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Genotype distribution of hepatitis C virus among Lebanese patients: Results from a major tertiary care center
Gene Reports 4 (2016) 190–193
Contents lists available at ScienceDirect
Gene Reports journal homepage: www.elsevier.com/locate/genrep
Genotype distribution of hepatitis C virus among Lebanese patients: Results from a major tertiary care center Fatmeh Abbas, Joseph Haddad, Rami Mahfouz ⁎ Department of Pathology and Laboratory Medicine, American University of Beirut Medical Center, Beirut, Lebanon
a r t i c l e
i n f o
Article history: Received 14 March 2016 Received in revised form 16 May 2016 Accepted 5 June 2016 Available online 16 June 2016 Keywords: Hepatitis C Virus Genotype Lebanon
a b s t r a c t Aims: The determination of hepatitis C virus (HCV) genotype is a crucial element in the therapeutic management of HCV infected patients. Studies showed that different treatment responses were obtained for different genotypes. The aim of this research study is to assess the distribution of hepatitis C virus genotypes in chronic HCV patients from a major tertiary care center in Lebanon. Materials and methods: For all HCV patients referred for viral load testing at the American University of Beirut Medical Center, we reviewed the HCV genotyping results for 203 patients positive for HCV RNA using the Linear Array Genotyping kit. Results: HCV genotypes were distributed as follows: Genotype 1 (39.6 42.86%), Genotype 3 (27.6 26.11%), Genotype 4 (26.0 24.63%), Genotype 2 (3.6 3.45%), and Genotype 5 (3.1 2.96%). Genotype 6 was not detected in any case from our patient population. Conclusion: Unlike previous studies in the country, our study interestingly showed a shift in viral genotype from Genotype 4 as the most predominant few years back to a current Genotype 1 predominance. Future epidemiological investigations, especially in light of these interesting results which differ from other populations in the Middle East region and show HCV viral profile changes, are warranted. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Since its discovery in 1989, hepatitis C virus (HCV) has been considered a global public health problem that affects about 3% of the world's population (about 185 million individuals worldwide) (Shepard et al., 2005; Mohd Hanafiah et al., 2013) and 3.4 to 4.4 million individuals in the United States (NIH consensus, 2002; Armstrong et al., 2006; Chak et al., 2011). In most European countries, the prevalence of HCV in the general population ranges from 0.5% to 2% (about 5 to 10 million infected individuals) (WHO, 2003). Studies in the Middle East showed that Genotype 4 predominates in the Arab countries (with the exception of Jordan), while Genotype 1 predominates in the non-Arab countries (Poustchi et al., 2010). For example, a recent study showed that the most predominant HCV genotype in East Turkey is Genotype 1b (İba Yilmaz et al., 2015). In addition, a study from Iran showed a high
Abbreviations: HCV, hepatitis C virus; RNA, Ribonucleic Acid; RT-PCR, Reversetranscriptase Polymerase Chain Reaction; DNA, Deoxyribonucleic Acid; IFN, interferon; RBV, ribavirin; PEG, pegylated; DAA, direct-acting antiviral; SVR, sustained viral response. ⁎ Corresponding author at: Molecular Diagnostics Laboratory, Department of Pathology and Laboratory Medicine, American University of Beirut Medical Center, Cairo Street, Beirut, Lebanon. E-mail address: [email protected] (R. Mahfouz).
http://dx.doi.org/10.1016/j.genrep.2016.06.002 2452-0144/© 2016 Elsevier Inc. All rights reserved.
prevalence for the HCV Genotype 1 (Liakina et al., 2015). However, another study in Libya reported a scattered HCV genotype prevalence that varied by geographical location, demographic, and risk factors (Daw et al., 2015). HCV can cause chronic hepatitis and is recognized to be a leading cause of end-stage liver disease with high progression to cirrhosis and hepatocellular carcinoma (Poynard et al., 2000; Kim, 2002; WHO, 2003). Approximately 80% of acutely infected HCV patients progress to chronic infection, 20% of whom develop cirrhosis within 25 years, with 25% of patients with a cirrhosis developing hepatocellular carcinoma (Freeman et al., 2001; Reddy et al., 2011). HCV is the primary cause of liver transplantation in the United States (Davis et al., 2010). Sequence heterogeneity in the viral genome of the hepatitis C virus was described. Okamoto et al. (1992) classified HCV into four different genotypes while Simmonds et al. (1993) classified it into six major types and a series of subtypes. The knowledge of the genotypes in chronic hepatitis C is a crucial element to be identified since treatment response varies among the different genotypes and subsequently therapeutic management options change according to the genotype, thus different treatments, treatment duration, and therapy dose are required in patient care (Hnatyszyn, 2005). The purpose of this study is to describe the distribution pattern of HCV genotypes in chronic hepatitis patients in a major tertiary care center in Lebanon.
