Development of Printable Graphene Electrochemical Biosensor for Environmental Monitoring and Medical Applications

Algal bloom is a type of harmful water pollution, which is mainly caused by the cyanobacteria or dinoflagellate that releases a variety of algal toxins into a water source. Among them, microcystins are often detected, of which microcystin-leucine-arginine (MC-LR) is known as one of the most toxic variants that has received a great amount of attention due to its serious consequences after ingestion such as irreversible organ damage or even death. Human cytomegalovirus (HCMV) is a type of herpes virus that can widely spread via mucous contact, resulting in many severe symptoms or even death especially for infants, pregnant women and immunocompromised patients if there is no timely diagnosis. Following these reasons, there is an urgent need to develop a commercially viable and sensitive monitoring system to reach a rapid identification on water quality or human health. This work mainly focuses on the development of vertically aligned graphene (VAG) electrodes through the novel use of flexographic printing and photonic annealing techniques for highly sensitive detection of biological targets using non-Faradaic electrochemical impedance spectroscopy (EIS). For the detection of MC-LR, the biosensor achieved an low limit of detection (LOD) of 1.2 ng/L via baseline method. In the baseline method, measurement was first performed using PBS. After that, measurement was then performed on antigen solution drop-casted on the biosensor. The biosensing response between PBS and antigen acquired at a specific frequency was dependent on the target concentration. The biosensor also exhibited excellent selectivity with high percentage of recovery (i.e., 91.8 %) and stability (i.e., 108.8 % and 99.4 % after one and three weeks, respectively). Moreover, similar good performance (i.e., 98.4%) was observed in tap water spiked with the antigen. As for the detection of CMV pp65-antigen, biosensing results showed a good linearity when tested on the control group (i.e., 0 ng/mL) up to 38,500 ng/mL of the antigen concentration using the same baseline measurement. The VAG biosensor showed a dynamic range of between 3.85 and 38,500 ng/mL for the detection of HCMV pp65-antigen, which matches with the clinically relevant range of 102 ~ 106 genomes/mL based on measurement performed on viral loaded urine samples using PCR technique. Measurements on the target concentration using the biosensor were also performed using a non-baseline method. In this method, only the antigen solution was used throughout the measurement, where the biosensing result was determined by the difference in the response recorded at the start of measurement and after a certain incubation duration at a specific frequency. In particular, the change in phase showed a strong correlation against the target concentration. The biosensing response for the control group (i.e., 0.38°±0.191°) up to 38,500 ng/mL (i.e., 2.26°±0.543°) antigen concentration was highly comparable to those (i.e., 0.16°±0.0854° for the control group and 2.21°±0.105° for 38,500 ng/mL) derived from the baseline method, implying the strong feasibility of the non-baseline testing.

Discordance between the predicted vs. the actually recognized CD8+ T cell epitopes of HCMV pp65 antigen and aleatory epitope dominance

CD8+ T cell immune monitoring aims at measuring the numbers and functions of antigen-specific CD8+ T cell populations engaged during immune responses, providing insights into the magnitude and quality of cell-mediated immunity operational in a test subject. The selection of peptides for ex vivo CD8+ T cell detection is critical, however, because for each restricting HLA class I molecule present in a human individual there is a multitude of potential epitopes within complex antigens, and HLA diversity between the test subjects predisposes CD8+ T cell responses to individualized epitope recognition profiles. We report here on a brute force CD8+ T cell epitope mapping approach for the human cytomegalovirus (HCMV) pp65 antigen on ten HLA-A*02:01-matched HCMV infected human subjects. In this approach, in each test subject, every possible CD8+ T cell epitope was systematically tested; that is 553 individual peptides that walk the sequence of the HCMV pp65 protein in steps of single amino acids. Highly individualized CD8+ T cell response profiles with aleatory epitope recognition patterns were observed. We compared the actually detected epitope utilization in each individual with epitope prediction ranking for the shared HLA-A*02:01 allele, and for additional HLA class I alleles expressed by each individual. No correlation was found between epitopes’ ranking on the prediction scale and their actual immune dominance. The data suggest that accurate CD8+ T cell immune monitoring might depend on the agnostic reliance on mega peptide pools, or brute force mapping, rather than individualized epitope predictions.

