ABSTRACT Parvovirus B19 (B19V), a member of the genus Erythroparvovirus within the Parvoviridae family, infects human erythroid progenitor cells (EPCs) of bone marrow and fetal liver and causes various hematological disorders. The minor capsid protein VP1 of B19V contains a unique N-terminal region (VP1u) that facilitates virus binding and internalization into EPCs via its receptor-binding domain (RBD). We previously identified tyrosine-protein kinase receptor UFO (AXL) as a proteinaceous receptor for B19V infection of EPCs. In this study, we employed an ascorbate peroxidase 2 (APEX2)-based proximity labeling method to identify host proteins that are associated with B19V VP1u during entry. This analysis revealed human transferrin receptor 1 (hTfR) as a key host protein associated with VP1u. hTfR knockdown in UT7/Epo-S1 cells, a B19V-permissive human megakaryoblastoid leukemia cell line, showed significantly reduced B19V internalization and replication. Biolayer interferometry (BLI) assays confirmed a direct interaction between B19V VP1u and hTfR extracellular domain (ECD). Inhibition of VP1u interaction with hTfR ECD, either by a monoclonal antibody targeting the apical domain of the ECD or human ferritin, a natural ligand of hTfR that binds the apical domain, significantly reduced VP1u binding to hTfR, as well as B19V internalization and B19V replication in ex vivo -expanded EPCs. Furthermore, mutant RBD proteins that bear amino acid substitutions in the three helical domains nearly abolished RBD binding to hTfR and significantly reduced the ability to inhibit B19V infection of EPCs. Collectively, our findings establish hTfR as a B19V entry co-receptor that mediates B19V internalization into its natural host EPCs. IMPORTANCE B19V causes severe hematological disorders, including transient aplastic crisis, chronic pure red cell aplasia, and hydrops fetalis, by selectively infecting erythroid progenitor cells (EPCs). Despite its clinical impact, no approved antivirals or vaccines exist, largely due to limited understanding of viral entry mechanisms. A unique feature of B19V is the externalization of the VP1 unique region (VP1u) from the viral capsid, which mediates receptor engagement. Our prior studies identified AXL as a co-receptor for B19V infection of EPCs. Here, we identify that human transferrin receptor 1 (hTfR) as a critical co-receptor that directly binds VP1u and promotes viral internalization. Inhibition of the VP1u-hTfR interaction by competitive binding of hTfR with either an anti-hTfR monoclonal antibody or human ferritin significantly reduces B19V internalization and replication in ex vivo -expanded EPCs, highlighting a link between VP1u binding to the apical domain of hTfR and viral internalization. RBD mutants that disrupt its interaction with hTfR barely inhibited B19V infection in EPCs. These findings support a model in which hTfR directly engages VP1u and promotes B19V internalization into EPCs. Defining these mechanisms provides a foundation for developing antiviral strategies targeting B19V entry into EPCs.

