Frequency of contamination of coagulation factor concentrates with novel human parvovirus PARV4.

Human parvovirus, PARV4 was identified in a plasma sample from a patient presenting with symptoms resembling acute HIV infection. Further strains of PARV4 and those of a closely related variant virus, were identified in plasma pools used in the manufacture of blood derivatives. DNA sequence analysis of these strains demonstrated two distinct PARV4 genotypes. It has subsequently been proposed that transmission of PARV4 occurs by parenteral routes. To investigate the risk of contamination of plasma-derived coagulation factor concentrates, we analysed 169 lots for PARV4 DNA by polymerase chain reaction. Positive samples were confirmed by nucleotide sequence analysis and quantification of the viral load. Twenty-one lots, representing eight different products were administered until the beginning of the 1980s and were not virally inactivated. Two lots examined were used in 1997, and 146 lots representing 13 products had been administered between October 2000 and February 2003. PARV4 DNA was detected in 7(33%) of the formerly administered lots, in one lot used in 1997, and in 13(9%) recently used lots. PARV4 genotype 2 DNA was predominantly present in the older concentrates, whilst genotype 1 was found more frequently in recently used lots. In three lots, both PARV4 genotypes were detected. Viral loads ranged between <100 and 10(5.8) copies mL(-1) of product, with higher viral loads in the older concentrates. The results show that PARV4 contamination can be detected in an appreciable proportion of clotting factor concentrates. Further studies are needed to determine whether or not PARV4 contamination of coagulation factors causes harm to the product recipients.

Human parvovirus PARV4 in clotting factor VIII concentrates.

BACKGROUND AND OBJECTIVES: Parvoviruses are small non-enveloped DNA viruses, relatively resistant to virus inactivation procedures. The recently identified human parvovirus PARV4, including a related genotype 2 virus (also termed PARV5), has been found to be a contaminant of pooled plasma used in the manufacture of plasma-derived products. This report describes an investigation to determine whether PARV4 is present in clotting factor concentrates. MATERIALS AND METHODS: Factor VIII concentrates manufactured in the past 30-35 years were screened for PARV4 and human parvovirus B19 (B19V) sequences. Viral loads in products testing positive for PARV4 were quantified using a consensus TaqMan assay designed to a highly conserved region. DNA sequence analysis was performed to confirm the genotypes present. RESULTS: From a total of 175 lots of factor VIII concentrate, 28 of these contained PARV4 sequences, and in two lots both genotypes 1 and 2 were found to be present. The highest viral loads observed exceeded 10(5) copies per ml. The majority of factor VIII concentrates testing positive for PARV4 were manufactured in the 1970s and 1980s. Human B19V was also a frequent contaminant of these products. CONCLUSIONS: PARV4 was detected in 16% of factor VIII concentrates, particularly in older batches from the 1970s and 1980s. The significance in terms of the viral safety and potential transmission to recipients of these products is not yet known.

Parvovirus B19 genotypes 1 and 2 detection with real-time polymerase chain reaction assays.

BACKGROUND AND OBJECTIVES: Parvovirus B19 (B19V) DNA screening has been introduced to comply with European regulations for certain plasma products. Current commercial and some in-house B19V DNA assays fail to detect or under-quantify the recently identified genotypes 2 and 3. In this report, we describe 2-year experience with B19V DNA screening using the commercial assay from Roche (detecting only genotype 1) combined with an in-house assay (detecting genotypes 1, 2 and 3). This dual testing approach enables the identification of molecular variants of B19V. MATERIALS AND METHODS: Between 2005 and 2007, approximately 2.6 million plasma donations were screened for B19V DNA loads exceeding 10(6) IU/ml using the Roche and the in-house real-time polymerase chain reaction assay. RESULTS: A total of 232 plasma units were identified with B19V DNA loads above 10(6) IU/ml. Concordant results were observed for the majority of B19V positive samples; however, three of these showed discrepant results between the two assay systems. One was a B19V genotype 2 strain not detected by the Roche assay; another was a B19V genotype 1 strain with a mismatch in the 3'-end of the reverse primer and therefore under-quantified by the Roche assay; and the third one was also a B19V genotype 1 strain that gave an unusual amplification plot in the in-house assay due to a mismatch in the probe-binding site. CONCLUSIONS: New, high viral load, B19V genotypes 2 and 3 infections are rare in blood donors tested by Sanquin. One case was found while testing 2.6 million donations. The prevalence of B19V genotype 1 variants not detected by commercial or in-house assays might be in the same range or even higher than the prevalence of B19V genotype 2 viruses, which remain undetected.

Quantitation of porcine cytomegalovirus in pig tissues by PCR.

A quantitative-competitive PCR for the quantification of porcine cytomegalovirus (PCMV) was developed. The virus was detected in a variety of pig organs (including potential xenotransplant donations), with viral loads ranging from <10 to 97 genome copies/microg of DNA. This assay will have significant utility for studying the activation and replication of PCMV and in swine models for allo- and xenotransplantation.