Genetic and behavioral modification of hemoglobin and iron status among first-time and high-intensity blood donors.

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.

Lack of persistent microchimerism in contemporary transfused trauma patients.

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.

Genomewide association study of HLA alloimmunization in previously pregnant blood donors.

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 < 10-6 , for women with anti-Class I alloantibodies only. Little is currently known about the function of NXPH2, although gene family members have been shown to impact immunity. SNPs in the E2F7 gene, a transcription factor related to cell cycle control and cellular proliferation, also approached genomewide significance (p = 2.5 × 10-7 ). CONCLUSION: Further work to extend the GWAS approach and to characterize variants in NXPH2 and E2F7 in the context of alloantibody formation is warranted.

Minimal infectious dose and dynamics of Babesia microti parasitemia in a murine model.

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.

Extensive virologic and immunologic characterization in an HIV-infected individual following allogeneic stem cell transplant and analytic cessation of antiretroviral therapy: A case study.

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.

Development and validation of a real-time reverse transcriptase PCR assay for sensitive detection of SFTSV.

BACKGROUND: Severe fever with thrombocytopenia syndrome bunyavirus (sftsv) is an emerging tick-borne rna virus recently identified as the pathogen that causes severe fever with thrombocytopenia syndrome (sfts) in china. the existing commercial nucleic acid testing (comnat) assay with a relatively high claimed limit of quantitative detection (loqd) is not capable of sensitive detection and quantitation of sftsv. Thus, a new real-time reverse transcriptase (rt)-pcr assay with improved sensitivity is needed for clinical diagnosis; it could also be used to screen blood donors if necessary. MATERIALS AND METHODS: We developed a new sftsv rt-pcr nat assay (newnat). About 129 plasma samples from 93 suspected sfts patients with typical clinical symptoms were tested using an anti-sftsv total antibody elisa and both comnat and newnat. The test performance of the two nat assays was evaluated and compared. RESULTS: The newnat had a lower limit for quantitative testing compared to comnat. Twelve samples were comnat negative but newnat positive. Out of 35 suspected sfts patients who were comnat negative and anti-sftsv total antibody negative, four tested positive by the newnat assay and one of these four seroconverted within 2-4 days after testing newnat positive. A high correlation was observed between the cts of the newnat and comnat assays. CONCLUSION: The newnat assay was sensitive for quantitative detection of sftsv and may be applicable to clinical diagnosis and studies of the need for blood donor screening.

Duration of Dengue Viremia in Blood Donors and Relationships Between Donor Viremia, Infection Incidence and Clinical Case Reports During a Large Epidemic.

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.

Transfusion-Transmitted Dengue and Associated Clinical Symptoms During the 2012 Epidemic in Brazil.

BACKGROUND: A linked donor-recipient study was conducted during epidemics in 2 cities in Brazil to investigate transfusion-transmitted (TT) dengue virus (DENV) by DENV RNA-positive donations. METHODS: During February-June 2012, samples were collected from donors and recipients and retrospectively tested for DENV RNA by transcription-mediated amplification. Recipient chart review, using a case (DENV positive)-control (DENV negative and not known to be exposed) design, was conducted to assess symptoms. RESULTS: Of 39 134 recruited blood donors, DENV-4 viremia was confirmed in 0.51% of donations from subjects in Rio de Janeiro and 0.80% of subjects in Recife. Overall, 42 DENV RNA-positive units were transfused into 35 recipients. Of these, 16 RNA-positive units transfused into 16 susceptible recipients were identified as informative: 5 cases were considered probable TT cases, 1 possible TT case, and 10 nontransmissions. The TT rate was 37.5% (95% confidence interval [CI], 15.2%-64.6%), significantly higher than the viremia rate of 0.93% (95% CI, .11%-3.34%) in nonexposed recipients (P < .0001). Chart review did not find significant differences between cases and controls in symptoms or mortality. CONCLUSIONS: During a large epidemic of DENV-4 infection in Brazil, >0.5% of donations were RNA positive, and approximately one third of components resulted in TT. However, no significant clinical differences were evident between RNA-positive and RNA-negative recipients.

Assessment of nucleic acid modification induced by amotosalen and ultraviolet A light treatment of platelets and plasma using real-time polymerase chain reaction amplification of variable length fragments of mitochondrial DNA.

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.

A prospective evaluation of chronic Babesia microti infection in seroreactive blood donors.

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.

Challenges in detecting HIV persistence during potentially curative interventions: a study of the Berlin patient.

