 plasmid universal primers (Eurofins MWG Operon, Ebersberg, Germany). When results were inconclusive or discordant with INNO-LiPA, data were completed with 10 additional clones and subsequently by the cloning of the INNO-LiPA-PCR DNA products. The analysis of 20 additional cloned INNO-LiPA sequences was performed to exclude a bias of PCR amplification. Sequences were analyzed as described elsewhere (15) with a set of 68 referenced HBV strains.
When tested in mixtures, both genotypes A and D were identified by INNO-LiPA when the minor genotype represented at least 20% of the total VL. Identical results were obtained by S-PCR DNA product sequencing after molecular cloning, whereas direct sequencing of the S-PCR DNA products identified only the major genotype when this genotype represented more than 70% of the mixture. Indeed, when the genotype represented less than 60% of the mixture, the genotype could not be determined due to ambiguities at genotype-specific nucleotides.
Of 200 blood donors, 187 were successfully genotyped by direct sequencing and 191 by INNO-LiPA (Table 1). Of these 191 samples, 163 (85.3%) carried a single genotype, whereas 28 (14.7%) had mixed-genotype infections. Samples identified as single genotype were not always reactive to the full set of genotype-specific probes. This was especially true for genotypes B and D: none of these samples hybridized to the second B-specific probe, and only 69% hybridized to the second D-specific probe. Moreover, even though not taken into account according to the interpretation chart, additional isolated positive signals with non-A probes were observed for 10 of the 48 genotype A samples. Of 28 mixed infections (Table 1), 25 were double infections and 3 were triple infections. The most frequent double infections were D + H (n = 6), A + D (n = 4), and A + E (n = 4). Finally, eight samples had an indeterminate profile with inconclusive hybridizations (six hybridized to one D probe and one H/F probe; one sample hybridized to one C probe and one E probe; and one sample hybridized to one B probe and one D probe).
Table 1.
Table 1. Comparison of HBV genotyping results obtained with direct sequencing and INNO-LiPA HBV genotyping assaya
Genotype determined by direct sequencingbNo. of samples with result in INNO-LiPA HBV genotyping assay
Successfully genotyped (n = 191)INDPCR NegTotal
Single genotype (n = 163)Mixed infection (n = 28)
ABCDEFGA + CA + DA + EB + DB+ EC + DD + ED + GD + HF + HA + D + EA + D + HB + D + H
A46       11       11   50
B 8                    8
C  10         1       1 12
D   61    1 1 1 16   16179
E    30    1 1 3        35
F     1          1     2
CGA rec      1               1
ND2  4   122  1       1 13
Total (n = 200)48810653011144113316111181200
a
CGA rec, CGA recombinant genotype; ND, not determined; IND, indeterminate; PCR Neg, PCR negative.
b
A total of 187 genotypes were identified as A to G; 13 genotypes were not determined.
Identical results were obtained with both INNO-LiPA and direct sequencing for 179 samples when one of the genotypes identified by INNO-LiPA in mixed-genotype infections was also identified by direct sequencing (n = 22) (Table 1). Of the 13 samples that were not genotyped by direct sequencing, 6 were double-genotype infections with INNO-LiPA, 6 were single genotype, and 1 was indeterminate (Table 1). Nine samples that were not genotyped by INNO-LiPA were genotyped by direct sequencing as D (n = 6) and C (n = 1). Finally, INNO-LiPA identified genotype H in 9 samples, in association with one or more genotypes. This was not confirmed by direct sequencing.
Of the 28 INNO-LiPA mixed infections investigated by sequencing of 10 S-PCR DNA clones (Table 2), 11 (39%) were confirmed, including a complete concordance for 9 samples and a partial concordance for 2 samples. In the latter 2 samples, one additional genotype (D or B) was identified by cloning; that result was in agreement with the INNO-LiPA isolated reactivity observed in one D probe and one B probe, respectively. The 17 remaining samples showing a single genotype after investigating 10 additional S-PCR clones were analyzed after the molecular cloning of the INNO-LiPA-PCR DNA products. As shown in Table 2, the sequence analysis of 20 clones per sample revealed (i) the presence of a second minor genotype in accordance with the INNO-LiPA profiles for 4 samples and (ii) the presence of a single genotype for 13 samples. Genotype H was never identified.
Table 2.
Table 2. Results of molecular cloning in the 28 INNO-LiPA HBV mixed-infection samples
Genotype (no. of isolates)Molecular cloning result (no. confirmed/no. tested)a
S-PCR productINNO-LiPA-PCR product
A + C (1)1c/1NT
A + D (4)3d/40/1
A + E (4)1/42/3
B + D (1)1/1NT
B + E (1)1/1NT
C + D (3)2/30/2
D + E (3)1/31/1
D + G (1)0/10/1
D + H (6)0/60/6
F + H (1)0/10/1
A + D + E (1)1/1NT
A + D + H (1)0/10/1
B + D + H (1)0/11e/1
Totalb11/284/17
a
NT, not tested.
b
Molecular cloning was initially performed in 10 S-PCR product clones in all INNO-LiPA mixed-infection samples (n = 28) and subsequently in 20 INNO-LiPA-PCR product clones in unconfirmed mixed-infection samples (n = 17).
c
Identified as A + C + D.
d
One sample identified as A + B + D.
e
Identified as B + D.
Sequencing of S-DNA clones derived from the 8 INNO-LiPA indeterminate profiles identified genotype D only (n = 6) or C only (n = 1) and a mixed infection of B + D (n = 1; note that INNO-LiPA revealed in this case a hybridization with one B-specific probe and one D-specific probe).
As confirmed here, direct sequencing, as opposed to the INNO-LiPA HBV genotyping assay, is not suitable for genotype identification in cases of mixed infection. In our hands, INNO-LiPA was capable of identifying more than one genotype in a given sample as long as its VL was greater than 20% of the total VL. The INNO-LiPA assay could detect genotypes A to G, with an excellent correlation with the direct sequencing analysis. Unfortunately, the detection of genotype H could not be tested due to the lack of such samples. Based on INNO-LiPA, and in accordance with a previous report (14), our study showed a 14.1% frequency (28 of 199) of mixed-genotype infections. However, this rate could have been overestimated, since the molecular cloning technique confirmed only 54% of them, even though this does not totally exclude the presence in minor amounts of an additional genotype that would have been identified by sequencing of a large number of clones or, alternatively, by using a more powerful sequencing technique, such as ultradeep pyrosequencing (8, 9, 16). Moreover, the high proportion of genotype H infection observed with INNO-LiPA and never confirmed by molecular cloning appears erroneous and has widely contributed to the overestimation of mixed infection. Indeed, genotype H is restricted to South and Central America (1, 2) and was not detected in a set of 940 French blood donors (15). If the 9 genotype H isolates are excluded, the frequency of mixed-genotype infection decreases from 14.1 to 10.6%, which is more in agreement with the 8% prevalence (7 of 93) observed in blood donors in Belgium (10). Interestingly, the proportion of mixed-genotype infection in the blood donor population, which mainly consists of asymptomatic HBV carriers, appears to be lower than those reported in patients suffering from chronic HBV infection (16% [41 of 262] [5]; 22% [23 of 103] [7]) or in intravenous drug users (16% [53 of 325] [4]). In addition to the overestimation of mixed infection, especially with regard to genotype H, INNO-LiPA probably also underestimates genotype mixtures, due to genotype-specific probe reactions which are not taken into account when isolated.
In conclusion, despite minimal significant improvement over the previous version, the modified version of INNO-LiPA remains a convenient and simple method for HBV genotyping. Our findings should convince the manufacturer to include genotype H-specific probes and to revise the interpretation criteria.

