Novel Hybrid Parvovirus-Like Virus, NIH-CQV/PHV, Contaminants in Silica Column-Based Nucleic Acid Extraction Kits

The discovery of a novel hybrid parvovirus-like virus, NIH-CQV, in non-A to-E (non-A-E) hepatitis virus samples by Xu et al. (1) as a possible causative agent of seronegative hepatitis prompted our investigation of samples collected from patients with a range of liver diseases, including acute hepatitis, chronic hepatitis, cirrhosis, hepatocellular carcinoma, and amoebic liver disease (n 48) as well as 40 healthy individuals. Nucleic acid from these 88 samples was extracted in 2009 using the silica col-umn-based QIAamp viral RNA Mini Kit (Qiagen). A further 112 samples from patients with non-A-C hepatitis, with nucleic acid extracted in 2012-2013 using the NucliSENS easyMag platform (bioMérieux), were also screened. A nested PCR, designed with primers targeting the rep gene of NIH-CQV (1), generated a 176-bp fragment. Inner primers used were those reported by Xu et al. (1); the outer primers used were OF (5=-GTCTTTTAGAAAG TTTCCACG) and OR (5=-CGATCGAAGAATCGGTTCTC). Screening of the liver disease samples showed that 46 of 48 (96%) were strongly positive for NIH-CQV, while 80 % of the healthy controls (32 of 40) also showed evidence of the novel virus. In contrast, only 4 of 112 non-A-C hepatitis samples (3.7%) were weakly positive. These results suggested that the samples ex-tracted using the Qiagen kit were contaminated, which was con-firmed when parallel extraction in 2013 of 12 random samples with Qiagen and easyMag kits showed that only samples extracted using the silica column-based method were positive. Reagents from three Qiagen kits were examined, and only water spun through the silica column was positive. Sequencing of PCR prod-ucts showed 100 % identity to NIH-CQV. Testing of an additional four silica-based magnetic plat-