Hepatitis B virus (HBV) affects more than 2 billion people worldwide and approximately 247 million chronic individuals are known to be chronic carriers (Caligiuri et al. 2016; WHO 2018). HBV can cause severe liver infections and cirrhosis may develop in 15–40% of individuals if they are not treated (Tang et al. 2018). There are several novel treatment strategies that have been used for the treatment of chronic hepatitis B infection in order to prevent the risk of developing liver failure, cirrhosis, and cancer (Lapiński et al. 2013). Interferon alpha (IFN-α) 2a- 2b, PEGylated interferon-α-2a (
HBV is characterized by its high replication capacity (> 1012 virion/day) and a lack of proofreading activity during replication (10−5 substitution/base/cycle). This means that each nucleotide on the HBV genome can be changed within a day and cause antiviral resistance-related mutations before treatment (Tezcan et al. 2015). Also, the S gene is completely overlapped by the
Cyprus is an island located in the Mediterranean Sea and has been divided into two communities (Northern and Southern Cyprus). Northern Cyprus, officially the Turkish Republic of Northern Cyprus, is a multinational society due to universities, casinos, luxury hotels, beaches, and other entertainment centers; thus, there are many people coming from foreign countries for education, business and/or tourism purposes. For HBV infection, the prevalence rate ranges from 1.2% (160/13 892) (Arikan et al. 2016) to 1.35% (339/25 442) (Guler et al. 2018). For Southern Cyprus, this rate was given as 0.77% and 1.01% (Altindis et al. 2016). Although there are few studies regarding the prevalence, up to now, there has been no publication on drug resistance in patients infected with HBV in Northern Cyprus (Altindis et al. 2006; Suer et al. 2014). Therefore, we aimed to show the presence of resistance mutations and their clinical significance in untreated chronic hepatitis B patients in Northern Cyprus.
In our study, the samples of 100 patients with HBsAg positive who had never been treated with NAs or IFN, were involved retrospectively. The study group consisted of 13 892 people who applied to the Near East University Microbiology Laboratory for assessment of hepatitis markers during three years. Of these individuals, 100 samples with positive HBsAg were included in the study for drug resistance analysis. The levels of viral markers [HBsAg, anti-hepatitis B core antigen (anti-HBc), anti-hepatitis B e antigen (anti-HBe), hepatitis B e antigen (HBeAg), alanine aminotransferase (ALT), and aspartate aminotransferase (AST)] were determined by using the chemiluminescent enzyme immunoassay kits according to the manufacturer instructions [(Architect i200, Abbott, USA), (Roche, Cobas E411), (Olympus AU680, Beckmann Coulter IFCC)]. HBsAg positive sera were stored at −80°C until use. The ethical approval of the study was taken from Near East University Scientific Researches Evaluation Ethics Committee (YDUBADEK, 20/06/2013 date and NEU/2013/16-88 decision number).
The viral loads of positive samples were determined by using real-time polymerase chain reaction (PCR) technique according to the manufacturer instructions (artus HBV QS RGQ Qiagen, Hilden, Germany). The HBV
The current study consisted of HBsAg positive-treatment naïve 100 CHB patients, of whom 13 were female and 87 were male, whose ages varied between 18–65 ages (with a median of 35 years). The origins of the patients were from Asia (68%), Africa (29%), North America (1%) and Europe (2%), and none of them have been taken antiviral therapy when their serum samples were collected. Ninety-six percent and 5% of the study group were positive for anti-HBc IgG and HBeAg, respectively. The serum HBV DNA level was calculated as a median of 1.0 + E7 (range: 1.9 + E1 – 2.8 + E8). ALT and AST levels were reported as 23 ± 19 and 28 ± 19, respectively. We sequenced only the samples of 68 patients in this study because HBV DNA was determined below 1000 IU/ml in 32 (32%) patients’ samples. The distribution of genotypes of the sequenced patients (68/100) was determined as D1 (n:48, 70.6%), D2 (n:4, 5.9%), D3 (n:1, 1.5%), A1(n:5, 7.3%), A2 (n:2, 2.9%), and E (n:8, 11.8%) (Arikan et al. 2016). Demographic and laboratory findings of the study are displayed in Table II.
Demographic and clinical characteristics of the study group.
