The impact of nucleic acid testing to detect human immunodeficiency virus, hepatitis C virus, and hepatitis B virus yields from a single blood center in China with 10-years review

Blood transfusion saves millions of lives annually across the globe. Nonetheless, transfusion transmissible infections (TTIs) remain a major problem. The main TTIs include hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), and Treponema pallidum (TP) [1‐3]. Notably, HIV, HBV, and HCV are causative agents of AIDS, hepatitis B, and C infection, respectively. Regardless of the low viral load, the risk of transmitting these viruses through transfusion of infected blood is markedly higher than through other routes [4]. The prevalence of these viral infections among blood donors varies by geography and nationality; it directly hinges on their prevalence in the general population. Based on the global estimates by the WHO (World Health Organization) till 2015, HBV and HCV chronically infected 257 million people and 71 million people, respectively. By the end of 2019, 38 million individuals were newly infected by HIV. Nevertheless, the prevalence of HBV, HCV, and HIV infections among blood donors in different countries and world regions varies from 0.003 to 5.54%, 0.002 to 2.23%, and 0.00 to 1.66%, respectively. For over 10 years, chronic hepatitis B is the leading among 27 infectious diseases reported by the Chinese government. Approximately 50% of the Chinese population has a history of HBV infection, out of which, 7.18% are chronic carriers of hepatitis B surface antigen (HBsAg) [5, 6]. Therefore, HBV is a major threat to blood safety in China.

Of note, advances in molecular screening for TTIs have significantly reduced the risk of infection transmission via blood transfusion. Nucleic acid amplification testing (NAT) is used to diagnose viral infections in transfusion medicine and is mandatory for blood services in China since 2016. The benefits of NAT include the capacity to directly detect viral genomes (DNA or RNA) with high specificity. Its sensitivity is several orders of magnitude greater than that of antigen and/or antibody immunological assays. Besides, NAT has markedly reduced the assay window for immunological assays [7]. In the present study, we assess the results of NAT over 10 years and analyze their effects on blood safety at the Blood Center of Zhejiang Province, China, where the infection rate of HBV is higher than that in the general population.

In China, NAT was first used as a pilot project in key blood centers in 2000 [8, 9], including the Blood Center of Zhejiang Province. Herein, we discovered that NAT yield rates for HBV, HCV, and HIV varied over time and between the seven blood service centers. Specifically, the NAT yield rates for HCV (1.54 per million) and HIV (2.31 per million) in Hangzhou were similar to other regions of China (NAT yield range: 0–3.4 per million for HCV [10], 0–3.55 per million for HIV [11]. In our study, the HCV NAT yield rate (0.97 per million) was lower than that in Mediterranean countries with high endemic HCV infection (2.15 per million in Spain, 5.97 per million in Greece, 2.5 per million in Italy, 4.27 per million in Slovenia) [12‐15]. HIV NAT yield rate (1.45 per million) was similar to that in the US (0.43 per million) [16] and European countries such as Italy (1.8 per million) and Germany (0.43 per million) [15, 17], but lower than that in HIV-1 endemic countries including South Africa (25.56 per million donations) [18].

In follow-up HCV NAT and serological testing, two HCV NAT-yield cases were negative. Nevertheless, all 3 HIV NAT yield donors were in the acute HIV infection phase. Reports indicate that 15–25% of HCV infections are self-limiting and vary depending on the HCV genotype. According to Lefrère et al., a few immunocompetent HCV-positive patients were found to be negative after self-limiting using ELISA, RIBA, and HCV-RNA test [19]. Therefore, we speculated that these two HCV NAT-yield donors may have had self-limiting HCV infection or were false positives upon HCV NAT tests. Moreover, Akuta et al. [20] reported HBeAg-negative and HBeAb-positive cases where chronic HBV infection persisted while acute HCV infection was spontaneously resolved. In this patient, HCV infection was interestingly accompanied by the appearance of PreC wild type (G1896); an increase in transiently suppressed HBV viral load at a level that was higher than that established before HCV infection. This case was similar to the BD2 case in our study, which was negative in HBV NAT and serological tests and HCV NAT reactive, but HCV-RNA was undetectable one year later and HBV-DNA positive. Therefore, NAT tests employing in HCV low risk population have low positive predictive value, results must be repeated to confirm.

