Background
Human parvovirus B19V is a small icosahedral, non-enveloped single-stranded DNA viral pathogen that can cause a variety of diseases, including erythema infectiosum (fifth disease), arthritis, transient aplastic crisis, chronic anemia (in immunocompromised patients), hydrops fetalis, and fetal death[
1‐
4]. The main route of B19V transmission is via the respiratory route, although it can also be transmitted vertically and via blood transfusion and organ transplantation[
5]. B19V infection usually happens during childhood; however, 40–60% of adults are still susceptible to primary infection[
6,
7]. Depending on assay sensitivity and epidemic incidence, the prevalence of B19V DNA in blood donors can be up to 1%, with virus titers reaching 1 × 10
14 geq/mL during early acute infection, although affected individuals are often asymptomatic. This level of prevalence is sufficient to contaminate most plasma pools used for fractionation[
8,
9], and, eventually, plasma derivatives that are usually prepared from pools of several thousand donations. One study demonstrated that, overall, 85% (60–100% depending on manufacturer) of plasma pools, 25% of albumin samples, 100% of factor VIII, 20% of IVIG, and 75% of intramuscular immunoglobulin preparations contained B19V DNA[
10]. Viral load in those samples ranged from 1 × 10
2 to 1 × 10
6 geq/mL. Another study reported a high prevalence (over 60%) of B19V DNA in factor IX, factor VIII, PCCs, and plasma pools with viral loads of 1 × 10
2 to 1 × 10
8 geq/mL[
11].
The small size (20–25 nm in diameter) and non-enveloped nature of B19V render it difficult to remove by filtration methods and very resistant to many virus inactivation procedures used in the production of plasma derivatives, including solvent/detergent (S/D) and heat treatment. The transmission of B19V through the administration of S/D-treated[
12] and certain dry heat-treated blood products has already been documented[
13‐
15]. B19V can also be transmitted by blood component[
16,
17], while one study indicated only high concentration containing component can cause infection[
18]. Transmission of B19V by blood and blood products and its resistance to common viral inactivation/removal methods raises the importance of detecting B19V prior to blood transfusion. The FDA has proposed a limit of 10
4 geq/mL for manufacturing pools destined for all plasma derivatives to reduce the potential risk of transmission[
19,
20]. Similarly, European Pharmacopoeia has imposed a limit of 10
4 IU/mL for levels of B19V in anti-D immunoglobulins and pooled virus inactivated plasma.
Many studies have demonstrated the presence of B19V DNA in plasma pools and plasma-derived products[
11,
21‐
24]; however, the prevalence of B19V DNA in Chinese blood products and plasma pools has not been extensively investigated. In this study, we aimed to determine the frequency and level of B19V DNA contamination in plasma pools collected during 2009–2011, and in plasma-derived products produced during two periods,1993–1995 (albumin, IVIG) and 2009–2011(albumin, IVIG, factor VIII, Fibrinogen), under license in China. Since no B19V Nucleic Acid Testing (NAT) regimen has previously been proposed for Chinese blood products manufacturers, the results of the present study will hopefully provide a significant advance in this area and have a positive impact on policy development with regard to blood safety in China.
Discussion
Blood products have been widely used for the prevention and treatment of a variety of life-threatening injuries and diseases; however, the contamination of these products with viruses also poses a great threat to patients. Due to the implementation of a variety of measures such as donor selection, virus inactivation/removal, and the testing of donations and of plasma pools, the risk of transmission of blood-borne viruses, especially hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV), through plasma and plasma products has been dramatically reduced in developed countries. In 2002, a guideline (the guideline of the technological methods and validation of viral removal/inactivation of blood products, No. 2002–160) was implemented in China with the aim of improving the virus safety of plasma derivatives. This guideline sets the Chinese standard for performing virus validation studies on medicinal products derived from human plasma. The removal/inactivation of viruses in these products requires the implementation of production procedures including S/D treatment, heat (pasteurization and dry heating), and filtration[
27]. For coagulation factor concentrates and immunoglobulin, one or several combinational methods should be used to inactivate/remove enveloped and non-enveloped viruses, and albumin manufactured by cold ethanol fractionation must be heat-treated (pasteurized) in the final container. These methods are very effective for the inactivation/removal of the aforementioned principal viruses (HIV, HBV, and HCV); however, non-enveloped viruses, such as B19V, cannot be efficiently removed by these methods and therefore pose a residual risk. This is because (1) plasma pools are prepared from a large number of donations (more than 5000), and it is likely that a high proportion of pools are contaminated with B19V, potentially including some highly contaminated donations (up to 10
14 geq/mL), and (2) B19V is extremely heat resistant and is small in size, which makes it difficult to remove or inactivate by heat treatment or filtration. B19V DNA contamination of plasma products has been reported in several studies[
28‐
32]; however, the risk posed by Chinese plasma derivatives has not previously been addressed. In this study, coagulation factor concentrates, such as plasma-derived factor VIII, fibrinogen and PCC were found to be highly contaminated with B19V DNA at levels of 10
2 to 10
7 geq/mL. IVIG and albumin were moderately contaminated with low levels of B19V DNA.
