Quantitative HBV-DNA and determination of drug- resistant mutants in patients with chronic hepatitis B virus (HBV) in Hai Duong province

JOURNAL OF SCIENCE OF HNUE Chemical and Biological Sci., 2014, Vol. 59, No. 9, pp. 139-147 This paper is available online at QUANTITATIVE HBV-DNA AND DETERMINATION OF DRUG- RESISTANT MUTANTS IN PATIENTS WITH CHRONIC HEPATITIS B VIRUS (HBV) IN HAI DUONG PROVINCE Le Thi Phuong Hai Duong Medical Technical University Abstract. The purposes of the paper are i) To survey quantitative HBV-DNA in patients with HBsAg + (Hepatitis B surface antigen positive) with antiretroviral drugs and ii) To determine drug resistant mutants in the Pre-S and RT regions in patients with quantitative HBV-DNA > 104 copies/mL 6 months after treatment. 195 patients with chronic hepatitis were treated with antiretroviral drugs, periodically examined and tested for biochemical indices; quantitative HBV-DNA was carried out 6 months before treatment and every 3 months after treatment. After being treated for 6 months, patients with quantitative HBV-DNA > 104 copies/mL would be determined to have drug resistant mutants in the RT and Pre-S regions. Group of patients with quantitative HBV-DNA levels > 106 copies/mL decreased from 67.7% (pre-treatment) to 4.6% after 3 months of treatment and 8.7% after 6 months of treatment; group of patients with quantitative HBV DNA positive under detection increased from 0% (pre-treatment) to 15.9% after 3 months of treatment and 78.5% after 6 months of treatment; 17/195 patients with quantitative HBV-DNA decreased but possibly flared up again. These samples carried mutations in the RT and Pre-S regions. The result showed that 9/17 specimens had mutations in the region RT. There were no mutations in Pre-S. Most of the patients were in serious disease condititon before treatment. This resulted in 67.7% of patients with quantitative HBV-DNA > 106 copies/mL. Keywords: Quantitative, HbeAg, HbsAg, HBV-DNA, real time PCR, chronic hepatitis. 1. Introduction The World Health Organization (WHO) estimates that over 350 and 250 million people worldwide are chronic carrier of hepatitis B virus (HBV) and hepatitis C virus (HCV) infection, respectively [1]. In Vietnam, the high rate of chronic HBV is due to HBV infection. It’s estimated that about 20% of the Vietnamese population has HBV. Among Received November 29, 2014. Accepted December 22, 2014. Contact Le Thi Phuong, e-mail address: phuongsinh@ymail.com 139 Le Thi Phuong them, about four to five million people have cirrhosis of the liver or liver cancer. Around 25 - 45% of those with chronic HBV are at risk of an early death [7]. In the past, clinicians diagnosed and treated people who showed clinical symptoms, doing biochemical indices tests or immune tests, however, results were not satisfactory because patients were affected by acute retroviral syndrome (ARS) HBV, the patients had a weakened immune system or the patients had types of mutant serums of hepatitis B virus. On the other hand, the diagnosis did not obtain quantitative results and it’s necessary to monitor and evaluate the efficacy of treatment for chronic HBV patients by determining the exact concentration of the virus in patients. Iloeje et al. [4] reported that if the HBV-DNA level was always at 106 copies/mL, 36.2% of the HBV patients would progress to cirrhosis of the liver after 10 years. If the HBV-DNA level decreased < 300 copies/mL, only 4.5% of patients would later have cirrhosis of the liver. The progression to cirrhosis in patients with chronic hepatitis B correlated strongly with the plasma level of HBV-DNA in blood, and it was independent of HBeAg status and level of ALT serum. Chen CJ et al. reported that that if HBV-DNA level was106 copies/mL, 14.9% of HBV patients would progress to HCC after 10 years. If the HBV-DNA level decreased < 3.102 copies/mL, only 1.3% of the patients would progress to HCC. An increase in the level of HBV-DNA serum ( 104 copies/mL) would be a strong risk predictor for HCC, independent of HbeAg status, the level of ALT serum and cirrhosis of the liver [2]. A real-time PCR technique has been applied to diagnose HBV which allows accurate quantification of HBV-DNA in the blood of patients and helps clinicians devise a reasonable treatment regimen with an adequate dose and patients with chronic hepatitis time and money [10]. 