Negative Donors Research Articles

Impact of utilization of hepatitis C positive organs in liver transplant: Analysis of united network for organ sharing database.

BACKGROUNDThe utility of hepatitis C virus (HCV) organs has increased after the Food and Drug Administration approval of direct acting anti-viral (DAA) medications for the HCV treatment. The efficacy of DAA in treating HCV is nearly 100%. AIMTo analyze the United Network for Organ Sharing (UNOS) database to compare the survival rates between the hepatitis C positive donors and negative recipients and hepatitis C negative donors and recipients. METHODSWe analyzed the adult patients in UNOS database who underwent deceased donor liver transplant from January 2014 to December 2017. The primary endpoint was to compare the survival rates among the four groups with different hepatitis C donor and recipient status: (Group 1) Both donor and recipient negative for HCV (Group 2) Negative donor and positive recipient for HCV (Group 3) Positive donor and negative recipient for HCV (Group 4) Both positive donor and recipient for HCV. SAS 9.4 software was used for the data analysis. Kaplan Meier log rank test was used to analyze the estimated survival rates among the four groups. RESULTSA total of 24512 patients were included: Group 1: 16436, Group 2: 6174, Group 3: 253 and Group 4: 1649. The 1-year (Group 1: 91.8%, Group 2: 92.12%, Group 3: 87%, Group 4: 92.8%), 2-year (Group 1: 88.4%, Group 2: 88.1%, Group 3: 84.3%, Group 4: 87.5%), 3-year (Group 1: 84.9%, Group 2: 84.3%, Group 3: 75.9%, Group 4: 83.2%) survival rates showed no statistical significance among the four groups. Kaplan Meier log rank test did not show any statistical significance difference in the estimated survival rates between Group 3 vs all the other groups. CONCLUSIONThe survival rates in hepatitis C positive donors and negative recipients are similar as compared to both hepatitis C negative donors and recipients. This could be due to the use of DAA therapy with cure rates of nearly 100%. This study supports the use of hepatitis C positive organs in the selected group of recipients with and without HCV infection. Further long-term studies are needed to further validate these findings.

Nucleic acid testing identifies high prevalence of blood borne viruses among approved blood donors in Mozambique.

BackgroundAlthough blood transfusion is an intervention that saves lives, it poses significant risks to the blood receivers, including the transmission of bloodborne pathogens. We aimed at determining the prevalence of Human Immunodeficiency Virus (HIV), Hepatitis B virus (HBV), and Hepatitis C virus (HCV) in candidates approved for blood donation, and in samples considered to be negative in reference blood banks in Mozambique.MethodsA cross-sectional study was performed between November 2014 and October 2015 in Maputo and Beira cities. Demographic information was obtained from all consenting blood donors using a structured questionnaire. Plasma samples were screened for HIVAb/Ag combinations, HBsAg and Anti-HCV. Blood donors considered to be negative by serological testing were re-tested in pools of six plasma samples using nucleic acid testing (NAT).ResultsMost blood donors were male 2,320 (83.4%) with an age range of 18 to 34 years. The overall seroprevalence of HIV, HBV and HCV infections among blood donors approved for donation was 4.6% (127; 95% CI 3.8–5.4), 4.5% (124; 95% CI 3.7–5.3) and 0.4% (11; 95% CI 0.2–0.7), respectively. The overall frequency by NAT of HIV RNA, HBV DNA, and HCV RNA in serologically negative blood donor samples was 2.6 per 1000 blood donors (7; 95% CI 1.1–5.4); 12.5 per 1000 blood donors (33; 95% CI 8.6–17.5) and 2.6 per 1000 blood donors (6; 95% CI 1.0–5.7), respectively.ConclusionOur results show high seroprevalence of HIV and HBV infections in blood donors approved for donation, and high frequency of molecular biomarkers of HIV, HBV, and HCV in blood considered to be safe. These results suggest the need for a new blood screening policy in Mozambique, including the use of NAT to detect infectious blood donations during the immunologically negative window.

The serum of COVID-19 asymptomatic patients up-regulates proteins related to endothelial dysfunction and viral response in circulating angiogenic cells ex-vivo

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has already caused 6 million deaths worldwide. While asymptomatic individuals are responsible of many potential transmissions, the difficulty to identify and isolate them at the high peak of infection constitutes still a real challenge. Moreover, SARS-CoV-2 provokes severe vascular damage and thromboembolic events in critical COVID-19 patients, deriving in many related deaths and long-hauler symptoms. Understanding how these processes are triggered as well as the potential long-term sequelae, even in asymptomatic individuals, becomes essential.MethodsWe have evaluated, by application of a proteomics-based quantitative approach, the effect of serum from COVID-19 asymptomatic individuals over circulating angiogenic cells (CACs). Healthy CACs were incubated ex-vivo with the serum of either COVID-19 negative (PCR −/IgG −, n:8) or COVID-19 positive asymptomatic donors, at different infective stages: PCR +/IgG − (n:8) and PCR −/IgG + (n:8). Also, a label free quantitative approach was applied to identify and quantify protein differences between these serums. Finally, machine learning algorithms were applied to validate the differential protein patterns in CACs.ResultsOur results confirmed that SARS-CoV-2 promotes changes at the protein level in the serum of infected asymptomatic individuals, mainly correlated with altered coagulation and inflammatory processes (Fibrinogen, Von Willebrand Factor, Thrombospondin-1). At the cellular level, proteins like ICAM-1, TLR2 or Ezrin/Radixin were only up-regulated in CACs treated with the serum of asymptomatic patients at the highest peak of infection (PCR + /IgG −), but not with the serum of PCR −/IgG + individuals. Several proteins stood out as significantly discriminating markers in CACs in response to PCR or IgG + serums. Many of these proteins particiArticle title: Kindly check and confirm the edit made in the articletitle.pate in the initial endothelial response against the virus.ConclusionsThe ex vivo incubation of CACs with the serum of asymptomatic COVID-19 donors at different stages of infection promoted protein changes representative of the endothelial dysfunction and inflammatory response after viral infection, together with activation of the coagulation process. The current approach constitutes an optimal model to study the response of vascular cells to SARS-CoV-2 infection, and an alternative platform to test potential inhibitors targeting either the virus entry pathway or the immune responses following SARS-CoV-2 infection.