Parvovirus B19V infection is relatively common worldwide. In immunocompetent individuals, the infection generally occurs without any serious consequences. However, in specific groups, such as pregnant women, patients with underlying hematological problems and patients with immunodeficiency, B19V infections can result in serious complications[
33‐
35]. It is estimated that 50% of all 15-year-olds in the western world have experienced an infection[
8]. However, in the elderly population, higher percentages, as high as 80 or 100%, have been observed[
36].
A study in our laboratory on blood donors from many Chinese regions reported the NAT testing of samples from 3957 donors using an in-house developed Q-PCR assay for the initial testing and a nested PCR assay for confirmation. The study indicated a 0.58% genome DNA prevalence rate among the blood donor population[
26], but the study was not large enough to define range of viral titers, as samples with very high titers are relatively rare. In this study, we investigated B19V DNA contamination in 142 plasma pools from two Chinese manufacturers. As indicated in Table
1, 77of 142 industrial plasma pools (54.2%) were positive and 33 contained B19V-DNA titers higher than 10
4 geq/mL. In the case of manufacturer A, 30% of plasma pools were positive, but in the case of manufacturer B, the majority (85.5%) were positive. This may reflect a regional difference in B19V infection prevalence. It is very interesting to found the prevalence of B19V in the mini-pool (each contain 90 donations) from a total of 49,680 plasma donations from manufacture A was 97%, much higher than that of other pools collected from both companies during 2009–2011. However, these individual plasmas were collected between August 2011 and March 2012 from company A which located in the south of China. Winter and spring are the epidemic periods of respiratory virus in this area. This may reflect a temporal difference in B19V infection. The quantity of B19V DNA varied from 10
2 to 3.07 × 10
8 geq/mL and approximately 43%(33 of 77 positive pools) of pools contained B19V DNA at titers higher than 10
4 geq/mL, which was sufficiently large to contaminate the products produced from these plasma pools. These data are consistent with previous reports indicating that B19V-DNA is detectable in more than 60% of pools used for the production of plasma products, although usually at relatively low levels[
28,
37].
In this study, we identified one donation with a B19V DNA titer of 1.09 × 10
10 geq/mL, which was IgG/IgM negative at the index time. Unexpectedly, B19V DNA and IgG were positive, although the levels were lower than that in a sample collected from the same individual two weeks previously (Table
3). This is not consistent with an acute B19V infection and probably indicates that the donor had a persistent B19V infection, which is usually associated with anomalies in immune status or an immune response that produces B19V antibodies that are ineffective in neutralizing the virus, this result support the data of a previously study[
25]. It is likely that, in the days prior to the index time, B19V was reactivated, which would have led to viral replication and a rapid increase in viral load, and, thereby, neutralization of the existing level of IgG. As indicated in Table
3, during the following two months, B19V-DNA decreased rapidly to 2.07 × 10
4 geq/mL, IgG gradually increased to a plateau, and IgM decreased to a barely detectable level. Other high titer donations are likely to have contributed to a higher prevalence of B19V DNA among plasma pools from manufacturer B. However, unfortunately, samples permitting further investigation were unavailable.
There are several limitations to this study. Firstly, the investigation was confined to the B19V genome content of Chinese plasma pools and plasma derivatives, and the infectivity of the virus in these samples was not determined. Nevertheless, although some inactivation/removal methods have been implemented, these data support the finding that B19V can be transmitted by blood products[
38‐
40] and that the B19V-DNA content reflects, at least partly, contamination with the infectious virus. Secondly, there are about 24 blood product manufacturers in China, and our investigation was restricted to plasma pools from two of these. Thus, the data obtained in this study does not reflect the nationwide status of B19V contamination of blood products in China and our results do not provide information about regional and temporal differences in contamination prevalence. Despite these limitations, our study represents the first investigation of B19V presence in Chinese blood products.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
WL designed, searched data and literature and gave a critical view of manuscript writing. ZW, LK, LC, ZY performed the experiments, collected and analyzed the data. All the authors’ read and approved the final manuscript.