Parvovirus B19 DNA and antibodies in Chinese plasma donors, plasma pools and plasma derivatives

Introduction

Human parvovirus B19 (B19V) is a non-enveloped virus with a liner single-stranded DNA genome. It belongs to Erythroparvovirus of the Parvoviridae family. B19V infection causes a variety of illnesses, including Fifth disease in children, aplastic crisis in patients with hemolytic disorders, fatal hydrops in pregnant women, arthropathy, cardiomyopathy, and inflammation of other various tissues (Qiu, Söderlund-Venermo & Young, 2017). Moreover, the epidemiology of B19V shows a wide geographic distribution and seasonal variations, and the peak season of infection is spring and winter. The transmission of B19V is primarily through the upper respiratory route, organ transplantation, and blood transfusion (Ganaie & Qiu, 2018; Parsyan & Candotti, 2007). Since the prevalence of B19V in blood donors is about 1% (Abdelrahman et al., 2021), there is a high probability that plasma pools can also be contaminated. When DNA in the blood donor population reaches 1%, their plasma could easily contaminate the entire pooled plasma. Once a single plasma donor with extremely high DNA concentrations exceeding 10¹⁴ IU/mL, the B19 viral DNA in the pooled plasma could reach 10¹⁰ IU/ml. Because of its special structure and small size, B19V is highly resistant to all commonly-used inactivation methods. Our preliminary research also indicated that plasma-derived clotting factor concentrates such as fibrinogen and PCC were found to be highly contaminated with B19V DNA (53.7%–85.7%), and IVIG and albumin were moderately contaminated (0–38.9%) (Zhang et al., 2012). Therefore, the safety of plasma products needs to be further evaluated.

In China, there has been no specific documentation or technical guidelines for monitoring B19V. To better ensure the safety of plasma derivatives, many governments request plasma manufacturers perform B19V screening in manufacturing plasma pools. For example, the US Food and Drug administration guidelines, US Pharmacopoeia, Plasma Protein Therapeutics Association, and European regulatory requirements recommend that the viral load of B19V in the manufacturing of pooled plasma should not exceed 10⁴IU/mL (The European Pharmacopoeia, 2020a; The European Pharmacopoeia, 2020b; Jin et al., 2010). Although the B19V-DNA prevalence among Chinese plasma donors is relatively low, asymptomatic plasma donors with high levels of B19V (up to 10¹² IU/mL) may present with a greater risk in plasma derivatives (Jia et al., 2019; Li et al., 2020; Marano et al., 2015; Schmidt et al., 2001; Siegl & Cassinotti, 1998; Zhang et al., 2012). This study’s major objective was to comprehensively and systematically determine the frequency and levels of B19V DNA in plasma donors, pooled plasma, and plasma derivatives from four Chinese blood products manufacturers. Additionally, reports on changes in B19V DNA in single–plasma donors are rare, so we also followed-up and evaluated changes in B19V DNA level in those B19V DNA-positive donors who were first confirmed after their subsequent donation. We continued to track and investigate contamination in the plasma derivatives produced by plasma pools with viral load of B19V DNA exceeding 10⁴ IU/mL. Moreover, B19V-specific immunoglobulin (IgG/IgM) antibodies in all of B19V DNA-positive samples were also determined. We expect that this study will provide a series of robust evidence on promoting the quality standards for plasma derivatives in China.

Materials & Methods

Results

Phylogenetic relationships among different B19V isolates

Seventeen 1,200-bp sample sequences were obtained within the NS1-VP1-U region. They were aligned with 35 reference sequences to reconstruct the phylogenetic tree under the NJ method. The tree is shown in Fig. 1 with the NJ bootstrap values depicted on the branches. All of the samples studied in this article formed a monophyletic group, and they fell in Genotype 1A.

