TRUGENE Sequencing Versus INNO-LiPA for Sub-Genotyping of HCV Genotype-4

Abdel Rahman N. Zekri,1 Hanaa M. Alam El-Din,1 Abeer A. Bahnassy,2 Amal M.R. El-Shehabi,3 Heba El-Leethy,1 Ashraf Omar,4 and Hussein M. Khaled5

1Virology and Immunology Unit, Cancer Biology Department, National Cancer Institute, Cairo, Egypt

2Tissue Culture Unit, Pathology Department, National Cancer Institute, Cairo, Egypt 3Biochemictry Department, Kasr El-Aini School of Medicine, Cairo, Egypt

4Tropical Medicine Department, Kasr El-Aini School of Medicine, Cairo, Egypt 5Medical Oncology Department, National Cancer Institute, Cairo University, Cairo, Egypt

Hepatitis C virus genotypes and subtypes determination is an important factor for understanding the epidemiologyof the virus, in the pre-treatment evaluation of the patients and in defining better treatment strategies. In the present study, we compared two commercially available assays for HCV genotyping: the reverse hybridization based Innogenetics INNO-LiPA HCV II and the direct sequencing by TRUGENE assay. The study included 31 HCV-RNA positive Egyptian patients; 18 patients with chronic active hepatitis, 8 with HCC, and 5 with cirrhosis. Using the TRUGENE genotyping test, all the samples had genotype 4 (100%) and subtyped as 4a in 18/31(58%), 4c in 10/31 (32%), 4e in 1/31 (3%), 4a/c in 1/31 (3%), and 4g in 1/31 (3%). Using the INNO-LiPA assay, 30 samples had genotype 4 (97%), and 1 sample had genotype 1e (3%). One sample showed mixed infection with type 4f and type 1. Only six samples were subtypable by INNO-LiPA, three were genotype 4c/d, and the other three were 4f, 4e, and 1e. Seven samples gave reactivity in the INNO-LiPA of lines 5, 6, 16, 17, 18, which are considered untypable by the interpretation chart but considered to be a rare HCV genotype 4 by the manufacturer. At the genotype level, there was a 97% concordance between TRUGENE sequencing and INNO-LiPA, but at the subtype level the concordance rate was 3% only. We conclude that the TRUGENE genotyping assay is a reliable test for HCV genotyping for the detection of major types and subtypes detection, while INNO-LiPA is a good test at the genotype level but unreliable for subtyping especially in the Egyptian population. This is mainly due to the high diversity of genotype 4, which is the most prevalent genotype in Egypt. J. Med. Virol. 75: 412-420, 2005. ©2005 Wiley-Liss, Inc. KEY WORDS: hepatitis C virus; sequence; Egypt; genotype 4 Introduction: Hepatitis C virus (HCV) is an enveloped positive single-stranded RNA virus that causes chronic hepatitis and end-stage liver disease worldwide. It is also a major cause of hepatocellular carcinoma [Sherlock and Dooley, 2002]. There is a high incidence of anti-HCV seropositivity in the Egyptian population, with an overall age-adjusted prevalence of HCV antibodies of 21.9% [Frank et al., 2000]. HCV demonstrates a high degree of sequence variability resulting in the formation of

