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Identi cation of occult hepatitis B virus infection in blood donors with non-discriminated reactive in southwest China


 Background

Since 2011, Chongqing Blood Center began routine screening of viral nucleic acid using a multiplex assay for simultaneous detection of HBV DNA, HCV RNA and HIV-1 RNA in blood donations. However, this multiplex nucleic acid testing (NAT) screening system introduced an intractable problem of non-discriminated reactive (NDR) donations, which were serologically nonreactive and reactive in multiplex NAT screening, but nonreactive in the following virus-specific discriminatory NAT assays. Some donations were considered to arise from the presence of low-level HBV DNA due to occult hepatitis B infection (OBI). We identified OBI among NDR donations in Chongqing area. In addition, rationality of the current blood screening strategy was evaluated.
Methods

From August 2017 to July 2018, 190 NDR blood donations were collected from 140577 volunteer blood donors at Chongqing Blood Center. Repeated discriminatory HBV (dHBV) testing, minipools-NAT (MPs-NAT), quantitative viral load determination and complementary serological testing were used to confirm OBI.
Results

Of 190 NDR donations, sixty-three (33.2%) were identified as OBI, sixty-one (32.1%) were repeatedly reactive in discriminatory HBV assay. Two (1.1%) were positive for HBV DNA in MPX assay on Roche Cobas s201 system. Of these sixty-three OBI donations, only thirteen (20.6%) were positive in Cobas AmpliPrep/TaqMan HBV DNA quantitative assay, which were all less than 20IU/ml. Anti-HBc and anti-HBs were detected in 85.3% and 58.4% of NDR donors, respectively. Anti-HBc and anti-HBs were detected in 96.8% and 47.6% of confirmed OBI donors in NDR group, respectively. Anti-HBc and anti-HBs were detected in 79.5% and 63.8% of unclassified donors in NDR group, respectively. There was no statistical difference in positive rate of anti-HBc between NDR donations and dHBV-initially-reactive donations (85.3% vs 90.0%). The male to female ratio was 2.2 of OBI donors in NDR group. The proportion of repeat-time OBI donors in NDR group was higher than that in dHBV-initially-reactive group (63.5% vs 50.0%). The median age of OBI donors was 45 years in NDR group.
Conclusion

A complicated OBI confirmatory procedure with labour-intensive and sophisticated equipment was implemented successfully. OBI with extremely low virus concentration is the main reason for discriminatory NAT nonreactive among blood donors in Chongqing area. The current blood screening strategy is a reasonable measure to reduce the risk of transfusion, which is able to maximize blood safety.

strategy is a reasonable measure to reduce the risk of transfusion, which is able to maximize blood safety.

Background Chronic hepatitis B virus (HBV) infection is a common cause of cirrhosis and hepatocellular carcinoma
globally. Although hepatitis B vaccine has been widely applied, there are still 257 million chronic hepatitis B patients (de ned as hepatitis B surface antigen [HBsAg] positive) worldwide [1]. Other than chronic HBV infection, occult hepatitis B virus infection (OBI) is another clinical presentation of HBV infection, it is de ned as HBsAg negative yet hepatitis B virus DNA positive in serum and/or liver, meanwhile HBV DNA level is usually less than 200IU/ml (about 1,000 copies/ml) [2]. OBI is most frequently observed in patients with hepatitis B core antibody (anti-HBc) reactive (with or without hepatitis B surface antibody (anti-HBs)) [3]. OBI has been reported as an additional risk factor for progression of nonalcoholic fatty liver disease, alcoholic liver cirrhosis, and HBV infection to hepatocellular carcinoma [4]. Through a retrospective study, some patients died of transfusion-transmissible HBV (TT-HBV) fulminant hepatitis caused by transfusion due to OBI-derived blood products [5]. Even though infection rate of such donations seems to be low, the prevalence of OBI in blood donors should be well evaluated, especially in HBV high-endemic areas.
Although HBsAg screening strategy has been carried out in all blood donations, blood centers in China have voluntarily implemented nucleic acid testing (NAT) to prevent TT-HBV infection since 2010. In 2015, NAT was included in the mandatory blood screening program. Chongqing is one of the four municipalities directly under the central government in China, and Chongqing Blood Center is the only blood center in Chongqing area. Since 2011, Chongqing Blood Center began routine screening of HBV/HCV/HIV-1 nucleic acids in blood donations using the Procleix Ultrio assay and the Procleix Ultrio Plus assay (in place since 2016). This multiplex NAT assay proves the presence of viral genomes in the donation by detecting a single signal that does not distinguish between these three viruses. Then virusspeci c discriminatory assays are used to identify the origin of the initial signal. However, this NAT screening system introduced an intractable problem of non-discriminated reactive (NDR) donations, which were serologically nonreactive and reactive in multiplex NAT screening, but nonreactive in the following discriminatory NAT assays, and which may be mainly observed in OBI donors [6,7]. Because the viral load of OBI is usually too low to be quanti ed or be sequenced [8]. It is reported that multiple repeated tests from the NDR donations' plasma bag may increase the chance of detecting viral nucleic acids present at levels around the assay's limit of detection (LOD) according to the Poisson distribution by alternative NATs [9]. Furthermore, repeated tests and different methods could increase the detection rate of OBI.
Since the implementation of the hepatitis B vaccine program in China, the seroprevalence of HBV in blood donors has declined [10,11]. OBI donors, who with mutations that may resulted in the absence of HBsAg production [7]. Moreover, the manufacturer of Ultrio Plus stated that the multiplex NAT assay was marginally more sensitive than the discriminatory assay for detecting HBV DNA [12], which is one of the causes of NDR results. The characteristic of OBI with low viral load is consistent with the occurrence of NDR results. This study set out with the aim of understanding the OBI status of discriminatory-NAT nonreactive donations in Chongqing, a municipality in southwest China. Furthermore, rationality of the current blood screening strategy was evaluated.

