Challenges to Differentiate Hepatitis C Genotype 1 and 6: Results from A Field-Study in Cambodia

Anja De Weggheleire, Irith De Baetselier, Sokkab An, Sylvie Goletti, Vanessa Suin, Sopheak Thai, Sven Francque, Tania Crucitti, Lutgarde Lynen, Steven Van Gucht, Benoît Mukadi Kabamba, Anja De Weggheleire, Irith De Baetselier, Sokkab An, Sylvie Goletti, Vanessa Suin, Sopheak Thai, Sven Francque, Tania Crucitti, Lutgarde Lynen, Steven Van Gucht, Benoît Mukadi Kabamba

Abstract

Introduction: We aim to report on results and challenges of different methods used for hepatitis C (HCV) genotyping in a Cambodian HCV/HIV coinfection project.

Methods: Samples of 106 patients were available. HCV genotyping was initially (63 samples) done by the LightPower Taqman real-time PCR method (Viet A Corp.) and quality controlled using the Versant 2.0 line probe assay (Siemens Healthcare). Next, following interim quality control results, all 106 samples were (re)genotyped with Versant 2.0, complemented with 5'UTR/core sequencing for uninterpretable/incomplete Versant results.

Results: Using Versant, 103 (97.2%) of the 106 HCV-coinfected patients had an interpretable genotype result: 1b (50.5%), 6 non-a/non-b (30.1%), 1a (6.8%), 6a or b (4.9%), 2 (3.9%), 1 (2.9%) and 3 (1.0%). For 16 samples that were interpreted as genotype 1 or 1b per Versant's current instructions, it could not be excluded that it concerned a genotype 6 infection as the core region line patterns on the Versant test strip were unavailable, inconclusive or atypical. Upon sequencing, seven of these were genotyped as 1b and nine as genotype 6. Combining Versant and sequencing results, a definitive genotype was assigned in 104 patients: 1b (44.2%), 6 non-a/non-b (39.4%), 1a (6.7%), 6a or b (4.8%), 2 (3.8%) and 3 (1.0%). Genotyping by LightPower and Versant was discordant for 23 (of 63) samples. The LightPower assay misclassified all genotype 6 non-a/non-b samples as genotype 1, which indicates that this assay is only using 5'UTR information.

Conclusions: HCV genotype 1b and genotype 6 non-a/non-b were most common. With Versant 2.0 (using 5'UTR and core information), genotype classification (1 or 6) remained inconclusive in 15% of samples. The locally available method (LightPower assay) failed to identify genotype 6 non-a/non-b, which highlights that methods using 5'UTR information only should not be used in Cambodia. Regional/national guidelines should be explicit about this.

Trial registration: This study was performed as part of a larger cross-sectional study on the burden of hepatitis C coinfection in HIV patients in Cambodia (Clinical.trials.gov: HCV-Epi NCT02361541).

Keywords: Diagnostic methods; Genotyping; Hepatitis C; Misclassification; Southeast Asia.

Figures

Fig. 1
Fig. 1
Genotype testing and quality control strategies applied during the study
Fig. 2
Fig. 2
Line pattern Versant 2.0 and results of 5′UTR and core sequencing for 16 samples scored genotype 1/1b by Versant 2.0 but for which genotype 6 could not be excluded. Faint colored bands are colored lighter gray
Fig. 3
Fig. 3
HCV genotype distribution in an HCV/HIV coinfected Cambodian cohort. Results of different testing strategies. The strategies are: (1) genotyping with the Versant 2.0 line probe assay (N = 103), (2) genotyping with the LightPower HCV test (N = 63) and (3) genotyping results of the Versant 2.0 complemented with sequencing for samples with uninterpretable/incomplete results by Versant 2.0 testing (N = 104)

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