F. Abbas et al. / Gene Reports 4 (2016) 190–193
191
The demographic data, mainly pertaining to age and gender, is shown in Table 2.
2. Materials and methods 2.1. Samples
4. Discussion This Institutional Review Board-approved study was performed at the American University of Beirut Medical Center (AUBMC), which is a major tertiary care center in Lebanon accommodating patients from the different districts and communities of the country. It is a retrospective analysis of HCV genotype testing performed in the Molecular Diagnostics Laboratory at AUBMC where we reviewed the HCV genotypes of 203 samples positive for HCV RNA in order to determine the distribution of genotypes among all patients. These patients have been referred to our center from all districts and ethnic communities of the country; however, no clinical data is available regarding the mode of transmission as it is not within the scope of this research. 2.2. HCV quantification and HCV genotyping Samples were tested for the presence of HCV RNA and its quantification by polymerase chain reaction in real time (RT-PCR) using Cobas Ampliprep/Taqman 48 platform (Roche Diagnostics GmbH, Germany). Then, HCV genotyping was performed using the Linear Array HCV Genotyping Test (Roche Diagnostics GmbH, Germany) and the Linear Array Detection Kit (Roche Diagnostics GmbH, Germany). The test utilizes reverse transcription of target RNA to generate complementary DNA (cDNA), amplification of target cDNA by the Polymerase Chain Reaction (PCR), and nucleic acid hybridization for the genotyping of HCV RNA in human serum or EDTA plasma. The Linear Array HCV Genotyping Test is based on five major processes: Specimen preparation; reverse transcription of the target RNA to generate complementary DNA (cDNA); PCR amplification of target cDNA using HCV specific complementary primers; hybridization of the amplified products to oligonucleotide probes specific to the target(s); and detection of the probe-bound amplified products by colorimetric determination. 2.3. Interpretation of results The Linear Array HCV Genotyping Strip is read visually by comparing the pattern of blue bands to a reference table of genotype patterns (as provided by the manufacturer). 2.4. Statistical analysis The HCV genotyping results of 203 patients were collected and the prevalence of the different genotypes was determined by direct counting methods. 3. Results Using the Linear Array Genotyping test, the distribution of different HCV genotypes was determined. It was found that HCV Genotypes 1, 3, and 4 are the most prevalent (39.6 42.86%, 27.6% 26.11, and 24.63%, respectively) and Genotypes 2 and 5 are less frequent (3.45% and 2.96%, respectively), however, Genotype 6 was not found (Table 1).
Hepatitis C virus genotypes differ in three major properties including their prevalence, pathogenicity, and response to treatment. According to the international literature, the prevalence of certain HCV genotypes is frequently associated with certain geographical areas. For example, HCV Genotype 1 is prevalent in North America and Japan, Genotype 3 is most common in the Indian subcontinent, Genotype 4 is most common in Africa and the Middle East, while Genotype 5 can be found in South Africa and Genotype 6 in Southeast Asia (Sy and Jamal, 2006). The countries located in the region closest to Lebanon (Arab and nonArab countries) showed a predominant prevalence of HCV Genotype 1 and 4. In Turkey and Iran, HCV Genotype 1b and 1 were the most dominant, respectively; while many Arab countries (Egypt, Saudi Arabia…) recorded a high prevalence of HCV Genotype 4 (İba Yilmaz et al., 2015; Liakina et al., 2015; Poustchi et al., 2010). Previous studies about the prevalence of HCV genotype in Lebanon showed that Genotype 4 was found to be the predominant genotype among 142 HCV-infected Lebanese patients followed by Genotype 1, these patients included 27 patients with β-thalassemia major or intermedia, 30 patients on haemodialysis, 32 multi-transfused patients and 15 IDUs (Sharara et al., 2007). The same distribution pattern was seen in a study among 395 thalassemics (Ramia et al., 2002). Another study conducted among 106 intravenous drug users (IDUs) in Lebanon found that Genotype 3 was the predominant HCV genotype followed by Genotypes 1 and 4, a situation similar to that among IDUs in Western Europe (Mahfoud et al., 2010). As shown in Table 3, the genotypic distribution is variable among the different studies so far conducted in Lebanon. Although the study by Mahfoud et al., is showing a predominance of Genotype 3, this only refers to the IDUs subgroup representing only 28 positive HCV-RNA in IDU cases. On the other hand, the study by Sharara et al., shows a wider variation of the predominant genotype among the different categories of patients included. Another hospital-based study showed a significantly different HCV genotype prevalence among dialysis and non-dialysis patients (a total of 77 infected cases). In the dialysis group (27 hemodialysis patients), Genotype 2 was predominant (80%, p b 0.001) while Genotype 1 (subtype 1b) was frequently detected in nondialysis cases (34.4%) whereas Genotype 1 was found in only 5% of dialysis cases (Irani-Hakime et al., 2003). Our study showed that HCV Genotypes 1, 3, and 4 are the most prevalent (42.86%, 26.11%, and 24.63%). This was surprising and in contrast with earlier findings where Genotype 4 was the predominant genotype (47.5%) in Lebanese patients (Ramia et al., 2002; Sharara et al., 2007). This might be due to the fact that this study was done among different groups of HCV-positive patients referred from all Lebanon, representing, thus more of a population-based sample. The pathogenicity of HCV infection also varies according to the different genotypes. For example, while HCV Genotype 3 infection is Table 2 Demographic data of HCV genotypes among the 203 hepatitis C patients.
