Nucleic acid test (NAT) methods to test plasma mini-pools for B19DNA are being used by some plasma fractionators as an in-process test to lower the viral load of manufacturing pools and thus to reduce or eliminate B19 in plasma derivatives. We formulated a parvovirus B19 DNA-positive working standard targeted to contain 106 geq/mL. It was prepared by diluting a window period donation, 3 x1012 geq/mL, with an anti-B19 negative, pooled plasma that was negative for HBsAg, anti-HIV1/2, anti- HCV, anti-B19, HIV RNA, HCV RNA, HBV DNA, B19 DNA, HAV RNA and HGV RNA. 3000 vials (1 mL/vial) were filled. Portion of the unique VP1 region of B19 genome has been sequenced. This standard is one of the four candidate samples in an international collaborative study to establish a WHO International Standard for B19 NAT assays. 26 laboratories participated and the results are being analyzed. We continued to investigate the extent of B19 contamination in both Immune Globulin Intravenous (IGIV) and Immune Globulin (IG) products. In addition, anti- D products, such as Rho(D)IGIV and Rho(D)IG were also tested because they are used to prevent hemolytic disease of the new born for Rh negative pregnant women, and B19 is known to cause fetal hydrops in pregnancy. Our preliminary results indicate that B19 is more prevalent in IG than IGIV lots assayed. The levels of B19 DNA detected in the IG lots were also higher than IGIV. These may be due to the lesser purification steps and the higher IgG concentrations in IG lots when compared to the IGIV lots. Although many more lots have yet to be tested, no anti-D lot was found positive thus far for B19 DNA. Investigation is on going for albumin products because many manufacturers' products are involved. Of the lots tested, only very low levels of B19 DNA were detected. In collaboration with scientists in OTRR, we participated in investigating whether a gene therapy product, which had been infused into quite a few patients, was contaminated with HCV. Using a PCR assay followed by a hybridization probe, a contract testing laboratory found that a master virus bank (MVB) for a gene therapy product was contaminated with HCV. We found no detectable HCV sequences in both MVB and the resulting product lots when assayed by a sensitive, in-house, nested PCR assay, and thus demonstrated that the initial results were most likely due to false positives by the PCR methodology. However, when tested by using another investigative PCR kit, one of the product lots was found positive in some replicate samples. Further studies are in progress to resolve whether the kit also detects false positives, i.e., detects non-specific cross- reactivities in vector or human genomic sequences, because of using a hybridization probe to detect the PCR amplified products. We also investigated whether a reported (MedWatch) case of B19 was causally related to two implicated lots of human plasma derived factor VIII concentrate (AHF). One lot contained a high level of B19 DNA. Serum specimen from a patient who received equivalent to 400,000 geq of B19 DNA were tested and found positive for B19 DNA. The B19 sequences from the patient were identical to those found in the implicated lot. However, because the pre-infusion serum specimen was found positive for B19 DNA, it was concluded that the patient had already infected with B19 prior to the AHF infusion. We continued to perform the lot release testing for HCV RNA in 2 mfrs' IGIM products not yet subjected to any viral inactivation procedure in the manufacture. No lot was positive for HCV RNA.
