Anti-HCV reactive volunteer blood donors distribution character and genotypes switch in Xi'an, China

To determinate the HCV infection rate in local blood donors, ELISA was performed according to the standard donor peripheral blood test procedure. From the January of 2000 to the December of 2009, about 0.45% (1151/273203) blood donors were found with anti-HCV positive reaction. As shown in Fig. 1, the infection rate is higher in the early of the study period. In the year of 2001, 0.81% donors were anti-HCV reactive. From the year of 2004, anti-HCV reactive rate in donors decreased to 0.42% and kept stable in the followed years. In the year of 2010, HCV infection rate is 0.54% hitherto (117/21578, up to 31^st May, 2010).Those data implied that anti-HCV reactive donors decreased in the latest 5 years in local region.

The relative value of sample absorbance and cutoff (S/CO) can somewhat reflect the degree of HCV infection. Thus we divided the 1151 anti-HCV reactive donors into different groups (Fig. 2A, black). More than 50% anti-HCV reactive donors' S/CO values were fewer than 2.0, while about 45% anti-HCV reactive donors' S/CO values were between 2.1 and 4.0. In ALT level measurement, more than 80% anti-HCV reactive donors' value was between 40 IU/L and 120 IU/L (Fig. 2B, black), while only a few donors' value was lower than 40 IU/L or higher than 120 IU/L. The 200 recruited donors for genotyping displayed similar distribution character (Fig. 2A and 2B, blank), which meant that the chosen sample pool, at least in part, could represent the total HCV infected donors in the current study. When HCV viral load detection was performed in the 200 recruited donors, about 61% samples' viral load were between 1 × 10² and 1 × 10³ copies/ml, while 16.3% samples' viral load fell into 1 × 10⁴ copies/ml (Fig. 2C). Altogether, these results suggested that most of the local anti-HCV reactive donors, without syndromes, had a low-grade infection when their blood collected.

To explore the HCV genotypes of local donors in the past 10 years, we chose 20 samples each year to perform genotyping. As shown in Table 1, 135 (67.5%) donors were 1b infection and 31 donors (15.5%) were 2a infection. The donor's age or sex was not associated with HCV infection. To be noted, 2 donors were mixed infection (1b and 2a) and 3 donors' infection genotype was not clarified. Furthermore, we compared 1b and 2a subtypes infection between donors and clinic confirmed patients. Results showed that 1b and 2a subtypes infection was no difference in these two groups (Fig. 3A). These data indicated that most of the local anti-HCV reactive donors infected with 1b and 2a subtypes virus.

Since 1b and 2a HCV virus were the prevailed strains in local infected donors, we are wondering whether the two main genotypes changed in the past years. As shown in Fig. 3, the cases of 1b genotypes decreased in local HCV infected donors, from 16 cases in the year of 2000 to 11 cases in the year of 2009 (Fig. 3B), while the cases of 2a genotypes increased in the last 10 years(Fig. 3C). Other genotypes did not change (data not shown). These results showed that in the past 10 years, the two main genotypes (1b and 2a) in local HCV infected donors underwent a switch, although not so markedly.

To monitor HCV infection progress after the preliminary analysis, we tracked some reactive donors, who donated blood from the year of 2000 to the year of 2007 and were genotyped also as mentioned above, in the following 2 years. These samples were negative also for other hepatitis virus. Consecutive ALT testing showed that the liver function of these donors infected with HCV, at least partly, impaired one year later or so after donation (Table 2). Especially, 1b genotype virus infected donors had a higher ALT level than that of other genotype virus infected donors in the follow up time (Table 2, P < 0.05). Virus load was serially quantitated by real-time PCR in the tracked donors. As shown in Table 3, the number of virus copies increased in all the donors. Once again, 1b genotype virus RNA was higher in the follow up period (P < 0.05). Altogether, these data suggested that 1b genotype infection led to more serious liver function impairment for local donors.

Volunteer blood donors from both urban and rural areas in Xi'an City, from January of 2000 to December of 2009, were recruited into current study. They were medically assessed and denied any known risk factors for viral infection listed in the questionnaire. 200 donors with HCV (20 donors each year), ruled out HBV infection, were asked to give peripheral blood samples for genotyping. The serum were isolated from the samples, then subpackaged and stored at -80°C before analysis. Ten samples from the chosen 200 donors (donated before the December of 2007 and negative for other hepatitis virus) were followed up in the next 2 years. The sampling, isolation and storage procedures were just like the mentioned above. The present study was approved by the Ethics Committee of Fourth Military Medical University and the informed consents were signed.

Test of the anti-HCV antibody were performed by ELISA using automatic enzyme detection system (Tecan GroupLtd., Mannedorf, Switzerland) and commercial kit (InTec Products, Xiamen, China). Briefly, 96-well plates were coated with antigen. Donors' peripheral blood serum was isolated and added into wells by automatic enzyme detection system before incubation. The plates were subsequently washed 5 times with PBST, and then the horseradish peroxidase labeled mono-antibody was added. After incubation, followed the manufacturer's instructions, washed plates and developed colorant to determine the results with absorbance reader (Thermo Scientific, Wohlen, Switzerland).

To measure the ALT level, donors' peripheral blood serum was isolated and employed to automatic biochemistry analyzer (Hitachi, Tokyo, Japan) with commercial Kit (Fousun Long March Medical Ltd., Shanghai, China).

RNA from donor's blood sample was prepared according to the manufacturers' manual (Qiagen, Hilden, Germany). In brief, 500 μl isolated serum was mixed with 500 μl TRIzol regent and extracted with chloroform and alcohol. After quantitation, reverse transcribed into cDNA using random primers was performed (Qiagen, Hilden Germany). After that, real time PCR was used to detect HCV RNA according the manufacture's manual (Daan Gene, Shenzhen, China) with standard controls [3, 26]. Briefly, extracted RNA was measured with fluorescence labeled and self-quench probe and Perkin Elme PCR analyzer (PTC-200, Perkin Elmer, Covina, USA). The viral load used the copies/ml as the units.

Genotyping was accomplished according to reported methods [7, 8, 27]. Briefly, Extracted RNA from the peripheral blood of 200 donors or 100 confirmed patients. Then the reverse transcription with random hexaprimers was carried out. After that, the seminested PCR of the 5' non-coding region with generic primers were performed. Amplicons were digested by restriction enzymes (Takara, Osaka, Japan). The digestion model, possible products and genotype categorization were schemed in Fig. 4.