FRI0440 RISK FACTORS FOR CYTOMEGALOVIRUS INFECTION IN PATIENTS WITH AUTOIMMUNE DISEASES

Background:The risk for opportunistic infections in patients with autoimmune diseases requiring intensive immunosuppressive therapy is high and cytomegalovirus (CMV) infection is one of the most common opportunistic infections. Since 2011, we have performed weekly CMV pp65 antigen testing for patients at risk of opportunistic infections owing to autoimmune diseases to ensure appropriate patient management.Objectives:To evaluate the risk factors that predict CMV infection in patients that received remission-induction therapy for autoimmune diseases.Methods:We enrolled 254 patients (93 male, 161 female) from our hospital with autoimmune disease and who received remission-induction therapy with prednisolone at a dose greater than 0.5 mg/kg/day between January 2011 and December 2018. We retrospectively analysed their clinical characteristics and laboratory data, including treatment regimens and CMV pp65 antigen test results. The presence of more than five CMV pp65 antigen-positive cells over two slides was considered a positive result. We conducted univariate and multivariate analyses to extract CMV risk factors.Results:Of the patients we evaluated, 60 suffered from systemic lupus erythematosus (SLE), 55 from anti-nucleolar cytoplasmic antibody-associated vasculitis (AAV), 31 from dermatomyositis (DM), 14 from interstitial pneumonia with anti-aminoacyl tRNA synthetase antibody, 14 from adult-onset Still’s disease (AOSD), 14 from rheumatoid arthritis (RA), 11 from mixed connective tissue disease (MCTD), 10 from Takayasu’s aortitis, and 45 suffered from other autoimmune diseases. Pulse therapy with methylprednisolone (mPSL) and immunosuppressive reagents were administered to 103 (40.6 %) and 97 (38.2 %), respectively. The median follow-up period was 61.0 days, and 66 patients became CMV pp65 antigen-positive during this period (SLE, 15; DM, 14; AAV, 9; AOSD, 8; and other, 20). Univariate analysis revealed that when compared to patients testing negative for the CMV pp65 antigen patients testing positive had lower total lymphocyte count (TLC) (825 /uL vs. 1220 /uL; p < 0.01), a lower serum albumin level (2.70 g/dL vs. 3.30 g/dL; p < 0.01), a higher HbA1c level (6.3 % vs. 5.9 %; p<0.01), and were older (66.0 vs. 59.5 year old; p < 0.01). Forty-nine of the 66 patients in the positive group received mPSL pulse therapy (p < 0.01), and 38 received immunosuppressive reagents (p < 0.01). Logistic regression analyses indicated that a higher age by decade (OR; 1.46 [95%CI 1.06 - 2.00]), a lower TLC per 100/uL (OR; 0.83 [95%CI 0.73 -0.94]), a higher HbA1c level per 1% (OR; 2.37 [95%CI 1.25-4.53]), and mPSL pulse therapy (OR; 3.92 [95%CI 1.33-11.5]) were risk factors for CMV pp65 antigen positivity.Conclusion:Higher age, lower TLC, higher HbA1c, and treatment with mPSL pulse therapy were risk factors for acquiring CMV infection, as measured by the presence of the CMV pp65 antigen, in patients receiving remission-induction therapy for autoimmune diseases. Careful monitoring of these, at risk, patients is necessary.Disclosure of Interests:None declared

Early Cytomegalovirus Reactivation and Donor Constellation Reduce Early and Late Relapse Incidence of Acute Myeloid Leukemia in a Long-Term Study