quasispecies. However, the levels of heterogenecity differ considerably among the various regions of the virus, ranging from 19% in the 50 untranslated region (50 UTR) to 50% or more in the E1 region [Chan et al., 1992; Honda et al., 1994; Bukh et al., 1995; Simmonds, 1995]. Based on the analysis of variability in the viral genome, many genotypes and subtypes are defined. The methods developed to investigate HCV genotypes are based on molecular biology or serological techniques. The first includes direct nucleic acid sequencing[Simmondset al., 1993], reverse hybridization line probe assay (LiPA) [Stuyver et al., 1996], subtype-specific reverse transcription [Okamoto et al., 1992], DNA restriction fragment length polymorphism [Nakao et al., 1991], whereas serotyping identifies type specific antibodies against HCV genotype dependant pitopes [Dixit et al., 1995]. The prevalent genotype in Egypt is type 4, with the presence of other genotypes. Genotype 4 was the commonest (73%) followed by genotype 1 (26%) whereas mixed infection was found in 15.7% of the studied samples [Zekri et al., 2000]. Many studies have used the sequence analysis derived from the 50 UTRofHCVto determine the genotype of the virus [Chen and Weck, 2002; Anderson et al., 2003; Haushofer et al., 2003]. The two most commonly used assays are the TRUGENE HCV 50 non-coding (50 NC) genotyping sequencing kit, which is a standard direct sequencing kit for genotyping determination, and the reverse hybridization Line Probe Assay (INNO-LiPA HCV II; INNOGENETICS, N.V; Zwijnaarde, Belgium). To date, there are only few comparison studies of these two tests, and most of them are reported from Europe [Ross et al., 2000; Ansaldi et al., 2001; Halfon et al., 2001; Nolte et al., 2001] and North America [Zheng et al., 2003] but none from Egypt where there is a high prevalence of HCV-4. Therefore, the present study was conducted to compare HCV genotyping by TRUGENE HCV direct sequencing analysis of the 50 NC region and the reverse hybridization by INNO-LiPA analysis of the same region for samples obtained from different parts of the Egyptian community. The concordance, accuracies, and sensitivities of both assays were assessed in this clinical evaluation. Patients And Methods The study included 31 subjects with anti-HCV positive and high levels of SGPT & SGOT for more than 6 months. All cases were subjected to complete history taking and thorough clinical examination. Liver function tests and liver biopsy were performed. Samples were tested for anti-HCV using a commercially available EIA. Detection of HCV 1-RNA extraction. HCVRNAwas extracted from the sera by the silica method as previously described [Boom et al., 1990]. 2-RT-PCR of HCV. RT and PCR were performed with a primer pair selected from the highly conserved 5-UTR of HCV genome [Choo et al., 1989]. All steps were done as previously described [Zekri et al., 1995]. The following sequences were used as antisense primers for c-DNA synthesis HCV-6 [5-ACC-TCC nucleotides (NT) 319-324]. The internal primers were RB6A and RB6B for amplification of 266 bp of the 5-UTR, RB6A [5-GTG AGG AAC TAC TGT CTT CAC G-3 (NT 47-68)], and RB6B [5-ACT CGC AAG CAC CCT ATC AGG (NT 292-312)-3] [Zekri et al., 1995]. All samples were analysed twice for HCV RNA by the RT-PCR on different days with identical results. Upon completion of the amplification reaction, 10 ml of each PCR reaction product was analyzed by electrophoresis through a 1.2% agarose gel in Tris-Acetate-EDTA buffer (pH 8.0) and ethidium bromide staining. DNA was

transferred from the gel onto nitrocellulose filter using alkaline buffer (4N NaOH). The transferred DNA was cross linked by incubation for 2-3 hr at 808Cand the blot was then hybridized with an internal probe [Zekri et al., 1995]. HCV Genotyping All clinical samples were genotyped with both the LiPA and TRUGENE 50 NCkits from aliquots of a single RT-PCR sequence analysis. 1-INNO-LiPA II, genotyping. The line probe assay was used to assess HCV genotypes using kits provided by INNOGENETICS, N.V. The 5-UTR region was amplified using nested PCR with biotinylated primers. The labeled amplicon was allowed to hybridize and mounted on a strip. After stringent washing, streptavidin labeled with alkaline phosphatase was used to trace the hybridized products, and nitroblue tetrazolium and 5-bromo-4-chloro-3-indoyl-phosphate were used as a substrate according to the manufacturer's instructions. The probe reactivity patterns were interpreted using the chart provided by the manufacturers [Zekri et al., 2000]. 2-HCV genotyping by sequencing. The TRUGENE HCV 50 NC genotyping kit, Visible Genetics, Inc. (Toronto, Ontario, Canada) was used in conjunction with the OpenGene DNA sequencing system. All HCV PCR products were run on a 1% agarose gel electrophoresis. A positive control of known standard sequence and a negative HCV control provided with the kit were utilized as controls in each run. The specific band was cut from the gel and purified by silica protocol [Boom et al., 1990], and sequenced by CLIP sequencing which allows both directions of the target amplicon to be sequenced simultaneously in the same tube using two different dye-labeled primers (Cy5.0 and Cy5.5) for each reaction. This method provides sequence information for both positive and negative DNA strands from a single reaction. The forward and reverse sequences are combined to form a query sequence. The query sequence is then compared to previously characterized isolates in the TRUGENE HCV 50 NC Module of the OpenGene software system in order to determine the HCV genotype of the sample. Gene Objects software analyses chromato-grams from each sample; the final 50 UTR sequence was obtained from the comparison of both sequenced strands. This information is compared with deposited HCV sequences by Gene LibrarianTM software with a minimal concordance of 98%. The genotype assignments of these samples were confirmed byBLAST searches. Nucleotide sequence accession numbers. The nucleotide sequences reported here are deposited in The National Center for Biotechnology Information/ National Institute of Health GenBank nucleotide sequence database (accession no. AY624965-AY624986).