Acknowledgments

This work was supported by ANRS (grant no. 2008-408-CSS7-A020082).
We thank the colleagues from the Etablissement Français du Sang (EFS) and the Centre de Transfusion Sanguine des Armées (CTSA), Josiane Pillonel from the Institut National de Veille Sanitaire (INVS), who participates in the HBV blood donor survey, and Catherine Jourdain and Annie Razer from the INTS for their technical assistance.

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Published In

cover image Journal of Clinical Microbiology
Journal of Clinical Microbiology
Volume 49Number 3March 2011
Pages: 1154 - 1156
PubMed: 21177906

History

Received: 16 September 2010
Returned for modification: 29 October 2010
Accepted: 11 December 2010
Published online: 21 December 2020

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Authors

Mélanie Mercier
National Reference Center for Hepatitis B and C in Blood Transfusion, Institut National de la Transfusion Sanguine, Paris, France
Syria Laperche [email protected]
National Reference Center for Hepatitis B and C in Blood Transfusion, Institut National de la Transfusion Sanguine, Paris, France
Annie Girault
National Reference Center for Hepatitis B and C in Blood Transfusion, Institut National de la Transfusion Sanguine, Paris, France
Camille Sureau
Laboratoire de Virologie Moléculaire, Institut National de la Transfusion Sanguine, Paris, France
Annabelle Servant-Delmas
National Reference Center for Hepatitis B and C in Blood Transfusion, Institut National de la Transfusion Sanguine, Paris, France

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