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Second Group IDs Contaminated Qiagen Sample Prep Products; Casts Doubt on Novel Virus Discovery

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Scientists from the University of Cape Town in South Africa have used nested PCR to discover that silica-based nucleic acid extraction products from Qiagen are contaminated with sequences linked to a purportedly novel virus previously identified in seronegative hepatitis patients by a group comprising researchers from China and the US National Institutes of Health.

The work, summarized in a letter to the editor in the January issue of the Journal of Virology, jibes at least in part with findings published last year by a University of California, San Francisco-led team. That group performed unbiased, ultra-deep sequencing on multiple clinical sample types to uncover what it first thought was essentially the same novel virus, but later discovered was also likely due to contaminated silica-based spin columns from Qiagen.

In addition, PCR Insider has learned that Baoyan Xu, the first author of the Chinese/NIH study — which was published last June in PNAS — has been found by the Office of Research Integrity of the US Department of Health and Human Services to have engaged in research misconduct by falsifying certain figures in that paper. The paper has been corrected but not retracted, since the NIH and ORI determined that Xu's contribution did not affect the overall findings of the research, according to the website Retraction Watch.

In the PNAS paper, the NIH researchers including Xu — a former NIH postdoc who is currently a scientist at the Institute of Infectious Disease of China's Third Military Medical University — described how they used unbiased deep sequencing to discover a virus that they called NIH-CQV in the blood samples of 92 Chinese people who had hepatitis that was not caused by any of the five known hepatitis viruses, also known as seronegative hepatitis.

The researchers from the University of Cape Town and South Africa's National Health Laboratory Service took a particular interest in the Xu paper because they were studying novel viruses, particularly the so-called non-A to -E hepatitis viruses, Heidi Smuts, corresponding author of the Journal of Virology letter, told PCR Insider in an email this week.

"Working with [co-author Michael] Kew, a prominent and internationally recognized expert on hepatitis B virus, I was given a selection of samples that may harbor non-A-E hepatitis viruses," Smuts said. "When the Xu et al. paper was published I designed primers for a nested PCR to screen these samples which had been extracted using the manual Qiagen spin column platform. My suspicions were raised when a high percentage of the healthy controls were positive for this hybrid virus, which did not fit the data published by Xu et al."

Specifically, Smuts and colleagues examined 48 samples collected from patients with a range of liver diseases, as well as 40 samples from healthy individuals. Nucleic acid from all 88 samples was extracted in 2009 using the QIAamp Viral RNA Mini Kit — not exactly the same as the QIAamp MinElute virus kit used by the Xu team, but using the same silica spin column technology.

Furthermore, Smuts' co-author Stephen Korsman had a large selection of non-A-C samples that he was investigating for the presence of hepatitis E virus. These samples, 112 in all, comprised nucleic acid extracted in 2012 and 2013 using the NucliSENS EasyMag platform from BioMérieux.

Screening of the liver disease samples using the nested PCR assay targeting the rep gene of NIH-CQV showed that 46 of 48, or 96 percent, were strongly positive for the virus, while 32 of 40, or 80 percent, of controls also showed evidence of the virus.

Meantime, only four of the 112 non-A-C hepatitis samples were weakly positive, suggesting that the samples extracted using the Qiagen kit were contaminated. Smuts and colleagues confirmed this by parallel extraction of 12 random samples in 2013 which used both the QIAamp and EasyMag kits and showed that only samples extracted using the Qiagen kit were positive. Subsequent sequencing of PCR products confirmed 100 percent identity to NIH-CQV.

"As we did not know where the contamination was coming from we screened all the reagents individually from three different lot-numbered Qiagen kits, using the BioMérieux platform for the extraction," Smuts wrote in her email. "We also screened water, Proteinase K, ethanol, and Eppendorf tubes washed with water and, of course, water spun through the spin column. Only water spun through the spin column gave a positive signal."

Finally, the group tested four additional silica-based magnetic platforms — Roche's MagNA Pure LC, MagNA Pure Compact, and Cobas AmpliPrep/TaqMan; and Abbott's m2000sp — and failed to amplify NIH-CQV from 10 replicates each of water.