There is intense interest in developing curative interventions for HIV. How such a cure will be quantified and defined is not known. We applied a series of measurements of HIV persistence to the study of an HIV-infected adult who has exhibited evidence of cure after allogeneic hematopoietic stem cell transplant from a homozygous CCR5Δ32 donor. Samples from blood, spinal fluid, lymph node, and gut were analyzed in multiple laboratories using different approaches. No HIV DNA or RNA was detected in peripheral blood mononuclear cells (PBMC), spinal fluid, lymph node, or terminal ileum, and no replication-competent virus could be cultured from PBMCs. However, HIV RNA was detected in plasma (2 laboratories) and HIV DNA was detected in the rectum (1 laboratory) at levels considerably lower than those expected in ART-suppressed patients. It was not possible to obtain sequence data from plasma or gut, while an X4 sequence from PBMC did not match the pre-transplant sequence. HIV antibody levels were readily detectable but declined over time; T cell responses were largely absent. The occasional, low-level PCR signals raise the possibility that some HIV nucleic acid might persist, although they could also be false positives. Since HIV levels in well-treated individuals are near the limits of detection of current assays, more sensitive assays need to be developed and validated. The absence of recrudescent HIV replication and waning HIV-specific immune responses five years after withdrawal of treatment provide proof of a clinical cure.

A study of seroprevalence and rates of asymptomatic viremia of severe fever with thrombocytopenia syndrome virus among Chinese blood donors.

BACKGROUND: Severe fever with thrombocytopenia syndrome virus (SFTSV), an emerging tick-borne pathogen that can cause fatal severe fever with thrombocytopenia syndrome, 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. STUDY DESIGN AND METHODS: From April 16 to October 31, 2012, a total of 17,208 plasma samples were collected from donors at Xinyang (located in an SFTSV-endemic area), Mianyang, and Luoyang Blood Centers. Assessment of anti-SFTSV total antibody was performed on all samples using enzyme-linked immunosorbent assay. Repeat-reactive samples were tested for SFTSV RNA using reverse transcription (RT)-real-time polymerase chain reaction (PCR) assay with Taqman probes. In addition, 9960 of the Xinyang samples were tested in pools of 4 by the same PCR method and each of the samples in a reactive pool was tested individually. RESULTS: Donor seroreactivity rates were as follows: Xinyang, 0.54% (80/14,752); Mianyang, 0.27% (3/1130); and Luoyang, 0.28% (3/1326). All seroreactive samples were negative on RT-PCR single-sample testing. Two RT-PCR-reactive donor samples were identified, both with estimated viral load of less than 20 plaque-forming units/mL. The RNA prevalence rate for SFTSV among donors in Xinyang was 0.02%. CONCLUSION: This was the first multiregion study of SFTSV sero- and viral prevalence among Chinese blood donors. Viral prevalence was low and no seroreactive sample was viremic, suggesting a limited impact of SFTSV on blood safety in China.

Antiretroviral-free HIV-1 remission and viral rebound after allogeneic stem cell transplantation: report of 2 cases.

BACKGROUND: It is unknown whether the reduction in HIV-1 reservoirs seen after allogeneic hematopoietic stem cell transplantation (HSCT) with susceptible donor cells is sufficient to achieve sustained HIV-1 remission. OBJECTIVE: To characterize HIV-1 reservoirs in blood and tissues and perform analytic antiretroviral treatment interruptions to determine the potential for allogeneic HSCT to lead to sustained, antiretroviral-free HIV-1 remission. DESIGN: Case report with characterization of HIV-1 reservoirs and immunity before and after antiretroviral interruption. SETTING: Tertiary care center. PATIENTS: Two men with HIV with undetectable HIV-1 after allogeneic HSCT for hematologic tumors. MEASUREMENTS: Quantification of HIV-1 in various tissues after HSCT and the duration of antiretroviral-free HIV-1 remission after treatment interruption. RESULTS: No HIV-1 was detected from peripheral blood or rectal mucosa before analytic treatment interruption. Plasma HIV-1 RNA and cell-associated HIV-1 DNA remained undetectable until 12 and 32 weeks after antiretroviral cessation. Both patients experienced rebound viremia within 2 weeks of the most recent negative viral load measurement and developed symptoms consistent with the acute retroviral syndrome. One patient developed new efavirenz resistance after reinitiation of antiretroviral therapy. Reinitiation of active therapy led to viral decay and resolution of symptoms in both patients. LIMITATION: The study involved only 2 patients. CONCLUSION: Allogeneic HSCT may lead to loss of detectable HIV-1 from blood and gut tissue and variable periods of antiretroviral-free HIV-1 remission, but viral rebound can occur despite a minimum 3-log10 reduction in reservoir size. Long-lived tissue reservoirs may have contributed to viral persistence. The definition of the nature and half-life of such reservoirs is essential to achieve durable antiretroviral-free HIV-1 remission. PRIMARY FUNDING SOURCE: Foundation for AIDS Research and National Institute of Allergy and Infectious Diseases.