Results All of the 31 HCV-RNA positive samples tested by the TrueGene sequencing were genotype 4 (100%). They were subtyped as 4a (65%), 4c (29%), 4a/c (3%), and 4g (3%) (Table I). Figure 1 shows 50 NC region sequence analysis and Fig. 2 showed the phylogenetic analysis of all studied samples in conjunction with three known GenBank nucleotide sequences with Blast database. Comparison between the sequences obtained in the present study and all previously reported 50 UTR sequences revealed a new 50 UTR mutation within HCV genotype-4 and confirm the heterogeneity of this type in the Egyptian population. On the other hand, most of these mutations were randomly distributed, however; they still maintained the postulated secondary structure of the 50 UTR genomic RNA and revealed the strong conservation of the genotypes. By the INNO-LiPA, the samples were found to be genotype 4 in 23 (74%) samples and genotype 1 in 1 (3%) sample. Seven samples (23%) gave reactivity of lines 5, 6, 16, 17, 18 which are considered untypable by the interpretation chart. However, by contacting the manufacturer, they considered them to be rare genotype 4 patterns, which will most probably be added to the future update of the interpretation chart. The genotype assignments of these samples were confirmed by the blast search. The concordance between TrueGene sequencing and INNO-LiPA was 74% (23/31 samples) at the main genotype level (Fig. 1A). By adding the seven untypable samples, which were considered by the manufacturer as type 4, the concordance rises to (97%). The cladograms of these sequences with known type 4 sequences from the GenBank are shown in Figure 1B. Subtyping was possible in six cases only by the INNOLiPA assay, of which three (9.7%) were 4c/d, one (3%) 4f, one (3%) 4e, and one (3%) 1e (Table I). Figure 1C shows the cladogram of five of these cases with type 4. Only one case showed mixed infection by INNO-LiPA, however it showed genotype-4a by TrueGene. One sample only (AY624963) showed discordance between the two tests at the genotype level. This sample was classified as genotype 1e by INNO-LiPA and as genotype 4a by TRUGENE. The cladogram of this sequence along with type 1 sequences from theGenBank is shown in Figure 1D. The demographic, clinical, and genotype features of the patients in relation to liver biopsy data are shown in Table II. The incidence of ascites, jaundice, and splenomegaly was significantly higher in HCC than in CAH patients (P¼0.000, 0.02, 0.03, respectively). HCC patients were mainly from Metropolitan Cairo Area (P¼0.03), whereas those with CAH were mainly from Delta area (P¼<0.001). Table II also shows the comparison between genotype 4a and 4c in relation to different study parameters. No significant abnormalities were detected in relation to the two subtypes.