Heart Transplant Outcomes by Donor HCV NAT Status: Intermediate Analysis

<h3>Purpose</h3> Donor hearts are a limited resource and inclusion of non-traditional donors such as those with hepatitis C (HCV) may help to increase transplantation rates. We sought to compare post-heart transplant outcomes of patients who received hearts from either HCV NAT positive or negative donors. <h3>Methods</h3> Recipients were identified from the UNOS Registry and stratified by donor HCV NAT status. Comparisons between cohorts were assessed using standard statistical methods, survival analysis was censored at 36 months using the Kaplan-Meier method, and multivariate Cox proportional hazard regression analysis (adjusted for age, sex, diabetes, race, ischemic time, dialysis, life support, waiting time and HLA mismatch. <h3>Results</h3> 18,828 recipients were identified of which 849 and 17,979 received HCV NAT positive (HCV+) and HCV NAT negative (HCV-) donors, respectively. HCV+ recipients were older (55.2 vs 53.5, p<0.001), male (77.4% vs 73.3%, p=0.008), and Caucasian (66.1% vs 62.1%, p=0.023). HCV+ recipients also had higher incidence of pre-transplant cardiac surgery (83.3% vs 77.9%, p<0.001), life support use (20.8% vs 16.5%, p<0.001), older donors (34.2 vs 32.1y, p<0.001), longer ischemic time (p<0.001), and with worse renal function (Cr 1.5 vs 1.4, p = 0.001). Notably, there was no difference in waitlist time. Recipients of HCV- donors were more likely to have diabetes (71.1% vs 66.4%, p = 0.004) and higher waitlist status (p<0.001). There was no difference in overall mortality between the two groups with cardiovascular (27.1% vs 26.4%) or non-cardiovascular (72.9% vs 73.6%, p=0.90) death. Unadjusted analysis demonstrated HR 0.98 (CI 0.78-1.22) and following adjustment HR 0.99 (0.78-1.24). <h3>Conclusion</h3> Utilization of HCV NAT positive donor hearts presents as a safe and effective way to expand the donor pool with no survival difference in this intermediate analysis. Further study is needed to understand the long-term outcomes of this population, particularly in the setting of widely available curative therapies.

Lung Transplant Recipients Developing Early DSAs within the First Month Are Characterized by a Higher Frequency of Naïve and a Lower Frequency of Memory B Cells

<h3>Purpose</h3> After lung transplantation (LuTx), the development of early donor-HLA-specific antibodies (eDSA) has been shown to be associated with antibody-mediated rejection (AMR) and poor graft survival. Since 2013, patients with eDSA within the first month after LuTx are treated with IgA/IgM-enriched intravenous immunoglobulins (IgGAM), in clinical AMR combined with anti-CD20 antibody (Rituximab). We hypothesize a different distribution and kinetics of IgD⁺CD27^- naïve B cells vs. IgD^-CD27^+/- memory B cell subsets in recipients with or without eDSA. <h3>Methods</h3> In a pilot study of 62 LuTx recipients, B cell subsets were by analyzed in blood pre, post (T0), 24 hours (T24) and 3 weeks after LuTx by flow cytometry using CD19, CD20, IgD, CD24, CD27, CD38 antibodies. Frequencies of B cell subsets were compared between two groups, eDSA-positive (n=14, 22,6%) and -negative (n=48, 77,4%) patients and healthy donors (n=30). <h3>Results</h3> Prior to LuTx, patients with end-stage lung disease showed significantly higher proprotions of IgD⁺CD27^- naïve B cells compared to HC (p<0.001). At T0, T1 and T24, patients with eDSAs showed significantly higher frequencies of naïve and lower frequencies of IgD^-CD27⁺ memory B cells (both p< 0.001) compared to eDSA-negative patients. These IgD⁺CD27^- naïve B cells displayed high CD24 and CD38 expression, which was stable also at 3 wks. A transient increase in IgD⁺CD27⁺ switch memory B cells was detected at T0 in both groups, returning to baseline levels at T24. <h3>Conclusion</h3> Lung transplant recipients show remarkable dynamics of naïve, memory and switch memory B cells within the first 3 wks. In both groups, we saw a shift in the B cell population: increased naïve B cells and decreased memory B cells, which was more pronounced in the eDSA group. Based on our preliminary data, a more refined B cell monitoring with additional markers may be able to identify patients with a higher risk for eDSA development. The impact of the treatment regimen on these B cell subsets is currently investigated.

BK Virus Nephropathy in HIV-Positive Heart-Kidney Transplant Recipient

<h3>Introduction</h3> BK viral reactivation is common in immunosuppressed states such as advanced HIV and organ transplantation. An important cause of kidney transplant allograft dysfunction and loss is BK reactivation leading to BK virus-associated nephropathy (BKVAN). We describe a problematic case of BKVAN in a combined heart-kidney transplant (HKT) recipient with well controlled HIV. <h3>Case Report</h3> A 46-year-old man with HIV (CD4 726 cells/mm³, HIV viral load <20 copies/mL) on dolutegravir/rilpivirine, non-ischemic cardiomyopathy, and HIV nephropathy underwent HKT with basiliximab induction and maintenance tacrolimus (goal 5-8 ng/mL), mycophenolate mofetil (MMF), and prednisone. Immediate post-transplant course was complicated by delayed renal graft function with acute tubular necrosis and urine leak. HIV has remained well controlled. Post-transplant nadir CD4 count was 639 cells/mm³. The patient developed BK viuria (90,891,555 copies/mL) and BK viremia (4,500 copies/mL) on post-transplant day (PTD) 55 and 62, respectively. Although MMF dose was reduced from 1,000 mg to 500 mg daily, renal allograft biopsy shortly after showed Class B1 BKVAN with no evidence of rejection. Patient began bi-weekly 5 mg/kg cidofovir infusions. Prednisone was decreased from 10 mg to 5 mg daily. Mycophenolate was further reduced to 250 mg daily and steroids were discontinued due to persistently high BK viral load. However, after decline in ejection fraction (EF) to 50% and suspicion for cardiac graft rejection, MMF dose was increased back to 500 mg daily. Endomyocardial biopsies have shown no evidence of rejection with negative donor specific anti-HLA antibodies. Renal function declined with serum creatinine (SCr) peaking at 2.28 mg/dL. Patient has remained on cidofovir infusions and reduced immunosuppression, on which SCr has stabilized to 1.8 mg/dL on PTD 694. Cardiac allograft function remains normal with an EF of 71% on PTD 644. BK viral load peaked at 16,672,298 copies/mL and remains elevated at 4,821 copies/mL on PTD 702. <h3>Summary</h3> We present a case of BKVAN in a HIV+ HKT recipient. This case demonstrates the difficulty of BKVAN management in dual organ transplant since heart allograft management requires higher levels of maintenance immunosuppression. It is unclear if HIV contributed to development of BKVAN in this recipient. Better antiviral therapies are needed for control of BK virus infections after transplant.

