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Summary Background Characterisation of the dynamics of Zika virus persistence following acute infection is needed to inform blood donor and diagnostic testing policies and understand the natural history of Zika virus infection. We aimed to characterise the natural history, persistence, and clinical outcomes of Zika virus infection through a prospective study in initially asymptomatic Zika virus RNA-positive blood donors. Methods Zika virus-infected blood donors identified through Zika virus nucleic acid amplification test (NAAT) screening at three blood collection organisations in the USA were enrolled into a 1-year follow-up study, with blood and body fluid samples and detailed symptom data collected at up to seven visits. All samples were tested for Zika virus RNA by real-time PCR (rtPCR); follow-up plasma, whole blood, and urine were also tested by replicate NAAT. Plasma was tested for flavivirus-specific IgM and IgG by ELISA. Zika virus RNA persistence for each assay or sample type and plasma antibody persistence from estimated date of plasma NAAT-detectable infection were calculated from follow-up data using survival statistical methods. Findings Between July 6, 2016 and March 7, 2017, we enrolled 53 participants. From the estimated date of plasma NAAT-detectable infection, Zika virus RNA was detectable in plasma for 9·9 days (95% CI 8·1–12·0), in red blood cells for 95·4 days (62·8–129·1), and in whole blood for 73·5 days (39·8–107·5). Replicate NAATs (one or more of eight replicates positive) extended detection of Zika virus RNA in plasma to 34·8 days (19·9–56·2) and in whole blood (at least one of two tests positive) to 104·8 days (76·7–129·9). Urine was rtPCR reactive up to 14·5 days (10·5–20·3) and saliva up to 26·4 days (19·7–38·7). Zika virus IgM persisted for 237·7 days (128·7–459·5) from estimated time since plasma NAAT-detectable infection. Zika virus RNA fell below detectable limits more rapidly in the saliva of participants with pre-existing dengue virus IgG than in those without. Of 25 donors identified pre-seroconversion with symptom data at the first or second study visit, 16 (64%) developed multiple Zika virus-related symptoms after asymptomatic index donations, compared with nine (36%) of 25 donors detected after seroconversion. Interpretation Determination of viral marker persistence is enhanced by follow-up of blood donors who are pre-symptomatic or asymptomatic, Zika virus RNA-positive, and antibody negative. Zika virus RNA persists in red blood cells for several months following clearance from plasma and body fluids, and replicate, highly sensitive NAATs extend RNA detection in all compartments. Whole blood testing can extend detection of acute infection for diagnostics and monitoring of pregnant women, sexual partners, and travellers. Funding National Heart, Lung, and Blood Institute, Biomedical Advanced Research and Development Authority.

Background Some people rapidly develop iron deficiency anemia following blood donation, while others can repeatedly donate without becoming anemic. Methods Two cohorts of blood donors were studied. Participants (775) selected from a 2-year longitudinal study were classified into six analysis groups based on sex, donation intensity, and low hemoglobin deferral. Associations with iron supplement use, cigarette smoking, and four genetic variants of iron metabolism were examined at enrollment and with longitudinal regression models. An unbiased assessment of genetic variability and ability to repeatedly donate blood without experiencing low hemoglobin deferral was conducted on participants (13,403) in a cross-sectional study who were examined by genome wide association (GWA). Results Behaviors and genetic variants were associated with differences in hemoglobin and ferritin change following repeated donation. At least weekly iron supplement use was associated with improved status in first-time donors, while daily use was associated with improved status in high-intensity donors. Cigarette smoking was associated with 0.5 g/dL increased hemoglobin in high-intensity donors. A736V in TMPRSS6 was associated with a rapid drop in hemoglobin and ferritin in first-time females following repeated donation. Conversely, the protective TMPRSS6 genotype was not enriched among high-intensity donors. H63D in HFE was associated with increased hemoglobin in female high-intensity donors. However, no differences in genotype between first-time and high-intensity donors were found in GWA analyses. Conclusion Behavioral and genetic modifiers contributed to first-time donor hemoglobin and iron status, while iron supplement use was more important than underlying genetics in high-intensity donors.

Background Human T-Cell Lymphotropic Viruses (HTLV) type 1 and type 2 account for an estimated 5 to 10 million infections worldwide and are transmitted through breast feeding, sexual contacts and contaminated cellular blood components. HTLV-associated syndromes are considered as neglected diseases for which there are no vaccines or therapies available, making it particularly important to ensure the best possible diagnosis to enable proper counselling of infected persons and avoid secondary transmission. Although high quality antibody screening assays are available, currently available confirmatory tests are costly and have variable performance, with high rates of indeterminate and non-typable results reported in many regions of the world. The objective of this project was to develop and validate a new high-performance multiplex immunoassay for confirmation and discrimination of HTLV-1 and HTLV-2 strains. Methodology/Principal findings The multiplex platform was used first as a tool to identify suitable antigens and in a second step for assay development. With data generated on over 400 HTLV-positive blood donors sourced from USA and French blood banks, we developed and validated a high-precision interpretation algorithm. The Multi-HTLV assay demonstrated very high performance for confirmation and strain discrimination with 100% sensitivity, 98.1% specificity and 100% of typing accuracy in validation samples. The assay can be interpreted either visually or automatically with a colorimetric image reader and custom algorithm, providing highly reliable results. Conclusions/Significance The newly developed Multi-HTLV is very competitive with currently used confirmatory assays and reduces considerably the number of indeterminate results. The multiparametric nature of the assay opens new avenues to study specific serological signatures of each patient, follow the evolution of infection, and explore utility for HTLV disease prognosis. Improving HTLV diagnostic testing will be critical to reduce transmission and to improve monitoring of seropositive patients., Author summary HTLV viruses are responsible for more than 10 million cases of infection worldwide. The infection is considered as a neglected disease due to lack of vaccines and treatments. Accurate diagnosis is crucial for counselling infected persons and prevention of secondary transmissions. In spite of the development of excellent serological screening assays, many cases of indeterminate and untyped results are still regularly reported and their infection status remain uncertain. To address the need of more precise diagnosis, we have developed a new cutting-edge in-vitro diagnostic confirmation test, named Multi-HTLV, which has been validated on a large panel of HTLV samples. The test is a multiplex immunoassay allowing powerful detection of antibodies against HTLV through combination of a set of selective and validated virus-specific antigens in a blood sample. The Multi-HTLV assay increases the reliability of HTLV diagnostics and strain typing thanks to a high precision mathematical algorithm.

Background Babesia microti is a parasite that infects red blood cells (RBCs) in mammals. It is transmitted to humans by tick bites, transfusion, organ transplantation, and congenital acquisition. Although the Babesia natural history and seroprevalence in donors have been well described, gaps in knowledge relevant to transfusion remain. Study design and methods Mice were infected with dilutions of parasitized blood to address the minimal infectious dose and the kinetics of parasitemia by quantitative polymerase chain reaction (qPCR) and of antibodies by enzyme immunoassay. Results In immunocompetent DBA/2 mice infected with 100 parasitized RBCs (pRBCs) and in immunodeficient NSG mice infected with 63 pRBCs, parasitemia was detectable in five of five mice each. Peak parasitemia up to 2 × 107 pRBCs/mL at 2 to 3 weeks or 5 × 108 pRBCs/mL at 6 weeks was observed for DBA/2 and NSG mice, respectively. Protracted fluctuating parasitemia was observed for 8 months in DBA/2 mice, whereas NSG mice exhibited a high-plateau parasitemia. Antibody titers continued to increase until 6 to 18 weeks in DBA/2 mice and remained high through 6 months. This study also investigated the analytical performance of Babesia assays that detect parasite DNA or RNA using a blinded panel. A Babesia assay targeting parasite RNA was approximately 10-fold more sensitive compared to qPCR targeting DNA. Conclusion The mice in this study were highly susceptible to Babesia infection using as few as 1 to 2 log pRBCs and maintained chronic parasitemia. If the infectious dose in human transfusion recipients is comparably low, a highly sensitive assay targeting parasite RNA may safeguard the blood supply, particularly before antibody detection.

