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[Investigation of Hepatitis C Virus (HCV) Genotypes by "Reverse Hybridisation Strip Assay" and DNA Sequence Analysis
Pelin Özmen1, Selma Gökahmetoğlu2
1Nevşehir Hacı Bektaş Veli University Faculty of Dentistry, Basic Medical Sciences, Department of Medical Microbiology, Nevşehir, Turkey.
Insights
Comparing Hepatitis C Virus (HCV) genotyping methods, this study found significant discrepancies between the widely used Line Probe Assay (LIPA) and sequence analysis, impacting treatment decisions. Further research with larger sample sizes is recommended to validate these findings.
Area of Science:
- Virology
- Infectious Diseases
- Molecular Diagnostics
Background:
- Hepatitis C Virus (HCV) affects 185 million globally, causing 350,000 deaths annually.
- Accurate HCV genotyping is crucial for effective treatment, influencing sustained virological response (SVR) rates.
- Current diagnostic methods require rigorous comparison to ensure reliable patient management.
Purpose of the Study:
- To compare the accuracy of the Line Probe Assay (LIPA) with sequence analysis for detecting HCV genotypes and subtypes.
- To evaluate the clinical implications of discrepancies in HCV genotyping results.
- To assess the reliability of LIPA for guiding direct-acting antiviral (DEA) treatment protocols.
Main Methods:
- HCV genotyping was performed on 212 chronic HCV patients with viral loads ≥10⁴ IU/ml using both LIPA and sequence analysis.
- LIPA targeted the 5'UTR and core regions, while sequence analysis focused on the NS5B gene region.
- Hemi-nested PCR amplified the NS5B region, followed by sequence analysis using an ABI 3500 Genetic Analyzer.
Main Results:
- Significant differences (p<0.001) were observed in HCV genotype and subtype detection between LIPA and sequence analysis.
- Genotype compatibility was 97.5% at the genotype level but only 19% at the subtype level.
- HCV genotype 1a exhibited the highest viral load (p<0.001) in patients.
Conclusions:
- The study highlights significant inconsistencies between LIPA and sequence analysis in HCV genotyping, particularly at the subtype level.
- These discrepancies may impact the efficacy of DEA treatment protocols, which rely on accurate genotype determination.
- Further studies with larger cohorts are recommended to confirm these findings and improve diagnostic accuracy for HCV management.
Abstract:
Hepatitis C Virus (HCV) is a virus that is estimated to infect approximately 185 million people worldwide and causes 350 000 deaths each year. The target in HCV treatment is the sustained virological response (SVR), and the most important virological factors determining SVR are genotype and baseline HCV-RNA levels. In this study, it was aimed to compare the HCV genotypes and subtypes detected by the "line probe assay (LIPA)" method with sequence analysis. HCV genotypes and subtypes were investigated by line probe assay (LIPA) (NLM analytica, Italy) and sequence analysis methods in 212 patients with chronic HCV diagnosis and with HCV RNA viral load of 10⁴ IU/ml and above. 5'UTR and core regions were studied in LIPA method and NS5B gene region was studied in the sequencing method. After amplifying 340 bp region in the NS5B gene region with the hemi-nested PCR method, sequence analysis (ABI 3500 Genetic Analyzer [Applied Biosystems, USA]) was performed. Statistical analysis of the study was determined by using TURCOSA Analytics program. In the study, HCV genotypes and subtypes that were determined in 212 patients by LIPA method were; 40 (18.8%) genotype 1a, 97 (45.75%) genotype 1b, 7 (3.3%) genotype 2, 12 (5.6%) genotype 2a/c, 2 (0.94%) genotype 2b, 19 (8.96%) genotype 3, 16 (7.55%) genotype 4, 1 (0.47%) genotype 4a, 15 (7.5%) genotype 4c/d, 1 (0.47%) genotype 4h and 2 (0.94%) genotype 5. The HCV genotypes and subtypes determined in 212 patients by sequence analysis were; 20 (9.43%) genotype 1a, 118 (55.6%) genotype 1b, 16 (7.55%) genotype 2a, 4 (1.89%) genotype 2b, 1 (0.47%) genotype 2k, 21 (9.91%) genotype 3a, 2 (0.94%) genotype 4a, 28 (13.21%) genotype 4d and 2 (0.94%) genotype 5a. The difference in results between the two methods was found to be statistically significant (p< 0.001). According to the viral load-genotype relationship, the highest viral load was detected in genotype 1a patients (p< 0.001). In conclusion, in order to determine HCV genotypes and subtypes in our study, LIPA and sequence analysis methods were compared and the genotype compatibility between the two methods were determined as 97.5% on the basis of genotype and 19% on the basis of subtype. Since the direct-acting antiviral (DEA) treatment protocol in chronic HCV patients is planned according to the genotype/subtype determination, the inconsistency of the results obtained in the LIPA method, which is routinely widely used, with the sequence analysis method is remarkable, and it was concluded that these results should be supported by studies containing more samples.