Smuts conceded that her group does not yet know why four of the 112 samples extracted using the BioMérieux kit were weakly positive. "There may have been environmental contamination, as the automated extractor is in the same room as the laminar flow hood in which manual extractions using the Qiagen spin columns are also performed," she said. "Another possibility is that the virus may infect humans and this could then explain the serology results from Xu et al."

At any rate, the findings of the South African group align with those of the UCSF scientists, who published a paper on their research also in the Journal of Virology in September.

The UCSF researchers used Illumina HiSeq sequencing to discover and de novo assemble what they thought was a novel, highly divergent DNA virus that they dubbed parvovirus-like hybrid virus, or PHV.

However, they subsequently found that the sequence of their virus was almost identical to that of the Chinese/NIH team's virus, a finding that, coupled with some other oddities, made them question their results.

They eventually narrowed down potential culprits for their results to Qiagen's QIAamp Viral RNA Mini — the same kit used by the South African researchers —and the QIAamp UltraSens Virus kits, both of which employ the silica spin column technology. And, similarly, they were able to detect sequences of their virus from water samples that had been run through the spin columns.

Some of the findings from Smuts et al. differ from those of the UCSF team — namely, the latter suggested that the contamination was only found in samples extracted from kits after 2011. However, the nucleic acids used by Smuts et al. had been extracted with Qiagen kits in 2009. In addition, the South African researchers screened stored nucleic acid from respiratory samples that had been extracted in 2004 and 2005 with silica column-based viral RNA kits manufactured by Italian company Talent, and these samples, too, showed similar contamination with NIH-CQV, with 21 of 24, or 88 percent of samples testing positive.

And, the UCSF group conducted a metagenomic survey of publicly available environmental databases and were able to find their specific virus sequences in databases from ocean waters of the Pacific coast of North America, suggesting that the silica contamination was geographically dependent. However, "unless Talent obtains their silica from the same source as Qiagen then there is also possibly no geographical specificity," Smuts said wrote in her email.

It is unclear where Talent obtained the silica for its spin columns; an internet search revealed that the company appears to be defunct.

A spokesperson for Qiagen this week told PCR Insider in an email that the QIAamp viral RNA Mini kits with silica-based spin columns used by the South African team "are designed for purification of viral RNA used in subsequent PCR and other applications detecting specific targets" but are not designed to be DNA-free.

"In standard PCR applications the described contamination will not impair the results," the spokesperson said. "For applications with enhanced sensitivity like nested PCR, however, as described in the study, a product from the QIAamp UCP product line (containing silica spin columns which have been
depleted of potential DNA contaminations) would be the appropriate sample preparation kit."

The spokesperson said that independent laboratories have confirmed that the QIAamp UCP spin columns do not contain the viral sequence in question.

The spokesperson provided a similar statement in October regarding the UCSF findings, noting that an ultrasensitive technique such as unbiased deep sequencing may pick up contaminants from the QIAamp viral RNA Mini kits but would likely not pick up any contaminants with the QIAamp UCP product.

Nevertheless, Qiagen "takes the issue very seriously and offers all customers who have purchased a QIAamp Viral RNA Mini Kit or a QIAamp UltraSens Virus Kit for their nested PCR applications to exchange the spin columns with the QIAamp UCP Mini columns at no cost."

He also stressed that the contamination incidents are not linked to Qiagen platforms used for clinical diagnostics and does not have an impact on reporting clinical results.

In general, both the South African and UCSF studies add to the growing body of evidence that researchers using ultra-sensitive molecular detection techniques such as PCR and sequencing need to be aware of the possibility of contamination, especially when trying to identify or detect new or potentially pathogenic organisms because of the possible public health implications.

As for Xu and the original PNAS paper, the subject of the ORI's judgment of research misconduct centered on a Western blot analysis of IgM and IgG antibodies from Chinese subjects in patients with non-A-E hepatitis, and not any of the molecular detection results, per se.

Specifically, Xu made a "limited admission" that a particular figure in the paper used images of the same strips several times to support some of the group's findings about immune reactivity toward their NIH-CQV virus.

Retraction Watch noted late last month that Xu has agreed to supervision of any NIH-funded research for three years, and to not serve on any peer-review committees for the same period of time. Retraction Watch also received word from the NIH that ORI decided not to retract the paper after determining that Xu's contribution "did not in any way effect the overall findings of the research. Since the findings still stand, only a correction is necessary and other papers should not be affected."

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