COVID-19 and humoral immune response in convalescent plasma donors in Pakistani cohort.

Current COVID-19 pandemic has affected the entire globe. While there was no vaccine neither any specific treatment, investigational use of convalescent plasma has been explored in clinical trials. A prospective multicenter study of convalescent plasma was conducted. Donors were tested for total Anti-SARS-CoV-2 antibodies by electrochemiluminescence (ECLIA) and RT-PCR for COVID-19. Enzyme Linked Immunosorbent Assay (ELISA) was used to detect semi-quantitative and quantitative IgG anti-SARS-COV-2 antibodies. IgG Immunofluorescence-based lateral flow immunoassay (LFIA) was used to recheck seronegative donors. A total of 400 donors were enrolled. Twelve donors were SARS-CoV-2 positive by RT-PCR. Nine of 12 donors had developed SARS-CoV-2 IgG antibodies, while in 3 donors antibodies were not developed. A total of 70 donors (17.5%) were deferred due to seronegative status; 64 (16%) of them did not develop antibodies when plasma collection was planned. The IgG semiquantitative ELISA was positive in 282 and quantitative in 284 of 330 donors with a mean value of &gt;1:160 and 44.10±39.22 IU/ml respectively. A total of 116 (29%) donors did not show IgG humoral response to COVID-19 even 28 days from the onset of illness. Subsequently, LFIA method was able to detect IgG antibodies in 20 of 48 (41.6%) seronegative donors and in 20 of 34 (58.8%) ECLIA positive ELISA negative donors. Viral RNA detection in recovered asymptomatic patients with concomitant IgG antibodies indicates recovery. Inability to detect antibodies by different testing kits may be due to their different antigenic targets or sensitivity. Significance of a positive COVID-19 RT-PCR in asymptomatic recovered patients is yet to be determined.

Tracing the SARS-CoV-2 infection on the ocular surface: Overview and preliminary corneoscleral transcriptome sequencing

COVID-19's impact on the ocular surface has already been recognized, however the molecular mechanisms induced by the infection on the ocular surface are still unclear. The aim of this paper is to provide a first overview of the transcriptional perturbations caused by SARS-CoV-2 on the ocular surface by analyzing gene expression profile of corneoscleral ring samples from post-mortem SARS-CoV-2 positive donors (PD). The presence of SARS-CoV-2 on the ocular surface, in tears and corneal tissues has rarely been detected in infected individuals in both the presence and the absence of ocular manifestations. In this preliminary study, 6 human corneoscleral tissues of 3 PD and two tissues from a negative donor (CTRL) were obtained at the local eye bank. The presence of genomic and sub-genomic SARS-CoV-2 RNAs was assessed by qRT-PCR, while transcriptome analysis (RNA-sequencing) was performed by Illumina. Principal Component Analysis (PCA), search for differentially expressed genes (DEGs) and Gene Ontology (GO)-enrichment analysis were performed. Three samples from PD were found positive for SARS-CoV-2 genomic RNA, although the absence of sub-genomic RNAs indicated an inactive virus. PCA analysis grouped 3 different clusters, one including CTRL, and the other two including, respectively, PD with undetected SARS-CoV-2 (PD-SARS-neg) and PD with detected SARS-CoV-2 (PD-SARS-pos). The DEGs in common with the 2 PD clusters included several genes associable to the interferon pathway, such as ADAMTS4, RSAD2, MMP1, IL6, ISG15 and proinflammatory cytokines. Among the down-regulated genes we found AQP5. GO analysis revealed 77 GO terms over-represented in PD-SARS-neg vs. CTRL, and 17 GO terms in PD-SARS-pos vs. CTRL.The presence of SARS-CoV-2 RNA and RNA-sequencing reads in ocular surface tissues supports the possibility that the eye acts as an entry route. The modulation of early responsive genes, together with several ISGs suggests a potential protective responsiveness of the ocular tissues to SARS-CoV-2.

Seroprevalence and Risk Factors of Trichinella spiralis Infection in Blood Donors from Western Romania.

Background and Objectives: Trichinellosis, a serious and sometimes fatal human disease, is a foodborne zoonotic disease with worldwide distribution caused by parasitic nematodes of the genus Trichinella. Humans are infected with Trichinella larvae through the ingestion of meat that has not been properly cooked. Romania reported most of the confirmed cases of trichinellosis among the EU countries. The aim of this cross-sectional study was to evaluate, for the first time, the seroprevalence and risk factors of Trichinella infection in blood donors from Western Romania. Materials and Methods: Serum samples of 1347 consecutive blood donors were investigated using an immunoenzymaticassay (ELISA) for the determination of specific IgG class antibodies against T.spiralis. A questionnaire interview was used to obtain information regarding the potential risk factors associated with T. spiralis infection. Mantel–Haenszel chi-squared test or the Fisher exact two-tailed test, as appropriate, were used for comparison between T. spiralis positive and T. spiralis negative blood donors. Student’s t-test was used to evaluate differences between means in studied groups and body mass index was calculated by dividing weight in kilograms by height in meters squared. Statistical analysis was performed using Epi Info Version 7.2 and Stata 16.1. Results: T. spiralis IgG antibodies were detected in 2.00% (27) of 1347 consecutive blood donors. Eating raw and/or undercooked meat, from pigs or wild boars, was found to be the main risk factor (p < 0.001). Strong alcoholic drink consumption was highly associated with T. spiralis infection (p = 0.009). Trichinella seroprevalence was higher among rural residents and males. Subjects identified as Trichinella seropositive were not previously diagnosed and have not been treated for Trichinella infection with any specific therapy. Conclusions: The demonstration of T. spiralis antibodies in healthy blood donors suggests that Trichinella infection may be detected in asymptomatic individuals that were not previously diagnosed with this zoonosis.