2. Content 2.1. Subjects and methods * Subjects We examined and treated 195 patients with chronic hepatitis B virus from December 2013 to August 2014 in Hai Duong Provincial General Hospital. They were aged 15 and older with the standards: HBsAg (+) over 6 months; Anti HBc (Hepatitis B core) IgG (+); Transaminases (AST, ALT- Alanine aminotransferase) increased more than twice the normal value; quantitative HBV-DNA > 104 copies/mL. * Methods Prospective study. Patients were monitored for treatment. They were periodically examined and tested to measure biochemical indices and quantitative HBV-DNA before treatment and every 3 months and 6 months. - Transaminase:As recommended by the IFCC (International Federation of Clinical Chemistry), normal values are AST: 0 - 40 U/L and ALT: 0 - 40 U/L. - Quantitative HBV-DNA: HBV-DNA samples extracted using a DNA extraction 140 Quantitative HBV-DNA and determination of drug-resistant mutants... kit of the Viet A Technology Joint Stock Company. After DNA extraction, it was stored at -20 C until the PCR was done. HBV-DNA was quantified in real time using PCR techniques on Stratagene real-time systems (Germany) using a TaqMan Probe and specific primer sets. Sequence of primers used for Real-time PCR [8]: + Forward primer: HBV-1 3’. . . CAA GGT ATG TTG CCC GTT TG. . . 5’ + Reverse primer: HBV-2 3’. . . AAA GCC CTG CGA ACC ACT GA. . . 5’ The temperature program of the Real time PCR cycle was 95 C for 5 min, (95 C for 15 s, 60 C for 1 min)  40 cycles. The real-time PCR results (HBV copies/mL of blood) are multiplied by 50. - Determination of drug-resistant mutants in the region Pre-S and RT In patients who used antiretroviral treatment for 6 months, quantitative HBV-DNA did not increase by 2 logs or, if HBVDNA did flare up, it was gene sequenced in the region Pre-S and RT to determine drug-resistant mutants. The results were compared with the standard sequences in Genbank. 2.2. Results and discussion 2.2.1. Tests before treatment in patients with chronic hepatitis B virus * Activity of transminase We tested transminase on patients with chronic hepatitis B virus 6 months before treatment and every 3 months after treatment. Table 1. Average activity of Transminase in chronic hepatitis B Transaminase Average activity AST 181.2  189.75 ALT 219.6  172.65 Transaminase increased 5 - 6 times comparing with the average value in patients with chronic hepatitis B virus. De Ritis rate (AST/ALT) < 1. This result is consistent with the study of Nguyen Thi Thu Oanh and Song LH [7, 8]. * Quantitative HBV-DNA Table 2. Results of quatitative HBV DNA for patients with chronic hepatitis B virus HBV-DNA (copies/mL) Numbers (n = 195) Rates (%) < 104 15 7.7 104 - 106 48 24.6 > 106 132 67.7 15 of the 195 patients with chronic hepatitis B virus were monitored (number of copies/mL < 10 ‘4’) rather than being givenantiretroviral drug treatment. Number of 141 Le Thi Phuong patients/mL > 106 was 67.7%. This showed that most patients were examined and treated at a late stage. This result is consistent with the study of Shao J. with 213 Chinese patients with chronic hepatitis B virus containing high quantitative HBV-DNA, on average of 6.6.  109 copies/mL [8]. Detected level: 3102 copies/mL Figure 1. Results of real-time PCR pre-treatment for patients with chronic hepatitis B virus * The relevance of quantitative HBV-DNA with sub-clinical characters Table 3. The relevant results of HBV-DNA with HBeAg in patients with chronic HBV HBV-DNA HBeAg (+); (n = 87) HBeAg (-); (n = 108) (copies/mL) n % n % <104 5 5,7 14 13.0 104 - 106 7 8,1 35 32.4 >106 75 86,2 59 54.6 P 2 = 115,58; p = 0.001 2 = 121,59; p = 0.01 75 patients were positive for HbeAg in the group of quantitative HBVDNA > 106 copies/mL (86.2%). It was found that 54.6% of the patients of quantitative HBV DNA > 106 copies/mL in the negative HbeAg group and 32.4% had a concentration of 104 - 106 copies/mL. This result is consistent with the study of Shao J. [4]. 2.2.2. Verification testing after 3 months and 6 months of treatment 195 patients with quantitative HBV DNA  104 copies/mL were monitored and periodically tested every 3 months and 6 months after treatment. 