West Nile virus nucleic acid persistence in whole blood months after clearance in plasma: implication for transfusion and transplantation safety.

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.

Cell-based measures of viral persistence are associated with immune activation and programmed cell death protein 1 (PD-1)-expressing CD4+ T cells.

BACKGROUND: Studies aimed at defining the association between host immune responses and human immunodeficiency virus (HIV) persistence during therapy are necessary to develop new strategies for cure. METHODS: We performed a comprehensive assessment of ultrasensitive plasma HIV RNA levels, cell-associated HIV RNA levels, proviral HIV DNA levels, and T cell immunophenotyping in a cohort of 190 subjects in whom HIV levels were suppressed by highly active antiretroviral therapy. RESULTS: The median CD4(+) T cell count was 523 cells/mm(3), and the median duration of viral suppression was 31 months. Cell-associated RNA and proviral DNA levels (but not ultrasensitive plasma HIV RNA levels) were positively correlated with frequencies of CD4(+) and CD8(+) T cells expressing markers of T-cell activation/dysfunction (CD38, HLA-DR, CCR5, and/or programmed cell death protein 1 [PD-1]) (P < .05). Having a low CD4(+) T-cell count despite receipt of virologically suppressive therapy was associated with high cell-associated RNA and proviral DNA levels (P < .01) and higher frequencies of CD4(+) T cells expressing CD38, HLA-DR, CCR5, and/or PD-1 (P < .0001). CONCLUSIONS: Cell-based measurements of viral persistence were consistently associated with markers of immune activation and the frequency of PD-1-expressing CD4(+) T cells. Treated patients with a low CD4(+) T-cell count had higher frequencies of PD-1-expressing CD4(+) T cells and cell-based measures of viral persistence, suggesting that HIV infection in these individuals may be more difficult to cure and may require unique interventions.

Antiretroviral therapy initiated within 6 months of HIV infection is associated with lower T-cell activation and smaller HIV reservoir size.

Background. CD4(+)/CD8(+) T-cell activation levels often remain elevated in chronic human immunodeficiency virus (HIV) infection despite initiation of antiretroviral therapy (ART). T-cell activation predicts early death and blunted CD4+ T-cell recovery during ART and may affect persistent HIV reservoir size. We investigated whether very early ART initiation is associated with lower on-therapy immune activation and HIV persistence. Methods. From a cohort of patients with early HIV infection (<6 months duration since infection) we identified persons who started ART early (<6 months after infection) or later (≥2 years after infection) and maintained ≥2 years of virologic suppression; at-risk HIV-negative persons were controls. We measured CD4(+)/CD8(+) T-cell activation (percent CD38(+)/HLA-DR(+)) and HIV reservoir size (based on HIV DNA and cell-associated RNA levels). Results. In unadjusted analyses, early ART predicted lower on-therapy CD8(+) T-cell activation (n = 34; mean, 22.1%) than achieved with later ART (n = 32; mean, 28.8%; P = .009), although levels in early ART remained elevated relative to HIV-negative controls (P = .02). Early ART also predicted lower CD4+ T-cell activation than with later ART (5.3% vs 7.5%; P = .06). Early ART predicted 4.8-fold lower DNA levels than achieved with later ART (P = .005), and lower cell-associated RNA levels (difference in signal-to-cutoff ratio (S/Co), 3.2; P = .035). Conclusions. ART initiation <6 months after infection is associated with lower levels of T-cell activation and smaller HIV DNA and RNA reservoir size during long-term therapy.

Development of a real-time polymerase chain reaction assay for sensitive detection and quantitation of Babesia microti infection.