BACKGROUND Alloimmunization through blood transfusion, transplantation, or circulating fetal cells during pregnancy is a significant concern. Some exposed individuals make alloantibodies while others do not, implying variation in genetic risk factors. STUDY DESIGN AND METHODS We conducted a genomewide association study (GWAS) of 9,427,497 single-nucleotide polymorphisms (SNPs) to identify genetic variants for HLA alloimmunization in previously pregnant blood donors with (n = 752) and without (n = 753) HLA Class I or II alloantibodies. RESULTS A SNP in the neurexophilin 2 (NXPH2) gene surpassed genome-wide significance (p = 2.06 × 10-8 ), with multiple adjacent markers p

People with sickle cell disease (SCD) are reported to have low rates of HIV infection, slower progression to AIDS and lower HIV-associated mortality compared to the general population. Mechanisms of potential resistance to HIV in SCD are incompletely understood. We retrospectively reviewed the Transfusion Safety Study to compare HIV status between people with SCD and other congenital anemias who were routinely exposed to blood products during the high-risk period before HIV screening implementation. Non-SCD congenital anemia diagnosis was associated with a higher risk of HIV acquisition compared to SCD (OR 13.1 95%CI 1.6-108.9). In addition, we prospectively enrolled 30 SCD cases and 30 non-SCD controls to investigate potential mechanisms of resistance to HIV in SCD. CCR5 and CCR7 expression was lower and CD4 expression was higher on CD4+ T cells from SCD cases compared to controls. Surface expression of CD4+ T cell CXCR4, CD38 and HLA-DR did not differ between the groups. SCD CD4+ T cells were not less susceptible to HIV infection than controls. Levels of multiple cytokines were elevated in the SCD plasma, but SCD plasma compared to control plasma did not inhibit HIV infection of target cells. In conclusion, our epidemiological data support people with SCD being resistant to HIV infection. Potential mechanisms include lower CD4+ T cell expression of CCR5 and CCR7, balanced by increased CD4 expression and cytokine levels, which did not result in in vitro resistance to HIV infection. Further study is needed to define the risk and pathophysiology of HIV in persons with SCD.

Background Following transfusion, donor white blood cells (WBCs) can persist long-term in the recipient, a phenomenon termed transfusion-associated microchimerism (TA-MC). Prior studies suggest TA-MC is limited to transfusion following traumatic injury, and is not prevented by leukoreduction. Study design and methods We conducted a prospective cohort study at a major trauma center to evaluate TA-MC following injury. Index samples were collected upon arrival, prior to transfusion. Follow-up samples were collected at intervals up to one year, and beyond for those testing positive for TA-MC. TA-MC was detected by real-time quantitative allele-specific polymerase chain reaction assays at the HLA-DR locus and several polymorphic insertion deletion sites screening for non-recipient alleles. Results A total of 378 trauma patients were enrolled (324 transfused cases and 54 non-transfused controls). Mean age was 42 ± 18 years, 74% were male, and 80% were injured by blunt mechanism. Mean Injury Severity Score was 20 ± 12. Among transfused patients, the median (interquartile range) number of red cell units transfused was 6 (3,12), and median time to first transfusion was 9 (0.8,45) hours. Only one case of long-term TA-MC was confirmed in our cohort. We detected short-term TA-MC in 6.5% of transfused subjects and 5.6% on non-transfused controls. Conclusions In contrast to earlier studies, persistent TA-MC was not observed in our cohort of trauma subjects. Short-term TA-MC was detected, but at a lower frequency than previously observed, and rates were not significantly different than what was observed in non-transfused controls. The reduction in TA-MC occurrence may be attributable to changes in leukoreduction or other blood processing methods.

BACKGROUND Babesia microti is the foremost infectious risk to the US blood supply for which a Food and Drug Administration (FDA)-licensed test is unavailable for donation screening. Characterization of the antibody response to B. microti and correlation with parasitemia is necessary to guide screening and donor management policies. STUDY DESIGN AND METHODS During an FDA licensure trial, blood donors were prospectively screened (July-November 2013) using a B. microti-specific antibody enzyme immunoassay (EIA, Immunetics) in highly endemic (New York [NY]; n = 13,688), moderately endemic (Minnesota [MN]; n = 4583), and nonendemic (New Mexico [NM]; n = 8451) regions. Blood donors with repeat-reactive (RR) results participated in a 12-month prospective cohort study using B. microti EIA, immunofluorescent assay, polymerase chain reaction (PCR), blood smear, and clinical questionnaire. RESULTS Thirty-seven (61.67%; 24 NY, seven MN, six NM) of 60 eligible RR donors enrolled in the study; 20 of 37 (54%) completed the 12-month follow-up visit of which 15 (75%) were still seroreactive. Nine PCR-positive donors were identified during index screening; five participated in the follow-up study, three were PCR positive at 6 months, and two remained positive at final follow-up (378 and 404 days). Most RR donors displayed low-level seroreactivity that was either stable or waning during follow-up. The level and pattern of reactivity correlated poorly with PCR positivity. CONCLUSION The findings indicate prolonged seropositivity in blood donors. Although rare, asymptomatic, persistent PCR positivity supports the current policy of indefinite deferral for donors with a history of babesiosis or positive test results. Repeat testing by PCR and serology will be necessary if reinstatement is to be considered.

Background The effectiveness of anti-parasite treatment with benznidazole in the chronic Chagas disease (ChD) remains uncertain. We evaluated, using data from the NIH-sponsored SaMi-Trop prospective cohort study, if previous treatment with benznidazole is associated with lower mortality, less advanced cardiac disease and lower parasitemia in patients with chronic ChD. Methods The study enrolled 1,959 ChD patients and abnormal electrocardiogram (ECG) from in 21 remote towns in Brazil. A total of 1,813 patients were evaluated at baseline and after two years of follow-up. Those who received at least one course of benznidazole were classified as treated group (TrG = 493) and those who were never treated as control group (CG = 1,320). The primary outcome was death after two-year follow-up; the secondary outcomes were presence at the baseline of major ChD-associated ECG abnormalities, NT-ProBNP levels suggestive of heart failure, and PCR positivity. Results Mortality after two years was 6.3%; it was lower in the TrG (2.8%) than the CG (7.6%); adjusted OR: 0.37 (95%CI: 0.21;0.63). The ECG abnormalities typical for ChD and high age-adjusted NT-ProBNP levels suggestive of heart failure were lower in the TrG than the CG, OR: 0.35 [CI: 0.23;0.53]. The TrG had significantly lower rates of PCR positivity, OR: 0.35 [CI: 0.27;0.45]. Conclusion Patients previously treated with benznidazole had significantly reduced parasitemia, a lower prevalence of markers of severe cardiomyopathy, and lower mortality after two years of follow-up. If used in the early phases, benznidazole treatment may improve clinical and parasitological outcomes in patients with chronic ChD. Trial registration ClinicalTrials.gov, Trial registration: NCT02646943., Author summary Chagas disease remains as one of the most neglected infectious diseases of the world, with millions of persons infected and dozens of thousands of deaths every year. The disease has a very long natural history and few options of etiologic treatment, including antiparasitic [1] drugs benznidazole and nifurtimox. Benznidazole has been recommended for treatment of the disease based mostly in physiopathological assumptions and relatively small observational studies and, recently, a negative trial was published, showing the absence of benefit of benznidazole in patients with Chagas cardiopathy. Thus, there is an urgent need not only of new therapeutic options but also of further evidence of the utility of benznidazole, at least in those patients with less severe disease. We conducted an observational study in the NIH SaMi-Trop cohort comparing patients with self-reported previous treatment with benznidazole with non-treated ones, using as primary outcome death in the follow-up of 2 years, and as secondary outcomes markers of severity based in the ECG and NT-proBNP levels, as well as parasitemia by PCR for Trypanosoma cruzi DNA in blood. Patients previously treated with benznidazole had significantly reduced parasitemia, a lower frequency of markers of severe cardiomyopathy and reduced mortality. If used in the early phases of Chagas disease, benznidazole treatment may result in better clinical and parasitological outcomes. It is reasonable to consider treating all Chagas disease patients without advanced cardiopathy with benznidazole, especially those less than 50 years-old of age.

The diagnosis of Chagas disease is based on the detection of Trypanosoma cruzi (T. cruzi)-specific antibodies. Nonetheless, there is concern about the sensitivity of current serological assays due to reports of T. cruzi PCR positivity among seronegative individuals. The aim of this study was to evaluate if T. cruzi seronegative infections occur in endemic areas. We recruited 2,157 individuals that were identified as having Chagas disease in a public health system database of an endemic region in Brazil. All participants were interviewed and 2,091 had a sample collected for serological and PCR testing. From these, 149 (7.1%) had negative serological results. PCR was positive in 610 samples (31.4%) of the 1,942 seropositive samples but in none of the 149 samples from seronegative participants. True T. cruzi seronegative infections seem to be rare (95% CI 0-3.7) and should not be a concern for blood supply, which relies on antibody screening.