BACKGROUND:Cytomegalovirus T-cell mediated immunity (CMV CMI) is essential for CMV control after allogeneic hematopoietic cell transplantation (HCT). Clinical experience with commercially available CMV CMI assays in letermovir (LTV) recipients is limited. OBJECTIVE:To evaluate the performance of the CMV inSIGHT T-cell immunity panel (Eurofins Viracor, Lenexa, KS) and factors associated with positive CMV CMI in LTV recipients. We also correlated the CMV inSIGHT with an in-house CMV-induced T-cell activation assay. STUDY DESIGN:In this prospective, observational study, the CMV inSIGHT was performed at day (D) 100 post-HCT. Adult recipients of peripheral blood HCT were eligible if they received LTV through D 100. Exclusion criteria included (i) clinically significant (cs) CMV infection by D 100; (ii) acute graft-versus-host disease grade ≥3; and (iii) corticosteroids within 3 weeks prior to CMV CMI testing. Fifty-three adult HCT recipients treated at Memorial Sloan Kettering Cancer Center from May 2020 to December 2024 were enrolled. The primary endpoint was positive CMV CMI at D 100 using the manufacturer's cutoff. The results were not reported to the clinicians. The association between CMV CMI and sub-clinical CMV DNAemia (blips), graft-versus-host disease prophylaxis regimen and lymphocyte subset counts were evaluated in univariate models. RESULTS:Of 53 patients included in the analyzes, 43% received post-transplant cyclophosphamide; and 48% had blips prior to D 100. At D 100, 38 (73%) patients had positive CMV CMI. Blips were associated with positive CMV CMI: odds ratio (OR), (95% confidence interval [95% CI]) 4.89 (1.06-31.73); P = 0.03. Rates of positive CMV CMI were similar between patients on post-transplant cyclophosphamide and calcineurin inhibitors (P = NS). Absolute CD4 and CD8 counts were not correlated with positive CD4 or CD8 CMV CMI respectively. Positive CD4 CMV CMI correlated with positive CD8 CMV CMI; OR (95% CI) 7.16 (1.66-36.41); P = 0.004. CONCLUSIONS:Seventy-three percent of letermovir recipients had positive CMV CMI by D 100, indicating CMV-specific immune reconstitution despite letermovir prophylaxis. Interventional studies are needed to assess the utility of the CMV inSIGHT T-cell immunity panel in guiding treatment decisions after discontinuation of letermovir prophylaxis.

KEY POINTS:Two-stage urine chemokine screening improved discrimination and positive predictive value versus blood-only testing while preserving high negative predictive value. In validation, urine chemokine C-X-C motif ligand 9/creatinine screening referred only 79% of samples for donor-derived cell-free DNA testing, reducing blood test use. Standalone blood biomarkers were more consistent for antibody-mediated rejection than cellular rejection; urine chemokine screening improved diagnostic yield. BACKGROUND:After kidney transplantation, subclinical acute rejection occurs in 20%-25% of clinically stable recipients; surveillance biopsies are invasive and often indicate no rejection. Moreover, the widespread use of noninvasive biomarkers is limited by cost. METHODS:We developed and sought to validate two-stage diagnostic models integrating urine chemokine assays (chemokine C-X-C motif ligand 9 [CXCL9] and C-X-C motif ligand 10) with confirmatory blood-based gene expression profiling (GEP) and donor-derived cell-free DNA (dd-cfDNA). Data from two studies, 476 biopsy-paired samples from 226 recipients (discovery) and 144 samples from 134 recipients (validation), were analyzed retrospectively. Rapid, low-cost urine chemokine assays were used for stage 1 screening; recipients with elevated urine chemokines underwent additional stage 2 testing with GEP and/or dd-cfDNA. RESULTS:In the validation cohort, dd-cfDNA outperformed GEP for overall rejection and antibody-mediated rejection, whereas neither test validated significant discrimination for cellular rejection. CXCL9-based two-stage testing improved the area under the receiver operating characteristic curve and positive predictive value versus standalone stage-2 testing, while maintaining similar negative predictive value and reducing stage 2 test use. At a probability screening threshold of 0.10, the CXCL9/creatinine-based logistic regression, adjusted for BK virus, urinary tract infection, sex, age, time post-transplant, and donor type, followed by dd-cfDNA validated a higher positive predictive value (0.39 versus 0.34), lower sensitivity (0.56 versus 0.62), and similar negative predictive value around 0.94, compared with dd-cfDNA alone, while recommending dd-cfDNA testing in only 0.79 (95% confidence interval, 0.72 to 0.85) of validation cases. Combined GEP and dd-cfDNA testing validated higher area under the receiver operating characteristic curves than dd-cfDNA as a second-stage test for overall (0.85 [0.79 to 0.94] versus 0.82 [0.74 to 0.92]), antibody-mediated (0.96 [0.93 to 1.00] versus 0.90 [0.79 to 1.00]), and cellular rejections (0.69 [0.61 to 0.89] versus 0.63 [0.53 to 0.81]). CONCLUSIONS:This staged approach reduces unnecessary biopsies and guides the efficient use of costly blood-based biomarkers while maintaining good clinical performance, particularly for subclinical antibody-mediated rejections. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER:ClinicalTrials.gov, NCT01289717 .