Introduction After allogeneic cell transplantation (HCT) for acute myeloid leukemia (AML) relapse remains the main cause of morbidity and mortality. Relapse rates directly translate into patients' overall survival. The connection between early human cytomegalovirus (CMV) replication and reduced leukemic recurrence has first been established by this group (Elmaagacli et. al. Blood 2011), using a pp65-antigenemia assay and confirmed by Green et al. (Blood 2013), Takenaka et al. (BBMT 2015) and others. In a more recent study by Teira et al. (Blood 2016) using PCR to detect reactivation, this effect has been questioned and the controversy remains probably due to methodological differences between studies. Methods Patients with AML received allogeneic HCT at the Department of Bone Marrow Transplantation of the University Hospital Essen between 10/1997 and 10/2017. All patient-, donor-, HCT- and virology data was prospectively documented and retrospectively analyzed. The immunosuppressive regimen consisted of cyclosporine A and methotrexate. The conditioning was according to physician's choice and included only non-anti-thymocyte globuline (ATG) conditioning regimens. Early supportive and follow-up care was identical for all patients. Patients were followed for up to 60 months. Surviving patients were censored at last follow-up date. Competing risk analysis was performed for calculating cause-specific relapse-incidences with non-relapse mortality as competing risk. CMV titers were measured at the Department of Virology using quantitative PCR (qPCR) and a pp65 antigenemia assay. CMV reactivation was defined as a replication of >500 CMV copies per ml EDTA blood or as >25 pp65 antigen expressing cells per 5×105 white blood cells. If several CMV reactivation episodes occurred, only the interval to the first episode after HCT was analyzed. Results A total of 420 consecutive adult patients (median age, 48 years; range, 16-73 years) with AML and HCT were included for analysis. Over 60 months of follow-up, cumulative incidence of CMV reactivation was detected in 28% of patients and AML relapsed in 34%. In this cohort, the sensitivity of qPCR or pp65 detection methods did not differ (p=0.35) with regard to the incidence of CMV reactivation. CMV reactivation associated with significantly reduced AML relapse risk. The relapse incidence (RI) for patients with CMV reactivation was 0.18 (95% Confidence interval (CI), 0.12-0.25) compared to 0.41 (95% CI, 0.35-0.46) for patients without CMV reactivation (p<0.0001). This significant difference was reproduced for prognostic AML subgroups in first complete remission (RI, 0.14 with CMV reactivation versus 0.35 without; p=0.02) and for higher risk AML (RI, 0.20 versus 0.45; p<0.0001). Notably, donor-recipient serostatus constellation significantly (p=0.005) associated with AML relapse reduction, independently of CMV reactivation. CMV positive donors had a RI of 0.40 (95% CI, 0.33-0.46) compared to 0.28 in CMV negative donors (95% CI, 0.22-0.34). Discussion This new PCR dataset confirms previous reports (Elmaagacli et al. 2011; Takenaka et al. 2015) of an independent reduction of AML relapse risk after CMV reactivation, using qPCR detection in a homogeneous population of AML patients. While the association between CMV reactivation and early relapse (< day+100) has also been confirmed by Green et al. (Blood 2013) and relapse (>day +100) by Takenaka et al. (BBMT 2015), our data shows that late relapse (>24 months) may occur in patients without CMV reactivation. While the aforementioned studies exclusively used pp65 antigenemia titers as a determinant of CMV reactivation, Teira et al. (Blood 2016) analyzed PCR samples. The sensitivity of CMV reactivation using PCR or pp65 antigen titers was identical in our dataset. With regard to the on-going controversy on the role of CMV reactivation and AML relapse, this data shows a reduction of relapse in the quantitative PCR age. Disclosures Turki: Neovii Biotech: Other: subsidies for the costs of travel. Beelen:Medac: Consultancy, Other: Travel Support.

Early abnormal liver enzyme levels may increase the prevalence of human cytomegalovirus antigenaemia after hematopoietic stem cell transplantation

Objective Human cytomegalovirus (HCMV) infection is common after bone marrow transplantation (BMT), and it increases morbidity and mortality for transplant recipients. HCMV infection may cause hepatitis and elevate the liver enzymes aspartate transferase (AST) and alanine transferase (ALT). This study aimed to analyse the associations between liver enzyme levels and infection with HCMV antigenaemia after BMT. Methods Data from 30 patients after BMT were collected at different time points (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0, and 6.0 months post-transplantation). The patients were divided into the HCMV antigenaemia-positive and HCMV antigenaemia-negative groups according to a peripheral blood pp65 antigen assay. Immunohistochemistry was used to identify HCMV pp65 antigen and conventional methods were used to detect liver enzyme levels. Results Twelve patients were pp65 antigenaemia-positive and 10 patients were positive in the first 3 months post-transplant. Liver enzyme levels were increased after positivity for HCMV antigenaemia (p = 0.034 and p = 0.018 for ALT and AST, respectively). One month before antigenaemia, AST levels were higher in the HCMV antigenaemia-positive group compared with the negative group (p = 0.006). Conclusion HCMV antigenaemia mostly occurs in the early stage of post-BMT and early abnormal liver enzyme levels may increase the chance of HCMV antigenaemia after BMT.