Healthy pregnancy is the most successful form of graft tolerance, whereas preterm labor (PTL) may represent a breakdown in maternal-fetal tolerance. Although maternal immune responses have been implicated in pregnancy complications, fetal immune responses against maternal antigens are often not considered. To examine the fetal immune system in the relevant clinical setting, we analyzed maternal and cord blood in patients with PTL and healthy term controls. We report here that the cord blood of preterm infants has higher amounts of inflammatory cytokines and a greater activation of dendritic cells. Moreover, preterm cord blood is characterized by the presence of a population of central memory cells with a type 1 T helper phenotype, which is absent in term infants, and an increase in maternal microchimerism. T cells from preterm infants mount a robust proliferative, proinflammatory response to maternal antigens compared to term infants yet fail to respond to third-party antigens. Furthermore, we show that T cells from preterm infants stimulate uterine myometrial contractility through interferon-γ and tumor necrosis factor–α. In parallel, we found that adoptive transfer of activated T cells directly into mouse fetuses resulted in pregnancy loss. Our findings indicate that fetal inflammation and rejection of maternal antigens can contribute to the signaling cascade that promotes uterine contractility and that aberrant fetal immune responses should be considered in the pathogenesis of PTL.

BACKGROUND Pathogen inactivation methods are increasingly used to reduce the risk of infections after transfusion of blood products. Photochemical treatment (PCT) of platelets (PLTs) and plasma with amotosalen and ultraviolet A (UVA) light inactivates pathogens and white blood cells through formation of adducts between amotosalen and nucleic acid that block replication, transcription, and translation. The same adducts block the amplification of nucleic acids using polymerase chain reaction (PCR) in a manner that correlates with the number of adducts formed, providing a direct quality control (QC). Current QC measures for PCT rely on indirect methods that measure the delivered UVA dose or percent residual amotosalen after illumination, rather than directly measuring nucleic acid modification. STUDY DESIGN AND METHODS Endogenous mitochondrial DNA (mtDNA), which is detectable in PLT and plasma units, was chosen as a target for the quantification of photochemically induced modifications. DNA was extracted from untreated or amotosalen and UVA–treated PLTs or plasma, and mtDNA fragments of variable lengths were quantified using a real-time PCR inhibition assay. RESULTS PCT induced increasing real-time PCR inhibition of mtDNA amplification for larger amplicon sizes. Amplification was unaffected by treatment with amotosalen or UVA alone, whereas up to 3 log inhibition was observed after PCT. Blinded PCR testing of a panel of 110 samples each, from PLT or plasma components prepared for routine use within a blood center, allowed 100% discrimination between untreated and treated units. CONCLUSION Our initial findings indicate that an adequately sensitive, quantitative real-time PCR inhibition assay targeting mtDNA could provide a valuable tool to confirm and monitor PCT.

Background Trypanosoma cruzi parasite, the causative agent of Chagas disease, infects about six million individuals in more than 20 countries. Monitoring parasite persistence in infected individuals is of utmost importance to develop and evaluate treatments to control the disease. Routine screening for infected human individuals is achieved by serological assays; PCR testing to monitor spontaneous or therapy-induced parasitological cure has limitations due to the low and fluctuating parasitic load in circulating blood. The aim of the present study is to evaluate a newly developed antibody profiling assay as an indirect method to assess parasite persistence based on waning of antibodies following spontaneous or therapy-induced clearance of the infection. Methodology/Principal findings We designed a multiplex serology assay, an array of fifteen optimized T. cruzi antigens, to evaluate antibody diversity in 1654 serum samples from chronic Chagas patients. One specific antibody response (antibody 3, Ab3) showed a strong correlation with T. cruzi parasite persistence as determined by T. cruzi PCR positive results. High and sustained Ab3 signal was strongly associated with PCR positivity in untreated patients, whereas significant decline in Ab3 signals was observed in BZN-treated patients who cleared parasitemia based on blood PCR results. Conclusion/Significance Ab3 is a new surrogate biomarker that strongly correlates with parasite persistence in chronic and benznidazole-treated Chagas patients. We hypothesize that Ab3 is induced and maintained by incessant stimulation of the immune system by tissue-based and shed parasites that are not consistently detectable by blood based PCR techniques. Hence, a simple immunoassay measurement of Ab3 could be beneficial for monitoring the infectious status of seropositive patients., Author summary Chagas disease is a zoonosis caused by Trypanosoma cruzi infection. In this study, we sought to identify a simple serologic biomarker that can supersede PCR techniques in characterizing patients for parasite persistence, whether circulating or shielding in tissues. A multiparametric assay was designed to evaluate antibody diversity in untreated and benznidazole-treated chronic cardiomyopathy patients. Out of 15 different antigens we identified a distinct antibody specificity that strongly correlates with parasite persistence. This putative biomarker could be used to monitor active infection in T. cruzi seropositive patients and identify patients that responded to anti-parasitic therapy.

Background Chikungunya virus (CHIKV) is the most common alphavirus infecting humans worldwide, causing acute and chronically debilitating arthralgia at a great economic expense. Methodology/Principal findings To facilitate our study of CHIKV, we generated a mCherry tagged replication-competent chimeric virus, CHIKV 37997-mCherry. Single particle cryoEM demonstrated icosahedral organization of the chimeric virus and the display of mCherry proteins on virus surface. CHIKV 37997-mCherry is attenuated in both IFNαR knockout and wild-type mice. Strong anti-CHIKV and anti-mCherry antibody responses were induced in CHIKV 37997-mCherry infected mice. Conclusions/Significance Our work suggests that chimeric alphaviruses displaying foreign antigen can serve as vaccines against both aphaviruses and other pathogens and diseases., Author summary Chikungunya virus (CHIKV) is an alphavirus capable of causing long term debilitating joint and muscle pain at a great economic expense. Currently there are no licensed vaccines or treatment for CHIKV infection. We generated a modified version of the virus, termed CHIKV 37997-mCherry, stably expressing a fluorescent tag on the surface of the virus. To achieve this, a red fluorescent protein, mCherry, was fused to the virus envelope protein E2. Structural studies demonstrated the presence of mCherry on the virus surface. Infection of mice with CHIKV 37997-mCherry caused less severe disease in the animals compared to wild-type virus. Infection with CHIKV 37997-mCherry induced immune responses against both the mCherry protein and the virus. Furthermore, CHIKV 37997-mCherry is as attenuated as the vaccine strain CHIKV 181/clone 25 in different mouse models, causing less joint swelling and reduced persistence of viral genomes in tissue. Our work suggests that chimeric alphaviruses carrying foreign antigen on virus particles may serve as vaccines against both aphaviruses and other pathogens and diseases.

Author(s): Sabino, EC; Ribeiro, AL; Lee, TH; Oliveira, CL; Carneiro-Proietti, AB; Antunes, AP; Menezes, MM; Ianni, BM; Salemi, VM; Nastari, L; Fernandes, F; Sachdev, V; Carrick, DM; Deng, X; Wright, D; Goncalez, TT; Murphy, EL; Custer, B; Busch, MP; Chagas Study Group of the NHLBI Retrovirus Epidemiology Donor Study-II, International Component | Abstract: BackgroundThe significance of detection of Trypanosoma cruzi DNA in blood of antibody-positive patients for risk of development of Chagas heart disease is not well established. The objective of this study was to compare detection of T. cruzi DNA with known clinical and laboratory markers of Chagas cardiomyopathy (CC) severity.MethodsThis is a case-control study nested within a retrospective cohort developed in Brazil to understand the natural history of Chagas disease. The study enrolled 499 T. cruzi seropositive blood donors (SP-BD) and 488 frequency matched seronegative control donors (SN-BD) who had donated between 1996 and 2002, and 101 patients with clinically diagnosed CC. In 2008-2010 all enrolled subjects underwent a health questionnaire, medical examination, electrocardiograms and echocardiograms and polymerase chain reaction (PCR) analyses. A blinded panel of three cardiologists adjudicated the outcome of CC. Trypanosoma cruzi kinetoplast minicircle sequences were amplified by real-time PCR using an assay with a sensitivity of one parasite per 20 mL of blood. All testing was performed on coded samples.ResultsRates of PCR detection of T. cruzi DNA were significantly (P = 0.003) higher in CC patients and SP-BD diagnosed with CC (79/105 [75.2 %]) compared with SP-BD without CC (143/279 [51.3%]). The presence of parasitaemia was significantly associated with known markers of disease progression such as QRS and QT interval duration, lower left ventricular ejection fraction, higher left ventricular index mass, and elevated troponin and NTpro-BNP levels.ConclusionTrypanosoma cruzi PCR positivity is associated with presence and severity of cardiomyopathy, suggesting a direct role of parasite persistence in disease pathogenesis.

