Minipool Nucleic Acid Testing Research Articles

The persistence of hepatitis C virus transmission risk in China despite serologic screening of blood donations

A total of 2%-2.9% of the population in China is infected with hepatitis C virus (HCV). This study estimated the prevalence and incidence of HCV among Chinese blood donors. We examined whole blood and apheresis platelet donations at five Chinese blood centers in 2008 to 2010. All donations were screened using two rounds of testing for alanine aminotransferase, antibody to human immunodeficiency virus Types 1 and 2, hepatitis B surface antigen, anti-HCV, and syphilis. Screening reactivity is defined by a reactive result in one or both rounds of screening tests. Confirmatory tests (Ortho third-generation HCV enzyme immunoassay, Johnson & Johnson) were performed on anti-HCV screening-reactive samples. Confirmatory positive rates among first-time donors (prevalence) and repeat donors (incidence) were calculated by blood center and demographic categories. Donor characteristics associated with HCV confirmatory status among first-time donors were examined using trend test and multivariable logistic regression analysis. Among 821,314 donations, 40% came from repeat donors. The overall anti-HCV screening-reactive rate was 0.48%. Estimated HCV prevalence was 235 per 100,000 first-time donors; incidence was 10 per 100,000 person-years in repeat donors. In multivariable logistic regression analysis, first-time donors older than 25 years displayed higher HCV prevalence than the younger donors. Less education is associated with higher HCV prevalence. Donors 26 to 35 years old and those above 45 years displayed the highest incidence rate. High prevalence and incidence in donors indicate high residual risks for transfusion-transmitted HCV in Chinese patients. Implementation of minipool nucleic acid testing in routine donation screening may prevent a substantial number of transfusion-transmitted HCV infections.

Infectivity of occult hepatitis B from two different points of view

Infectivity of occult hepatitis <scp>B</scp> from two different points of view

West Nile virus infection transmitted by granulocyte transfusion

To the editor: Eleven cases of transfusion-transmitted West Nile virus (WNV) infection and associated illness have been reported from 9 blood donors since the introduction of minipool nucleic acid testing (MP-NAT) in 2003.[1][1][⇓][2]–[3][3] This number of breakthrough infections is against a

Évolutions technologiques en qualification biologique du don et leur impact sur le risque résiduel transfusionnel

During the two last decades, the risk of viral transfusion transmission for some infectious agents (HIV, HCV, HBV and HTLV) has been markedly reduced by improved donor screening, improvements of serological assays and the implementation of minipool nucleic acid testing for HIV-1 and HCV viruses. However, implementation during the year 2010 of nucleic acid testing for the detection of HIV RNA, HCV RNA and HBV DNA in a single triplex assay may provide additional safety, especially after acute infection during the window period. New Procleix(®) Tigris(®) technology (Novartis) allow the French blood screening laboratories to answer their actual requirements in terms of security and throughput and to implement nucleic acid testing in case of emergent risks requiring a direct detection of viruses. Furthermore, Tigris(®) is a fully automated system with high level of security during the analytical process, reducing the number of invalid or non-available results observed with the previous semi-automated technologies. Moreover, renewal of material by fully automated and secure systems, especially for the critical step of sample pipeting, may provide additional safety in blood screening laboratories.

Recipients potentially infected with parvovirus B19 by red blood cell products

Since 2000, blood donor screening for parvovirus B19 (B19) by nucleic acid testing (NAT) at the Ulm Institute has been conducted 6 to 8 weeks postdonation, that is, after transfusion of cellular blood products, whereas at the Frankfurt Institute all donations are screened before releasing any blood product. In this study, we evaluated the infectivity of B19-positive blood products in relation to the virus concentration in the transfused blood component. Recipients were classified into two groups (A, transfused with blood products with B19 virus load less than 10(5) IU/mL; and B, transfused with blood products with B19 virus load greater than 10(5) IU/mL). Phylogenetic analyses were done for B19 DNA-positive donor and recipient pairs in the variant VP-1u genome region. All samples were investigated for immunoglobulin (Ig)M and IgG B19 antibodies. B19 DNA was detected in 9 of 18 recipients of red blood cells (RBCs) from Group B, whereas none of the 16 recipients of RBCs from Group A were positive for B19 DNA (p=0.016). Phylogenetic analysis demonstrated identical genomic sequences between the donors and recipients. Because recipient B19 DNA and antibody results were not available before transfusion, we interpret our overall data to indicate equivocal evidence of B19 transmission by RBC transfusion. B19 transmission by cellular blood products correlates with the virus concentration and the concentration of neutralizing antibodies. Thus, blood donor screening for B19 by minipool NAT should be done to supply at-risk patients (e.g., immunosuppressed patients) with B19-negative blood components.