KEY POINTS:There is an independent association between two independent noninvasive biomarkers and all-cause graft loss. Time-varying analysis demonstrated that the independent noninvasive biomarkers can provide more information than either one alone. BACKGROUND:The primary objective of this analysis was to assess the association between noninvasive biomarker results within the first 2 years postkidney transplant and 10-year all-cause graft loss (ACGL). METHODS:Clinical Trials in Organ Transplantation 08 (CTOT-08) study was linked with the Scientific Registry of Transplant Recipients to assess 10-year ACGL. Cox regression was used to determine the association between gene expression profile (GEP) and donor-derived cell-free DNA (dd-cfDNA) results and ACGL. A Cox regression with time-varying covariates was used to test associations with ACGL with simultaneous and repeated biomarker results. RESULTS:The final landmarked Cox model included clinical acute rejection (adjusted hazard ratio [aHR], 2.58 [1.30 to 5.13] for one rejection; aHR, 3.41 [1.07 to 10.85] for two rejections), diabetes (aHR, 1.71 [0.99 to 2.96]), recipient age (aHR, 1.02 [1.00 to 1.04]), the highest quartile of positive GEP (aHR, 2.44 [1.45 to 4.10]), and the highest quartile of positive dd-cfDNA (aHR, 2.91 [1.77 to 4.78]). In the time-varying model, having both biomarkers positive at the same time carried the greatest association with ACGL (aHR, 4.29 [2.10 to 8.76]). CONCLUSIONS:In this multicenter, prospective analysis of GEP and dd-cfDNA on patient and graft outcomes, an independent association with long-term ACGL was demonstrated. Furthermore, a time-varying analysis demonstrated that two biomarkers with independent methodologies tested at the same time can provide more information than either one alone. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER:ClinicalTrials.gov, NCT01531257 .

Streptococcus pneumoniae is estimated to cause approximately 400,000 hospitalizations annually. Early mortality rate is as high as 26% for immunocompromised patients. In June 2024 a Pneumococcal 21-valent Conjugate Vaccine (P21-vCV) was approved by the FDA and includes serotypes not found in prior vaccines (Danish nomenclature): 15A, 15C, 16F, 23A, 23B, 24F, 31, and 35B. Detection of these new and existing serotypes is critical to determine immunity for immunocompromised patients.Figure 1.Donors 4 Week P21-vCV Response - All 32 SerotypesPost P21-vCV 4 week MFI IgG responses for all 32 serotypes for immunocompromised and immunocompetent donors.Figure 2.Immunocompetent Donor 1 - Vaccine Response to P21-vCVImmunocompetent Donor 1's MFI IgG response at Predose and 4 week post P21-vCV vaccination. 6B is not included in the P21-vCV vaccine, but the donor produced reactive IgG antibodies to both 6A and 6B. The international standard NIBSC 007sp and 23 unvaccinated reference range serum were tested. Serum from 2 immunocompetent and 2 immunocompromised individuals were collected at predose and 4 weeks after P21-vCV vaccination. IgG was detected using commercially available polysaccharides (1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20A, 22F, 23A, 23B, 23F, 24F, 31, 33F, and 35B) activated with DMTMM and individually coupled to Luminex magnetic bead regions. A 32-plex bead pool was created. Samples were pre-processed for specificity, incubated with the bead pool, incubated with an anti-human IgG phycoerythrin antibody, and analyzed on a Intelliflex.Figure 3.Immunocompromised Donor 2 - P21-vCV Predose and 4 Week Results µg/mLImmunocompromised donor 2 did not have a response to the vaccine from predose to 4 week. However, this patient was vaccinated to an earlier pneumococcal vaccine prior to immunosuppressive treatment and still has a >1.3 µg/mL IgG result