142 Quantitative HBV-DNA and determination of drug-resistant mutants... * Results after 3 months of treatment Table 4. Results of transaminase activity change after 3 months treatment Transaminase AST ALTn % n % Normal limits 109 55.9 98 50.2 Above normal limits 86 44.1 97 49.8 Average activity 44.7  21.64 52.7  37.96 The rate of ALT normalization of 195 patients after 3 months of treatment was 50.2% or 98 patients. Average transaminase activity of patients with chronic HBV after 3 months of treatment dropped 4 - 5 times. HBV-DNA change Detected level: 3102 copies/mL Figure 2. The results of real-time PCR after 3 months of treatment in patients with chronic hepatitis Table 5. Results of HBV-DNA change after 3 months of treatment HBV - DNA (copies/mL) Numbers (n = 195) Rates (%) Non - decrease 9 4.6 Decrease < 102 15 7.7 Decrease  102 140 71.8 Decrease under detective (< 3102) 31 15.9 143 Le Thi Phuong 140/195 patients (71.8%) with quantitative HBV-DNA decreased 102 copies/mL, including 31 patients (15.9%) with a decrease under the detectable level (< 3102 copies/mL) after 3 months of treatment with antiretroviral drugs. Change from HBeAg (+) to HBeAg (-): Table 6 shows the chane in the 195 patients that had HBV-DNA 104 copies/mL, including 87 patients (44.6%) with HBeAg (+). After 3 months of treatment, 25/87 patients (22.2%) changed from HBeAg (+) to HBeAg (-). Table 6. Results of change from HBeAg(+) to HBeAg(-) HBeAg Before treatment After 3 months of treatment n % n % HBeAg(+) 87 44.6 62 31.8 HBeAg(-) 108 55.4 133 68.2 Thus, the sub-clinical tests improved significantly after 3 months of treatment. The average transaminase activity of patients with chronic HBV, after 3 months of treatment, reduced 4 - 5 times compared with the upper limit of normal, and we did not detect the presence of ALT after 3 months of treament; 140/195 patients (71.7%) with HBV-DNA concentration decreased 102 copies/mL after 3 months of treatment, 31 patients (15.9%) decreased under the detectable level; the rates of patients with HBeAg (+) decreased from 100% (87 patients) to 71.2% (62 patients) after 3 months of treatment. This result suggests that our treatment regimen is completely feasible. * Results after 6 months of treatment Transaminase activity: Table 7. The results of transaminase activity change after 6 months of treatment Transaminase AST ALT Numbers (n) Rates (%) Numbers (n) Rates (%) Normal limits 158 81.0 132 67.7 Above normal limits 16 19.0 63 32.3 Average activity 36.5  14.1 38.2  27.52 The rate of normalization of liver enzymes (ALT) increased after 6 months of treatment in 132 patients (67.7%) comparing with 50.2% (after 3 months of treatment). Normal transaminase activity in patients with chronic HVB after 6 months of treatment was in the normal limits (4 - 40 U/L). This result is consistent with the study of Yu MW et al. [12]. 144 Quantitative HBV-DNA and determination of drug-resistant mutants... HBV-DNA change: Detected level: 3102 copies/mL Figure 3. Results of real-time PCR in patients with chronic HBV after 6 months of treatment Table 8. Results of HBV-DNA change after 6 months of treatment Quantitative HBVDNA (copies/mL) Numbers (n = 195) Rates (%) Increase 17 8.8 Decrease 27 13.8 Decrease under detective level (< 3102) 151 77.4 There were 151 patients (77.4%) with positive quantitative HBV-DNA under the detectable level (< 3102 copies/mL) after 6 months of treatment, including 17 patients (8.8%) with increasing quantitative HBVDNA during the treatment period. This result is consistent with the study of Xu Xh et al. [11]. Change from HBeAg (+) to HBeAg (-): Table 9. Results of the change from HBeAg(+) to HBeAg(-) after 6 months of treatment HBeAg Before treatment After 3 months of treatment After 6 months of treatment n % n % n % HBeAg(+) 87 44.6 62 31.8 25 12.9 HBeAg(-) 108 55.4 133 68.2 170 87.1 62/87 patients (71.3%) changed from HBeAg (+) to HbeAg (-). This result is a bit higher than the result in the study of Nguyen Thi Thu Oanh with HbeAg (+) which was 145 Le Thi Phuong 45.9%, with HbeAg (-) being 54.1% [7]. The study of Yu MW et al. in Taiwan showed that 150/272 patients (55%) with chronic HBV were positive for HBeAg [12]. The study of Mojiri in northwestern Iran showed that the rate of those testing negative for HbeAg in the study group was 52.7% [6]. Table 10. Summary of quantitative HBV-DNA before and after treatment HBV-DNA Before treatment After 3 months of treatment After 6 months of treatment (copies/mL) n % n % n % >106 132 67.7 9 4.6 17 8.7 104-106 48 24.6 15 7.7 7 3.6 <104 15 7.7 140 71.8 18 9.2 Positive under detective level 0 0 31 15.9 153 78.5 2.3. The results identify drug resistant mutants in the RT gene and pre-S of HBV 17/195 patients with quantitative HBVDNA increased after 6 months of treatment. This suggests that it’s inefficient to use antiretroviral drugs and this could be a sign of the presence of drug-resistant mutants. These specimens were sequenced to drug resistant mutants in the Pre-S and RT regions. 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