Transfusion‐transmitted human immunodeficiency virus (HIV) from seroconverting donors is rare in England and Wales: results from HIV lookback, October 1995 through December 2008

Lookback is considered when human immunodeficiency virus (HIV) is detected in a repeat blood donor in case the immediately previous negative donation was donated in the infectious window period (IWP) or the assay(s) produced a false-negative result. HIV lookback investigations undertaken by NHS Blood and Transplant and the Welsh Blood Service between October 1995 and December 2008 are described. Investigations were undertaken into the previous negative donations of 113 HIV-infected donors, including retrospective testing of archive samples, tracing of components, and identification of recipients who were offered HIV testing when appropriate. Data were collated on HIV seroconverters and outcome of the lookback was summarized. Two previous negative donations given before the introduction of minipool nucleic acid testing (MP-NAT) screening were confirmed positive by individual retrospective polymerase chain reaction (PCR) testing of the archive specimen. Red blood cell components had been transfused from both donations. One recipient died after transfusion, and the other was alive and tested HIV positive. All 23 (20%) donations previously testing negative by MP-NAT were confirmed to be PCR negative on individual testing of an archive specimen and none of the tested recipients of these donations had evidence of transfusion-transmitted HIV. The yield of lookback was low with one positive recipient identified over 13 years of surveillance: HIV transmission occurred from a window period donation given before the introduction of MP-NAT screening and would have been detected using current testing methods. Current residual risk estimates for the United Kingdom predict that HIV lookback will be of limited benefit, as demonstrated by our data.

Development of multiple primer, HIV mini-pool NAT and its application in detecting acute infection of MSM

Objective To establish a mini-pool nucleic acid testing (NAT) assay using multiplex RT-nested PCR for the detection of HIV RNA, and apply it in screening for acute HIV infection among MSM. Methods Frozen EDTA plasma samples collected between Oct. 2008 and Mar. 2009 from 3 HIV infectors during window-period, a total of 30 HIV chronically infected individuals and 97 healthy subjects were used to develop the NAT assay. Plasma samples from 10 cases were pooled into one tube and centrifuged at high speed for the collection of viruses. HIV RNA was extracted. Two pairs of primers were designed according to two conserved regions of HIV RNA ( HXB2 nt 5783-nt 6228 and nt 1235-nt 2012).Multiplex RT-PCR and nested PCR were performed. Individual NAT-reactive samples were confirmed by commercially available NAT assays. The sensitivity and performance efficacy were also evaluated. The assay was then applied to 1 005 plasma specimens from MSM with negative or uncertain HIV antibody test results.These were collected in the same period as the other samples. Results ( 1 ) Two fragments of HIV were amplified successfully with the low detection limit of 162 copies/ml plasma; (2) Results of the mini-pool HIV NAT validation with samples from 3 HIV infectors during window-period were consistent with the expected values; (3) All 30 plasma samples from MSM with positive HIV antibody, which were tested by multiplex RT nested PCR, were found to be HIV RNA positive; (4) One out of 1 005 plasma samples was found to be HIV RNA positive, for this case acute infection was followed-up and sero-conversion was found. Conclusion Mini-pool NAT has good sensitivity, and may be applied to screening HIV RNA among MSM during window-period. Key words: HIV infections; Homosexuality, male; HIV-1; Nucleic acid amplification techniques

Refinement of a viral transmission risk model for blood donations in seroconversion window phase screened by nucleic acid testing in different pool sizes and repeat test algorithms

