Michael P. Busch

Approximately 20 percent of persons infected with hepatitis C virus (HCV) clear viremia. Factors associated with resolution of viremia are not well defined. Implementation of routine nucleic acid testing (NAT) of blood donors has yielded a large data set for analysis of demographic correlates of resolved viremia. HCV antibody and NAT data, liver enzyme (alanine aminotransferase [ALT]) results, and donor demographic characteristics were compiled for 2,579,290 allogeneic donations given at five large blood centers after NAT implementation in 1999 through December 2001. Donation HCV RNA status was compared between first-time donors categorized by ALT levels, sex, age, race and/or ethnicity, country of birth, level of education, blood center location, and blood group, with chi-square tests and multivariable logistic regression methods. Of 35 confirmed-seropositive repeat donors, 19 (54.3%) tested negative for the presence of HCV RNA; there was no association between RNA status and preseroconversion intervals (p = 0.74). Of 2105 RIBA-positive, first-time donors, 402 (19.1%) tested negative for the presence of HCV RNA by NAT (presumptive resolved infections). There were significant differences in the frequency of RNA negativity among first-time donors categorized by ALT levels and by race and/or ethnicity. ALT levels were more likely to be elevated in RNA-positive, first-time donors (p < 0.0001). Viremia was less likely to resolve in Asian (8.2%) and black non-Hispanic (14.4%) donors than in white non-Hispanic (20.7%), Hispanic (22.1%), and other race and/or ethnicity (22.1%) donors (p = 0.02). No significant associations were found for age, sex, country of origin, level of education, blood type, and donor center location. These results confirm that the frequency of HCV RNA negativity among seropositive persons differs by race and/or ethnicity. Follow-up studies of donors with resolved viremia are warranted to further elucidate viral, immunologic, and genetic factors underlying spontaneous viral clearance.

BACKGROUND: Screening donors for human immunodeficiency virus (HIV) and hepatitis C virus (HCV) RNA is primarily performed on minipools (MPs) with one of two commercial nucleic acid amplification tests (NAT; Roche Molecular Systems; or Gen‐Probe/Chiron). We compared these assays with respect to detection of RNA in early HIV and HCV infection.STUDY DESIGN AND METHODS: Twelve HIV plasma donor panels (116 serial samples) and 12 HCV panels (180 serial samples) were selected to optimally represent early viremia. Initial testing was performed in singlicate or triplicate on separately coded aliquots, both neat and at dilutions corresponding to MP screening (1:16 for Gen‐Probe; 1:24 for Roche); 20 additional replicates were performed when discordant results were observed. Odds ratios (ORs) comparing detection of RNA by different assays were derived with logistic regression models. Differences in window‐period closure and yields of assays in MP or individual‐donation (ID) format were estimated.RESULTS: Differences in detection rates between Roche and Gen‐Probe NAT assays were small and only observed with samples with very‐low‐level viremia. ORs for detecting RNA by the Gen‐Probe versus the Roche assay were significant for HIV if conducted on MPs (1.8; 95% confidence interval [CI], 1.3‐2.5) but not neat (1.0; 95% CI, 0.72‐1.4). Odds of detecting HCV RNA were higher if the Gen‐Probe assay was conducted either neat (2.3; 95% CI, 1.6‐3.2) or on MPs (4.0; 95% CI, 2.8‐5.8). These differences translated to CONCLUSIONS: Differences in sensitivities of licensed NAT assays for HIV and HCV are very small and clinically insignificant, particularly when compared to differences of MP versus ID NAT screening.

BACKGROUND: It is important to characterize viral dynamics in early hepatitis C virus (HCV) infection to further our understanding of viral pathogenesis and the potential for secondary transmission in acute infection through blood transfusion or other routes.STUDY DESIGN AND METHODS: Serial units given by 77 source plasma donors who had evolved from HCV RNA–negative to HCV RNA–positive by nucleic acid amplification technology (NAT) screening with 512‐unit pool‐NAT or were followed from RNA detection to antibody conversion were tested by individual NAT and quantitative RNA assays.RESULTS: During the ramp‐up phase when exponential growth occurs, HCV viral load doubled every 10.8 hours (95% confidence interval [CI], 9.9‐12.0). Intermittent viremia was observed before the ramp‐up phase in 37 of 50 panels with the earliest detectable viremic bleed occurring 63 days before the estimated onset of ramp‐up. The plateau phase or high‐titer viremic period that occurs between ramp‐up and seroconversion was estimated to last 56.3 days (95% CI, 44.8‐67.8).CONCLUSIONS: Intermittent low‐level HCV viremia can occur as much as 2 months before the periods of exponential increase in viral load and the high‐titer plateau‐phase viremia that usually precede seroconversion. Animal inoculation studies are in progress to evaluate if transfusion of low‐level viremic plasma can transmit HCV infection.