Subjects and samples
During the period from August 2017 to July 2018, a total of 140577 blood donations were collected from Chongqing Blood Center. Among them, 67622 (48.1%) blood donations were from rst-time donors, and the rest of 72955 (51.9%) blood donations were from repeat-time donors. In this study, we de ned a rsttime donor to be someone with no history of blood donation at Chongqing Blood Center, and a repeattime donor was de ned as a person who has donated blood at least twice at Chongqing Blood Center. The blood donations with HBsAg, anti-HCV, HIV Ag & anti-HIV ELISA nonreactive and initially reactive in Ultrio Plus HBV/HCV/HIV-1 multiplex NAT assay, among which were nonreactive in the following virusspeci c discriminatory assays de ned as non-discriminated reactive (NDR). We collected 190 NDR donations in the experimental group and 100 dHBV-initially-reactive donations in the control group.
Routine serological and nucleic acid testing All donations in this study were tested for HBsAg, anti-HCV, HIV Ag & anti-HIV with enzyme-linked immunosorbent assay (ELISA) using two reagents from different manufacturers and multiplex NAT assay for HBV DNA, HCV RNA and HIV-1 RNA based on transcription-mediated ampli cation (TMA) was used (Procleix Ultrio Plus on Tigris platform; Grifols, Spain) for individual-donation-nucleic acid testing (ID-NAT), multiplex NAT-initially-reactive donations were discriminated with three virus-speci c discriminatory NAT assays. Regardless of the discriminatory results, as long as multiplex NAT was initial reactivity, the related blood products would be eliminated.
Repeated discriminatory HBV testing 190 NDR donations were retested ve times with Ultrio Plus dHBV assay. Any reactive result was identi ed to be positive for HBV DNA.

Minipools nucleic acid testing (MPs-NAT)
PCR Cobas multiscreen (COBAS s201, Branchburg, USA) technology in pools of six samples was used to con rm the status of donations, which were still nonreactive after ve repetitions with dHBV assay. If a positive result is obtained, a resolution test will be performed to identify the speci c positive one from the six donations according to the manufacturer's instruction. Donation that was reactive in both MPs-NAT and resolution test was considered to be positive for HBV DNA.

Complementary serological testing
All donations in this study were subjected to detected anti-HBc and anti-HBs with ELISA (Wantai Diagnostics, Beijing, China) according to the manufacturer's procedures.

De nition of occult HBV infections
In this study, OBI was de ned as HBV DNA positive, anti-HBc positive and/or anti-HBs positive but HBsAg negative [13].

Statistical analysis
The data in the research were analyzed using SPSS statistic version 22.0. Chi-square test was applied to determine the association between nominal data, A p-value less than 0.05 was used as the cutoff level of statistical signi cance.

