Determination of Babesia microti seroprevalence in blood donor populations using an investigational enzyme immunoassay.

BACKGROUND: Transfusion-transmitted babesiosis caused by Babesia microti has emerged as a significant risk to the US blood supply. This study estimated the prevalence of B. microti antibodies in blood donors using an investigational enzyme immunoassay (EIA). STUDY DESIGN AND METHODS: A peptide-based EIA that detects both immunoglobulin (Ig)G and IgM antibodies to B. microti was developed and validated. Donor samples randomly selected from areas defined as high-risk endemic, lower-risk endemic, and nonendemic for B. microti were deidentified and tested using the investigational EIA. Samples that were EIA repeat reactive were further tested by B. microti immunofluorescent assay (IFA), polymerase chain reaction (PCR) on red blood cell lysates, and peripheral blood smear examination. A random subset of 1272 samples from high-risk endemic areas was tested by IFA, PCR, and peripheral blood smear in parallel with EIA. RESULTS: Among 15,000 donations tested with the investigational B. microti EIA, EIA repeat-reactive rates were 1.08% (54/5000) in a high-risk endemic area, 0.74% (37/5000) in a lower-risk area, and 0.40% (20/5000) in a nonendemic area. After application of a revised cutoff, these values were reduced to 0.92%, (46/5000), 0.54% (27/5000), and 0.16% (8/5000). Overall concordance between EIA and IFA among donor samples was 99.34%. One seropositive sample was positive by PCR. CONCLUSION: The seroprevalence of B. microti in blood donors in a high-risk area measured by an investigational EIA was approximately 1%. The EIA shows promise as an efficient high-throughput blood donor screening assay for B. microti.

Epidemiologic and laboratory findings from 3 years of testing United States blood donors for Trypanosoma cruzi.

BACKGROUND: At most blood centers in the United States routine testing of donations for Trypanosoma cruzi using an enzyme-linked immunosorbent assay (ELISA) is followed by supplemental testing by radioimmunoprecipitation assay (RIPA). The objective of this study was to report the results of routine testing and risk factor data from allogeneic blood donors. STUDY DESIGN AND METHODS: T. cruzi testing data from January 2007 through December 2009 were analyzed, and risk factor interviews and follow-up studies were conducted on seroreactive donors. Prevalences of confirmed infection and risk factors associated with infection were assessed using logistic and multivariable logistic regression. RESULTS: Of 2,940,491 allogeneic donations from 1,183,076 donors, 305 (0.01% per donation tested and 0.026% per blood donor) were repeat reactive (RR) and 89 of those were confirmed positive by RIPA, yielding an overall seroprevalence of 1 per 33,039 donations and 1 per 13,292 donors. Country of birth and US blood center location differences in the seroprevalence of T. cruzi were evident. The odds of confirmed infection were highest if the donor reported having been bitten by the reduviid (kissing) bug (odds ratio [OR], 76.1; 95% confidence interval [CI], 11.1-3173) followed by having lived in a rural area of Latin America (OR, 38.6; 95% CI, 15.1-102.5). In multivariable analyses, having spent 3 months or more in Mexico or Central and/or South America was associated with the highest odds of RIPA-confirmed infection (OR, 8.5; 95% CI, 2.7-26.5). Polymerase chain reaction (PCR) testing of ELISA RR donors exhibited low sensitivity (1/22 [4%] RIPA-confirmed donors was PCR positive). CONCLUSION: Risk factors for confirmed infection in US blood donors are consistent with the known epidemiology of Chagas disease. Blood donors or transfusions do not substantially contribute to the burden of T. cruzi infection in the United States.

West Nile virus testing experience in 2007: evaluation of different criteria for triggering individual-donation nucleic acid testing.