Impact of donor multidrug-resistant organisms on solid organ transplant recipient outcomes.

The impact of donor colonization or infection with multidrug-resistant organisms (MDROs) on solid organ transplant (SOT) recipient outcomes remains uncertain. We thus evaluated the association between donor MDROs and risk of posttransplant infection, graft failure, and mortality. A multicenter retrospective cohort study was performed. All SOT recipients with a local deceased donor were included. The cohort was divided into three exposure groups: recipients whose donors had (1) an MDRO, (2) a non-MDRO bacterial or candidal organism, or (3) no growth on cultures. The primary outcomes were (1) bacterial or invasive candidal infection within 3 months and (2) graft failure or death within 12months posttransplant. Mixed effect multivariable frailty models were developed to evaluate each association. Of 658 total SOT recipients, 93 (14%) had a donor with an MDRO, 477 (73%) had a donor with a non-MDRO organism, and 88 (13%) had a donor with no organisms on culture. On multivariable analyses, donor MDROs were associated with a significantly increased hazard of infection compared to those with negative donor cultures (adjust hazard ratio [aHR] 1.63, 95% CI 1.01-2.62, p=.04) but were not associated with graft failure or death (aHR 0.45, 95% CI 0.15-1.36, p=.16). MDROs on donor culture increase the risk of early posttransplant infection but do not appear to affect long-term graft or recipient survival, suggesting organ donors with MDROs on culture may be safely utilized. Future studies aimed at reducing early posttransplant infections associated with donor MDROs are needed.

The Frequencies of ABO and Rh Phenotypes among Male Blood Donors in Northwestern Saudi Arabia.

Transfusion-dependent patients in Saudi Arabia are numerous because of the existence of life-threatening inherited diseases such as sickle cell anemia and thalassemia. Thus, analysis of the frequencies of the ABO and rhesus (Rh) phenotypes is vital. This study sought to evaluate the frequencies of the ABO and Rh phenotypes among male blood donors in Hail region. One hundred and twenty-six (126) blood samples were collected from male donors living in Hail region and were screened for ABO and Rh phenotypes. The collected data were statistically analyzed using GraphPad Prism (version 9.3.1). Among 126 blood donors, 43.6% were classified into the O blood group. Additionally, Rh antigen e was predominantly detected in this study (99.2%). A total of 103 blood donors exhibited D antigen, whereas 23 were negative for D antigen. The DCe/dce (R1r) phenotype was observed in 29.1% of RhD positive donors, while 73.9% of RhD negative blood donors expressed the dce/dce (rr) phenotype. The O phenotype and e antigen were most frequently observed in male blood donors from Hail province. RhD positive samples took advantage of total blood donor samples over RhD negative samples. CcDee (R1r) phenotype was commonly identified in the RhD positive population, whereas ccddee (rr) phenotype was consis¬tently detected in the RhD negative male donors.

Effectiveness of the HCV blood screening strategy through eighteen years of surveillance of HCV infection in blood donors in France.

The question of maintaining blood screening based on both Hepatitis C virus (HCV) infection antibodies (Ab) and Nucleic Acid Testing (NAT) has been raised in several countries. The French blood donor surveillance database was used to address this issue. In France, HCV-NAT was implemented in mini pools (MP) in 2001 and in individual testing (ID) in 2010. HCV-positive donations are further investigated including detection of RNA with an alternative polymerase chain reaction assay: Amplicor HCV v2.0 (Roche; LOD95 50 IU/mL) from 2001 to 2006 and CobasTaqMan (CTM) HCV 2.0 assay (Roche; LOD95 9.3 IU/mL) since 2007. From 2001 to 2018, 3,058/48.8 million donations were confirmed HCV positive: 64.4% were Ab+/NAT+, 35.1% Ab+/NAT- and 0.5% Ab-/NAT+. From 2001 to 2018, the NAT yield decreased from 0.65 per million donations to 0, and NAT+ donations dropped from 77% to 46% of the total of HCV donations. 2,491/3,058 were further tested for HCV-RNA: 1,032 (816 NAT+, 216 NAT-) with Amplicor and 1,459 (897 NAT+, 562 NAT-) with CTM. Four (3 MP and 1 ID-NAT, 0.5%) of the 778 NAT negative donations had low viral loads. The decline in HCV-NAT yield cases raises the question of the relevance of NAT. Conversely, the increase in Ab+/NAT-donors, suggesting a growing number of resolved infections, argue for Ab discontinuation. In our experience, at least 0.5% of Ab+/NAT-donations had low RNA level when retested. Although the risk of viral transmission by such donations is probably low, the uncertainty associated with their infectivity goes against the removal of Ab in blood screening in our country.

Implications of the accumulation of CXCR5+ NK cells in lymph nodes of HIV-1 infected patients.