Gender Table 1 Distribution of HCV genotypes among the 192 hepatitis C patients.
Age group
Genotype
# of patients
Frequency (%)
1 2 3 4 5 6 Total
87 7 53 50 6 0 203
42.86 3.45 26.11 24.63 2.96 0.00 100
Female Male 0–10 11–20 21–30 31–40 41–50 51–60 61–65 N65 Missing Total
# of patients
Frequency (%)
47 156 2 12 57 29 37 36 6 17 7 203
23.2 76.8 1.0 5.9 28.1 14.3 18.2 17.7 3.0 8.4 3.4 100.0
192
F. Abbas et al. / Gene Reports 4 (2016) 190–193
Table 3 Comparison of Genotype frequencies between different studies performed among Lebanese patients. Genotype
1 2 3 4 5 6 Mixed genotype (1/3) Total
Sharara et al. (2007)
Mahfoud et al. (2010)
Abbas et al. (this study)
Number
Number
Number
Frequency (%)
Frequency (%)
60 7 9 65 1 0 0
42.25 4.93 6.34 45.77 0.70 0.00 0.00
6 0 16 5 0 0 1
21 0 57 18 0 0 4
87 7 53 50 6 0 0
142
100.00
28
100
203
Frequency (%) 42.86 3.45 26.11 24.63 2.96 0 0
100
associated with a higher degree of liver steatosis (Rubbia-Brandt et al., 2001), Genotype 1 is associated with a higher risk of hepatocellular carcinoma development (Bruno et al., 2007). The response rates to interferon (IFN)-based therapy vary significantly between the different HCV genotypes with IFN representing the backbone of hepatitis C therapeutic options. Pegylated-IFN and ribavirin (PEG-IFN/ RBV) combination therapy is the standard of care for the treatment of chronic hepatitis C infection of different HCV genotypes. Recently, new direct-acting antivirals (DAAs) have been introduced to the treatment of HCV Genotype 1 infection where the genotype is an important determinant of both treatment strategy and outcome (Sherman et al., 2011). Treatment efficacy is measured by the sustained virologic response which is the treatment goal of undetectable plasma HCV RNA 12 or 24 weeks after therapy completion (Kohli et al., 2014). A systematic review of treatment of Hepatitis C showed that hepatitis C virus Genotype 1 is more difficult to cure than Genotype 2 or Genotype 3. Patients with HCV Genotype 1 should receive treatment with sofosbuvir, pegylated interferon, and ribavirin because of the shorter duration of therapy and high rates of sustained viral response (SVR, 89%–90%). Simeprevir with pegylated interferon and ribavirin is an alternative for patients with HCV Genotype 1 (SVR, 79%–86%). Patients with HCV Genotypes 2 and 3, representing 20% to 29% of HCV infections in the United States, should receive therapy with sofosbuvir and ribavirin alone (SVR for Genotype 2, 12 weeks' duration: 82%–93%; SVR for Genotype 3, 24 weeks' duration, 80%–95%) (Kohli et al., 2014). These differences in the SVR rates observed among different HCV genotypes have suggested that viral genome variability could play a role in determining treatment outcome (Pang et al., 2009). In non-transplant patients with chronic hepatitis C virus infection, HCV genotype has been linked to a differential response to antiviral therapy, risk of steatosis, fibrosis and all-cause mortality and correlated with outcomes in post-liver transplant patients (Campos-Varela et al., 2014). In post–liver transplant, Genotype 1 has the highest risk of advanced fibrosis and the lowest rate of SVR while compared to Genotypes 2 and 3 (Lai et al., 2011). The determination of HCV genotype is a crucial element in the therapeutic management of HCV infected patients and for post-transplant recipients. Treatment regimen, optimal dose, and treatment duration are guided by the determined genotype of HCV infection. In light of this information, the interesting data showing in this report should be alarming for investigators and clinicians in terms of the higher prevalence of Genotype 1 in our patients' population, while Genotype 4 has been previously reported to be the most prevalent in the Middle East region. This has major implications for clinical outcome, therapy response, as well as for potential vaccine development. Accumulated data show that there are different patterns for HCV genotype prevalence and our study showed a new prevalence pattern with predominance of Genotype 1. Future clinical studies revealing the modes of transmission,
like blood transfusions and injecting drug users, may be needed to better reflect on the distribution of the HCV RNA positive cases and assess if it is a genuine change in genotype as opposed to, probably, a different sample population in question at large.