BACKGROUND: In 2007, clients served by Blood Systems Laboratories used variable approaches for triggering West Nile virus (WNV) RNA individual-donation (ID) nucleic acid testing (NAT). These included two minipool (MP) NAT-reactive donations and a greater than 1:1000 rate in a 7-day interval (primary trigger), criteria based on one MP-NAT-reactive donation when there was WNV activity in overlapping and/or adjacent geographic areas (neighbor trigger), or zero MP-NAT-reactive donation (self-trigger). STUDY DESIGN AND METHODS: The Procleix WNV assay was used in either a 16-sample MP or an ID format. NAT-repeat reactivity or anti-immunoglobulin M (IgM) positivity defined true positives (TPs). TPs that were negative on 1:16 dilution testing were considered ID-NAT yield cases. RESULTS: WNV NAT performed on 1,217,929 donations identified 162 TPs; 87 were detected by MP (rate of 0.008%) and 75 by ID (rate of 0.10%; p < 0.0001). There were 34 ID-NAT yield cases, including 4 IgM/immunoglobulin G (IgG)-negative and 9 IgM-positive/IgG-negative donations. Rates of yield cases by primary, neighbor, and self-triggering were 0.077, 0.052, and 0.004% (p = 0.0003). None of 11 ID-NAT yield cases detected by the neighbor trigger would have been detected if the primary trigger had been used. CONCLUSIONS: Primary triggering criteria identified 21 viremic donations that would have been missed by MP testing; however, 11 other low-level viremic donations required more stringent criteria (e.g., neighbor trigger) for detection. It is reasonable to adopt more stringent ID-NAT triggers, including elimination of the rate criterion and triggering on one NAT-reactive donation for regions adjacent to centers which have already triggered.

Genetic variability of West Nile virus in US blood donors, 2002-2005.

West Nile virus (WNV) was detected in the United States in 1999, has reoccurred every summer since, and has become endemic. Transfusion transmission was documented in 2002, and screening of blood donations for WNV began in 2003. We investigated genetic variation of WNV in human isolates obtained from specimens collected from 30 infected blood donors who tested positive for WNV RNA during 2002-2005. Complete genomic sequences of 8 isolates and structural gene sequences from 22 additional isolates were analyzed. We found some genetic diversity in isolates from different geographic regions and genetic divergence from reported sequences from epidemics in 1999-2001. Nucleotide divergence of structural genes showed a small increase from 2002 (0.18%) to 2005 (0.37%), suggesting absence of strong selective pressure and limited genetic evolution of WNV during that period. Nevertheless, WNV has continued to diverge from precursor isolates as geographic distribution of the virus has expanded.

Screening the blood supply for West Nile virus RNA by nucleic acid amplification testing.

BACKGROUND: The use of nucleic acid amplification tests of "minipools" of 16 samples to screen blood donors for West Nile virus RNA began in July 2003. We report the yield and characteristics of positive donations and the incremental yield and safety of nucleic acid amplification tests of individual donations. METHODS: Reactive minipools were analyzed to identify the individual reactive donations. For the regions with the highest yield on minipool testing, retrospective nucleic acid amplification testing was performed on individual donations that were negative on minipool testing. Reactive donations were confirmed by alternative nucleic acid amplification tests and IgM and IgG tests, and donors were followed to document seroconversion. RESULTS: From July 1 through October 31, 2003, 677,603 donations were prospectively screened for West Nile virus by minipool testing, yielding 183 confirmed viremic donations (0.027 percent, or 1 in 3703 donations). Retrospective individual testing of 23,088 donations from high-prevalence regions that were negative on minipool testing yielded 30 additional units with a low level of viremia, with 14 additional viremic units detected by prospective testing of individual donations late in the 2003 transmission season. Of all the viremic units detected, 5 percent were detected only by individual testing and were negative for IgM antibody, 29 percent were detected by individual testing after IgM seroconversion, and 66 percent were detected by minipool testing. West Nile virus infection was confirmed in both recipients of IgM-negative units that were reactive on individual testing, whereas neither recipient of antibody-positive blood components that were reactive on individual testing was infected. In 2004, prospective testing of individual donations in regions that yielded donations that were reactive on minipool testing resulted in a 32 percent incremental yield of units with a low level of viremia that would have been missed by minipool testing. CONCLUSIONS: Although nucleic acid amplification testing of minipools of blood donations prevented hundreds of cases of West Nile virus infection in 2003, it failed to detect units with a low level of viremia, some of which were antibody-negative and infectious. These data support the use of targeted nucleic acid amplification testing of individual donations in high-prevalence regions, a strategy that was implemented successfully in 2004.

Testing strategy to identify cases of acute hepatitis C virus (HCV) infection and to project HCV incidence rates.

