Hepatitis A virus (HAV) is a non-enveloped virus and difficult to be inactivated. Some fractionators use minipool screening of plasma by NAT methods to limit the HAV viral load in the manufacturing pools. Similar to parvovirus B19, FDA also considers such HAV NAT screening as an in-process control test and hence a working standard to compare the sensitivity of various NAT assays is needed. We developed a sensitive, in-house NAT method and formulated a CBER HAV RNA working standard. The standard was included as one of the five candidate preparations for the WHO collaborative study to characterize an international standard for HAV RNA nucleic acid amplification assays. We were one of the testing laboratories participated. The collaborative study has been completed. A lyophilized plasma preparation will be recommended to the WHO as the International Standard. It has an assigned potency of 10e5 IU of HAV RNA/mL. As a result of the study, CBER HAV working standard has a consensus level of 10e4 PCR detectable units/mL and an assigned value of 10e3.8 IU/mL. We continued to investigate whether a commercial NAT kit utilizing a hybridization probe to detect the RT-PCR amplified products for HCV RNA might have detected false positives, i.e., detected non-specific, cross-reactivities in vector or human genomic sequences used in a gene-therapy product. A further study is still needed to resolve the issues. We continued to evaluate both HCV RNA and B19 DNA quantitation utilizing TaqMan real-time PCR methods. We also continued to monitor the viral safety of plasma derivatives with respect to parvovirus B19. Most manufacturers have voluntarily implemented the B19 minipool screening of plasma to limit the viral levels in the manufacturing pools. We are in progress in monitoring the effectiveness of such screening on the extents of B19 contamination in various products. In collaboration with the CDC, we investigated whether a reported MedWatch case of B19 was causally related to two implicated lots of human plasma derived factor VIII concentrates (AHF) made by a manufacturer. One AHF lot was derived from the B19 screened pools and no B19 DNA was found in the final container product. The other implicated lot, however, was from mostly unscreened pools and was found positive for B19 DNA. The recipient's pre-infusion serum specimen was available and no B19 infection marker was detected. The 4-week post-infusion specimen was, however, positive for both anti-B19 and B19 DNA. Based on the sequencing of VP 1 region, B19 sequences detected from the post-infusion specimen were more closely related to the B19 sequences detected from the implicated AHF lot and a high-titer, unscreened, plasma pool, than to other B19 isolates. Hence, our data strongly suggest that the reported MedWatch case of B19 transmission was causally related to the infusion of the implicated lot of AHF and that the case could have prevented if the B19 minipool screening of plasma implemented.