HBV NAT yield rate was much higher than that of HCV and HIV, ranging from 883.58 to 2582.83 per million at different blood service centers (1:387 in Jiande to 1:1132 in Hangzhou). This rate was a little higher than the average figure of China (1:1482, range:1:1861 to 1:1269) [21], and much higher than that in other low HBV endemic countries including USA, Canada, Germany, Switzerland, and New Zealand [22‐26], as well as Mediterranean countries with moderate endemism [12‐14]. We found that despite common routes of transmission and similar risk factors, the HBV NAT yield rate is higher than that of HCV and HIV, possibly because HBV is highly prevalent in China [5]. Conversely, the extremely low TTI residual risks for HCV and HIV may be attributed to their low prevalence in the population and short window periods of HCV and HIV testing using ID-NAT [16, 27].

Several studies have compared the sensitivity of NAT systems for HBV, HCV, and HIV [28‐34]; as a consequence, differing findings have been reported. Using PROCLEIX ULTRIO (Ultrio) assay and TaqScreen multiplex (Cobas MPX) test, Margaritis et al. reported equal HBV NAT yields rate in donations from Hong Kong [29]. However, Phikulsod et al. in Thailand reported that TaqMan MP6 was more sensitive than Ultrio in ID format [30]. Using the Ultrio Plus assay relative to the Ultrio ID-NAT and TaqMan MP6, Marion et al. in South Africa found a significantly higher proportion of replicate assays on HBV NAT yields [28]. In this work, we compared NAT yields rates in five different assays, including Ultrio, Ultrio Plus, and Ultrio Elite assays using the TMA method in ID format, as well as MPX and MPX2.0 using the PCR method in 6MP-format. Consequently, there were no statistically significant differences in HBV, HCV, and HIV NAT yield rates between the 2 NAT methods. Nonetheless, among the 5 assays, Ultrio Plus was effective at non-discriminating reactive and HCV detection, whereas Ultrio Eilte exhibited the highest HBV NAT yield in the screening test. Interestingly, MPX2.0 was slightly but not significantly more sensitive in detecting low viral load OBI samples using the ID format. Collectively, these results indicate that besides reagents sensitivity, the capacity of NAT methods to detect HBV, HCV, and HIV, particularly at low viral loads depends on pool size.

In addition to HBV, HCV, and HIV, some samples were screening tests-positive, but non-discriminating reactive using the TMA method. The reasons for these non-resolved results were potential because of a sensitivity gap between screening and discriminatory reagents in the TMA method, or the viral loads in the donations may have been too low to be detected by discriminatory reagents. Some non-resolved results were found with HBV DNA positive through increasing number of tests, concentrating with high-speed centrifugation, and using other NAT methods [35‐37]. In China, Ye et al. [37, 38] found that 91.1% of non-discriminated reactive donors were anti-HBc reactive OBI with low viral loads. Thus, non-discriminating reactive donations have a great risk for HBV transmission and should be excluded. Also, we found that the HBV NAT yields risk factors included male gender, older age, low education level, lower technology work, and first-time donors. Therefore, NAT screening for TTIs and higher sensitivity screening, specifically for HBV, improve the safety of blood supply. Differences in NAT yield at different blood service centers may be attributed to NAT screening methods and virus prevalence in the general population.

In conclusion, high HBV NAT yield rates were discovered in an analysis of NAT yield rates at seven Chinese blood service centers. Besides, the efficiency of HBV, HCV, and HIV NAT yield was similar for TMA and PCR methods but different in the 5 reagent assays. NAT screening at blood donation reduces the risk of transfusion-transmitted infections, shortens the duration of serological tests, and increases blood safety. Nonetheless, NAT yields rates varied across blood services and hinged on the NAT detection mode and blood donor features.