Surveillance for hepatitis C virus (HCV) is limited by the challenge of differentiating between acute and chronic infections. In this study, we evaluate a cross-sectional testing strategy that identifies individuals with acute HCV infection and we estimate HCV incidence. Anti-HCV-negative persons from four populations with various risks, i.e., blood donors, Veterans Administration (VA) patients, young injection drug users (IDU), and older IDU, were screened for HCV RNA by minipool or individual sample nucleic acid testing (NAT). The number of detected viremic seronegative infections was combined with the duration of the preseroconversion NAT-positive window period (derived from analysis of frequent serial samples from plasma donors followed from NAT detection to seroconversion) to estimate annual HCV incidence rates. Projected incidence rates were compared to observed incidence rates. Projected HCV incidence rates per 100 person-years were 0.0042 (95% confidence interval [95% CI], 0.0025 to 0.007) for blood donors, 0.86 (95% CI, 0.02 to 0.71) for VA patients, 39.8 (95% CI, 25.9 to 53.7) for young IDU, and 53.7 (95% CI, 23.4 to 108.8) for older IDU. Projected rates were most similar to observed incidence rates for young IDU (33.4; 95% CI, 28.0 to 39.9). This study demonstrates the value of applying a cross-sectional screening strategy to detect acute HCV infections and to estimate HCV incidence.

Chagas disease and the US blood supply.

PURPOSE OF REVIEW: To describe new developments in blood-bank screening and management of patients with chronic Trypanosoma cruzi infection in the United States. RECENT FINDINGS: The first US Food and Drug Administration licensed serological test for T. cruzi blood screening went into widespread usage in January 2007. More than 500 confirmed T. cruzi-infected donations were detected by mid-June 2008. Until recently, drug therapy was recommended for acute and congenital infections, but seldom for chronic infections, which were believed to respond poorly. However, in the 1990s, efficacy was demonstrated in two placebo-controlled trials of benznidazole in children with chronic T. cruzi infection. In 2006, a nonrandomized, nonblinded trial demonstrated that benznidazole treatment may slow progression of cardiomyopathy and decrease mortality risk in infected adults. SUMMARY: Blood-bank screening will continue to detect T. cruzi-infected donors. Based on recent data, antitrypanosomal treatment is recommended for all acute and congenital T. cruzi infections, reactivated infection, and chronically infected children. In adults aged 19-50 years without advanced heart disease, treatment should generally be offered; management should be individualized for older adults. Less toxic, more effective drugs, a sensitive, specific assay for response to treatment, and improved healthcare access would promote more effective management.

Detection of HIV-1 and HCV infections among antibody-negative blood donors by nucleic acid-amplification testing.

BACKGROUND: Testing of blood donors for human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) RNA by means of nucleic acid amplification was introduced in the United States as an investigational screening test in mid-1999 to identify donations made during the window period before seroconversion. METHODS: We analyzed all antibody-nonreactive donations that were confirmed to be positive for HIV-1 and HCV RNA on nucleic acid-amplification testing of "minipools" (pools of 16 to 24 donations) by the main blood-collection programs in the United States during the first three years of nucleic acid screening. RESULTS: Among 37,164,054 units screened, 12 were confirmed to be positive for HIV-1 RNA--or 1 in 3.1 million donations--only 2 of which were detected by HIV-1 p24 antigen testing. For HCV, of 39,721,404 units screened, 170 were confirmed to be positive for HCV RNA, or 1 in 230,000 donations (or 1 in 270,000 on the basis of 139 donations confirmed to be positive for HCV RNA with the use of a more sensitive HCV-antibody test). The respective rates of positive HCV and HIV-1 nucleic acid-amplification tests were 3.3 and 4.1 times as high among first-time donors as among donors who gave blood repeatedly. Follow-up studies of 67 HCV RNA-positive donors demonstrated that seroconversion occurred a median of 35 days after the index donation, followed by a low rate of resolution of viremia; three cases of long-term immunologically silent HCV infection were documented. CONCLUSIONS: Minipool nucleic acid-amplification testing has helped prevent the transmission of approximately 5 HIV-1 infections and 56 HCV infections annually and has reduced the residual risk of transfusion-transmitted HIV-1 and HCV to approximately 1 in 2 million blood units.

Virus and antibody dynamics in acute west nile virus infection.