Discussion The identification of different HCV genotypes, subtypes, and isolates is helpful in understanding the evolution and the epidemiology of the virus in relation to patient age, area, risk factors, and degree of liver disease [Icardi et al., 1997]. Although sequence analysis is considered the ''gold standard'' for HCV genotype determination, it is expensive, time consuming, and inconvenient for routine use. Analysis of the 50 UTR by the INNO-LiPA HCV II provides a fast and easy method for determination of theHCVgenotype [Chen and Weck, 2002]. To date, there are only few comparison studies of these two tests and most of them are reported from Europe [Ross et al., 2000; Ansaldi et al., 2001; Halfon et al., 2001; Nolte et al., 2001] and North America [Zheng et al., 2003]. Our study is the first comparison between the two methods using clinical samples from Egypt. The results of the present study demonstrate a good concordance between the two genotyping methods at the genotype level, but the possibility to analyze the sequence allows an absolute precision in the classification of the subtypes. Sequencing analysis by TRUGENE method allowed subtyping of all samples, whereas only five samples could be subtyped by the INNO-LiPA test. We found a very high level of agreement (97%) between TrueGene sequencing system and INNO-LiPA at the genotype level. Other studies showed similar results [Chen and Weck, 2002; Gargiulo et al., 2003; Nolte et al., 2003; Zheng et al., 2003]. At the subtype level there was only one sample (3%) that showed concordance between the two methods. This disagreement was reported by others [Chen and Weck, 2002; Nolte et al., 2003]. The lack of agreement reported in this study at the subtype level is not surprising considering the high diversity of genotype 4, especially in the Egyptian population as previously shown by others [Zekri et al., 2001] comparing sero- and geno-typing methods, The detection of a large number of individual mutated strains by hybridization assay such as INNO-LiPA in a single test is difficult since it requires very high stringent conditions [Ansaldi et al., 2001]. Also the lack of agreement between the subtypes is not unusual considering that both methods target the 50 NCregion. The 50 NCregion is among the most conserved regions of the viral genome, and in several cases, only one or two nucleotide changes distinguish unique subtypes [Smith et al., 1995]. It has been suggested that clinical laboratories should not call HCV subtypes from analysis of the 50 NC regardless of the method employed because of the inherent inaccuracy of the calls [Nolte et al., 2003]. Also they stated that there are no recognized subtype-specific differences in disease progression or response to therapy that would

warrant these designations. In our study, subtypes 4a and 4c were the most prevalent; this was similar to that previously reported [Zekri et al., 2000]. We could not correlate between the different subtypes and clinical picture, gender, residence, or liver biopsy results due to the low number of cases studied. In Egypt, the response of genotype 4 to interferon treatment was reported in many studies [El-Zayadi et al., 1999; Esmat et al., 2002; Thakeb et al., 2003], however there are no available data of the response of subtypes 4a and 4c to interferon treatment. Regarding the patient with cirrhosis that showed non-concordant results, it was classified as type 1e by INNO-LiPA and genotype 4a by the Visible Genetics sequencing system. A similar result was previously obtained [Ansaldi et al., 2001]. The mistyping could be ascribed to the close phylogenetical similarity between genotype 1 and 4, since the difference in the region analyzed is only six nucleotides [Ansaldi et al., 2001]. From this study, we concluded that TRUGENE sequencing for HCV genotyping and INNO-LiPA provide reliable results at the type level and have almost similar analytical sensitivities regarding genotype 4. TRUG-ENE method cannot, however, detect mixed infection as the INNO-LiPA method. Although, the TRUGENE 50 NC method is more technically complex to perform than LiPA, it is more detailed informative method in the level of the subtype especially in Egyptian populations with a very high heterogeneity of the sutype-4. It is also the best method for the epidemiological investiga-tions. In addition, TRUGENE 50 NC system can be easily updated, as new HCV sequence information becomes available of the HCV genome. As treatment of HCV is dependent on main genotype and not subtype, INNO-LiPA test can be used for genotyping of HCV until more studies can detect significant difference in response to interferon in different subtypes in genotype 4, the most prevalent in Egypt. Future analysis is needed in order to correlate subtyping with TRUGENE with response to interferon treatment in Egyptian patients with type 4, since all previously published data in this field was done by INNO-LiPA sequencing method. References 1. Anderson J, Simonetti J, Fisher D, Williams J, Yamamura Y, Rodriguez N, Sullivan DG, Gretch DR, McMahon B, Williams KJ. 2003. Comparison of different HCV viral load and genotyping assays. J Clin Virol 28:27-37.

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