Transfusion-transmitted West Nile virus (WNV) infections were first reported in 2002, which led to rapid development of investigational nucleic acid amplification tests (NAT). A study was conducted to evaluate sensitivities of WNV screening and supplemental NAT assays first employed in 2003. Twenty-five member-coded panels were distributed to NAT assay manufacturers. Panels included five pedigreed WNV standards (1, 3, 10, 30, and 100 copies/mL), 15 or 16 donor units with very-low-level viremia identified through 2003 screening, and four or five negative control samples. Samples were tested neat in 10 replicates by all assays; for NAT screening assays, 10 replicates were also performed on dilutions consistent with minipool (MP)-NAT. The viral load distribution for 142 MP-NAT yield donations was characterized, relative to the analytical sensitivity of MP-NAT systems. Analytical sensitivities (50% limits of detection [LoD] based on Poisson model of detection of WNV standards) for screening NAT assays ranged from 3.4 to 29 copies per mL; when diluted consistent with MP pool sizes, the 50 percent LoD of screening NAT assays was reduced to 43 to 309 copies per mL. Analytical sensitivity of supplemental assays ranged from 1.5 to 7.7 copies per mL (50% LoD). Detection of RNA in donor units varied consistent with analytical LoD of assays. Detection of low-level viremia after MP dilutions was particularly compromised for seropositive units, probably reflecting lower viral loads in the postseroconversion phase. Based on the viral load distribution of MP-NAT yield donations (median, 3519 copies/mL; range, < 50-690,000), 13 to 24 percent of units had viral loads below the 50 percent LoD of screening NAT assays run in MP-NAT format. WNV screening and supplemental assays had generally excellent analytical sensitivity, comparable to human immunodeficiency virus-1 and hepatitis C virus NAT assays. The presence of low-level viremic units during epidemic periods and the impact of MP dilutions on sensitivity, however, suggest the need for further improvements in sensitivity as well as a role for targeted individual-donation NAT in epidemic regions.

In some patients multiply transfused to treat severe trauma, white blood cells (WBCs) from a single blood donor can persist for years, constituting up to 5 percent of all circulating WBCs. The immunologic mechanisms responsible for this are not known but, if understood, might allow manipulation of the human immune system to induce microchimerism for a variety of therapeutic purposes. To better characterize these mechanisms, a murine transfusion model was developed with a panel of immunologic knockouts as transfusion recipients. By conducting a systematic series of transfusion experiments, the purpose was to determine which recipient immune cell population, when abrogated, could lead to prolonged survival of donor cells (microchimerism).Blood was transfused from normal donors to knockout recipients in syngeneic, allogeneic, and xenogeneic settings. Donor WBC survival was evaluated by quantitative polymerase chain reaction, and recipient lymphocyte subsets by fluorescence-activated cell sorting.In the syngeneic setting, donor WBCs persisted in C2ta, RAG-1, and TCR knockout recipients. Allogeneic donor WBCs persisted in RAG-2 and RAG-2/Common gamma knockout recipients. Xenogeneic donor WBCs required RAG-2/Common gamma and RAG-2/Pfp double knockouts to persist.It is concluded that donor-recipient major histocompatibility complex (MHC) concordance alone is not sufficient to achieve microchimerism. Further, the degree of recipient immune compromise necessary to achieve persistent microchimerism is directly proportional to the degree of donor-recipient MHC disparity.

BACKGROUND:  Estimates for human immunodeficiency virus (HIV)‐1 and hepatitis C virus (HCV) transfusion‐transmitted risks have relied on incidence derived from repeat donor histories and imprecise estimates for infectious, preseroconversion window periods (WPs).STUDY DESIGN AND METHODS:  By use of novel approaches, WPs were estimated by back‐extrapolation of acute viral replication dynamics. Incidence was derived from the yield of viremic, antibody‐negative donations detected by routine minipool nucleic acid testing (MP‐NAT) of 37 million US donations (1999‐2002) or from sensitive/less‐sensitive HIV‐1 enzyme immunoassay (S/LS‐EIA) results for seropositive samples from 6.5 million donations (1999). Incidences and WPs were combined to calculate risks and project yield of individual donation (ID)‐NAT.RESULTS:  The HIV‐1 WP from presumed infectivity (1 copy/20 mL) to ID‐NAT detection was estimated at 5.6 days, and the periods from ID to MP‐NAT detection and from MP‐NAT to p24 detection at 3.4 and 6.0 days, respectively; corresponding estimates for HCV were 4.9, 2.5, and 50.9 days (the latter represents period from MP‐NAT to HCV antibody detection). The HIV‐1 incidence projected from MP‐NAT yield or from S/LS‐EIA data was 1.8 per 100,000 person‐years, resulting in a corresponding HIV‐1 transfusion‐transmitted risk of 1 in 2.3 million. The HCV incidence from MP‐NAT yield was 2.70 per 100,000 person‐years with a corresponding risk of 1 in 1.8 million donations. Conversion from MP‐NAT to ID‐NAT was projected to detect two to three additional HIV‐1 and HCV infectious units annually.CONCLUSIONS:  MP‐NAT yield and S/LS‐EIA rates can accurately project transfusion risks. HCV and HIV‐1 risks, currently estimated at 1 per 2 million units, could be reduced to 1 in 3 to 4 million units by ID‐NAT screening.

Background and Objectives  Blood banks in the USA have recently introduced minipool nucleic acid amplification testing (MP‐NAT) of blood products to reduce the transmission of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) by transfusions. However, MP‐NAT is limited in its ability to detect preseroconversion samples with very low viral RNA loads.Materials and Methods  To determine whether a red blood cell unit, from an MP‐NAT‐negative donation, transmitted HIV when transfused to a patient, we compared the viral sequences from the blood donor and recipient. The implicated donation was also tested by commercially available NAT assays at a range of dilution factors to determine whether the infectious unit could have been detected using individual‐donation NAT (ID‐NAT).Results  Phylogenetic linkage of HIV sequences in the blood donor and recipient confirmed the transmission of HIV by blood transfusion, the first such case identified since introduction of MP‐NAT screening in 1999. Viral RNA was reliably detected by ID‐NAT, but only inconsistently detected by MP‐NAT.Conclusions  Even following the introduction of MP‐NAT, a preseroconversion donation with a viral load of ≤ 150 copies of RNA/ml went undetected and resulted in an HIV transmission. Implementation of ID‐NAT will further reduce such rare transmissions, but at a considerable cost per infectious unit interdicted.