BACKGROUND: The dynamics of the early stages of West Nile virus (WNV) infection can be assessed by follow-up studies of viremic blood donors. METHODS: A total of 245 donors with WNV viremia were followed up weekly for 4 weeks and then monthly for up to 6 additional months or until seroconversion. Plasma samples were tested for WNV RNA by transcription-mediated amplification (TMA) and for WNV-specific IgM and IgG antibodies. RNA persistence was investigated by 6 replicate TMA tests; samples that were viremic for >40 days were tested for WNV-neutralizing activity. Follow up of 35 additional viremic donors for up to 404 days was conducted to evaluate persistence of WNV-specific antibody. RESULTS: The median time from RNA detection to IgM seroconversion was 3.9 days; to IgG seroconversion, 7.7 days; to RNA negativity by single-replicate TMA, 13.2 days; and to RNA negativity by 6-replicate TMA, 6.1 additional days after results of single-replicate TMA are negative. For 4 donors in whom RNA persisted for >40 days after the index donation, all samples obtained after this threshold were also positive for WNV IgG and neutralizing activity. The mean times to IgM and IgA negativity were 156 and 220 days, respectively. CONCLUSIONS: IgM and IgG develop rapidly after viremia and before RNA levels become undetectable, which occurred a mean of 13.2 days after the index donation among donors in this study. WNV RNA detection by replicate TMA rarely persists for >40 days after the index donation and is accompanied by WNV-specific neutralizing antibody, consistent with an absence of WNV transmission via transfusion of seropositive blood components.

Phylogenetic analysis of WNV in North American blood donors during the 2003-2004 epidemic seasons.

West Nile Virus (WNV) collected from 179 human blood donors in 25 US states and three Canadian provinces during the 2003 and 2004 epidemic seasons were genetically analyzed. The evolution of WNV during its Western spread was examined by envelope (E) gene sequencing of all 179 cases and full open reading frame sequencing of a subset of 20 WNV to determine if geographic and temporal segregation of distinct viral variants had occurred. Median joining network analysis was used to examine the genetic relationship between E gene variants and identified four large genetic clusters showing the gradual accumulation of mutations during the virus' western expansion. Two related WNV variants and their descendents, undetected in prior years, expanded in frequency. Apparent founder effects were observed in some regional outbreaks possibly due to local WNV colonization by a limited number of viruses. Amino acid mutations associated with newly expanding genetic variants reflect either selectively neutral mutational drift and/or mutations providing replicative advantages over the previously dominant forms of WNV.

Integration of nucleic acid amplification test results into hepatitis C virus supplemental serologic testing algorithms: implications for donor counseling and revision of existing algorithms.

BACKGROUND: The routine use of hepatitis C virus (HCV) nucleic acid amplification testing (NAT) donor screening assays has provided an opportunity for revision of the current HCV supplemental testing algorithm, which requires that recombinant immunoblot assay (RIBA) be performed on every HCV enzyme immunoassay (EIA)-repeat-reactive donation. The FDA has approved variance requests to use a new algorithm that eliminates the need to perform RIBA when HCV NAT results are reactive. Data are provided in support of this new algorithm. STUDY DESIGN AND METHODS: HCV EIA (including signal-to-cutoff optical density ratio), RIBA, and NAT data were compiled from 33.2 million donations screened over an approximately 4-year period by the American Red Cross and Blood Systems Laboratories. Further, donations having specific combinations of HCV EIA, RIBA, and minipool (MP) NAT results were evaluated, with more sensitive individual-donation (ID) NAT, to construct improved counseling messages for donors. RESULTS: Of 47,041 EIA-repeat-reactive donations, 49.3 percent were RIBA-positive, 17.1 percent RIBA-indeterminate, and 33.5 percent RIBA-negative. NAT-reactive rates were 79.2, 2.5, and 0.18 percent for RIBA-positive, -indeterminate, and -negative donations, respectively. The new algorithm classified an additional 1 percent of donations as HCV-infected while at the same time detecting all infections classified as HCV-infected under the current algorithm. An additional 2.4 percent of RIBA-positive, MP NAT-nonreactive donations were reactive when a frozen-thawed aliquot was retested by ID NAT. CONCLUSION: Integrating HCV NAT results with RIBA results for purposes of donor notification allows more appropriate counseling messages to be given to EIA-repeat-reactive donors. The new HCV supplemental algorithm is an acceptable alternative to the current algorithm because it provides equivalent or superior accuracy in formulating donor counseling messages and may also result in reduced costs and more timely notification of infected donors.

A new strategy for estimating risks of transfusion-transmitted viral infections based on rates of detection of recently infected donors.

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.

Triggers for switching from minipool testing by nucleic acid technology to individual-donation nucleic acid testing for West Nile virus: analysis of 2003 data to inform 2004 decision making.