In minipool nucleic acid test (MP-NAT) screening protocols, the donations implicated in reactive test pools are released for transfusion when they are nonreactive in a repeat test on the individual samples, but in individual-donation (ID)-NAT screening algorithms the release of nonrepeatable reactive (NRR) donations is under discussion. A previously developed window phase (WP) transmission risk model for NAT-screened blood transfusions has been refined to take the effect of repeat tests of initially reactive (IR) MP- or ID-NAT results into account. The model has then been applied to simulate the effect of different screening algorithms with ULTRIO and the new-generation ULTRIO Plus assay (Novartis Diagnostics) on transmission risk for hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV). We calculated WP risk-day equivalents for MP16-, MP8-, and ID-NAT with and without duplicate retesting of IR results of 3.1, 2.7, 1.5, and 1.3 days for HCV; 6.3, 5.5, 3.3, and 2.9 days for HIV; and 24.4, 22.2, 15.6, and 14.1 days for HBV, respectively. These latter infectious HBV WPs reduced to 20.4, 18.2, 11.6, and 10.3 days, respectively, with the more sensitive ULTRIO Plus assay. ULTRIO Plus ID-NAT screening reduces the virus transmission risk in the WP by 54% to 58% compared to ULTRIO MP16-NAT, while the incremental risk caused by releasing donations with duplicate ID-NAT NRR results is 5% to 6%. To achieve maximum safety and specificity a similar repeat test algorithm can be applied to ID-NAT as used for serologic assays.

First transmission of human immunodeficiency virus Type 1 by a cellular blood product after mandatory nucleic acid screening in Germany

In February 2007, a 63-year-old man underwent surgery. Retrospective testing with nucleic acid testing (NAT) showed that the patient was human immunodeficiency virus Type 1 (HIV-1) positive 10 days after transfusion. The transfusion-transmitted infection had been identified by a donor-related lookback started in April 2007 after anti-HIV seroconversion. Sequence analysis was performed in the gag-pol region as well as in the V3 loop env region. Archived plasma from the transmitting donation was investigated for the individual-donation NAT with the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 test (Roche CAP/CTM HIV-1 test) and for HIV antigen/antibody combination testing (Abbott Architect). Additional testing was done on the donor's follow-up sample and on the recipient's sample. The Roche CAP/CTM HIV-1 test failed to detect viral RNA by minipool NAT in the index donation (April 2007) as well as in the donation that caused the infection (January 2007). Phylogenetic analysis showed a very high genetic similarity among viral sequences from both donor and recipient, proving the HIV-1 transmission by sequence data. This case represents the first documented HIV-1 transmission by transfusion of red blood cells after mandatory introduction of HIV-1 NAT for blood screening in Germany. Low viral load and mismatches in the primer/probe region might explain the detection failure of the NAT screening assay. A certain risk remains that new virus variants contain mutations at positions critical for amplification or detection of viral genomes. An option to reduce the risk of a detection failure by NAT is the simultaneous use of several conserved regions as amplification targets.

Blood donor screening with cobas s 201/cobas TaqScreen MPX under routine conditions at German Red Cross institutes

In 1997 the German Red Cross (GRC) blood donor services introduced mini-pool nucleic acid testing (NAT) for human immunodeficiency virus (HIV)-1, hepatitis C virus (HCV) and hepatitis B virus (HBV) to increase blood safety. With the new cobas s 201/cobas TaqScreen MPX, a fully automated extraction method and a multiplex amplification system specifically adapted to the needs of blood donation services is available. The cobas s 201 system was evaluated at the GRC BTS locations Hagen, Springe and Frankfurt. In phase A, the analytical sensitivity for the detection of HBV, HCV and HIV-1 was investigated and in phase B, at least 60,000 samples at each test site were screened in parallel with the MPX test on s 201 system and the existing routine mini-pool NAT system to compare the diagnostic specificity and the diagnostic sensitivity. Comparable analytical sensitivities in a range of 1.6-3.6 IU/ml, 4.9-10.9 IU/ml and 14.7-26.6 IU/ml for HBV, HCV HIV, respectively, for the MPX test on s 201 system (95% probability based on probit analysis) were determined at all test sites. The diagnostic sensitivity was 99.8% and the diagnostic specificity was 99.85%. The MPX test on s 201 system is a fully automated NAT system suitable for routine blood donor screening. The analytical sensitivity as well as the diagnostic sensitivity fulfilled all requirements of the Paul Ehrlich Institute for blood donor screening in mini-pools up to 96 donations per pool. A major benefit of the automated NAT system is the reduced personnel time and the extensive complete barcode-controlled process documentation.