Discussion

Because of its special raw resource, the safety of plasma derivatives is always an important and concerning issue (Di Minno et al., 2016). As a potential contaminant of blood transfusion disease, B19V can cause serious complications in some high-risk patients, such as those with hemophilia, potential hematological malignancy, and immunodeficiency. Due to B19V’s pathogenicity and risk of transmission through plasma derivatives, great attention has been focused on it. Japan, Germany, and the Netherlands screen for B19V DNA or B19V specific antibodies in blood donors (Groeneveld & van der Noordaa, 2003; Sakata et al., 2013; Schmidt et al., 2007). In regards to plasma, the US Food and Drug Administration (FDA) and European Pharmacopoeia have proposed a limit of 10⁴IU/mL for B19V DNA levels in plasma pools when manufacturing all kinds of plasma derivatives (The European Pharmacopoeia, 2020a; The European Pharmacopoeia, 2020b; Jin et al., 2010). Although there are no regulations for monitoring B19V DNA in China, great efforts have been made to investigate the epidemic and characterization of B19V in plasma donors, plasma pools, and plasma derivatives in China over the last several years. Jia et al., (2015) found that the contamination of B19V in plasma pools was serious in China. In her study, 71.91% (169/235) of plasma pools were contaminated with B19V, with concentrations of 5. 18 × 10²–1.05 × 10 ⁹IU/mL. Approximately 31.95% of the DNA-positive plasma pools were only moderately contaminated (<10⁴IU/mL), while 68.05% contained >10⁴IU/mL. These data were consistent with those of our study, which demonstrated a relatively high prevalence of B19V in Chinese plasma pools. According to the limit standard of 10⁴ IU/mL established by the US FDA, European Pharmacopoeia, and the Plasma Protein Therapeutics Association (PPTA), and based on our results, approximately 60% plasma pools should be discarded in China, which would be a great waste of plasma. In order to use raw plasma optimally, we recommend B19V DNA screening for individual plasma donors instead of plasma pools and plasma derivatives.

Moreover, we also monitored the contamination of plasma derivatives produced by plasma pools with a viral load of B19V DNA exceeding 10⁴ IU/mL. After considering business privacy, only one company was willing to continue in this study. Therefore, there were only a total of 20 batches of plasma derivatives that were continually collected and monitored. These products were factor VIII, PCC, IVIG, and albumin made from plasma pools with high B19V DNA (>10⁴IU/mL). The results indicated that B19V DNA was not detected in any batch of albumin and IVIG, except for plasma-derived clotting factor concentrates. The contamination of B19 DNA in plasma-derived clotting factor concentrates was high, indicating that different manufacturing procedures were the key factors. The results were consistent with those of Geng et al. (2007). These contaminated products may impose risks to the patients who received them. There are few clinical data on the transmission of B19V through plasma derivatives. Previous studies found that the seroprevalence of IgG antibodies to B19V was much higher among very young (age 2–7 years) hemophiliac patients exposed to plasma-derived products compared to those not exposed (Azzi, Morfini & Mannucci, 1999; Santagostino et al., 1997; Soucie et al., 2004). Another study also found that young children with bleeding disorders exposed only to plasma-derived factor concentrates were 70% more likely to have antibodies to B19V than those who were not exposed to other products (Soucie et al., 2013). In that respect, we propose paying more attention to avoid the contamination of plasma-derived clotting factor concentrates.

Previous studies demonstrated that the prevalence of B19V DNA in Chinese plasma donors ranged between 0.03% and 0.09% (Han et al., 2015). However, we found that 0.29% (42/14187) of specimens were positive for B19V DNA. This was higher than what was mentioned before, but lower than 0.58%, which Ke et al. (2011) found in whole blood donors. The results may be related with the plasma donors’ geographic differences and methodological differences in diagnostic procedures. Additionally, only two plasma donors from Guangxi Province were infected with B19V at levels higher than 10⁴IU/mL (5 ×10¹² IU/mL). In some reports, the peak virus titer reached 10¹³ IU/mL (Frickhofen & Young, 1989). Therefore, B19V NAT testing for single Chinese plasma donor screening is necessary. In addition, 11 plasma donors who were B19V DNA-positive at the first screening time were followed up with until the COVID-19 pandemic. One of the plasma donors was a classic example. He was B19V DNA-positive at his first donation, with a virus titer of 5.09 × 10¹² IU/mL. After his second and third donation (about 14 days later), he was B19V DNA-positive with a lower virus titer (1.86 ×10⁵IU/mL, 1.09 ×10⁴IU/mL), and IgG and IgM-positive. This plasma donor may have been in the viremia stage at his first donation. After 36 days, the donors were still B19V DNA and IgG- positive and IgM-negative. Those variations were in accordance with epidemiological trends of B19V in the blood phase. IgM antibodies develop 10–14 days post-infection followed by the development of IgG antibodies directed toward viral capsid components. Meanwhile, this result also indicated that the plasma donor who was infected with B19V could not be permanently refused.