BACKGROUND: Concern about West Nile virus (WNV) transfusion-transmitted infections missed by minipool (MP) nucleic acid testing (NAT) has prompted consideration of the use of individual-donation (ID) NAT. Strategies were investigated for the application of limited ID-NAT capacity in 2004. STUDY DESIGN AND METHODS: Patterns of WNV MP-NAT-reactive donations tested by the Blood Systems Laboratory each week for 79 blood centers from June 29 to November 23, 2003 (196 MP-NAT repeat-reactive [RR] donations among 801,697 units), were analyzed. ID-NAT initiation strategies were developed consisting of counts of RR donations and/or weekly RR rates, together with three ID-NAT discontinuation strategies, and ID testing burden was assessed based on these combined start and stop strategies. RESULTS: The effectiveness, reported as the percentage of MP-RR donations that would trigger ID-NAT based on each initiation strategy, ranged from 57 to 100 percent. The addition of a 1- or 2-week no-yield requirement for ID discontinuation substantially increased testing burden. Combined strategies resulted in projected ID-NAT of between 10 and 50 percent of donations for a 10- to 20-week period. For this organization, the most feasible ID-NAT initiation strategy was 2 MP-reactive donations and a weekly rate of 1 in 1000, which had an effectiveness of 81 percent and led to peak weekly ID-NAT of 20 to 25 percent of donations depending on the discontinuation rule. CONCLUSION: This new approach of targeted ID-NAT based on ongoing monitoring of MP-NAT yield may prove to be a rational policy for agents like WNV that cause seasonal and regional epidemics.

Clinical significance of nondiscriminated reactive results with the Chiron Procleix HIV-1 and HCV assay.

BACKGROUND: A NAT was developed (Procleix multiplex, Chiron Corporation) to simultaneously detect HIV-1 and HCV RNA with multiplex transcription-mediated amplification (mTMA) on pooled or single donations. HIV-1 and/or HCV RNA discriminatory probes confirm infection and discriminate the virus type. When a multiplex reactive sample does not react in discriminatory assays, the result is considered nondiscriminated reactive (NDR) and the donor status is uncertain. This study was designed to determine the clinical significance of NDR results. STUDY DESIGN AND METHODS: Three years of donor NAT and serology results were reviewed to determine HCV and HIV-1 infection rates as well as NDR events. Index NDR and seronegative donations were retested by mTMA and serology. Follow-up samples were tested by serology, mTMA, and PCR (selected samples). RESULTS: From April 1999 through April 2002, 5.1 million donations were screened with Procleix HIV-1 and HCV mTMA. The observed NDR rate declined from 0.014 percent (1 in 7242) to 0.0053 percent (1 in 18,795). None of the 462 donors with index NDR donations and additional NAT and serology data (from 724 donation and follow-up samples) were found infected. CONCLUSION: In most NDRs, the original mTMA reactivity is not reproducible and likely related to technical errors (mTMA cross-contamination). The retest and clinical follow-up data, combined with published evidence that the two discriminatory assays have the same sensitivity as multiplex HIV-1 and HCV, support the recommendation that donors with an NDR result for whom the multiplex reactivity cannot be reproduced should either not be notified or be deferred or should be counseled that they are not infected and expeditiously reinstated.

Clinical specificity and sensitivity of a blood screening assay for detection of HIV-1 and HCV RNA.

BACKGROUND: An HIV-1 and HCV NAT blood screening assay (Procleix HIV-1/HCV, Gen-Probe, Inc.) simultaneously detecting HIV-1 and HCV RNA) has been implemented. Donor plasma samples reactive in the Procleix HIV-1/HCV assay are tested with the HIV-1 and HCV discriminatory assays to resolve whether HIV-1 RNA, HCV RNA, or both are present. STUDY DESIGN AND METHODS: To determine the specificity of the Procleix HIV-1/HCV assay, data were analyzed for samples from 192,288 donations, tested in 16-member pools. To determine sensitivity, data were analyzed for 2014 commercial samples known to contain HIV-1, HCV, or both, as well as 10 HIV-1 and 10 HCV commercial seroconversion panels. RESULTS: The specificity of the Procleix HIV-1/HCV assay was 99.7 percent. The HIV-1 and HCV discriminatory assays showed similar specificity. The sensitivity of the Procleix HIV-1/HCV assay was 99.9, 99.6, and 100 percent, respectively, for samples containing HIV-1, HCV, or both. The Procleix discriminatory assays were comparably sensitive. The Procleix discriminatory assays detected all tested samples of known HIV-1 subtype or HCV genotype. Procleix HIV-1/HCV testing of seroconversion panels showed that the median times to a positive reaction for HIV-1 and HCV were reduced by 3 and 25 days, respectively, compared to serologic tests. CONCLUSION: These studies support the use of the Procleix HIV-1/HCV assay for routine blood donor screening.