References Armstrong, G.L., Wasley, A., Simard, E.P., McQuillan, G.M., Kuhnert, W.L., Alter, M.J., 2006. The prevalence of hepatitis C virus infection in the United States, 1999 through 2002. Ann. Intern. Med. 144, 705–714. http://dx.doi.org/10.7326/0003-4819-144-10200605160-00004 (PMID: 16702586). Bruno, S., Crosignani, A., Maisonneuve, P., Rossi, S., Silini, E., 2007. Mondelli MU. Hepatitis C virus genotype 1b as a major risk factor associated with hepatocellular carcinoma in patients with cirrhosis: a seventeen-year prospective cohort study. Hepatology 46, 1350–1356. http://dx.doi.org/10.1002/hep.21826 (PMID: 17680653). Campos-Varela, I., Lai, J.C., Verna, E.C., O'Leary, J.G., Stravitz, R.T., L.M, F., Trotter, J.F., Brown, R.S., Terrault, N.A., 2014. For the consortium to study health outcomes in HCV liver transplant recipients (CRUSH-C). Hepatitis C genotype influences post-liver transplant outcomes. Transplantation (00: 00Y00). Chak, E., Talal, A.H., Sherman, K.E., Schiff, E.R., Saab, S., 2011. Hepatitis C virus infection in USA: an estimate of true prevalence. Liver Int. 31 (8), 1090–1101. Davis, G.L., Alter, M.J., El-Serag, H., Poynard, T., Jennings, L.W., 2010. Aging of hepatitis C virus (HCV)-infected persons in the United States: a multiple cohort model of HCV prevalence and disease progression. Gastroenterology 138 (2), 513–521. Daw, M., El-Bouzedi, A., Dau, A., 2015. Geographic distribution of HCV genotypes in Libya and analysis of risk factors involved in their transmission. BMC Res. Notes 8, 367. Freeman, A.J., Dore, G.J., Law, M.G., et al., 2001. Estimating progression to cirrhosis in chronic hepatitis C virus infection. Hepatology 34 (4 pt 1), 809–816. Hnatyszyn, H.J., 2005. Chronic hepatitis C and genotyping: the clinical significance of determining HCV genotypes. Antivir. Ther. 10, 1–11. İba Yilmaz, S., Erol, S., Özbeks, A., Parlak, M., 2015. Distribution of viral genotypes and extrahepatic manifestations in patients with chronic hepatitis C in eastern Turkey. Turk. J. Med. Sci. 45 (1), 70–75. Irani-Hakime, N., Samaha, H., Almawi, W., Nasr, E., Mokhbat, J., Abou Jaoude, M., Daccache, J., Rahal, E., 2003. Prevalence of hepatitis C virus isolate genotypes from chronically infected Lebanese patients: a hospital-based study. J. Med. Liban. 51 (3), 121–126 (Jul-Sep). Kim, W.R., 2002. The burden of hepatitis C in the United States. Hepatology 36, S30–S34. http://dx.doi.org/10.1053/ jhep.2002.36791 (PMID: 12407574). Kohli, A., Shaffer, A., Sherman, A., Kottilil, S., 2014. Treatment of hepatitis C: a systematic review. J. Am. Med. Assoc. 312 (6), 631–640. http://dx.doi.org/10.1001/jama.2014. 7085. Lai, J.C., Verna, E.C., Brown, R.S., et al., 2011. Hepatitis C virus-infected women have a higher risk of advanced fibrosis and graft loss after liver transplantation than men. Hepatology 54, 418. Liakina, V., Hamid, S., Tanaka, J., Olafsson, S., Sharara, A.I., Alavian, S.M., et al., 2015 Decc. Historical epidemiology of hepatitis C virus (HCV) in select countries - volume 3. J. Viral Hepat. 22 (Suppl. 