Variable | Study Group |
---|---|
Patient, n | 100 |
Gender, F/M, n (%) | 13 (13%)/87 (87%) |
Age, median year (range) | 35 (18-65) |
Nationality, region/country, n (%) | |
| |
Turkey | 43 (63) |
Northern Cyprus | 14 (21) |
Pakistan | 3 (4) |
China | 3 (4) |
Turkmenistan | 3 (4) |
Syria | 1 (2) |
Georgia | 1 (2) |
| |
Nigeria | 28 (97) |
Benguela | 1 (3) |
| |
Mexican | 1 (100) |
| |
Azerbaijan | 1 (50) |
Bulgaria | 1 (50) |
Anti HBc IgG positivity, n (%) | 96 (96) |
HBeAg positivity, n (%) | 5 (5) |
ALT (average ± SD) (U/L) | 23 ± 19 |
AST (average ± SD) (U/L) | 28 ± 19 |
HBV DNA median IU/ml (range) | 1.0 + E7 |
Genotype/subgenotype of HBV, n (%) | |
D1 | 48 (70.6) |
D2 | 4 (5.9) |
D3 | 1 (1.5) |
A1 | 5 (7.3) |
A2 | 2 (2.9) |
Treatment status, n (%) | |
Naive | 100 (100%) |
Under treatment | - |
F - female, M - male, ALT - alanine aminotransferase, AST - aspartate aminotransferase, SD - standard deviation
Mutation characteristics of the HBV
HBV virion | Mutation characteristic | Mutation pattern | Nucleos(t)ide analogues | n (%) |
---|---|---|---|---|
Primary resistance mutation | ND | ND | - | |
Compensatory mutation | rtL91I | LdT related | 25 (36.8) |
ND - not determined, LAM - lamivudine, ADV - adevofir, LdT - telbivudine
In the
In the
Typical HBsAg escape and combined mutations of the study patients (n = 68).
Typical HBsAg escape mutation | Mutation patterns | n (%) | Combined mutations | Mutation patterns | n (%) |
---|---|---|---|---|---|
Immune escape | sY100C, sI110L, sP120L/R, sTl23N, sT127L, sP127T, sA128V, sT131N, sS132P, sY134F/H, sT140I/S, sS143T, sD144E, sS144T, sP210S | 16 (24) | Immune response -vaccine | sI110L + sS193L | 1 (1.5) |
Vaccine escape | sT126I, sD144A/E, sG145A/R, S193L, sP210T | 7 (10) | Vaccine-HBIg | sP120L + sT123N + sT126I + sA128V + sY134H + sD144E + sG145A | 1 (1.5) |
HBIg* escape | sT118A, sP120T, sD144A/E, sG145A/E/R | 4 (6) | Immune response -HBIg | sT118A + sP127T | 1 (1.5) |
Diagnostic escape | sT118A, sT131I, sP120T, sC121Y, sD144A, sG145R | 3 (4) | Immune response-vaccine-HBIg | sI110L + sP120T + sD144A + sG145R sP120R + sC121Y, sT131I + sS132P | 1 (1.5) |
Total | 30 (44) | 6 (9) |
HBIg - Hepatitis B immunoglobulin
Patients may have more than one mutation pattern
There were also six different combined mutations. The mutation patterns were sI110L + sS193L, sP120L + + sT123N + sT126I + sA128V + sY134H + sD144E + +sG145A, sT118A+sP127T, sI110L+sP120T+sD144A+ +sG145R and sP120R+sC121Y+sT131I + sS132P. The resistance mutation patterns are given in Table III.
Even in treatment naïve patients because of the high replication capacity and the overlapping reading frames, antiviral resistance mutations may occur throughout the HBV genome (Yano et al. 2015). To our knowledge, this is the first study that focused on the resistance mutations in the HBV
In the current study, we detected rtV173M partial resistance mutation in one patient according to Geno2pheno Drug Resistance Program, however, the rtV173L substitution has been reported more commonly in the literature (Lin et al. 2012; Gürsoy et al. 2019). Both amino acid substitutions are regarded as compensatory mutations and enhance viral fitness (Asan et al. 2018). The single compensatory mutation profiles are generally associated with low-level resistance; however, they may cause high-level resistance when they combine with other patterns (Lazarevic 2014). Thus, screening to detect resistance may be regarded to be a benefit in the control of CHB. Sayan has shown in one of his studies that rtV173L mutation combined with sD144E produced HBV vaccine escape +HBIG escape (Sayan and Bugdaci 2013). LAM-associated resistance triple mutation pattern (rtV173L + rtL180M + rtM204V) has also been shown to enhance viral replication compared with rtL180M + rtM204V (Sheldon and Soriano 2008).
In the current study, among 68 patients, there were 25 (37%) secondary/compensatory resistance mutations (rtL91I, rtQ149K, rtQ215H/P/S, rtN238D) which are related to LdT, LAM, and ADV resistance (Saran et al. 2017; Asan et al. 2018). It may be a result of naturally emerging mutations due to the biology of HBV. The most common compensatory mutations have been reported to be rtQ149K, rtQ215H/P/S and rtL91I (Asan et al. 2018). These compensatory mutations are important as they assist viral replication and fitness, and hence are associated with drug resistance (Ahn 2015). In our study, we determined one of the most commonly detectable compensatory mutation rtQ215/H/P/S, which is associated with LAM and ADV resistance (Shaw et al. 2006; Altindis 2016; Asan et al. 2018).
Mutations in the
In conclusion, these findings on drug resistance mutations show that treatment-naïve CHB patients have low HBV polymerase resistance mutation (1.5%) rate. Hence, primary drug resistance analysis may not be necessary to be performed before the initiation of antintiviral therapy; however, we have discovered a novel compensatory mutation, rt173M that may act as a primary mutation together with other pattern