Effectiveness of various minipool nucleic acid test systems for hepatitis B virus detection in blood donors

Effectiveness of various minipool nucleic acid test systems for hepatitis B virus detection in blood donors

A modeling framework for evaluation and comparison of trigger strategies for switching from minipool to individual‐donation testing for West Nile virus

To decrease the likelihood of transmission from donations containing West Nile virus (WNV) levels below minipool nucleic acid test (MP-NAT) detection limits, blood centers switch from MP-NAT to individual-donation testing (ID-NAT) after detection of MP-NAT-positive donations. The effectiveness of strategies to trigger or discontinue ID-NAT screening is largely unknown. Twenty-seven strategies to trigger and discontinue ID-NAT screening were evaluated with a statistical model based on known dynamics of WNV infection and historical data on WNV prevalence among blood donations. Breakthroughs were defined as WNV immunoglobulin M antibody-negative, viremic (RNA-positive) donations that could only be identified by ID-NAT, but were screened by MP-NAT. Effectiveness (proportional reduction of breakthroughs relative to MP-NAT screening alone) and efficiency (absolute reduction of breakthroughs relative to the number of tests performed) were estimated by simulating donation years of varying outbreak severities over a range of blood collection frequencies. Most strategies were effective (>75% reduction in breakthroughs) when daily donations exceeded 560. In larger centers (1008 donations daily), effectiveness of trigger-on strategies based on absolute number of MP-NAT-positive donations improved, but worsened for strategies using rate-based criteria. Effectiveness increased slightly by triggering on one MP-NAT-positive rather than two and increased substantially by increasing the duration from 7 to 14 days that no ID-NAT-positive donations are detected before resuming MP-NAT. Most trigger strategies become effective when test results from at least 560 donations daily are considered. A 14-day ID-NAT period may improve safety relative to the increase in the number of tests performed.

Two HBV DNA+/HBsAg− blood donors identified by HBV NAT in Shenzhen, China

Background In order to further improve blood safety, mini-pool (MP) nucleic acid testing (NAT) was implemented to screen samples negative for hepatitis B surface antigen (HBsAg), anti-hepatitis C virus (anti-HCV), anti-human immunodeficiency virus (anti-HIV), syphilis (anti-Treponemal antibody) and with normal ALT. Study design and methods From August 2006 to February 2008, 41,301 donations were screened using commercial HIV/HCV RNA and HBV DNA Real-Time PCR NAT assays in pools of 8. Reactive pools were re-tested as individual samples using the appropriate screening test and confirmed using an alternate commercial NAT assay. Donors reactive on both NAT assays were considered ‘confirmed’ positive for the virus concerned and recalled for additional follow-up testing and counseling. Results Of the 41,301 samples screened, no HIV or HCV RNA-positive/seronegative donations were detected but two HBV DNA positive/HBsAg negative blood donors (Donors 1 and 2) were identified. Their respective hepatitis immunological markers were: Donor 1 – anti-HBc positive/anti-HBe positive/HBeAg negative/ALT normal and HBV DNA viral load of 112 IU/ml; Donor 2 – anti-HBc positive/anti-HBe negative/HBeAg negative/ALT normal and HBV DNA viral load 2750 IU/ml. Conclusions MP NAT identified two HBsAg negative donors with presumed occult infection but no HIV or HCV seronegative/NAT positive (yield) donors. The HBV yield rate of 1 in 20,650 (95%CI – 1 in 5663 to 1 in 75,303) is comparatively high, exceeds the predicted rate based on previous modeling for the population and demonstrates the incremental blood safety value of NAT in countries where HBV is highly epidemic. The low viral load of the two yield samples underscores the importance of optimizing the sensitivity of the HBV NAT assay selected for screening.