4), 4–20. Mahfoud, Z., Kassak, K., Kreidieh, K., Shamra, S., Ramia, S., 2010. Distribution of hepatitis C virus genotypes among injecting drug users in Lebanon. Virol. J. 7, 96. Mohd Hanafiah, K., Groeger, J., Flaxman, A.D., Wiersma, S.T., 2013. Global epidemiology of hepatitis C virus infection: new estimates of age-specific antibody to HCV seroprevalence. Hepatology 57 (4), 1333–1342. NIH Consensus, 2002. Statement on management of hepatitis C. NIH Consens. State Sci. Statements 19, 1–46 (2002, PMID:14768714). Okamoto, H., Sugiyama, H., Okada, S., Kurai, K., Akahane, Y., Sugai, Y., Tanaka, T., et al., 1992. Typing hepatitis C virus by polymerase chain reaction with type-specific primers: application to clinical surveys and tracing infection sources. J. Gen. Virol. 73, 673–679. Pang, P.S., Planet, P.J., Glenn, J.S., 2009. The evolution of the major hepatitis C genotypes correlates with clinical response to interferon therapy. PLoS ONE 4, e6579. http:// dx.doi.org/10.1371/journal.pone.0006579 (PMID: 19668364). Poustchi, H., Sepanlou, S.G., Esmaili, S., Mehrabi, N., Ansarymoghadam, A., 2010. Hepatocellular carcinoma in the world and the Middle East. Mid. East J. Digest. Dis. 2 (1/ January). Poynard, T., Ratziu, V., Benhamou, Y., Opolon, P., Cacoub, P., Bedossa, P., 2000. Natural history of HCV infection. Baillieres Best Pract. Res. Clin. Gastroenterol. 14, 11–228. Ramia, S., Koussa, S., Taher, A., Haraki, S., Klayme, S., Sarkis, D., et al., 2002. Hepatitis C virus genotypes and hepatitis G virus infection in Lebanese thalassemics. Ann. Trop. Med. Parasitol. 96, 197–202. Reddy, K.R., Bruno, S., Rossaro, L., et al., 2011. Predictors of sustained virologic response among treatment-naive patients with hepatitis C virus genotype 1 when treated with boceprevir (boc) plus peginterferon alfa-2b/ribavirin (pr). J. Hepatol. 54, S190. Rubbia-Brandt, L., Leandro, G., Spahr, L., Giostra, E., Quadri, R., Malé, P.J., Negro, F., 2001. Liver steatosis in chronic hepatitis C: a morphological sign suggesting infection with HCV genotype 3. Histopathology 39, 119–124. http://dx.doi.org/10.1046/j. 1365-2559.2001.01208.x (PMID: 11493327). Sharara, A.I., Ramia, S., Ramlawi, F., Eid Fares, J., Klayme, S., Naman, R., 2007. Genotypes of hepatitis C virus (HCV) among positive Lebanese patients: comparison of data with that from other middle eastern countries. Epidemiol. Infect. 135 (3), 427–432 (Apr). Shepard, C.W., Finelli, L., Alter, M.J., 2005. Global epidemiology of hepatitis C virus infection. Lancet Infect. Dis. 5, 558–567. http://dx.doi.org/10.1016/S1473-3099(05)70216-4 (PMID: 16122679).
F. Abbas et al. / Gene Reports 4 (2016) 190–193 Sherman, K.E., Flamm, S.L., Afdhal, N.H., Nelson, D.R., Sulkowski, M.S., Everson, G.T., Fried, M.W., Adler, M., Reesink, H.W., Martin, M., Sankoh, A.J., Adda, N., Kauffman, R.S., George, S., Wright, C.I., Poordad, F., 2011. Response-guided telaprevir combination treatment for hepatitis C virus infection. N. Engl. J. Med. 365, 1014–1024. http://dx. doi.org/10.1056/NEJMoa1014463 (PMID: 21916639). Simmonds, P., Holmes, E.C., Cha, T.A., Chan, S.W., McOmish, F., Irvine, B., Beall, E., et al., 1993. Classification of hepatitis C virus into six major genoptypes and a se-
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ries of subtypes by phylogenetic analysis of the NS-5 region. J. Gen. Virol. 74, 2391–2399. Sy, T., Jamal, M.M., 2006. Epidemiology of hepatitis C virus (HCV) infection. Int. J. Med. Sci. 3, 41–46. http://dx.doi.org/10.7150/ijms.3.41 (PMID: 16614741). WHO, 2003. Global alert and response (GAR). Letter: Hepatitis C Virus (Available from: URL: http://www.who.int/csr/disease/hepatitis/whocdscsrlyo2003/en/index4.html).