SummaryBackgroundB cell follicles are immune-privileged sites where intensive HIV-1 replication and latency occur, preventing a permanent cure. Recent study showed that CXCR5+ NK cells in B cell follicles can inhibit SIV replication in African green monkeys, but this has not been reported in HIV-1 infected patients.MethodsLymphocytes and tissue sections of lymph node were collected from 11 HIV-1 positive antiretroviral therapy (ART)-naive and 19 HIV-1 negative donors. We performed immunofluorescence and RNA-scope to detect the location of CXCR5+ NK cells and its relationship with HIV-1 RNA, and performed flow cytometry and RNA-seq to analyze the frequency, phenotypic and functional characteristics of CXCR5+ NK cells. The CXCL13 expression were detected by immunohistochemistry.FindingsCXCR5+ NK cells, which accumulated in LNs from HIV-1 infected individuals, expressed high levels of activating receptors such as NKG2D and NKp44. CXCR5+ NK cells had upregulated expression of CD107a and β-chemokines, which were partially impaired in HIV-1 infection. Importantly, the frequency of CXCR5+NK cells was inversely related to the HIV-1 viral burden in LNs. In addition, CXCL13—the ligand of CXCR5—was upregulated in HIV-1 infected individuals and positively correlated with the frequency of CXCR5+ NK cells.InterpretationDuring chronic HIV-1 infection, CXCR5+ NK cells accumulated in lymph node, exhibit altered immune characteristics and underlying anti-HIV-1 effect, which may be an effective target for a functional cure of HIV-1.

Allogeneic Stem Cell Transplantation in Patients Aged ≥70 Years: Epidemiology, Outcomes, and Risk Factors Based on the German Registry for Stem Cell Transplantation (DRST)

Introduction. Malignant diseases treated with allogeneic hematopoietic stem cell transplantation (alloHSCT) predominantly occur beyond the 7 th decade of life. Numerical age per se is not regarded an adverse risk factor in alloHSCT. In an aging society, interventions historically deemed high risk are increasingly used in elder patients.Methods. Epidemiology, outcomes and risk factors of patients aged ≥70 years undergoing alloHSCT in Germany 1999-2019 and registered with the DRST/EBMT database were analyzed retrospectively. Baseline patient, disease, and transplant data were collected from MED-A forms. Centers were contacted to provide additional treatment and follow-up information.Results. Between 1999 and 2019, 1648 patients aged ≥70 years (median 72, range 70-79.7; 585 female) were transplanted in 50 German centers. More than 90% of all patients were transplanted 2010-2019. Centers transplanted between 2 and 192 patients, with 14 centers contributing <10 and 4 centers contributing >100 patients each. Most patients suffered acute leukemia (1084, 65.8%) or MDS/MPN (410, 24.9%). Karnofsky index before start of conditioning was 100% (n=230, 14%), 90% (n=651, 39.5%), 80% (n=480, 29.1%), 70% (n=94, 5.7%), <70% (n=55, 3.3%). Myeloablative conditioning was chosen in 25.6%. Total body irradiation was used for 305 patients (18.6%). Conditioning contained antithymocyteglobulin in 49.6%. Donors were unrelated for 85.5%. Median donor age was 37 (18-79) years. Patient CMV IgG was positive in 63.1% and the constellation ‘negative donor, positive patient’ was present in 19.9%. Median overall survival (OS) and disease free survival (DFS) was 408/ 344 days. With a median follow up of 536 days for surviving patients, Kaplan Meier estimates of OS/ DFS were 52.6%/ 48.5% and 40.9%/ 38.6% at 1 and 2 years. In a competing risk analysis, cumulative incidence of non-relapse-mortality (NRM)/ relapse (RI) was 22.2%/ 29.3% at 365 days. Frequency of acute graft versus host disease (GvHD) II-IV was 25.1% and chronic limited/ extended GvHD 11.7%/ 14.8%. Karnofsky performance score, CMV IgG matching, acute and chronic GvHD and stem cell source showed a prognostic impact on OS, DFS, RI and/ or NRM (Table 1). Underlying disease did not impact outcome, neither did age amongst patients at an age of 70-80 years. To compare with outcome in the decade below (60-69 years), an analysis after matching for underlying disease, CMV relation, and Karnofsky index included 2728 patients (each 1364 patients 60-69 and ≥70 years of age). For each year of life, univariate HR for OS and DFS were 1.01 [95%CI 1.001-1.023, p=0.035] and 1.01 [95%CI 0.99-1.02, p=n.s.], respectively, in this matched-pair analysis. The cumulative HR (OS, DFS) for both age groups was 1.16 [95%CI 1.05-1.28, p<0.01] and 1.13 [95%CI 1.02-1.24, p=0.016] for patients ≥70 years.Conclusion. AlloHSCT is increasingly used to treat elder patients in Germany with a sharp increase during the last decade. Age per se is a modest adverse risk factor for adult patients after alloHSCT with slightly increased mortality in patients 70-80 versus those at 60-69. Further research might concentrate on patient selection and further reduction of procedural toxicity. [Display omitted] DisclosuresSchetelig: Roche: Honoraria, Other: lecture fees; Novartis: Honoraria, Other: lecture fees; BMS: Honoraria, Other: lecture fees; Abbvie: Honoraria, Other: lecture fees; AstraZeneca: Honoraria, Other: lecture fees; Gilead: Honoraria, Other: lecture fees; Janssen: Honoraria, Other: lecture fees . Einsele: Janssen, Celgene/BMS, Amgen, GSK, Sanofi: Consultancy, Honoraria, Research Funding. Stelljes: Pfizer: Consultancy, Research Funding, Speakers Bureau; Medac: Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Celgene/BMS: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; MSD: Consultancy, Speakers Bureau; Kite/Gilead: Consultancy, Speakers Bureau. Dreger: AbbVie: Consultancy, Speakers Bureau; Bluebird Bio: Consultancy; Novartis: Consultancy, Speakers Bureau; Janssen: Consultancy; AstraZeneca: Consultancy, Speakers Bureau; Gilead Sciences: Consultancy, Speakers Bureau; BMS: Consultancy; Riemser: Consultancy, Research Funding, Speakers Bureau; Roche: Consultancy, Speakers Bureau. Wulf: Takeda: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Clinigen: Consultancy, Honoraria. Scheid: Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Honoraria; Roche: Consultancy; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Bethge: Novartis: Consultancy, Honoraria, Speakers Bureau; Kite-Gilead: Consultancy, Honoraria, Speakers Bureau; Miltenyi Biotec: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau.