Babesia microti is a parasite that infects red blood cells (RBCs) in mammals. It is transmitted to humans by tick bites, transfusion, organ transplantation, and congenital acquisition. Although the Babesia natural history and seroprevalence in donors have been well described, gaps in knowledge relevant to transfusion remain.Mice were infected with dilutions of parasitized blood to address the minimal infectious dose and the kinetics of parasitemia by quantitative polymerase chain reaction (qPCR) and of antibodies by enzyme immunoassay.In immunocompetent DBA/2 mice infected with 100 parasitized RBCs (pRBCs) and in immunodeficient NSG mice infected with 63 pRBCs, parasitemia was detectable in five of five mice each. Peak parasitemia up to 2 × 10The mice in this study were highly susceptible to Babesia infection using as few as 1 to 2 log pRBCs and maintained chronic parasitemia. If the infectious dose in human transfusion recipients is comparably low, a highly sensitive assay targeting parasite RNA may safeguard the blood supply, particularly before antibody detection.

Background Notwithstanding 1 documented case of HIV-1 cure following allogeneic stem cell transplantation (allo-SCT), several subsequent cases of allo-SCT in HIV-1 positive individuals have failed to cure HIV-1 infection. The aim of our study was to describe changes in the HIV reservoir in a single chronically HIV-infected patient on suppressive antiretroviral therapy who underwent allo-SCT for treatment of acute lymphoblastic leukemia. Methods and findings We prospectively collected peripheral blood mononuclear cells (PBMCs) by leukapheresis from a 55-year-old man with chronic HIV infection before and after allo-SCT to measure the size of the HIV-1 reservoir and characterize viral phylogeny and phenotypic changes in immune cells. At day 784 post-transplant, when HIV-1 was undetectable by multiple measures—including PCR measurements of both total and integrated HIV-1 DNA, replication-competent virus measurement by large cell input quantitative viral outgrowth assay, and in situ hybridization of colon tissue—the patient consented to an analytic treatment interruption (ATI) with frequent clinical monitoring. He remained aviremic off antiretroviral therapy until ATI day 288, when a low-level virus rebound of 60 HIV-1 copies/ml occurred, which increased to 1,640 HIV-1 copies/ml 5 days later, prompting reinitiation of ART. Rebounding plasma HIV-1 sequences were phylogenetically distinct from proviral HIV-1 DNA detected in circulating PBMCs before transplantation. The main limitations of this study are the insensitivity of reservoir measurements, and the fact that it describes a single case. Conclusions allo-SCT led to a significant reduction in the size of the HIV-1 reservoir and a >9-month-long ART-free remission from HIV-1 replication. Phylogenetic analyses suggest that the origin of rebound virus was distinct from the viruses identified pre-transplant in the PBMCs., Andrew Badley and colleagues study the viral reservoir in an individual with HIV infection after allogeneic stem cell transplantation., Author summary Why was this study done? Currently there is no cure for HIV infection. The only previously documented case of HIV cure occurred in the setting of stem cell transplantation for acute myeloid leukemia. However, other similar cases have not resulted in HIV cure. This observational study was done to further describe in detail the effects of allogeneic stem cell transplantation on residual HIV in a patient being treated for acute lymphoblastic leukemia. What did the researchers do and find? We prospectively collected blood and tissue samples from before and after stem cell transplantation and measured the HIV reservoir size using multiple complementary techniques. We found that the size of the HIV reservoir decreased substantially after transplantation to levels at or below the limit of detection of most assays. We observed a prolonged remission from HIV rebound after antiretroviral treatment interruption in the post-transplant period. The genetic sequence of the rebounding virus in the blood clustered closely with sequences from blood prior to treatment interruption. What do these findings mean? These findings affirm that allogeneic stem cell transplantation can profoundly decrease the size of the HIV reservoir. However, current technologies for measuring reservoir size in blood are insufficiently sensitive to predict HIV cure. Until new biomarkers of HIV cure are developed, the decision to discontinue antiretroviral therapy after allogeneic stem cell transplantation to assess a possible cure should be undertaken cautiously.

Alloimmunization through blood transfusion, transplantation, or circulating fetal cells during pregnancy is a significant concern. Some exposed individuals make alloantibodies while others do not, implying variation in genetic risk factors.We conducted a genomewide association study (GWAS) of 9,427,497 single-nucleotide polymorphisms (SNPs) to identify genetic variants for HLA alloimmunization in previously pregnant blood donors with (n = 752) and without (n = 753) HLA Class I or II alloantibodies.A SNP in the neurexophilin 2 (NXPH2) gene surpassed genome-wide significance (p = 2.06 × 10Further work to extend the GWAS approach and to characterize variants in NXPH2 and E2F7 in the context of alloantibody formation is warranted.

Background SFTSV, an emerging tick-borne pathogen that can cause fatal severe fever with thrombocytopenia syndrome (SFTS), was first identified in China in 2009. Limited evidence suggests that SFTSV can be transmitted between humans via blood contact raising concerns over transfusion safety. A study of donor samples from three Chinese blood centers was conducted to investigate the seroprevalence and rate of SFTSV viremia among Chinese blood donors.

Background and Objectives Transfusion is associated with a risk of infection andalloimmunization. Pathogen reduction using riboflavin and UV light (Mirasoltreatment) inactivates pathogens and leucocytes. With increasing adoption of thetechnology in clinical use, regulatory agencies have recommended the introduc-tion of quality control measures to monitor pathogen reduction efficacy. Wesought to develop a real-time PCR-based assay to document the impact of patho-gen reduction on the mitochondrial genome in blood components.Materials and Methods DNA was extracted from platelet and plasma componentsbefore and after treatment with riboflavin and UV light. Inhibition of PCR ampli-fication of mitochondrial DNA (mtDNA) in short- and long-amplicon targetregions, ranging from under 200 base pairs (bp) to over 1800 bp, was measuredin treated relative to untreated components.Results Pathogen reduction of platelets using riboflavin and UV light resulted ininhibition of PCR amplification of long-amplicon mtDNA targets, demonstratingapproximately 1 log reduction of amplification relative to untreated products.Amplification of short-amplicon mtDNA targets was not affected by treatment.Evaluation of 110 blinded platelet samples from the PREPAReS clinical trialresulted in prediction of treatment status with 100% accuracy. Pathogen reduc-tion of plasma components resulted in similar levels of PCR inhibition, whiletesting of 30 blinded plasma samples resulted in prediction of treatment statuswith 93% accuracy.Conclusion A differential sized amplicon real-time PCR assay of mitochondrialDNA effectively documents nucleic acid damage induced by Mirasol treatment ofplatelets. The use of the assay for plasma product pathogen reduction requiresfurther investigation.Keywords: mitochondrial DNA, pathogen reduction, plasma, platelets, quantita-tive PCR.

Background Previous reports of West Nile virus (WNV) RNA persistence in blood compartments have raised concerns around the remaining risk of WNV transfusion transmission. This study characterized the dynamics of WNV viremia in blood compartments in a longitudinal cohort of 54 WNV-infected blood donors. Study Design and Methods Blood samples were collected throughout the year after WNV RNA–positive blood donation (index) and characterized for WNV immunoglobulin (Ig)M and IgG antibodies and for WNV RNA by real-time reverse transcription–polymerase chain reaction. WNV viral loads were compared in plasma and whole blood samples and correlated with blood groups and clinical outcomes. Results WNV RNA persisted in the red blood cell (RBC) compartment up to 3 months postindex in 42% of the donors. Donors with the highest WNV RNA levels in plasma at index maintained the highest WNV RNA levels in whole blood over the 3 months postindex. Blood group A donors maintained higher postindex WNV viral load in whole blood than blood group O individuals (p = 0.027). Despite a trend for WNV RNA to persist longer in whole blood from symptomatic subjects, no significant association was found between WNV RNA levels in whole blood and disease outcome. Conclusion This study confirmed that WNV RNA persists in the RBC fraction in whole blood and further suggested that the level of persistence in whole blood may be a reflection of initial viral burden in plasma. The association with blood groups suggests that WNV adherence to RBCs may be mediated by molecules overrepresented at the surface of blood group A RBCs.

Chronic inflammation may contribute to human immunodeficiency virus (HIV) persistence through a number of potential pathways. We explored the impact of immunosuppressant therapy on peripheral blood measures of HIV persistence following kidney transplantation. Stored plasma and peripheral blood mononuclear cells prior to transplantation and at weeks 12, 26, 52 and 104 posttransplant were obtained from 91 transplant recipients. In a multivariate model, higher pretransplant plasma HIV RNA level (p < 0.0001) and a longer duration of follow-up posttransplant (p = 0.09) were associated with higher posttransplant plasma HIV RNA levels. A higher baseline HIV DNA (p < 0.0001) was significantly associated with higher HIV DNA levels posttransplant, while higher CD4+ T cell count (p = 0.001), sirolimus use (p = 0.04) and a longer duration of follow-up (p = 0.06) were associated with lower posttransplant HIV DNA levels. The association between sirolimus exposure and lower frequency of cells containing HIV DNA levels posttransplant suggest that the immune-modifying drugs may affect the level of HIV persistence during effect therapy. Future studies of sirolimus as a reservoir-modifying agent are warranted.