BACKGROUND: Babesia microti, the most frequently implicated pathogen in transfusion-transmitted babesiosis, is widely endemic in the Northeast and upper Midwestern United States. High seroprevalence in endemic areas limits antibody-based donor screening. A high-performance molecular test is needed to identify donors in the preseroconversion window phase as well as to discriminate past serologic exposure with parasite clearance from continued parasitemia. STUDY DESIGN AND METHODS: Frozen Babesia-spiked whole blood was microcentrifuged, and the supernatant transferred and microcentrifuged again to concentrate the parasite. The DNA was extracted and amplified using real-time polymerase chain reaction (PCR) using Babesia-specific primers. The assay was employed in three series of experiments: 1) a validation and optimization spiking experiment, 2) a blinded serial dilution probit analysis to determine the limit of detection, and 3) evaluation of two blinded panels of clinical samples from possible babesiosis cases. RESULTS: At a decreasing inoculum of 445, 44.5, and 4.45 copies/mL, the assay had positive rates of 100, 97.5, and 81%, respectively. The blinded probit analysis demonstrated a detection rate of 95 and 50% at 12.92 and 1.52 parasites/2 mL of whole blood, respectively. Evaluation of clinical samples showed 13 of 21 samples to be positive, with a range of 85 to 4.8 million parasites/mL. There were no positives detected among 48 healthy donors CONCLUSION: We have developed a highly sensitive and specific, quantitative real-time PCR-based assay for detection of B. microti that could have a useful role in blood screening. It can also be employed broadly to understand Babesia epidemiology, disease pathogenesis, and host immunology.

Antibody levels correlate with detection of Trypanosoma cruzi DNA by sensitive polymerase chain reaction assays in seropositive blood donors and possible resolution of infection over time.

BACKGROUND: The clinical significance of anti-Trypanosoma cruzi low-level reactive samples is incompletely understood. Polymerase chain reaction (PCR)-positive rates and antibody levels among seropositive blood donors in three countries are described. STUDY DESIGN AND METHODS: Follow-up samples were collected from T. cruzi-seropositive donors from 2008 through 2010 in the United States (n = 195) and Honduras (n = 58). Also 143 samples from Brazil in 1996 to 2002, originally positive by three serologic assays, were available and paired with contemporary follow-up samples from these donors. All samples were retested with Ortho enzyme-linked immunosorbent assay (ELISA). PCR assays were performed on coded sample panels by two laboratories (Blood Systems Research Institute [BSRI] and American Red Cross Holland Laboratory [ARC]) that amplified kinetoplast minicircle DNA sequences of T. cruzi. RESULTS: PCR testing at BSRI yielded slightly higher overall sensitivity and specificity (33 and 98%) compared with those at the ARC (28 and 94%). Among seropositive donors, PCR-positive rates varied by country (p < 0.0001) for the BSRI laboratory: Brazil (57%), Honduras (32%), and the United States (14%). ELISA signal-to-cutoff ratios (S/CO) were significantly higher for PCR-positive compared to PCR-negative donors (p < 0.05 for all comparisons). Additionally, PCR-negative Brazilian donors exhibited greater frequencies of antibody decline over time versus PCR-positive donors (p = 0.003). CONCLUSION: For all three countries, persistent DNA positivity correlated with higher ELISA S/CO values, suggesting that high-level seroreactivity reflects chronic parasitemia. Significant S/CO declines in 10% of the PCR-negative Brazilian donors may indicate seroreversion after parasite clearance in the absence of treatment.

Fetal production of growth factors and inflammatory mediators predicts pulmonary hypertension in congenital diaphragmatic hernia.

BACKGROUND: Congenital diaphragmatic hernia (CDH) represents a spectrum of lung hypoplasia, and consequent pulmonary hypertension (PH) 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 PH. 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 fetus was quantified using quantitative real-time PCR for nonshared alleles. Biomarker profiles were then correlated with CDH severity on the basis of the degree of PH. RESULTS: Cord blood levels of epidermal growth factor, platelet-derived growth factor, and several inflammatory mediators increased significantly as the severity of CDH increased, whereas maternal levels of growth factors and mediators decreased significantly with CDH severity. Maternal cells were increased in fetuses with severe CDH as compared with controls, with elevated levels of the CXC chemokine ligand-10 in patients with the highest trafficking. CONCLUSION: Patients with CDH demonstrate proinflammatory and chemotactic signals in fetal blood at the time of birth. Because some of these molecules have been implicated in the development of PH, prenatal strategies targeting specific molecular pathways may be useful adjuncts to current fetal therapies.

The impact of HFE mutations on haemoglobin and iron status in individuals experiencing repeated iron loss through blood donation*.

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.