Somatic STAT3 Mutations in CD8+ T Cells of HTLV-2 Positive Blood Donors

Introduction: T-cell large granular lymphocyte (T-LGL) leukemia is a rare lymphoproliferative disorder with recurrent somatic STAT3 mutations. It has been suggested that viral antigens act as the initial stimuli resulting in clonal expansion of CD8+ cells in the disease. However, less is known whether chronic exposure to viral antigens is associated with acquisition of somatic mutations in CD8+ T cells among individuals without clinically detectable lymphoproliferations.Human T-cell leukemia virus type 2 (HTLV-2) antibody positivity has been detected in patients with T-LGL leukemia. Here, we examined whether CD8+ T cells from HTLV-2 positive healthy blood donors harbor somatic mutations in STAT3 or other immune-associated genes, potentially identifying individuals at risk of subsequent lymphoproliferative diseases.Methods: We analyzed HTLV-2 infected (n=30) and uninfected (n=35) healthy blood donor samples obtained from University of California San Francisco and Vitalant Research Institute, which were enrolled in the United States-based HTLV Outcomes Study (HOST) cohort. All cases had serologic evaluation for HTLV-2 infection at the time of sampling. We examined somatic mutations of STAT3 in CD4+ and CD8+ T-cell populations using ultra-deep targeted amplicon sequencing. In addition, we applied a custom sequencing panel covering the coding regions of 2,533 immune-related genes to characterize a larger spectrum of somatic mutations in CD8+ T cells.Results: Somatic STAT3 mutations were detected in CD8+ but not in CD4+ T cells of four (13.3%, 4/30) HTLV-2 positive healthy blood donors (p.Y640F, p.N647I, p.D661Y, and p.Y657_K658insY with variant allele frequencies of 11.9%, 0.5%, 4.9%, and 1.2%, respectively) using amplicon sequencing. The detected STAT3 mutations have been previously described and reported in T-LGL leukemia. Total white blood cell and lymphocyte counts were similar between STAT3 mutated and non-mutated cases. No STAT3 mutations were discovered in HTLV-2 negative donors with amplicon sequencing.Of the 28 HTLV-2 positive cases, 19 had at least one somatic variant in CD8+ T cells based on the immunogene panel sequencing (n=28). 8 cases had variants in genes previously identified in T-LGLL (STAT3, KMT2D, TYRO3, DIDO1, BCL11B, CACNB2, KRAS, LRBA and FANCA), and 5 cases had variants in genes involved in JAK-STAT signaling (NFKBIA, PIK3R5, MAPK14, EP300, MPL, IFNAR1, IL6ST and IL20RA). Three recurrently mutated genes were detected: VWF, SMAD7 and MXRA5. The four HTLV-2 positive blood donors harboring STAT3 mutations had more somatic mutations (median=6) than HTLV-2 positive donors without STAT3 mutations (median=1, p=0.06).Conclusion: We report the presence of somatic gain-of-function STAT3 mutations in CD8+ T cells of 13% of HTLV-2 positive healthy blood donors. We identified additional somatic mutations in genes associated with JAK-STAT signaling, immune regulation and lymphoproliferation in CD8+ T cells of HTLV-2 positive cases. While STAT3 activation, with or without mutations, is considered as a hallmark of T-LGLL, our results reveal the presence of STAT3 mutations in CD8+ T cells of healthy blood donors harboring HTLV-2 without clinical history of lymphoproliferative disease. Additional research is warranted to elucidate whether HTLV-2 carriers harboring STAT3 and other mutations are at increased risk of subsequent T-LGL leukemia or other lymphoproliferative diseases. DisclosuresMustjoki: Pfizer: Research Funding; BMS: Research Funding; Novartis: Research Funding; Janpix: Research Funding.

Letermovir Prophylaxis Versus Pre-Emptive Therapy for Cytomegalovirus after Hematopoietic Stem-Cell Transplantation

IntroductionCytomegalovirus (CMV) infection is one of the most common complication after allogeneic hematopoietic stem-cell transplantation (HSCT) still associated with significant morbidity and mortality. Although pre-emptive therapy (PET) are routinely used in treatment of CMV after SCT, their prophylactic use is limited by clinically unacceptable myelosuppression and nephrotoxicity. Letermovir, available since 2019 in Italy, is the first antiviral agent approved by FDA and EMA which is indicated for the prophylaxis of CMV infection in CMV seropositive (R+) patients undergoing SCT. Presently, cost and drug interactions are the main disadvantages of Letermovir use. We performed a single-center observational retrospective study to evaluate the efficacy of primary Letermovir prophylaxis for CMV infection among high-risk patients (R+) receiving HSCT from serological negative donor (D-).MethodsWe evaluated a cohort of R+/D- patients transplanted from January 2017 to December 2020 (86/235 transplanted patients): among those eligible for Letermovir prophylaxis (N=70), 29 patients (transplanted after 2019) received Letermovir until day +100, whereas 41 patients (the historical control group transplanted before 2019) received CMV PET only in case of increased viral load (CMV reactivation). Patients unable to take oral therapy at day +7 from HSCT or assuming drugs for concomitant clinical conditions bringing about major pharmacokinetic interaction were excluded (N=16). We compared day +100 and day +200 cumulative incidence of clinically significant CMV infection (CS-CMVi), defined according to drug registration trial: Letermovir discontinuation before day +100, CMV reactivation (CMV-DNAemia leading to PET), CMV tissue invasive disease, disease relapse and death from any causes. Survival functions between groups were estimated by the Kaplan-Meier method and compared using log-rank test. Moreover, the overall survival (OS), disease free survival (DFS), non-relapse mortality (NRM) and cumulative incidence of II-IV grade acute graft-versus-host disease (aGVHD) was compared in the two cohorts. Finally, we analyzed the number of accesses in day hospital from initial discharge to day +180, as an indirect cost-effectiveness evaluation of letermovir prophylaxis.ResultsNo severe adverse events related to the therapy were observed in the letermovir group. Letermovir prophylaxis started at a median of 11 days (range, 5-27) after HSCT. The median duration of Letermovir administration was 89 days (range, 40-113). The only early stop was due to patient death, not related to CMV or drug toxicity. CS-CMVi at day +100 occurred in 13.8% vs 61.0% in letermovir and historical group, respectively (p <0.001). Of note, none of the events in letermovir group was related to CMV reactivation whereas 24/25 in the historical group were. A trend toward lower CS-CMVi in the letermovir group was observed also at day +180 (44,8% vs 65,9%, p =0.080), with 6 late reactivations in patients who received prophylaxis. Moreover, at follow-up one patient in the experimental group and 3 in the control group developed CMV disease. Of note, in vivo T-cell depletion was used in 86% of patients in letermovir and 83% in historical group, and most of the CMV reactivations occurred after development of aGVHD: in 83% and 54% of patients, respectively. No differences in OS and DFS were observed between the two cohorts. Finally, a trend toward lower number of day hospital admissions was shown in patients who received letermovir prophylaxis (median 2 admissions, IQR 0-8, vs median 4.5, IQR 0-16), suggesting higher quality of life and costs reduction.ConclusionsOur real-life experience demonstrated the efficacy of Letermovir in reducing the incidence of CMV reactivation. Longer follow-up is needed to clarify advantages in terms of disease-free and overall survival. Further studies are needed to investigate the role of prophylaxis beyond day 100 in high risk patients, such as those who receive a T-depleted transplant or who develop aGVHD. The role of Letermovir prophylaxis on immuno-reconstitution and its cost-effectiveness remains to be evaluated. DisclosuresMarco: Jazz: Consultancy; Insight,: Consultancy; Janssen: Consultancy. Ferrero: EUSA Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Gilead: Research Funding, Speakers Bureau; Morphosys: Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees; Clinigen: Membership on an entity's Board of Directors or advisory committees; Servier: Speakers Bureau.