Background Congenital diaphragmatic hernia (CDH) represents a spectrum of lung hypoplasia and consequent pulmonary hypertension is an important cause of postnatal morbidity and mortality. We studied biomarkers at the maternal-fetal interface to understand factors associated with the persistence of pulmonary hypertension. Methods Maternal and cord blood samples from fetuses with CDH and unaffected controls were analyzed using a human 39plex immunoassay kit. Cellular trafficking between the mother and the fetu was quantified using quantitative real-time PCR for non-shared alleles. Biomarker profiles were then correlated with CDH severity based on the degree of pulmonary hypertension. Results Cord blood levels of epidermal growth factor, platelet-derived growth factor, and several inflammatory mediators increased significantly as the severity of CDH increased, while maternal levels growth factors and mediators decreased significantly with CDH severity. Maternal cells were increased in fetuses with severe CDH compared to controls, with elevated levels of the chemokine CXCL-10 in patients with the highest trafficking. Conclusion Patients with CDH demonstrate pro-inflammatory and chemotactic signals in fetal blood at the time of birth. Since some of these molecules have been implicated in the development of pulmonary hypertension, prenatal strategies targeting specific molecular pathways may be useful adjuncts to current fetal therapies.

Background Dengue viruses (DENV-1-4) pose a transfusion-transmission risk. This study estimated the dengue RNA detection period in asymptomatic blood donors and relationships between donor viremia and dengue incidence during a large epidemic. Methods Donor samples from the 2012 dengue transmission season in Rio de Janeiro, Brazil, were tested for DENV RNA by a transcription-mediated amplification (TMA) assay, with DENV types and viral loads determined by polymerase chain reaction. Samples collected during the first and last weeks of enrollment were tested for DENV immunoglobulin (Ig) G and IgM to estimate incidence during the study period, which was analyzed relative to nucleic acid amplification technology (NAT) yield to estimate the duration of NAT-detectable viremia and compared with reported clinical dengue cases in Rio. Results Samples from 16 241 donations were tested; 87 (0.54%) were confirmed as DENV-4 RNA positive. Dengue IgM-positive/IgG-positive reactivity increased from 2.8% to 8.8%, indicating a 6.2% incidence (95% confidence interval [CI], 3.2%-9.1%) during the study period. Based on these data, we estimated a 9.1-day period (95% CI, 4.4-13.9 days) of RNA detectable with TMA. With 100 475 reported cases of clinical dengue, 1 RNA-positive donation was identified per 800 DENV cases. Conclusions These parameters allow projections of dengue incidence from donor NAT yield data and vice versa, and suggest that viremic donations will be rare relative to clinical disease cases.

Stressful events early in life have been widely linked to behavioral phenotypes and have been implicated in the development of psychiatric disorders. Using a maternal separation paradigm, we investigated phenotypic and epigenetic changes following early life stress in two inbred strains of mice, C57BL/6J and DBA/2J. We found an increase in the corticosterone response to stress in male, C57BL/6J mice that had undergone maternal separation compared to controls. In addition, early life stress induced a number of mild but significant behavioral changes, many of which were sex and strain dependent. Following maternal separation anxiety was decreased in males but increased in DBA/2J females, DBA/2J males displayed reduced exploration of a novel object, and baseline activity was altered in males of both strains. Finally, we examined DNA methylation levels in the hippocampus across promoter regions of Nr3c1, Avp, and Nr4a1, and found altered levels at several CpG sites in maternally separated male mice compared to controls. This study contributes to a growing body of recent literature suggesting that epigenetic changes may mediate the impact of early life stress on behavior. In particular, we establish that the phenotypic and epigenetic responses to an adverse environment differ as a function of genetic background.

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.

BACKGROUND: Xenotropic murine leukemia virus (MLV)-related virus (XMRV) and other related MLVs have been described with chronic fatigue syndrome and certain types of prostate cancer. In addition, prevalence rates as high as 7% have been reported in blood donors, raising the risk of transfusion-related transmission. Several laboratories have utilized microneutralization assays as a surrogate marker for detection of anti-MLV serologic responses—with up to 25% of prostate cancer patients reported to harbor neutralizing antibody responses. STUDY DESIGN AND METHODS: We developed a high-throughput microneutralization assay for research studies on blood donors using retroviral vectors pseudotyped with XMRV-specific envelopes. Infection with these pseudotypes was neutralized by sera from both macaques and mice challenged with XMRV, but not preimmune serum. A total of 354 plasma samples from blood donors in the Reno/Tahoe area were screened for neutralization. RESULTS: A total of 6.5% of donor samples gave moderate neutralization of XMRV, but not control pseudotypes. However, further testing by Western blot revealed no evidence of antibodies against MLVs in any of these samples. Furthermore, no evidence of infectious virus or viral nucleic acid was observed. CONCLUSION: A microneutralization assay was developed for detection of XMRV and can be applied in a high-throughput format for large-scale studies. Although a proportion of blood donors demonstrated the ability to block XMRV envelope-mediated infection, we found no evidence that this inhibition was mediated by specific antibodies elicited by exposure to XMRV or MLV. It is likely that this moderate neutralization is mediated through another, nonspecific mechanism.

Frequent blood donors become iron deficient. HFE mutations are present in over 30% of donors. A 24-month study of 888 first time/reactivated donors and 1537 frequent donors measured haemoglobin and iron status to assess how HFE mutations impact the development of iron deficiency erythropoiesis. Donors with two HFE mutations had increased baseline haemoglobin and iron stores as did those with one mutation, albeit to a lesser extent. Over multiple donations haemoglobin and iron status of donors with HFE mutations paralleled those lacking mutations. The prevalence of HFE mutations was not increased in higher intensity donors. Thus, in general, HFE mutations do not temper donation-induced changes in haemoglobin and iron status. However, in Black donors there was an increase of H63D carriers at baseline, from 3·7% in first time/reactivated donors to 15·8% in frequent donors, suggesting that the relative effects of HFE mutations on iron absorption may vary between racial/ethnic groups. In secondary analyses, venous haemoglobin decreased more slowly in donors with ferritin ≥12μg/l; and haemoglobin recovery time was shorter in donors with reticulocyte haemoglobin (CHr) ≥32·6pg, indicating that these biochemical measures are better indicators of a donor's response to phlebotomy than their HFE mutation status.

BACKGROUND Transfusion-associated microchimerism (TA-MC), the persistence of significant levels of donor leukocytes in blood recipients for prolonged periods, has been demonstrated following non-leukoreduced and leukoreduced transfusion to patients with severe traumatic injury. Development of TA-MC has not been rigorously studied in settings that do not involve massive trauma where the blood is leukoreduced and irradiated.

BACKGROUND: Despite implementation of targeted individual-donor nucleic acid test (NAT) screening of blood donors for West Nile virus (WNV), three “breakthrough” WNV transfusion transmission cases were reported (2004-2008), suggesting that current plasma-based assays are unable to detect all WNV-infectious donations. A 2007 report found that 19 of 20 red blood cell components from WNV-infected donors contained 1 log higher viral load than plasma components. This study's aim was to further establish the value of screening whole blood relative to plasma for WNV RNA by generating differential viral loads on paired samples derived from blood screening tubes. STUDY DESIGN AND METHODS: WNV RNA–positive donors identified by routine NAT screening were enrolled and quantitative viral data were generated using cross-sectional (index-donation) and longitudinal (follow-up) specimens. A real-time reverse transcription–polymerase chain reaction viral load assay was used on both study sample sets and replicate qualitative NAT screening assays were also used on the longitudinal study samples. RESULTS: For the cross-sectional study, seronegative index donations (n = 29) had WNV RNA concentrations fourfold higher in plasma than in whole blood, whereas for seropositive donations (n = 13), the WNV RNA concentrations were 10-fold higher in whole blood than in plasma. All 10 longitudinal study participants were seropositive throughout the follow-up study; whole blood viral load was consistently greater than plasma viral load (mean difference, 343 copies; p

Highly active antiretroviral therapy (HAART) has been effective in decreasing morbidity and mortality associated with human immunodeficiency virus (HIV) infection [1]. However, a significant proportion of individuals are unable to achieve a normal CD4+ T cell count despite prolonged viral suppression with effective HAART. In one study, ∼25% of patients who started HAART at a CD4+ T cell count of 500 cells/mm3 even after more than 7–10 years of HAART [2]. Moreover, having a suboptimal CD4+ T cell response has been associated with significant clinical consequences, including increased AIDS-related and non–AIDS-related morbidity and mortality [3–6]. The mechanisms of suboptimal immune recovery are not completely understood. Persistent T cell activation may be causally related to the inability to reconstitute normal CD4+ T cell counts, perhaps owing to its effect on lymphoid tissue architecture [7–11]. Moreover, ongoing low-level viral replication may be the proximal cause of persistent activation during HAART [12–15]. Several studies have attempted to address this issue [15–18], although none have focused on the host responses to HIV in both blood and gut-associated lymphoid tissue (GALT). We conducted a randomized, double-blinded, placebo-controlled study of raltegravir intensification to assess whether ongoing viral replication contributes to immune activation and suboptimal immune recovery during HAART. Our secondary objective was to explore the host determinants of viral persistence in both blood and GALT.