Contents lists available at ScienceDirect
Gene Reports journal homepage: www.elsevier.com/locate/genrep
Genotype distribution of hepatitis C virus among Lebanese patients: Results from a major tertiary care center Fatmeh Abbas, Joseph Haddad, Rami Mahfouz ⁎ Department of Pathology and Laboratory Medicine, American University of Beirut Medical Center, Beirut, Lebanon
a r t i c l e
i n f o
Article history: Received 14 March 2016 Received in revised form 16 May 2016 Accepted 5 June 2016 Available online 16 June 2016 Keywords: Hepatitis C Virus Genotype Lebanon
a b s t r a c t Aims: The determination of hepatitis C virus (HCV) genotype is a crucial element in the therapeutic management of HCV infected patients. Studies showed that different treatment responses were obtained for different genotypes. The aim of this research study is to assess the distribution of hepatitis C virus genotypes in chronic HCV patients from a major tertiary care center in Lebanon. Materials and methods: For all HCV patients referred for viral load testing at the American University of Beirut Medical Center, we reviewed the HCV genotyping results for 203 patients positive for HCV RNA using the Linear Array Genotyping kit. Results: HCV genotypes were distributed as follows: Genotype 1 (39.6 42.86%), Genotype 3 (27.6 26.11%), Genotype 4 (26.0 24.63%), Genotype 2 (3.6 3.45%), and Genotype 5 (3.1 2.96%). Genotype 6 was not detected in any case from our patient population. Conclusion: Unlike previous studies in the country, our study interestingly showed a shift in viral genotype from Genotype 4 as the most predominant few years back to a current Genotype 1 predominance. Future epidemiological investigations, especially in light of these interesting results which differ from other populations in the Middle East region and show HCV viral profile changes, are warranted. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Since its discovery in 1989, hepatitis C virus (HCV) has been considered a global public health problem that affects about 3% of the world's population (about 185 million individuals worldwide) (Shepard et al., 2005; Mohd Hanafiah et al., 2013) and 3.4 to 4.4 million individuals in the United States (NIH consensus, 2002; Armstrong et al., 2006; Chak et al., 2011). In most European countries, the prevalence of HCV in the general population ranges from 0.5% to 2% (about 5 to 10 million infected individuals) (WHO, 2003). Studies in the Middle East showed that Genotype 4 predominates in the Arab countries (with the exception of Jordan), while Genotype 1 predominates in the non-Arab countries (Poustchi et al., 2010). For example, a recent study showed that the most predominant HCV genotype in East Turkey is Genotype 1b (İba Yilmaz et al., 2015). In addition, a study from Iran showed a high
Abbreviations: HCV, hepatitis C virus; RNA, Ribonucleic Acid; RT-PCR, Reversetranscriptase Polymerase Chain Reaction; DNA, Deoxyribonucleic Acid; IFN, interferon; RBV, ribavirin; PEG, pegylated; DAA, direct-acting antiviral; SVR, sustained viral response. ⁎ Corresponding author at: Molecular Diagnostics Laboratory, Department of Pathology and Laboratory Medicine, American University of Beirut Medical Center, Cairo Street, Beirut, Lebanon. E-mail address: [email protected] (R. Mahfouz).
http://dx.doi.org/10.1016/j.genrep.2016.06.002 2452-0144/© 2016 Elsevier Inc. All rights reserved.
prevalence for the HCV Genotype 1 (Liakina et al., 2015). However, another study in Libya reported a scattered HCV genotype prevalence that varied by geographical location, demographic, and risk factors (Daw et al., 2015). HCV can cause chronic hepatitis and is recognized to be a leading cause of end-stage liver disease with high progression to cirrhosis and hepatocellular carcinoma (Poynard et al., 2000; Kim, 2002; WHO, 2003). Approximately 80% of acutely infected HCV patients progress to chronic infection, 20% of whom develop cirrhosis within 25 years, with 25% of patients with a cirrhosis developing hepatocellular carcinoma (Freeman et al., 2001; Reddy et al., 2011). HCV is the primary cause of liver transplantation in the United States (Davis et al., 2010). Sequence heterogeneity in the viral genome of the hepatitis C virus was described. Okamoto et al. (1992) classified HCV into four different genotypes while Simmonds et al. (1993) classified it into six major types and a series of subtypes. The knowledge of the genotypes in chronic hepatitis C is a crucial element to be identified since treatment response varies among the different genotypes and subsequently therapeutic management options change according to the genotype, thus different treatments, treatment duration, and therapy dose are required in patient care (Hnatyszyn, 2005). The purpose of this study is to describe the distribution pattern of HCV genotypes in chronic hepatitis patients in a major tertiary care center in Lebanon.
F. Abbas et al. / Gene Reports 4 (2016) 190–193
191
The demographic data, mainly pertaining to age and gender, is shown in Table 2.