Haploidentical Natural Killer Cell Therapy As an Adjunct to Stem Cell Transplantation for Refractory Acute Myeloid Leukemia

Refractory acute myeloid leukemia (AML), defined as failure of 2 cycles of induction therapy at diagnosis or of 1 cycle at relapse, represents a subgroup with poor clinical outcomes. In our transplant cohort, the 5-year overall survival in this subgroup was 16% (Ganapule at al. JGO 2017). Haploidentical natural killer cell (NK) therapy is a strategy that is being explored in refractory malignancies. Our in-vitro and animal model data suggest that exposure to arsenic trioxide (ATO) results in enhanced NK cytotoxicity (Alex AA et al. Front. Immunol 2018). Historically, at our center, patients with refractory AML have been treated with cytoreductive therapy (FLAG ± idarubicin or mitoxantrone + etoposide for 3 to 5 days) followed by 1-week rest and then a reduced-intensity transplant with fludarabine + melphalan conditioning while in peak cytopenia. From February 2019, we initiated a phase II single arm clinical trial (CTRI/2019/02/017505) enrolling patients with refractory AML planned for a stem cell transplant in peak cytopenia. Patients received CD56-positive cells from an HLA haploidentical related family donor (other than the stem cell donor, wherever feasible) following cytoreductive chemotherapy. The NK cell donor preference strategy included presence of KIR ligand mismatch, greater number of KIR B motifs (or the B score), lower donor age, and negative donor specific antibodies tested using flowcytometry crossmatch (Figure 1a). CD56-positive selection was done using CliniMACS prodigy system. This was followed by overnight incubation of the CD56 positive cells in autologous plasma with 2 micromolar ATO and 500 U/mL of interleukin-2. The CD56 positive cells were then infused to the patient 1-day after the completion of cytoreductive chemotherapy. This was followed by a reduced intensity stem cell transplant (Figure 1b). The primary outcome variable was 1-year relapse free survival. From February 2019, 14 patients with median age 28 years (IQR: 15.75-31.5) were enrolled in this trial. Six were females. Six had primary-refractory AML while 8 had relapsed-refractory AML. The cytoreductive chemotherapy was FLAG ± idarubicin (n=7), Mitoxantrone + Etoposide (n=6) and GCLAC (n=1). The median blast percentage on flowcytometry MRD testing prior to NK infusion was 15.9% (IQR: 9.1%-54.5%) (n=11). The median B score for the NK cell donors was 2 (IQR: 1-3). The median age of the NK cell donor was 43 years (IQR: 36-49.5). KIR ligand mismatch with the patient was noted in 2 donors. The median CD56-cell dose infused was 46.16 x 10 6/kg (IQR: 25.06-70.36) (Figure 1c). Pre-defined release criteria, including sterile cultures, and endotoxin negativity were met in all cases. There was no infusion related toxicity. The median blast percentage on flowcytometry MRD testing done following NK cell infusion was 11.9% (IQR: 4.9%-47.6%) (n=8). One patient withdrew consent after NK cell infusion and did not undergo transplant. For the remaining 13 patients, the stem cell donor was HLA matched (n=4), HLA 9/10 matched (n=1) or HLA haplomatched (n=8). The median CD34 cell dose infused was 10 x 10 6/kg (IQR:7.51-11.6). Five (38.5%) patients died of immediate post-transplant complications (sepsis (n=3) on days 1, 2 and 28, cerebral venous sinus thrombosis (n=1) on day 1 in a patient treated with hormonal contraceptives for menorrhagia, and veno-occlusive disease (n=1) on day 15 in a patient undergoing a second transplant) while 2 (15.4%) did not engraft (both subsequently died of infective complications following engraftment post-second transplant). Of the remaining 6 (46.2%) patients who engrafted and survived beyond 1 month of the transplant, the day 28 post-transplant MRD was negative for 5 patients while it was positive in 1 patient (0.13%). On follow up, 2 (15.4%) patients developed disease relapse (on days 54 and 218 respectively) and died. The remaining 4 (30.8%) patients are alive and relapse free at last follow up (mean follow up of surviving patients is 16 months). One patient received a CD34 cell boost on day 96 (cell dose - 8.55 x 10 6/kg) for poor graft function. For the entire cohort, the estimated 1-year event free survival is 28.8% ± 13.1%(Figure 1d). Whereas, acute GVHD was noted in 3 patients (50%; out of 6 evaluable patients) and chronic GVHD was noted in 3 patients (50%; out of the 6 evaluable patients). Thus, haploidentical NK cell therapy as an adjunct to transplant is safe and merits further evaluation in patients with AML. [Display omitted] DisclosuresMathews: Christian Medical College: Patents & Royalties: US 2020/0345770 A1 - Pub.Date Nov.5, 2020; AML: Other: Co-Inventor.