Objective: Transient HIV infections have been invoked to account for the cellular immune responses detected in highly virus-exposed individuals who have remained HIV-seronegative. We tested for very low levels of HIV RNA in 524 seronegative plasma samples from 311 highly exposed women and men from three longitudinal HIV cohorts. Design: Two thousand and seventy-three transcription-mediated amplification (TMA) HIV RNA tests were performed for an average of 3.95 TMA assays per plasma sample. Quadruplicate TMA assays, analyzing a total of 2 ml of plasma, provided an estimated sensitivity of 3.5 HIV RNA copies/ml. Results: Four samples from individuals who did not seroconvert within the following 6 months were positive for HIV RNA. For one sample, human polymorphism DNA analysis indicated a sample mix-up. Borderline HIV RNA detection signals were detected for the other three positive samples but further replicate TMA testing yielded no positive results. Nested PCR assays (n = 254) for HIV proviral DNA in peripheral blood mononuclear cells (PBMCs) from these three individuals were negative. Conclusion: Transient viremia was not reproducibly detected in highly HIV-exposed seronegative men and women. If transient infections do occur, plasma HIV RNA levels may remain below the detection limits of the sensitive assay used here, be of very short duration, or viral replication may be restricted to mucosal surfaces or their draining lymphoid tissues.

Introduction Because the receptor for Parvovirus B19 (B19V) is on erythrocytes, we investigated B19V distribution in blood by in-vitro spiking experiments and evaluated viral compartmentalization and persistence in natural infection.

Background: Fetal microchimerism (F-MC), the persistence of fetal cells in the mother, is frequently encountered following pregnancy. The high prevalence of F-MC in autoimmune disease prompts consideration of the role for immune tolerance and regulation. This study examines the association between F-MC and multiple sclerosis (MS), an autoimmune disorder, of undetermined etiology. Study Design and Methods: Seventy-three female subjects were assigned to 3 groups according to disease status and pregnancy history: (1) MS positive (+) women with a history of one male pregnancy before symptom onset (n=27); (2) MS negative (-) female siblings of MS+ women with a history of one male pregnancy (n=22); and (3) MS+ women that reported never having been pregnant (n=24). Ten micrograms of genomic DNA obtained from peripheral blood leukocytes of each subject were analyzed for F-MC using allele-specific real-time PCR targeting the SR-Y sequence on the Y-chromosome. MC classification was dichotomous (positive vs. negativ...

HAART can effectively reduce plasma HIV RNA levels to below the level of detection in most HIV-infected patients. The degree to which residual low-level viremia persists during HAART remains unclear.We identified 180 individuals (median duration of HIV infection 12 years) who had at least two consecutive plasma HIV-1 RNA levels below the level of detection (50-75 copies/ml) while taking antiretroviral drugs; 36 of 180 had been virologically suppressed for more than 5 years. Longitudinal plasma samples that were taken from these individuals during periods of viral load suppression were selected and analyzed. The isothermal transcription-mediated amplification (TMA) (limit of detection3.5 copies RNA/ml) assay was used to measure persistent viremia. A 'detuned' EIA assay was used to obtain quantitative HIV antibody levels.A total of 1606 TMA assays were performed on 438 specimens in 180 HAART-suppressed individuals (median 3 replicates per specimen). In the first year of viral suppression, plasma RNA levels declined significantly (P = 0.001), but after month 12 there was no evidence for a continued decline (P = 0.383). In the first year of viral suppression, HIV antibody levels also declined (P = 0.054), but after month 12 there was no evidence for a continued decline (P = 0.988).Viremia continued to decline during the first 12 months after viremia became undetectable using conventional methods, and then remained stable. HIV antibody levels also decreased in the first year of viral suppression and then remained stable. Viremia and the HIV-associated host response appear to achieve a steady-state 'set-point' during long-term combination therapy.

Background: The mechanisms of HIV transmission from mothers to infants are poorly understood. A possible mechanism of in utero transmission is transplacental transfer of HIV-infected maternal leukocytes into the fetal circulation during pregnancy. Objective: To determine if the frequency of in utero HIV infection correlates with presence or levels of maternal cells (MCs) in placenta-derived cord blood. Methods: DNA was extracted from dried cord blood spots (DBS) from newborns born to HIV+ mothers and corresponding maternal DBS specimens. Paired mother-infant samples were probed to identify unique maternal sequences targeted by 24 allele-specific real-time polymerase chain reaction assays. Infant DBS-derived DNA was then probed in replicate analyses for noninherited maternal allelic sequences. Rates of detection and levels of MCs in DBS samples of HIV(+) and HIV(−) newborns were compared. Results: Of 114 mother-infant pairs with informative alleles, 38 newborns were HIV(+) and 76 HIV(−), based on detection of HIV DNA/RNA at birth. MC were detected in 23 of 38 HIV(+) newborns (60.5%) and in 47 of 76 HIV(−) newborns (61.8%). The mean and median concentrations of nucleated MCs in DBS for the HIV(+)/MC(+) newborns (n = 23) were 0.33% and 0.27%, respectively, compared with 0.09% and 0.10% for the HIV(−)/MC(+) newborns (n = 47) (2-sample T test for means: P = 0.78). Conclusions: There was no significant difference in rates of detection or concentrations of MC in DBS between HIV(+) and HIV(−) newborns. Therefore, we could not demonstrate a correlation between MC in DBS, assumed to reflect levels of in utero maternal-fetal cell trafficking, and the risk of in utero HIV transmission.

Parvovirus B19V infection can be a serious infection for hematology patients with underlying hemolysis or compromised erythropoiesis syndromes. Although case reports of B19V transmission by blood component transfusion (as contrasted to manufactured plasma derivatives) are rare, no studies have systematically determined a rate of transmission to recipients transfused with B19V DNA–positive components. We used a linked donor and recipient repository and a sensitive, quantitative B19V DNA polymerase chain reaction (PCR) assay to assess such transmission in B19V-susceptible (ie, anti-B19V immunoglobulin G [IgG] negative) recipients. We assessed 112 B19V DNA–positive components from 105 donors (of 12 529 tested donations) transfused into a population of surgical patients with a pretransfusion B19V IgG seroprevalence of 78%. We found no transmission to 24 susceptible recipients from transfusion of components with B19V DNA at concentrations less than 106 IU/mL (upper 95% confidence interval, 11.7%). We found an anamnestic IgG response in one pretransfusion seropositive recipient transfused with a component containing greater than 1010 IU/mL B19V DNA. These findings show either that transmission from components with less than 106 IU/mL does not occur, or, if it does, it is an uncommon event. These data do not support the need to routinely screen blood donations with a sensitive B19V DNA nucleic acid assay.

There is increasing use of highly sensitive testing with polymerase chain reaction (PCR) to study white cell microchimerism after transfusion and transplantation. This study investigated possible artifactual sources of allogeneic sample contamination before PCR testing.Quantitative Y-chromosome PCR was used to study microchimerism among transfused patients with sickle cell disease (SCD) and thalassemia by using residual specimens from the clinical laboratory. High levels of circulating male white cells among transfused patients with SCD but not thalassemia led to concern over the artifactual origin of male cells. To investigate, paired specimens were collected from 26 female SCD patients: one specimen underwent processing only for PCR, while the other underwent testing in the clinical laboratory before PCR as a process control. All laboratory instruments were also assessed for their ability to impart male allogeneic cells to aliquots of female blood.Thirty-three (31%) of 107 SCD samples, but 0 of 20 thalassemia samples, gave a high-level PCR signal. One of 26 paired samples that was not exposed to clinical laboratory equipment had low-level PCR positivity while 10 of the 26 became strongly positive after testing on a blood cell analyzer and a reticulocyte analyzer. Sixteen of 32 female samples became positive after reticulocyte analysis, while none became positive after blood cell analysis. Samples from thalassemia patients tested PCR-negative because reticulocyte counts had not been performed.Allogeneic cell contamination is common with clinical laboratory equipment. These samples may not be suitable for microchimerism studies. In addition to method controls, process controls should be employed where appropriate.