Cytomegalovirus Infection in Pediatric Haploidentical Hematopoietic Stem Cell Transplantation at a Single Center

Introduction : Clinical aspects and risk factors of Cytomegalovirus (CMV) reactivation and diseases are an emerging interest in recent investigation fields of hematopoietic stem cell transplantation (HSCT). Several risk factors and clinical manifestations of CMV infection have been reported in numbers of previous reports. Monitoring of viral replication and timely given antiviral agents have resulted in a decreased incidence of CMV-related death recently. However, CMV in still a major cause of infectious complications after HSCT. Specially in haploidentical HSCT setting, clinical manifestations and associated risk factors have been rarely reported. This study is conducted to identify risk factors and clinical aspects of CMV reactivation and disease in pediatric haploidentical HSCT. Methods : We retrospectively reviewed the medical records of 92 pediatric patients who underwent haploidentical HSCT using ex vivo T cell-depleted grafts at Asan Medical Center between December, 2004 and June, 2016. After haploidentical HSCT, all recipients were monitored for CMV infection by CMV pp65 antigen and/or CMV-PCR. Both recipient and donor were screened for CMV serology before the HSCT. During the study period, methodologic changes in ex vivo T cell depletion and CMV prophylaxis policy have been modulated with the course of time. During the 1st study period (Group I, n=19), CD3+ T cell was depleted without CMV prophylaxis while ganciclovir for CMV prophylaxis was given with CD3+ T cell depletion during the 2nd period (Group II, n=16). The T cells expressing alpha-beta T cell receptor (TCRαβ) was depleted during the 3rd period (Group III, n=57) with ganciclovir prophylaxis. Results : Of the 92 patients, 49 (53.3%) developed CMV reactivation after haploidentical HSCT. CMV reactivation significantly reduced in group II and III compared to group I (84±8.4% vs 46±5.9%; p<0.001). Patients with chronic graft-versus-host-disease (GVHD) also experienced more reactivation (p=0.041). In the multivariate analysis, difference of study group was the only variable predicted the occurrence of CMV reactivation (Group I vs Group III; hazard ratio, 3.728; 95% confidence interval (CI), 1.910-7.278). CMV disease was developed in 16/92 (17.4%) patients during the study period (16/49, 32.7% among CMV reactivation patients). The peak CMV pp65 antigen level, peak CMV DNA-PCR level, and duration of CMV antigenemia were significantly higher in patients with CMV disease (p=0.024, p=0.009, p=0.046, respectively). In univariate analysis, the peak CMV pp65 antigen level (p=0.020), and duration of CMV antigenemia (p=0.05) were associated with increased occurrence of CMV disease. In multivariate analysis, CMV disease significantly increased in patients with high CMV pp65 antigenemia greater than 40/200,000 cells (hazard ratio, 3.568; 95% CI 1.062-12.594). CMV diseases included retinitis in 12 (75%), pneumonitis in 3 (25%), gastritis, colitis, and encephalitis in 1 (8.3%) each, respectively. Of the 16 patients, 3 patients (2 pneumonitis, 1 encephalitis) died of CMV disease. Conclusions : Our study demonstrated that ganciclovir prophylaxis has effectively reduced the occurrence of CMV reactivation in ex vivo T cell depleted haploidentical HSCT. In addition, close monitoring of CMV pp65 antigen and timely preemptive management is important to prevent CMV related death. Disclosures No relevant conflicts of interest to declare.