Comparison of different methods of bacterial detection in blood components

Background Over the last two decades, the residual risk of acquiring a transfusion‐transmitted viral infection has been reduced to less than 1 : 1 000 000 via improvements in different techniques (e.g. donor selection, leuco‐depletion, introduction of 3rd or 4th generation enzyme‐linked immunosorbent assays and mini‐pool nucleic acid testing (MP‐NAT). In contrast, the risk for transfusion‐associated bacterial infections has remained fairly stable, and is estimated to be in a range between 1 : 2000 and 1 : 3000. Platelets are at an especially higher risk for bacterial contamination, because they are stored at room temperature, which provides good culture conditions for a broad range of bacterial strains. To improve bacterial safety of blood products, different detection systems have been developed that can be divided into culture systems like BacT/ALERT or Pall eBDS, rapid detection systems like NAT systems, immunoassays and systems based on the FACS technique. Culture systems are used for routine bacterial screening of platelets in many countries, whereas rapid detection systems so far are mainly used in experimental spiking studies. Nevertheless, pathogen‐reduction systems are currently available for platelet concentrates and plasma, and are under investigation for erythrocytes.Methods In this review, the functional principles of the different assays are described and discussed with regard to their analytical sensitivity, analytical specificity, diagnostic sensitivity, diagnostic specificity and clinical efficiency. The detection methods were clustered into three groups: (i) detection systems currently used for routine screening of blood products, (ii) experimental detection systems ready to use for routine screening of blood products, and (iii) new experimental detection systems that need to be investigated in additional spiking studies and clinical trials.Results A recent International Society of Blood Transfusion international forum reported on bacterial detection methods in 12 countries. Eight countries have implemented BacT/ALERT into blood donor screening, whereas in three countries only quality controls were done by culture methods. In one country, shelf‐life was reduced to 3 days, so no bacterial screening was implemented. Screening data with culture methods can be used to investigate the prevalence of bacterial contamination in platelets. Differing results between the countries could be explained by different test definitions and different test strategies. Nevertheless, false‐negative results causing severe transfusion‐related septic reactions have been reported all over the world due to a residual risk of sample errors. Rapid screening systems NAT and FACS assays have improved over the last few years and are now ready to be implemented in routine screening. Non‐specific amplification in NAT can be prevented by pre‐treatment with Sau3AI, filtration of NAT reagents, or reduction of the number of polymerase chain reaction cycles. FACS systems offer easy fully automated handling and a handling time of only 5 min, which could be an option for re‐testing day‐5 platelets. New screening approaches like immunoassays, detection of bacterial adenosine triphosphate, or detection of esterase activity need to be investigated in additional studies.Conclusion Bacterial screening of blood products, especially platelets, can be done with a broad range of technologies. The ideal system should be able to detect one colony‐forming unit per blood bag without a delay in the release process. Currently, we are far away from such an ideal screening system. Nevertheless, pathogen‐inactivation systems are available, but a system for all blood components will not be expected in the next few years. Therefore, existing culture systems should be complemented by rapid systems like NAT or FACS especially for day‐5 platelets.

Sensitivity of two hepatitis B virus, hepatitis C virus (HCV), and human immunodeficiency virus (HIV) nucleic acid test systems relative to hepatitis B surface antigen, anti‐HCV, anti‐HIV, and p24/anti‐HIV combination assays in seroconversion panels

Accurate determination of the infectious window period (IWP) that remains with individual-donation (ID) or minipool (MP) NAT compared to those with serology assays is essential for residual risk estimations. The relative sensitivity of the Procleix Tigris system (Gen-Probe/Chiron) used in ID-NAT format and cobas s 201 (Roche Molecular Systems) applied in 1:6 diluted samples to mimic six-minipool (MP6) nucleic acid test (NAT) was assessed by quadruplicate testing of five seroconversion panels per marker. A mathematical analysis based on the log-linear increase of viremia in the ramp-up phase, as established with bDNA 3.0 assays enabled estimation of the IWP for human immunodeficiency virus (HIV) and hepatitis B virus (HBV) assays. The mean IWPs were Tigris HIV RNA 5.5 days, s 201 (1:6) HIV RNA 7.4 days, GenScreen Plus p24/anti-HIV 17.8 days, PRISM anti-HIV 19.0 days, Tigris HBV DNA 20.6 days, s 201 (1:6) HBV DNA 22.6 days, Bio-Rad hepatitis B surface antigen (HBsAg) 37.8 days, and PRISM HBsAg 35.5 days. At estimated 50 percent NAT seroconversion rates, s 201 (1:6) and Tigris showed mean window-period reduction times (WPRTs) of 30.5 to 35.5 days to hepatitis C virus antibody (anti-HCV) assays, 10.4 to 13.5 days to anti-HIV, or combination p24/anti-HIV assays and 12.8 to 17.2 days to HBsAg assays. Tigris ID-NAT detected HIV RNA 2 days earlier than s 201 MP6-NAT, but the difference in sensitivity between the two NAT systems was not significant in HBV seroconversion panels. Insufficient seroconversion samples were available for reliable modeling of WPRT in early HCV infection, but 1.4 to 2.0 days could be predicted by translating analytical sensitivity data. Both multiplex NAT systems demonstrate significant WPRTs compared to (combined) antigen and antibody assays.

Hepatitis B virus traceback and lookback: factors to consider

Hepatitis B virus traceback and lookback: factors to consider

Dengue and Chikungunya viruses in blood donations: risks to the blood supply?

Dengue and Chikungunya viruses in blood donations: risks to the blood supply?