Fresh whole blood (FrWB) is routinely used in the resuscitation of combat casualties in Operation Iraqi Freedom and Operation Enduring Freedom. However, studies have shown high rates (20%-40%) of transfusion-associated microchimerism (TA-MC) in civilian trauma patients receiving allogenic red blood cell (RBC) transfusions. We explored the incidence of TA-MC in combat casualties receiving FrWB compared with patients receiving standard stored RBC transfusions.Prospective data on TA-MC ator=14 days posttransfusion were collected on 26 severely injured combat casualties admitted to the National Naval Medical Center between December 2006 and March 2007. Demographic variables included age, sex, Injury Severity Score, and transfusion history. Data are expressed as mean +/- SD.The mean age of the study cohort was 24 +/- 7; mean Injury Severity Score was 17 +/- 12. All were men and suffered penetrating injury. Average hospital length of stay was 46 +/- 35 days. TA-MC was present in 45% (10 of 22) patients who were transfused at least 1 unit of blood. The four nontransfused patients all tested negative for TA-MC. Among six patients who received 4 to 43 units of FrWB, five also received RBCs and one aphaeresis platelets. The remaining 16 transfused patients who received RBCs (no FrWB) included seven who also received platelets in theater. The prevalence of TA-MC was 50% (3 of 6) in FrWB patients, 50% in patients given platelets (4 of 8), and 38% (3 of 8) in those given only RBCs as a cellular component (p = 0.61).Although these preliminary data do not demonstrate a significantly increased rate of TA-MC in FrWB or apheresis platelets recipients compared with RBC recipients, the overall 45% (10 of 22) rate of TA-MC in transfused soldiers warrants further study to ascertain possible clinical consequences such as graft-versus-host or autoimmune disease syndromes.

Blood transfusion is a newly recognized cause of microchimerism, the stable persistence of a minor population of allogeneic cells. Relatively recent advances in polymerase chain reaction technology have spawned new information about the frequency and aetiology of transfusion-associated microchimerism (TA-MC). Although conceptually related to fetal-maternal microchimerism, TA-MC is a distinct and separate entity. Evidence of TA-MC has been strongest among patients with severe traumatic injuries who receive relatively fresh blood products shortly after an episode of massive haemorrhage. The presence of a focal deficit in the cellular immunologic repertoire prior to transfusion that happens to match a blood donor's human leucocyte antigen type also appears to be an important predisposing factor. TA-MC seems to be common (affecting approximately 10% of transfused injured patients), enduring (lasting years to decades) and pronounced (involving up to 5% of circulating leucocytes and multiple immunophenotypic lineages suggestive of haematopoietic engraftment). Further study of this topic may reveal important information regarding potential clinical consequences of TA-MC, as well as basic haematologic and immunologic processes.

The presence of a population of allogeneic cells underlies and partially defines both microchimerism (MC) and graft-versus-host disease (GVHD). Although the relationship between these two apparently distinct clinical entities is not well understood, it is plausible that progenitor cell dose, histocompatibility, recipient immune status and genetic profile all help determine whether allogeneic cells die, persist as a minor population or proliferate in a GVHD response. Although MC has been described, at low levels, in association with pregnancy and autoimmune disease, much higher levels of MC have recently been found in association with blood transfusion. This transfusion-associated microchimerism (TA-MC) has thus far been identified only in patients multiply transfused for severe traumatic injury, which is well documented to induce broad short-term suppression of both the innate and adaptive arms of the host immune system. In this review, we will summarize the limited available data concerning the clinical, immunologic and genetic settings in which TA-MC and GVHD develop and examine the factors that may influence their development. We will not address humoral alloimmunization as a result of transfusion here because its relationship to donor leucocyte persistence is unknown.

Microchimerism, the stable persistence of an allogeneic cell population, can result from allogeneic exposures including blood transfusion. Transfusion-associated microchimerism (TA-MC) appears to be a common but newly recognized complication of blood transfusion. Thus far TA-MC has been detected when severely injured patients are transfused. Injury induces an immunosuppressive and inflammatory milieu in which fresh blood products with replication-competent leukocytes can sometimes cause TA-MC. TA-MC is present in approximately half of transfused severely injured patients at hospital discharge and is not affected by leukoreduction. In approximately 10% of patients, the chimerism from a single blood donor may increase in magnitude over months to years, reaching as much as 2% to 5% of all circulating leukocytes. In this review, we discuss recent studies of TA-MC in the civilian trauma population and the potential for study of TA-MC in the military population, where the severity of injury and freshness of blood products suggest that TA-MC may be even more prominent. We also discuss the need for future studies to address the immunology of TA-MC, its stem cell biology, and its clinical manifestations that have the potential to be either pathologic (autoimmunity, graft-versus-host disease) or therapeutic (tolerance induction, various cell and gene therapies).

Background and Objectives Damage-associated molecular patterns (DAMPs) are found in transfusion products, but their potential impacts are not fully understood. We examined the influence of manufacturing method on levels of mitochondrial (mt) DNA and extracellular vesicle (EV) DAMPs in red cell concentrates (RCCs). Materials and Methods Eighty-seven RCCs were prepared using nine different methods (6–15 units/method), including three apheresis, five whole blood (WB)-derived leucoreduced (LR) and one WB-derived non-LR method. On storage days 5 and 42, levels of mtDNA (by PCR) and number and cell of origin of EVs (by flow cytometry) were assessed in RCC supernatants. Results There was a 100-fold difference in mtDNA levels among methods, with highest levels in non-LR, followed by MCS+ and Trima apheresis RCCs. There was a 10-fold difference in EV levels among methods. RBC-derived CD235a+ EVs were found in fresh RCCs and increased in most during storage. Platelet-derived CD41a+ EVs were highest in non-LR and Trima RCCs and did not change during storage. WBC-derived EVs were low in most RCCs; CD14+ EVs increased in several RCCs during storage. Conclusion DAMPs in RCCs vary by manufacturing method. MtDNA and EV could be informative quality markers that may be relevant to RCC immunomodulatory potential.

Author(s): Kwaan, Nicholas; Lee, Tzong-Hae; Chafets, Daniel M; Nass, Catharie; Newman, Bruce; Smith, James; Garratty, George; Murphy, Edward L; HTLV Outcomes Study (HOST) Investigators | Abstract: BackgroundThe human T lymphotropic virus (HTLV)-I or -II proviral load (VL) may be linked to viral pathogenesis, but prospective data on VL and disease outcomes are lacking.MethodsUsing data from a prospective cohort study of HTLV disease outcomes, we examined baseline VLs with real-time quantitative polymerase chain reaction in 122 HTLV-I- and 319 HTLV-II-infected subjects and serial VLs over the course of 6 visits in a subset of 30 HTLV-I- and 30 HTLV-II-infected subjects. Cox and logistic-regression models were used to test baseline associations, and repeated-measures analysis was used to study variations in VL over time.ResultsOver the course of a median of 10.4 years, HTLV-I VLs decreased slightly (slope, -0.017 log(10) copies/10(6) peripheral blood mononuclear cells [PBMCs]/year; P=.042) and HTLV-II VLs did not change (slope, -0.019 log(10) copies/10(6) PBMCs/year; P=.165). Changes in VL over time were associated positively with alcohol use (P=.07) and negatively with black race (P=.03) for HTLV-I and positively with smoking (P=.08) for HTLV-II. In the larger group, there was no association between baseline VL and disease outcomes. In the smaller group with serial VL data, there was an association between increasing VL and bladder or kidney infections for both HTLV-I (P=.005) and HTLV-II (P=.022).ConclusionsHTLV VLs are stable over time, but alcohol and tobacco intake may affect the progression of VLs. The association between increasing VLs and bladder/kidney infection may be explained by early HTLV-related neuropathologic progression.

