To increase blood safety, we introduced minipool NAT screening in our blood donor service in 1997 for hepatitis B virus (HBV), hepatitis C virus (HCV), and HIV-1 and in 2000 for hepatitis A virus (HAV) and parvovirus B19 (4). For these purposes, 100-μL aliquots of up to 96 blood samples were pooled. The complete pool of up to 9.6 mL was centrifuged at 58,000× g for 60 min at 4°C. Viruses were extracted by using spin columns, and nucleic acid was eluted in a total volume of 75 μL. Only 60 μL of extract is needed for routine NAT screening. A residual volume of 15 μL can then be used for additional NAT testing (5) for influenza viruses.

The real-time quantitative amplification of influenza/H5 was performed according to the manufacturer’s instructions (Artus Influenza/H5 LC RT-PCR Kit, QIAGEN, Hamburg, Germany) by using a thermocycler (LightCycler; Roche Applied Science, Mannheim, Germany). The test consists of 2 individual amplification reactions. In the first step, a generic influenza PCR is performed. The specificity of this reaction was demonstrated for all subtypes of influenza A (H1–H15, N1–N9) and all subtypes of influenza B. Samples with a positive test result in the first PCR were analyzed in a second PCR with influenza (H5N1)–specific primers and probes. Therefore, the assay allows differentiation between avian influenza (H5N1) and other influenza virus strains.

To mimic a situation like an H5-positive donation, a purified culture supernatant of Vero cells infected with influenza (H5N1) (strain A/Thailand/1 (KAN-1)/2004) (6) was used as an external quantification standard. Virion integrity in this preparation was confirmed by electron microscopy. The viral RNA concentration was determined in an external laboratory by multiple quantitative real-time PCR determinations (7). Different dilutions of the external influenza (H5N1) subtype quantification standard (0.0, 0.91, 1.96, 3.91, 7.81, 15.63, 31.25, 62.5, 125, and 250 PFU/mL) were prepared, and 100 μL of each dilution was spiked into 9.5-mL negative plasma pools. Each dilution was repeatedly spiked and tested in 8 minipools. Five microliters of the extract was analyzed with the generic influenza NAT as well as with the specific influenza (H5N1) NAT. Results are shown in Tables 1 and 2. Probit analysis of these data yielded a detection probability of >95% in parallel tests when an average of at least 13.4 PFU/mL (95% confidence interval [CI] 8.3–184 PFU/mL) and 7.4 PFU/mL (95% CI 5.2–14.7 PFU/mL) for influenza generic assay and for the influenza (H5N1)–specific test, respectively, were present in individual plasma samples before pooling.

A total of 117 routine minipools, representing 10,272 blood donor samples, containing an average of 88 ± 8 samples per pool, had previously been tested for HIV-1, HBV, HCV, HAV, and parvovirus B19. All pools were negative for influenza virus when tested with the generic influenza PCR and the influenza (H5N1)–specific PCR. One pool had invalid results (failed amplification of internal control RNA, representing 0.01% of all analyzed runs).

As reported by AuBuchon et al. (8), NAT significantly increased the safety of blood products. At the German Red Cross, look-back examinations showed only 1 transfusion had transmitted HIV-1 (1998) after the introduction of NAT testing. Blood donor screening by NAT was made technically and financially feasible by creating minipools of up to 96 individual samples per pool. Roth et al. demonstrated an efficient enrichment for all tested viruses in plasma samples (9). In the absence of an infective donor, different concentrations of the new influenza genotype H5N1 were spiked into minipools of 95 samples. As shown in Table 2, the influenza (H5N1) subtype was detected by the generic influenza primers as well as by the influenza (H5N1)–specific primers when our routine minipool screening procedure was used. Sensitivity was expressed as PFU/mL and can be converted into viral genome copy number according the calculation of Yoshikawa et al. (7). Therefore, the analytical sensitivity was ≈804 geq/mL and 444 geq/mL for a generic influenza and for the influenza (H5N1) subtype, respectively.

After screening 10,272 samples by minipool NAT, none of the samples were found to be infected by influenza, which corresponds with the low EISS Index (European Influenza Surveillance Scheme index) of <20 during the study period (February–April, 2006) (10). An EISS index >80 is expected during an influenza epidemic, as was seen in 2005. Therefore, blood screening should be repeated during the next acute influenza season.

Accepted incubation periods for influenza range from 2 to 10 days (11,12). As with other viruses, a viremic phase of infection can be assumed to precede clinical symptoms such as fever (13,14). Recently Chutinimitkul et al. (15) detected influenza (H5N1) virus (3,080 copies/mL) in the plasma of a 5-year-old boy, which indicates a viremic phase of influenza (H5N1) infection. Those donors may be infective, especially to immunosuppressed patients. In addition to quarantine of infected patients, treatment with antiviral drugs, and development of avian influenza vaccines, blood donors should be tested during a pandemic to avoid transfusion-transmitted infections. Our study demonstrates that NAT screening could be incorporated into blood testing without delay and that the influenza virus could be sufficiently enriched by centrifugation. Sensitivity of our influenza-screening method would have been sufficient to detect recently reported virus concentrations in plasma of infected persons (15). However, as with all minipool methods, infections can be transmitted to transfusion recipients on rare occasions because the viremia level in the donor is below the analytical sensitivity of the screening assay.

To reduce this risk, a selective infectious dose NAT strategy (e.g., triggering of infectious dose NAT testing when at least 1 viremic donation is collected per week with the standard minipool screening algorithm), as performed for West Nile virus (WNV) screening in the United States might be necessary. Implementation of WNV-NAT in the United States in 2003 interdicted well over 1,000 donations from persons infected with WNV and is a good example of successful implementation of NAT screening for emerging viruses.

The collective fight against new viruses such as severe acute respiratory syndrome virus, WNV, or influenza (H5N1) presents an immense challenge for the whole community, but new molecular-biologic methods offer opportunities to overcome this challenge. NAT screening tests are now available soon after the sequencing of new viruses. In the absence of a general pathogen inactivation method for all blood products (erythrocytes, platelets, and plasma), the NAT screening procedure allows testing for new viruses to ensure blood safety.