2. Materials and methods 2.1. Samples
4. Discussion This Institutional Review Board-approved study was performed at the American University of Beirut Medical Center (AUBMC), which is a major tertiary care center in Lebanon accommodating patients from the different districts and communities of the country. It is a retrospective analysis of HCV genotype testing performed in the Molecular Diagnostics Laboratory at AUBMC where we reviewed the HCV genotypes of 203 samples positive for HCV RNA in order to determine the distribution of genotypes among all patients. These patients have been referred to our center from all districts and ethnic communities of the country; however, no clinical data is available regarding the mode of transmission as it is not within the scope of this research. 2.2. HCV quantification and HCV genotyping Samples were tested for the presence of HCV RNA and its quantification by polymerase chain reaction in real time (RT-PCR) using Cobas Ampliprep/Taqman 48 platform (Roche Diagnostics GmbH, Germany). Then, HCV genotyping was performed using the Linear Array HCV Genotyping Test (Roche Diagnostics GmbH, Germany) and the Linear Array Detection Kit (Roche Diagnostics GmbH, Germany). The test utilizes reverse transcription of target RNA to generate complementary DNA (cDNA), amplification of target cDNA by the Polymerase Chain Reaction (PCR), and nucleic acid hybridization for the genotyping of HCV RNA in human serum or EDTA plasma. The Linear Array HCV Genotyping Test is based on five major processes: Specimen preparation; reverse transcription of the target RNA to generate complementary DNA (cDNA); PCR amplification of target cDNA using HCV specific complementary primers; hybridization of the amplified products to oligonucleotide probes specific to the target(s); and detection of the probe-bound amplified products by colorimetric determination. 2.3. Interpretation of results The Linear Array HCV Genotyping Strip is read visually by comparing the pattern of blue bands to a reference table of genotype patterns (as provided by the manufacturer). 2.4. Statistical analysis The HCV genotyping results of 203 patients were collected and the prevalence of the different genotypes was determined by direct counting methods. 3. Results Using the Linear Array Genotyping test, the distribution of different HCV genotypes was determined. It was found that HCV Genotypes 1, 3, and 4 are the most prevalent (39.6 42.86%, 27.6% 26.11, and 24.63%, respectively) and Genotypes 2 and 5 are less frequent (3.45% and 2.96%, respectively), however, Genotype 6 was not found (Table 1).
Hepatitis C virus genotypes differ in three major properties including their prevalence, pathogenicity, and response to treatment. According to the international literature, the prevalence of certain HCV genotypes is frequently associated with certain geographical areas. For example, HCV Genotype 1 is prevalent in North America and Japan, Genotype 3 is most common in the Indian subcontinent, Genotype 4 is most common in Africa and the Middle East, while Genotype 5 can be found in South Africa and Genotype 6 in Southeast Asia (Sy and Jamal, 2006). The countries located in the region closest to Lebanon (Arab and nonArab countries) showed a predominant prevalence of HCV Genotype 1 and 4. In Turkey and Iran, HCV Genotype 1b and 1 were the most dominant, respectively; while many Arab countries (Egypt, Saudi Arabia…) recorded a high prevalence of HCV Genotype 4 (İba Yilmaz et al., 2015; Liakina et al., 2015; Poustchi et al., 2010). Previous studies about the prevalence of HCV genotype in Lebanon showed that Genotype 4 was found to be the predominant genotype among 142 HCV-infected Lebanese patients followed by Genotype 1, these patients included 27 patients with β-thalassemia major or intermedia, 30 patients on haemodialysis, 32 multi-transfused patients and 15 IDUs (Sharara et al., 2007). The same distribution pattern was seen in a study among 395 thalassemics (Ramia et al., 2002). Another study conducted among 106 intravenous drug users (IDUs) in Lebanon found that Genotype 3 was the predominant HCV genotype followed by Genotypes 1 and 4, a situation similar to that among IDUs in Western Europe (Mahfoud et al., 2010). As shown in Table 3, the genotypic distribution is variable among the different studies so far conducted in Lebanon. Although the study by Mahfoud et al., is showing a predominance of Genotype 3, this only refers to the IDUs subgroup representing only 28 positive HCV-RNA in IDU cases. On the other hand, the study by Sharara et al., shows a wider variation of the predominant genotype among the different categories of patients included. Another hospital-based study showed a significantly different HCV genotype prevalence among dialysis and non-dialysis patients (a total of 77 infected cases). In the dialysis group (27 hemodialysis patients), Genotype 2 was predominant (80%, p b 0.001) while Genotype 1 (subtype 1b) was frequently detected in nondialysis cases (34.4%) whereas Genotype 1 was found in only 5% of dialysis cases (Irani-Hakime et al., 2003). Our study showed that HCV Genotypes 1, 3, and 4 are the most prevalent (42.86%, 26.11%, and 24.63%). This was surprising and in contrast with earlier findings where Genotype 4 was the predominant genotype (47.5%) in Lebanese patients (Ramia et al., 2002; Sharara et al., 2007). This might be due to the fact that this study was done among different groups of HCV-positive patients referred from all Lebanon, representing, thus more of a population-based sample. The pathogenicity of HCV infection also varies according to the different genotypes. For example, while HCV Genotype 3 infection is Table 2 Demographic data of HCV genotypes among the 203 hepatitis C patients.