BACKGROUND: Transfusion of trauma patients can result in long-term survival of donor white blood cells (WBCs) or “transfusion-associated microchimerism” (TA-MC). The aim was to determine whether leukoreduction of blood transfusions, advocated to reduce the immunomodulatory effect of transfusion, decreases the likelihood of developing TA-MC. STUDY DESIGN AND METHODS: A subgroup of trauma patients from a randomized trial was examined, evaluating the risk of infection following leukoreduced versus nonleukoreduced blood transfusion. Patients’ blood was sampled at least 1 month after hospital discharge, and TA-MC was assessed with quantitative allele-specific polymerase chain reaction detection of differences at the HLA-DR locus or a panel of insertion-deletion polymorphism loci distributed throughout the chromosomal complement. At the time of blood sampling, a scripted interview was used to ascertain symptoms suggestive of chronic graft-versus host disease (cGVHD). RESULTS: For 67 patients evaluated, the mean age was 43 ± 17 years and mean Injury Severity Score was 24 ± 12. Median time from injury to blood sampling for TA-MC was 240 (interquartile range, 116-360) days. Nine of 32 patients (28%) in the nonleukoreduced transfusion group developed TA-MC compared to 13 of 35 patients (37%) in the leukoreduced group (p = 0.43). Subjects with TA-MC were no more likely than subjects without TA-MC to have at least one symptom suggestive of cGVHD (64% vs. 76%, respectively). CONCLUSIONS: TA-MC seems to be a prevalent condition among injured patients at the second of two regional trauma centers evaluated, suggesting that it is a common phenomenon after transfusion in the setting of injury. Although leukoreduction removes greater than 99.9 percent of donor WBCs, it fails to prevent or even substantially reduce the likelihood of developing TA-MC. TA-MC does not appear to be strongly associated with symptoms suggestive of cGVHD several months after transfusion.

Cellular immune responses to Kaposi sarcoma-associated herpesvirus (KSHV), the etiological agent of KS and several other malignancies, are incompletely characterized. We assessed KSHV-specific interferon- gamma enzyme-linked immunospot responses in a cohort of 154 individuals, using overlapping peptide sets spanning the KSHV-encoded latency-associated nuclear antigen (ORF73) and the minor capsid glycoprotein (ORF65). Among KSHV-seropositive subjects, ORF73-specific responses dominated over responses to ORF65 and were preferentially detected in human immunodeficiency virus-coinfected individuals who had elevated levels of cell-associated KSHV DNA, indicating that the viral antigen burden may have been driving these responses. Responses to both ORF73 and ORF65 were also detected in several KSHV-seronegative subjects who were at increased risk for KSHV infection, which demonstrates that cellular immunity can be found in the absence of detectable humoral responses. These data have implications for the reliable identification of KSHV infection and may help guide the design of immune-based therapeutic and prophylactic interventions.

BACKGROUND: Long-term white blood cell (WBC) microchimerism (MC), of at least 2 years, has been reported in trauma patients receiving fresh nonleukoreduced (non-LR) blood. It is unknown, however, whether this occurs with LR blood products that are nearly devoid of WBCs. Twenty-seven patients transfused with LR and non-LR blood products were studied after severe traumatic injury. A secondary aim was to explore donor-recipient mixed lymphocyte reactivity in vitro. STUDY DESIGN AND METHODS: To quantify MC, allele-specific real-time polymerase chain reaction assays were developed targeting HLA Class II sequence polymorphisms. Extensive validation showed that these assays reliably detect a single copy of target sequence in a complex allogeneic background without false positivity. RESULTS: At a median follow-up of 26 months (range, 24-39 months), long-term MC was observed in 3 of 20 patients (15%) who received non-LR blood products and 2 of 7 (29%) who received LR blood products. The maximum MC ranged from 0.40 to 4.90 percent of circulating WBCs and appeared, by Class II genotype analysis, to be attributable to a single donor. CONCLUSION: It is concluded that robust levels of long-term MC, apparently traceable to a single donor, occur at similar frequency despite leukoreduction of transfused blood products. Exploratory analysis of donor-recipient mixed lymphocyte reactivity suggests that long-term MC may require a state of bidirectional tolerance before transfusion.

Author(s): Roucoux, Diana F; Wang, Baoguang; Smith, Donna; Nass, Catharie C; Smith, James; Hutching, Sheila T; Newman, Bruce; Lee, Tzong-Hae; Chafets, Daniel M; Murphy, Edward L; HTLV Outcomes Study Investigators | Abstract: BackgroundCross-sectional studies support sexual transmission of human T lymphotropic virus (HTLV)-I/II; however, prospective incidence data, particularly for HTLV-II, are limited.MethodsA cohort of 85 HTLV-positive (30 with HTLV-I and 55 with HTLV-II) blood donors and their stable (gor=6 months) heterosexual sex partners were followed biannually over the course of a 10-year period.ResultsFour of 85 initially seronegative sex partners of HTLV-I and -II carriers seroconverted, for an incidence rate (IR) of 0.6 transmissions/100 person-years (py) (95% confidence interval [CI], 0.2-1.6). This includes 2 HTLV-I transmissions/219 py (IR, 0.9 transmissions/100 py [95% CI, 0.1-3.3]) and 2 HTLV-II transmissions/411 py (IR, 0.5 transmissions/100 py [95% CI, 0.06-1.8]), with no significant difference by HTLV type. There were 2 male-to-female (IR, 1.2 transmissions/100 py [95% CI, 0.1-4.3]) and 2 female-to-male (IR, 0.4 transmissions/100 py [95% CI, 0.05-1.6) transmissions. HTLV-I or -II proviral load was 2 log10 lower in newly infected partners than in index positive partners who transmitted HTLV (P=.007).ConclusionsThe incidence of sexual transmission of HTLV-II may be similar to that of HTLV-I, and female-to-male transmission may play a more important role than previously thought. HTLV-I and -II proviral load may be lower in sexually acquired infection, because of a small infectious dose.

Background: Blood transfusion can result in long-term survival of donor leukocyte subpopulations, or microchimerism, in the peripheral blood of injured patients. Neither injury severity nor the number of transfusions is associated with its occurrence. We sought to determine whether changes in general or antigen-specific lymphocyte activation may be associated with the subsequent development of microchimerism. Methods: We evaluated 63 transfused trauma patients, which we compared with 10 non-transfused trauma patients and 10 healthy control subjects. Of the 63 transfused patients, 31 were known to have evidence of microchimerism at hospital discharge with real-time quantitative PCR for non-recipient HLA DR alleles. We assessed lymphocyte response to phytohemagglutinin (PHA) using blood sampled upon arrival to the hospital (before transfusion) and at discharge. We performed one-way mixed leukocyte cultures (MLC) with pre-transfusion recipient specimens to assess recipient lymphocyte response to mitomycin-C treated donor cells and vice versa. Results: Lymphocyte response to PHA in microchimeric transfusion recipients was lower at admission (before transfusion) and discharge than in non-microchimeric recipients. Lymphocytes from microchimeric patients had less response to donor cells than did lymphocytes from non-microchimeric patients. Microchimeric patients also more frequently had diminished lymphocyte response to a single blood donor on MLC. Conclusions: Transfusion-associated microchimerism is correlated with diminished response to mitogen challenge as well as to specific alloantigenic challenges. This microchimerism is predated by diminished lymphocyte response to a specific blood donor in many instances. The blood donor associated with this diminished alloantigenic lymphocyte response may be the source of microchimeric cells present in the recipient.

High human T lymphotropic virus (HTLV)-I provirus load (VL) has been associated with an increased risk of HTLV-associated myelopathy, but little is known about variation in HTLV-I or -II VLs by demographic characteristics and risk behaviors.We measured HTLV-I and HTLV-II VLs in a large cohort of 127 HTLV-I-seropositive and 328 HTLV-II-seropositive former blood donors, by use of real-time polymerase chain reaction using tax primers. Multivariable linear regression was used to control for confounding by relevant covariates.The mean VLs were 3.28 log(10) copies/10(6) peripheral blood mononuclear cells (PBMCs) (range, 0.5-5.3 log(10) copies/10(6) PBMCs) for HTLV-I and 2.60 log(10) copies/10(6) PBMCs (range, 0.05-5.95 log(10) copies/10(6) PBMCs) for HTLV-II (P.0001). HTLV-II VLs were higher in those subjects with subtype A infection (mean, 2.82 log(10) copies/10(6) PBMCs) than in those with subtype B infection (mean, 2.29 log(10) copies/10(6) PBMCs) (P=.005). Higher HTLV-I VL was associated with previous receipt of a blood transfusion (P=.04), and lower HTLV-II VL was associated with female sex (P=.007). These associations persisted in virus-specific multivariate linear regression models controlling for potential confounding variables.VL was significantly higher in HTLV-I than in HTLV-II infection and was higher in HTLV-II subtype A than in HTLV-II subtype B infection. Chronic HTLV VLs may be related to the infectious dose acquired at the time of infection, with higher VLs following acquisition by blood transfusion and lower VLs following sexual acquisition.