The presence of B19V-specific IgM antibodies suggested recent infection. A survey reported that the seroprevalence of B19V-specific IgM antibodies was commonly below 2% in healthy people. The presence of only B19V-specific IgG antibodies is indicative of past exposure. Both B19V-specific IgG and IgM may be present at or soon after the onset of illness and reach peak titers within 30 days. The prevalence of B19V IgG antibodies increases with age. Other research reported that approximately 2% of children under the age of 5 and 80% of blood donors 18–65 years of age had IgG antibodies (Ke et al., 2011; Kelly et al., 2000; Manaresi et al., 2004). In other studies, about 30% of 18 to 30 year-old donors had detectable IgG, while about 60% of 50 year-old donors were seropositive (Zaaijer, Koppelman & Farrington, 2004). A study in our laboratory on the characteristics of Chinese plasma donors showed that most plasma donors (78.5%) were aged 46–55 years old (Sun et al., 2021). This may explain the prevalence of B19V-specific IgG in Chinese plasma donors. The Health Council of Netherlands found that blood with persistent anti-B19V IgG (B19V-specific IgG were detected in two separate blood samples, one taken at least 6 months after the other) might be B19V-safe blood (Groeneveld & van der Noordaa, 2003). B19V-specific IgM antibodies are detectable 10 to 14 days after infection and can generally persist for 5 months. The prevalence of B19V IgM aided in the assessment of the rate of recent B19V infection in donors. In this study, only one individual B19V DNA-positive plasma donor was detected with B19V-specific IgM. There was no association between B19V DNA levels and the titer of IgM/IgG yet.

These data recommended the implementation of B19V screening for plasma donors and plasma pools in order to contract the transmission of B19V via plasma derivatives in China. Moreover, the National Medical Products Administration prepared the national reference standard for B19V DNA detection and explored the quantitative real-time detection of B19V kits. Whether B19V NAT screening of plasma and plasma derivatives are launched in China or not, Chinese plasma fractionation industries should be encouraged to spontaneously perform B19V NAT screening in plasma donors and mini-pool plasma. These measures can ensure that samples with high titer B19V DNA are discarded in order to control prevention and control of this transfusion-transmitted virus.

B19V is subdivided into three distinct genotypes. Jia et al. (2016) found that there were at least three subtypes (1a, 1b, and 3b) of B19V circulating in China. In this study, the prevalent genotypes in 118 B19V-DNA-positive source plasma pool samples attributed 61.86% to genotype 1a, 10.17% to genotype 1b, and 17.80% to genotype 3b. Our study indicated that the prevalent genotypes in the four provinces’ plasma donors belonged to B19V genotype 1. These differences may be related to sample resources.

Additionally, our study had some limitations. The plasma derivatives were distributed all around the country and administrated to different recipients, making it difficult to integrate the many resources to perform such large-scale systematic research on the recipients of these blood products. Moreover, participants may have been dropped from the program at any time, and they may have been treated with other blood products. These unfavorable factors may have greatly limited the study. In view of this, B19V NAT screening is recommended in plasma donors and mini-pool plasma to better protect the recipients of plasma derivatives.

Conclusion

In this study, we found that only 0.29% (42/14,187) of plasma donors were positive for B19V DNA, but the contamination of B19V in pooled plasma (64.8%) and plasma-derived clotting factor concentrates (100%) was significant in China. B19V NAT screening is recommended for plasma donors and mini-pool plasma to ensure that samples with high titer B19V DNA are discarded in order to increase the prevention and control of this transfusion-transmitted virus.

Supplemental Information