Routine screening of blood donations
During the period from August 2017 to July 2018, a total of 140577 volunteer blood donations were collected from Chongqing Blood Center. The 140577 blood donors were 51.8% (72765) males and 48.2% (67812) females, respectively. 190 NDR donors were distributed in 60.5% (115) males and 39.5% (75) females. There was a statistical difference in gender distribution between blood donor population and NDR donors ( χ2 =10.6, p<0.05). Figure 1 shows the blood screening tests and the OBI con rmatory algorithm.

< Fig. 1>
Repeated discriminatory HBV testing Of 190 NDR donations, sixty-one (32.1%) were positive for HBV DNA at least once in ve repeated dHBV tests. In addition, more than half (55.7%) of these donations were reactive for dHBV only once out of ve repetitions. Out of the ve repeated tests, up to three more times were reactive ( Table 1). The rest of 129 (67.9%) donations were identi ed as negative for repeated dHBV testing. The positive rate of HBV DNA increased with the number of repetitions (Table 2).
Minipools nucleic acid testing (MPs-NAT) Two donations that were negative for repeated dHBV testing were positive for HBV DNA on Roche Cobas s201 system in MPs of six samples, which were identi ed as HBV DNA-positive. The cycle threshold (Ct) values were all less than 38, and the minimum Ct value was 47.2 (Table 3).

<Table 3>
Quantitative viral load determination Of sixty-three HBV DNA-positive donations, only thirteen (20.6%) were positive in Cobas AmpliPrep/TaqMan HBV DNA quantitative assay based on real-time uorescent qPCR. However, the viral loads of these quantitatively-positive donations were all less than 20IU/ml, which were lower than the 95% LOD of this assay.

Discussion
Occult hepatitis B virus infection (OBI) has been identi ed 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][15][16]. OBI shows undetectable HBsAg, but a sustained low or uctuating 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 de ned as NDRs, which was similar to the result (68%) previously reported by a national research in China [17].
Due to the Poisson distribution of virus particles in the sample [18], 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 con rm low-level HBV DNA such as OBI. Sixty-one NDR donations were identi ed to be HBV DNA-positive in repeated dHBV tests, all were con rmed as seropositive-OBI donors. Moreover, it presented the characteristic of non-repetitive reactivity, which might be related to extremely low and uctuating levels of viral nucleic acids and was mainly observed in OBI donors [7]. Only 55.7% of HBV DNA-positive donations showed once reactivity in ve 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 ampli cation (TMA). However, MPX is a NAT assay in MPs of six samples format based on real-time uorescent 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 recon rmation 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 unclassi ed donations. Therefore, the actual proportion of OBI should be greater than 33.2% (63/190) con rmed this time.
Only 20.6% of HBV DNA-positive donations were detected of HBV DNA in virus load quanti cation, and all virus loads were less than 20 IU/ml, which could not be accurately quanti ed. 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 signi cantly 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 signi cantly higher than that in unclassi ed donations (79.5%). In contrary, the percent of anti-HBs positive in HBV DNA-positive donations (47.6%) was signi cantly lower than that in unclassi ed 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 signi cantly 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 signi cant 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 signi cantly 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 [24]. 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 [25]. 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 [26]. The Chinese government has implemented infant vaccination from 1992, and resulted in a signi cant reduction of carrier rate in children from 10 to < 1% over the two decades, which de nitely improve the blood safety [27]. .
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-reactivedonation needs to carry out repeated testing in South Africa [28]. 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 con rm 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 lowtiter anti-HBs and high-titer anti-HBc are sifted out [29].
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 uncon rmed 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 con rmatory reagents. Thus, the data would be underestimated.

Conclusion
In summary, we used a variety of complementary experiments and methods to con rm OBI in the donations with nonreactive results in virus-speci c discriminatory assays. However, the con rmation process is complicated, time-consuming, labour-intensive and required sophisticated equipment, which is not feasible for general screening laboratory. In Chongqing area, OBI with extremely low virus concentration is the main reason for the occurrence of NDR donations. Therefore, the current screening strategy is a reasonable measure to reduce the risk of blood transfusion and ensure maximal blood safety. At the same time, we should also be aware that even if more sensitive NAT assays are implemented, it is still challenging to distinguish true positive from low-level viral loads blood donors.    Figure 1 Ultrio Plus NAT screening and con rmatory testing ow

Supplementary Files
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