Occult hepatitis B virus infection (OBI) has been identified as one of the potential risk factors that threaten blood transfusion safety [14, 15]. OBI is related to these factors such as HBV gene mutation, virus replication and low expression levels, and host's immunity status [14–16]. OBI shows undetectable HBsAg, but a sustained low or fluctuating level of HBV DNA in serum or liver. This study was based on the previous research of Chongqing Blood Center. Firstly, Ultrio Plus multiplex ID-NAT assay for simultaneous detection of HBV DNA, HCV RNA, and HIV-1 RNA was adopted, following discriminatory assays of these three viruses were carried out if the initial result was reactive. We have noted that only about a third were reactive in discriminatory assays among NDR donations. About two-thirds donations were nonreactive in discriminatory assays were defined as NDRs, which was similar to the result (68%) previously reported by a national research in China .
Due to the Poisson distribution of virus particles in the sample , the randomness of sampling leads to non-repetitive reactivity in NAT, and the lower the virus load in the sample, the lower the probability of capturing virus particles. Five duplicate tests with Ultrio Plus dHBV assay were implemented to improve chance of sampling virus particles to confirm low-level HBV DNA such as OBI. Sixty-one NDR donations were identified to be HBV DNA-positive in repeated dHBV tests, all were confirmed as seropositive-OBI donors. Moreover, it presented the characteristic of non-repetitive reactivity, which might be related to extremely low and fluctuating levels of viral nucleic acids and was mainly observed in OBI donors . Only 55.7% of HBV DNA-positive donations showed once reactivity in five repeated tests, which was also related to extremely low viral load of OBI [7, 19]. The HBV DNA-positive rate increased with the increase of repetitions, indicating that if the number of repetitions increased again, more HBV DNA-positive donations would be detected. Although increasing the number of repeated tests can improve the detection probability of samples with low viral load, the time and cost also increase, which is not feasible for general blood screening laboratory.
Different methodologies can complement each other to improve the chance of detecting HBV DNA. Ultrio Plus is an ID-NAT format assay based on transcription-mediated amplification (TMA). However, MPX is a NAT assay in MPs of six samples format based on real-time fluorescent PCR. Two donations that were negative in repeated dHBV testing were positive for HBV DNA on Cobas s201 NAT platform, all Ct values were lower than 38 and the minimum value was 47.2, further illustrating that the extremely low viral load of OBI in DNR donations. However, the reconfirmation of OBI in repeated-dHBV-negative donations and a higher proportion of anti-HBc positive involved in NDRs, indicating that there was a possibility of the presence of OBI with low-level HBV DNA in unclassified donations. Therefore, the actual proportion of OBI should be greater than 33.2% (63/190) confirmed this time.
Only 20.6% of HBV DNA-positive donations were detected of HBV DNA in virus load quantification, and all virus loads were less than 20 IU/ml, which could not be accurately quantified. This might be due to the different sensitivity of the reagents. Ultrio Plus assay has a 95% LOD of 3.4 IU/ml for HBV DNA, while Cobas Ampliprep/Taqman quantitative assay has a 95% LOD of 20 IU/ml for HBV DNA. The reason for the rest of 50 donations were not detected quantitatively might be that the virus loads were below the 95% LOD of the quantitative assay, or it might be that the virus loads were just around the 95% LOD. We have performed HBV DNA quantitative determination on 63 OBI donations which initially reactive in dHBV assay, obtained a median of 108.6 IU/ml, which is significantly higher than this study. This further suggests that OBI in NDR donors has a very low viral load.