Gender Table 1 Distribution of HCV genotypes among the 192 hepatitis C patients.
Age group
Genotype
# of patients
Frequency (%)
1 2 3 4 5 6 Total
87 7 53 50 6 0 203
42.86 3.45 26.11 24.63 2.96 0.00 100
Female Male 0–10 11–20 21–30 31–40 41–50 51–60 61–65 N65 Missing Total
# of patients
Frequency (%)
47 156 2 12 57 29 37 36 6 17 7 203
23.2 76.8 1.0 5.9 28.1 14.3 18.2 17.7 3.0 8.4 3.4 100.0
192
F. Abbas et al. / Gene Reports 4 (2016) 190–193
Table 3 Comparison of Genotype frequencies between different studies performed among Lebanese patients. Genotype
1 2 3 4 5 6 Mixed genotype (1/3) Total
Sharara et al. (2007)
Mahfoud et al. (2010)
Abbas et al. (this study)
Number
Number
Number
Frequency (%)
Frequency (%)
60 7 9 65 1 0 0
42.25 4.93 6.34 45.77 0.70 0.00 0.00
6 0 16 5 0 0 1
21 0 57 18 0 0 4
87 7 53 50 6 0 0
142
100.00
28
100
203
Frequency (%) 42.86 3.45 26.11 24.63 2.96 0 0
100
associated with a higher degree of liver steatosis (Rubbia-Brandt et al., 2001), Genotype 1 is associated with a higher risk of hepatocellular carcinoma development (Bruno et al., 2007). The response rates to interferon (IFN)-based therapy vary significantly between the different HCV genotypes with IFN representing the backbone of hepatitis C therapeutic options. Pegylated-IFN and ribavirin (PEG-IFN/ RBV) combination therapy is the standard of care for the treatment of chronic hepatitis C infection of different HCV genotypes. Recently, new direct-acting antivirals (DAAs) have been introduced to the treatment of HCV Genotype 1 infection where the genotype is an important determinant of both treatment strategy and outcome (Sherman et al., 2011). Treatment efficacy is measured by the sustained virologic response which is the treatment goal of undetectable plasma HCV RNA 12 or 24 weeks after therapy completion (Kohli et al., 2014). A systematic review of treatment of Hepatitis C showed that hepatitis C virus Genotype 1 is more difficult to cure than Genotype 2 or Genotype 3. Patients with HCV Genotype 1 should receive treatment with sofosbuvir, pegylated interferon, and ribavirin because of the shorter duration of therapy and high rates of sustained viral response (SVR, 89%–90%). Simeprevir with pegylated interferon and ribavirin is an alternative for patients with HCV Genotype 1 (SVR, 79%–86%). Patients with HCV Genotypes 2 and 3, representing 20% to 29% of HCV infections in the United States, should receive therapy with sofosbuvir and ribavirin alone (SVR for Genotype 2, 12 weeks' duration: 82%–93%; SVR for Genotype 3, 24 weeks' duration, 80%–95%) (Kohli et al., 2014). These differences in the SVR rates observed among different HCV genotypes have suggested that viral genome variability could play a role in determining treatment outcome (Pang et al., 2009). In non-transplant patients with chronic hepatitis C virus infection, HCV genotype has been linked to a differential response to antiviral therapy, risk of steatosis, fibrosis and all-cause mortality and correlated with outcomes in post-liver transplant patients (Campos-Varela et al., 2014). In post–liver transplant, Genotype 1 has the highest risk of advanced fibrosis and the lowest rate of SVR while compared to Genotypes 2 and 3 (Lai et al., 2011). The determination of HCV genotype is a crucial element in the therapeutic management of HCV infected patients and for post-transplant recipients. Treatment regimen, optimal dose, and treatment duration are guided by the determined genotype of HCV infection. In light of this information, the interesting data showing in this report should be alarming for investigators and clinicians in terms of the higher prevalence of Genotype 1 in our patients' population, while Genotype 4 has been previously reported to be the most prevalent in the Middle East region. This has major implications for clinical outcome, therapy response, as well as for potential vaccine development. Accumulated data show that there are different patterns for HCV genotype prevalence and our study showed a new prevalence pattern with predominance of Genotype 1. Future clinical studies revealing the modes of transmission,
like blood transfusions and injecting drug users, may be needed to better reflect on the distribution of the HCV RNA positive cases and assess if it is a genuine change in genotype as opposed to, probably, a different sample population in question at large.
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