Blood donor screening for parvovirus B19 in Germany and Austria

Although the main transmission pathway of parvovirus B19 (B19) is typically via the respiratory route, several transfusion-transmitted infections have been reported. To increase blood safety, all blood donations to our blood donor service have been screened by a B19 minipool real-time nucleic acid testing (NAT) since April 2000. Additional customers have been screened since the summer of 2003. In total, 2.8 million donations from Germany and Austria were screened for B19 by real-time minipool NAT. A subgroup of 50 B19 DNA-positive donors was screened for B19 immunoglobulin G (IgG) and IgM antibodies and B19 DNA over a 6-month period. Results were compared to those of 100 B19 DNA-negative donors. Data accumulated over the past 6 years indicate a high incidence period from May 2004 to January 2006. In total, the incidence was 12.7 and 261.5 per 100,000 donations with high virus loads equal to or above 10(5) and below 10(5) IU per mL, respectively. Median virus concentration in the case group was 4.85 x 10(7) IU per mL at Time Point T0 and was reduced to 4 x 10(2) IU per mL at the time of the next donation (3 months later). Neutralizing antibodies (VP2) were detected in all donations if virus load was reduced to less than 10(5) IU per mL. The release of B19 DNA-positive blood products with a concentration of less than 105 IU per mL is thought to be safe due to the high level of neutralizing VP2 antibodies and is currently examined in a donor recipient infectivity study. In contrast, blood products with a high B19 DNA concentration (> or =10(5) IU/mL), some of which did not contain neutralizing antibodies, were discarded to protect at risk individuals.

A multi–Chinese blood center study testing serologic‐negative donor samples for hepatitis C virus and human immunodeficiency virus with nucleic acid testing

A multi-blood center study was conducted to evaluate a human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) multiplex nucleic acid testing (NAT) donor screening test and to determine the residual risk for HIV-1 and HCV infection. A commercially available HIV-1 and HCV assay (Procleix, Chiron Corp.) was used for simultaneous detection of HIV-1 RNA and HCV RNA on 89,647 unlinked donor samples. NAT was performed with pools of 16 samples that had passed all routine screening tests. Single-donor NAT was performed for samples that had been disqualified by any reactive screening test result(s). Anti-HCV (Ortho third-generation HCV enzyme immunoassay [EIA]), alanine aminotransferase, and HCV NAT (Roche COBAS Amplicor HCV test) confirmatory tests were used for HCV EIA-nonreactive, HCV NAT-reactive samples. Three HCV NAT yield cases and no HIV-1 yield cases were detected. The yield rate for HCV NAT was 3.4 per 10(5) (95 percent confidence interval [CI], 0.7-9.8). The estimated incidence rate for HCV is 24.2 per 100,000 person-years (95% CI, 3.4-88.0). If minipool NAT is added to routine donor screening, the residual risk for HCV is estimated to be reduced to 1 in 20.4x10(4) (95% CI, 1 in 5.2x10(4)-1 in 165.5x10(4)). The residual risk for transfusion-transmitted HCV infection is still relatively high in China. Incorporating NAT technology into blood donor screening would be estimated to reduce the residual risk of HCV infections eightfold over current EIA screening.

West Nile Virus Adheres to Human Red Blood Cells in Whole Blood

Background. West Nile virus (WNV) is endemic in the United States. It is transmissible by blood transfusion, and the nation's blood supply is currently screened for WNV. Documented transmission of WNV infection through red blood cell (RBC) units in which the plasma co-component had a low viral load could be explained, in at least 1 instance, by cell-association of WNV; in this case, the RBC unit was released as negative by minipool nucleic acid testing (NAT) performed on plasma but was intermittently NAT-positive when subsequently tested as an individual sample. We hypothesized that a proportion of WNV bound to blood cells and was not measured by NAT performed on plasma samples. We have investigated whether WNV binds to RBCs, leading to reduction of WNV RNA detection by NAT performed on plasma samples.Methods. Equal volumes of leukoreduced RBCs and their corresponding plasma components from 20 blood donors with NAT results that were positive for WNV were tested in 5 replicates by reverse-transcriptase polymerase chain reaction TaqMan for WNV. In addition, aliquots from 8 of the RBC units were tested by infectivity assays using Vero cells.Results. The reverse-transcriptase polymerase chain reaction TaqMan assay showed that the viral load in the RBC components exceeded that in the corresponding plasma units by 1 order of magnitude. In addition, viruses associated with the RBCs were infectious in Vero cell cultures.Conclusions. These observations reinforce the notion that extraction of viral RNA from whole blood could improve assay sensitivity for blood donor screening and further reduce the residual risk of WNV transmission through transfusion.