HBV serological markers help us to identify HBV occult carriers in NDRs, especially anti-HBc and anti-HBs. In this study, the percent of anti-HBc positive in HBV DNA-positive donations (96.8%) was significantly higher than that in unclassified donations (79.5%). In contrary, the percent of anti-HBs positive in HBV DNA-positive donations (47.6%) was significantly lower than that in unclassified donations (63.8%). As is well-known, anti-HBc is considered as a vital marker related to HBV infection or exposure and anti-HBs is regarded as a kind of protective antibody, illustrating that a portion of repeated dHBV-nagative donations were false reactivity in multiplex NAT testing. Twelve donations without serological markers were also more likely to be false reactivity in multiplex NAT assay. Fourteen donations only carried anti-HBs might be false reactivity in multiplex NAT assay or they might be OBI with extremely low virus concentrations. The HBV DNA-positive donations were divided into two groups: anti-HBc positive and anti-HBc negative. The anti-HBc-positive rate in experimental group (96.8%) and control group (90.0%) was significantly higher than the anti-HBc-negative rate (3.2% and 10.0%), indicating that there was a close relationship between HBV DNA and anti-HBc. There was no statistically significant difference in anti-HBc-positive rate between experimental group and control group, indicating that most NDR donors have been exposed to HBV, which might be potential OBI. Moreover, this is in accord with previous studies reporting that 68-91% anti-HBc positive rate of Chinese NDR donors [17, 20, 21]. Based on the above results, we could draw the following conclusions: (1) anti-HBc-positive donors had a significantly higher risk of transmitting HBV through blood transfusion than anti-HBc-negative donors. However, China is a high-endemic area of HBV, the prevalence of anti-HBc in population is about 16-90% [22, 23]. Thus, the introduction of universal anti-HBc to donor screening would have inevitably strain the blood supply because of the high reactivity rate. (2) There was no statistical difference in the probability of previous HBV infection or exposure between experimental group and control group. Therefore, from the perspective of blood transfusion safety, the NDR donors posed a potential risk of transmitting HBV.
There were more male (68.3% and 65.2%) than female (31.7% and 34.8%) donors in experimental group and control group, respectively, with nearly identical proportions in two groups. This result is consistent with that reported by Ramachandran et al in the United States . The proportion of repeat-time donors in experimental group was higher than that in control group, indicating that the OBIs in experimental group with more lower viral loads than that in control group. These OBI donors are likely to be missed by multiplex NAT assay, which is determined by the Poisson distribution . The median age of OBI was 45 years in NDR group, which was similar to previous study reporting by Deng et al in Dalian, China . The Chinese government has implemented infant vaccination from 1992, and resulted in a significant reduction of carrier rate in children from 10 to < 1% over the two decades, which definitely improve the blood safety . .
The non-repetitive reactivity of such low viral load donations poses serious challenges for blood screening and transfusion safety. As a result, some regions have formulated more stringent blood screening strategies based on the local HBV prevalence. For example, multiplex NAT initially-reactive-donation needs to carry out repeated testing in South Africa . If the repeated result is nonreactive, then perform anti-HBc detection; if the repeated result is reactive, then carry out discriminatory tests, following plasma sample is subjected to multiplex NAT and discriminatory tests to confirm infection status of donation. However, the blood production will be eliminated regardless of the result of repeated multiplex NAT. In Japan, quantitative detection of anti-HBs and anti-HBc are adopted into blood screening, and donations with high-titer anti-HBs and low-titer anti-HBc are released. On the contrary, donations with low-titer anti- HBs and high-titer anti-HBc are sifted out .
It is worth noting that the HIV WP detection rate was 1.5 per 100,000 and there was no HCV WP donor since the implementation of NAT in Chongqing Blood Center. Therefore, in-depth research related to HCV and HIV has not been carried out. However, this study included some limitations. A total of 140577 blood donations were collected, and 525 (0.37%) donations were HBsAg, anti-HCV and HIV Ag & anti-HIV ELISA nonreactive and Ultrio Plus multiplex NAT initially reactive, among which 206 donations (0.15%) were both reactive in multiplex NAT and dHBV assay. Only one donation was reactive in multiplex NAT and dHIV assay, and the other 318 (0.23%) were nonreactive in discriminatory assays. The NDR rate obtained in Chongqing blood donors was similar to that reported in Shenzhen (0.21%), South China, but it was still higher than in Dalian, Northeast, China (0.10%) and New Zealand (0.09%) when using the same NAT assay [19, 20, 26]. The differences observed between studies might relate to differences in the regional prevalence of HBV infection. For some objective reasons, we only collected 190 DNR donations. According to the ratio, we infer that the proportion of OBI in NDRs is approximately 7.5 per 10000 donations. Since 79.5% of the NDR donors with unconfirmed HBV DNA were anti-HBc positive and some of these might have had viral loads below the 95% LOD of Ultrio Plus multiplex assay and confirmatory reagents. Thus, the data would be underestimated.