The Prevalence of Hepatitis D Virus in Reactive HBsAg Blood Donors at Department of Pathology, LUMHS Jamshoro

Background: The hepatitis delta virus (HDV) is a defective hepatotropic virus that only affects patients infected with the hepatitis B virus (HBV). Infection with the hepatitis delta virus can cause acute hepatitis, including the fulminant presentation or spontaneously resolving infection and chronic infection Aim: The present study's aim was to determine the prevalence of the Hepatitis D virus in reactive HBsAg blood donors at Diagnostic and Research Lab Hyderabad. Materials and Methods: This cross-sectional study was conducted on 434 blood donors at the Pathology department (Diagnostic and Research Laboratory) Civil Hospital, Hyderabad from January 2017 to December 2017. All the healthy individuals who visited at Diagnostic and Research Laboratory of Civil Hospital, LUMHS Hyderabad as blood donors with specific age groups of either gender were included in this study. All the individuals underwent Hepatitis screening. All of those cases that were noted with positive HBV further underwent HDV screening test. All the data was recorded in the proforma for the purposed of analysis. SPSS version 20 was used for data analysis. Results: Of the total 434 blood donors, 420 (96.8%) were male and 14 (3.2%) were female. The overall mean age was 31.65±4.67 years with an age range of 18 to 60 years. Out of 434, the incidence of positive delta virus was 62 (14.3%) while the remaining 372 (85.7%) were negative delta hepatitis blood donors. The prevalence of delta hepatitis blood donors with respect to age distribution were as follows; 18-30 years had 208 (48%), 31-40 years had 188 (27%), 41-50 years 69 (16%), and 51-60 years 39 (9%). Of the total 62 positive delta hepatitis, the prevalence of males and females was 98.4% and 1.6% respectively. All the donor’s blood group was divided into +O, +B, +A, +AB, -O, -A, and –B with their respective prevalence was 48.6%, 22.6%. 11.5%, 8.8%, 2.5%, 3.5%, and 2.5% whereas their frequency in positive tested delta hepatitis was 36 (58.1%), 7 (11.3%), 10 (16.1%), 6 (9.7%), 1 (1.6%), 2 (3.2%), and 0 (0%) respectively. Conclusion: It is concluded that the prevalence of HDV is 14.3% in Hepatitis B reactive healthy donors. This is a higher prevalence as compared to previously published studies. No such adequate recent data is available at the local level. More research is needed on this event, to provide adequate knowledge, which will be helpful to clinical and laboratory investigators, and physicians to reduce the burden of liver disease caused by HBV and HDV co-infection. Keywords: Hepatitis delta virus; HBsAg reactivity; Blood donors;

Novel variants in Krueppel like factor 1 that cause persistence of fetal hemoglobin in In(Lu) individuals

Beta-hemoglobinopathies become prominent after birth due to a switch from γ-globin to the mutated β-globin. Haploinsufficiency for the erythroid specific indispensable transcription factor Krueppel-like factor 1 (KLF1) is associated with high persistence of fetal hemoglobin (HPFH). The In(Lu) phenotype, characterized by low to undetectable Lutheran blood group expression is caused by mutations within KLF1 gene. Here we screened a blood donor cohort of 55 Lutheran weak or negative donors for KLF1 variants and evaluated their effect on KLF1 target gene expression. To discriminate between weak and negative Lutheran expression, a flow cytometry (FCM) assay was developed to detect Lu antigen expression. The Lu(a−b−) (negative) donor group, showing a significant decreased CD44 (Indian blood group) expression, also showed increased HbF and HbA2 levels, with one individual expressing HbF as high as 5%. KLF1 exons and promoter sequencing revealed variants in 80% of the Lutheran negative donors. Thirteen different variants plus one high frequency SNP (c.304 T > C) were identified of which 6 were novel. In primary erythroblasts, knockdown of endogenous KLF1 resulted in decreased CD44, Lu and increased HbF expression, while KLF1 over-expressing cells were comparable to wild type (WT). In line with the pleiotropic effects of KLF1 during erythropoiesis, distinct KLF1 mutants expressed in erythroblasts display different abilities to rescue CD44 and Lu expression and/or to affect fetal (HbF) or adult (HbA) hemoglobin expression. With this study we identified novel KLF1 variants to be include into blood group typing analysis. In addition, we provide further insights into the regulation of genes by KLF1.

A high-throughput microsphere-based immunoassay of anti-SARS-CoV-2 IgM testing for COVID-19 diagnostics.

The pandemic of novel coronavirus disease COVID-19 is rapidly expanding across the world. A positive result of antibody tests suggests that the individual has potentially been exposed to SARS-CoV-2, thus allowing to identify asymptomatic infections and determine the seroprevalence in a given population. The aim of this study was to evaluate the performances of a newly developed high throughput immunoassay for anti-SARS-CoV-2 IgM antibody detection on the Luminex MAGPIX platform. Clinical agreement studies were performed in 42 COVID-19 patient serum samples and 162 negative donor serum/plasma samples. Positive percent agreement (PPA) was 42.86% (95% CI: 9.90% to 81.59%), 71.43% (95% CI: 29.04% to 96.33%), and 28.57% (95% CI: 13.22% to 48.67%) for samples collected on 0-7 days, 8-14 days, and 2-8 weeks from symptom onset, respectively. Negative Percent Agreement (NPA) was 97.53% (95% CI: 93.80% to 99.32%). There was no cross-reactivity with the SARS-CoV-2 IgG antibody. Hemoglobin (200 mg/dL), bilirubin (2 mg/dL), triglyceride (250 mg/dL) and EDTA (10 mM) showed no significant interfering effect on this assay. In conclusion, an anti-SARS-CoV-2 IgM antibody assay with high sensitivity and specificity has been developed. With the high throughput, this assay will speed up the anti-SARS-CoV-2 IgM testing.