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NEJM Study of Gen-Probe Assay Backs Use of Triplex NAT in HBV Blood Screening


This article was originally published on Jan. 19.

By Ben Butkus

Nucleic acid testing of blood donations revealed a higher-than-expected rate of hepatitis B infection in donors, including those who had been previously vaccinated and who would not have been identified by routine antibody screening, according to results of one of the largest clinical trials of nucleic acid testing conducted to date.

The study suggests that triplex PCR-based tests such as Gen-Probe's and Novartis' Procleix Ultrio assay for the Tigris system can help identify potentially infectious HBV, HCV, and HIV in the window of time before seroconversion.

The data also lend further support to the routine use of such tests to screen the blood supply. Besides the Gen-Probe/Novartis test, Roche also markets a triplex test for screening blood donations for HBV, HCV, and HIV.

In the study, published today in the New England Journal of Medicine, scientists from the American Red Cross, University of Giessen, University of Cambridge, and Baylor College of Medicine analyzed and further collected data related to a one-year clinical trial conducted during 2008 at three of the five American Red Cross National Testing Laboratories.

The goal of the study, according to the researchers, was to assess the value of nucleic acid testing of donated blood for hepatitis B virus, which to that point had been detected by screening for hepatitis B surface antigen (HBsAg) and antibodies against hepatitis B core antigen (anti-HBc).

The scientists used the automated triplex Procleix Ultrio assay manufactured by Gen-Probe and marketed by Novartis Vaccines and Diagnostics to test for HIV RNA, HCV RNA, and HBV DNA in 3.7 million blood donations. Testing was performed in either mini-pools of six to 16 donations or individually; and the scientists further analyzed samples that were positive for HBV DNA but negative for HBsAg and anti-HBc.

Testing identified nine donors who were positive for HBV DNA but negative for the antigen and antibody, for a yield of one in 410,540 donations. In addition, Procleix Ultrio detected 26 total confirmed-positive HBV, HCV, and HIV infections during the pre-seroconversion window for a total study yield of one in 154,000 donations, suggesting that using the triplex assay may provide additional safety in identifying window-period cases of all three infections.

Susan Stramer, an investigator in the Scientific Support Office of the American Red Cross and lead author on the NEJM paper, told PCR Insider this week that other groups had performed smaller studies on triplex nucleic acid testing yield, with variable results.

"We wanted to do a large study to understand the yield of positive donations; and then when we found positives, to further characterize why they weren't detected by our routine serologic tests," Stramer explained.

According to Stramer, finding nine in 3.7 million blood donations positive for HBV DNA "was a little higher than we would have expected," which suggested that prior screening methods were missing a significant number of HBV infections.

In an e-mail to PCR Insider, Pat Gahan, vice president and head of commercial operations for the Americas at Novartis Diagnostics, noted that the study "further underscores the value of NAT testing and the Procleix Ultrio test to to provide highly sensitive detection not only for HIV-1 and hepatitis C virus, but also very low level hepatitis B virus that might be missed by traditional serology testing. This can be critical for intercepting blood from donors who have early stage, asymptomatic, or latent chronic infections."

Gahan added that the study "underscores the leadership of blood banks like the American Red Cross, which have pro-actively adopted NAT testing for hepatitis B virus. Even in the US, which has low rates of HBV infection and high rates of HBV vaccination, NAT testing and the Procleix Ultrio assay have played a key role in further increasing blood safety."

In another unexpected finding from the study, six of the nine yield donors previously had received the HBV vaccine and developed a subclinical, medically inconsequential HBV infection. Among those six subjects, five showed infection with an HBV subtype different than that used in the vaccine; and the sixth showed infection with the subtype used in the vaccine but had received the vaccine 27 years prior.

"In theory, they should not have been infected, because the vaccine should not only prevent clinical disease, but the theory is that it should also prevent infection," Stramer said. "So perhaps the vaccine doesn't provide as good protection from infection for genotypes that differ from the parent genotype. That was an unexpected finding, because we would expect that the vaccine would have equal protection to all genotypes."

Although the potential for transmission of these subtype HBV infections has not yet been resolved, Stramer said that "it's only an issue if these people develop clinical hepatitis, which would mean that the vaccine was not efficacious. But all of these individuals had low-level viral infection and cleared their viral infection rapidly."

As such, the finding "only tell us that with the vaccine, perhaps for different genotypes or after a long period of time following vaccination, you can still get a low-level infection; but that vaccine memory will still protect you from clinical disease," she added.

The study was funded in part by Gen-Probe and Novartis, which provided the Ultrio test kits and Tigris instruments. The clinical trial at the heart of the study was conducted to support an investigational protocol designed to determine the frequency of donors testing HBV-seronegative but DNA-positive so that an HBV DNA blood-donation screening claim could be justified for the Ultrio assay on the Tigris platform. Based in part on the results of the trial, the US Food and Drug Administration cleared the test to screen blood for HBV in 2008.

Procleix Ultrio is a single-tube assay that performs HIV-1 RNA, HCV RNA, and HBV DNA target amplification by transcription-mediated amplification; and detects amplification products using hybridization protection assay technology. The FDA first approved the test in May 2007 to screen donated blood, plasma, organs, and tissue for HIV-1 and HCV on the Tigris system, but did not approve the HBV screening claim at the time because the initial study did not demonstrate HBV yield.

Gahan said that currently, more than 80 percent of the US blood supply is screened with Procleix tests.

Roche also markets a triplex nucleic acid test for HIV, HBV, and HCV: the Cobas TaqScreen MPX test for use on the Cobas S 201 system. Roche's test was approved by the FDA in December 2008 to screen donated blood for HIV-1 group M RNA, hepatitis C RNA, and hepatitis B DNA in a single, automated assay.

Have topics you'd like to see covered in PCR Insider? Contact the editor at bbutkus [at] genomeweb [.] com.

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