Prospective Analysis of Cytomegalovirus Reactivation and the Immune State of Low-Grade B-Cell Lymphoma Patients Treated with Bendamustine

Bendamustine is an alkylator containing a benzimidazole ring; it only has partial cross-resistance with the other alkylating agents. Several studies have shown that bendamustine is effective as a single agent or as combined treatment with rituximab for low-grade B-cell lymphoma. Although bendamustine has a favorable safety profile, the occurrence of myelosuppression and lymphocytopenia is relatively frequent. In lymphocytopenia, CD4-positive (CD4+) T-cell recovery is specifically impaired. If this occurs over a prolonged period of time, patients are at risk of opportunistic infections, such as cytomegalovirus (CMV) reactivation. This is a risk for bendamustine-treated patients, although the actual frequency of CMV reactivation in these patients is not clear. In this study, we prospectively evaluated the occurrence of CMV reactivation in relapsed or refractory low-grade B-cell lymphoma patients who were treated with bendamustine alone or in combination with rituximab. We simultaneously investigated the immune state of the patients. We analyzed CMV pp65 antigen in leukocytes, immunoglobulin (Ig) G, IgM, and IgA levels, and the CD4+:CD8+ lymphocyte ratio prior to treatment and after the third and sixth courses of treatment. There were 29 patients enrolled in the study. The median age was 68 years old (range, 28–83 years). Patients were treated a median of 2 times prior to bendamustine treatment; these treatments included chemotherapy and radiotherapy. There were 15 patients (51%) who completed 6 courses of chemotherapy. The immune status of the patients is shown in Figure 1. The median CD4+ lymphocyte count prior to treatment was 218/µL, which decreased to 75/µL by the end of treatment. The median of IgG, IgM, and IgA concentrations before treatment were 909, 52, and 147 mg/dL, respectively; by the end of treatment, the IgG, IgM, IgA concentrations had decreased to 832, 30, and 110 mg/dL, respectively. The extent of CD4+ T-cell depression during treatment appeared to be more severe than the Ig depression. CMV pp65 antigen was detected in 3 patients after the third course of treatment, and in 2 patients after the sixth course of treatment. None of the patients had any symptoms and CMV pp65 antigen count returned to negative without any treatment. The median CD4+ lymphocytes count in patients with or without CMV reactivation is shown in Table 1. There is a trend that the depression of CD4+ cells after the 6th course of bendamustine increases the risk of CMV reactivation. This is the first study to prospectively analyze CMV reactivation in low-grade B-cell lymphoma patients treated with bendamustine. In this study, we found that CMV reactivation by bendamustine occurred in approximately 15% of patients. On the other hand, the risk of symptomatic CMV disease, which would require medical intervention, does not appear to be high in bendamustine-treated patients. Most patients with relapsed or refractory low-grade B-cell lymphoma who are administered bendamustine therapy are in an immunosuppressed condition at the beginning of treatment. This is due to heavy chemotherapy pretreatment, which is often performed using rituximab. In addition to this, the decrease in CD4+ lymphocytes observed during bendamustine treatment, is likely to increase the risk of opportunistic infections, such as CMV reactivation. In future studies, we aim to investigate the risk factors for symptomatic cytomegalovirus infection in patients treated with bendamustine, especially in combination with rituximab. We hope that our study, which evaluates the risk of opportunistic infections in bendamustine treated patients, will be beneficial in determining the optimal timing for a preemptive approach for CMV infection. This will be helpful in establishing safe bendamustine treatment regimens in relapsed or refractory low-grade B-cell lymphoma patients. Figure 1 Figure 1. Table 1. The median number of lymphocytes, CD4+ lymphocytes and CD4/8 ratio in patients with or without cytomegalovirus reactivation Parameter All patients CMV reactivation p value + - Before treatment (n=24) Lymph (/µl) 996 1518 (n=5) 987 (n=19) 0.24 CD4+ cells (/µl) 218 261 218 0.96 CD4/8 ratio 0.69 0.60 0.69 0.54 After the 3rd course (n=23) Lymph (/µl) 980 1330 (n=5) 944 (n=18) 0.39 CD4+ cells (/µl) 130 118 130 0.52 CD4/8 ratio 0.20 0.12 0.20 0.16 After the 6th course (n=15) Lymph (/µl) 628 579 (n=5) 902 (n=10) 0.27 CD4+ cells (/µl) 75 36 118 0.035 * CD4/8 ratio 0.20 0.17 0.21 0.35 *; significant Disclosures No relevant conflicts of interest to declare.