to certain serotypes. Immunocompetent donors had a median 12-fold increase in median fluorescent intensity (MFI) predose to 4 week. Immunocompetent donor 1 had a MFI response to 23 of 32 serotypes. Immunocompetent donor 2 had a MFI response to 22 of 32 serotypes. Immunocompromised donor 1 had a lower MFI response compared to immunocompetent donors, but did respond to 13 of the 32 serotypes, Figure1. Polysaccharides with similar conformation can produce cross-reactive IgG. In Figure 2, P21-vCV produced cross-reactive IgG to serotypes 6A and 6B. Immunocompromised Donor 2 did not have a response to the vaccine. Prior vaccine immunity was detected at >1.3 µg/mL baselined from NIBSC 007sp, Figure 3. The reference range median MFI was 1,208 and a median value of 0.21 µg/mL. MFI results for new serotypes were “Negative” at < 1,208, “Detected” was 1,208-6,039, and “Detected +” was >6,040. Some serotypes can have cross-reactive IgG detection after vaccination. IgG was detected for all 32 serotypes. However, a commercially available international standard for the new serotypes is needed for accurate quantification. Joe Douglas, MS, Eurofins Viracor: Employee Jerod Davidson, B.A., Eurofins Viracor: Employee Michael Church, PhD, Eurofins Viracor: Employee Jamie Nutt, n/a, Eurofins Viracor: Employee Pam Morris, M.S., PMP, Pam Morris: Employee Steve Kleiboeker, PhD, Eurofins Viracor: Employee

Abstract Background Mucormycosis are diseases with high morbidity and mortality caused by fungal species belonging to the order Mucorales. The intended use of a Mucorales qPCR assay is as an adjunct to microbiological, clinical, and radiologic means of diagnosing invasive mucormycosis (IM). Serum collection is a non-invasive alternative to BAL and biopsy. Our design process included sequences from all species within the Mucorales genera: Cunninghamella, Lichtheimia, Absidia, Apophysomyces, Mucor, Rhizomucor, Rhizopus and Saksenaea. The Mucorales qPCR assay is designed to detect and quantify Mucorales DNA extracted from human serum. Methods Primers and probes were designed to target the 18S regions of Mucorales species. Nucleic acid was extracted using the MagMAX™ Cell-Free DNA Isolation Kit and KingFisher™ Flex system. Amplification and detection were performed using TaqMan™ Fast Universal Master Mix and the Applied Biosystems™ QuantStudio 7 Pro. Quantification was performed using Rhizopus oryzae target-bearing linearized plasmid standards with results reported in copies/mL. Analytical validation was performed by spiking negative human serum with four different Mucorales species’ DNA: Rhizopus oryzae, Rhizomucor miehei, Lichtheimia corymbifera, and Cunninghamella bertholletiae. Results The limit of detection was 181 copies/mL, and the lower limit of quantification was determined to be 253 copies/mL as shown in Table 1. Linear regression of dilution data produced an R2=0.997, slope=1.029, and y-intercept=0.212 as show in Figure 1. The Mucorales target detection was 100% (60 of 60) of blinded positive samples with all observed log10 copies/mL results within ±0.5 log10 copies of expected values as shown in Table 2. All (20 of 20) blinded negative samples were negative for Mucorales and positive for Internal Control (IC). Intra-assay precision ranged from 5 – 22 %CV and inter-assay precision ranged from 12 – 17 %CV as Shown in Table 3. Conclusion The validated assay described here demonstrates excellent sensitivity, linearity, precision, and accuracy, providing a reliable means of detecting Mucorales DNA in human serum. This assay represents a non-invasive, rapid, and sensitive method to guide clinical decisions on patient populations at high risk of IM. Disclosures Jerod Davidson, B.A., Eurofins Viracor: Employee James Grantham, B.S., Eurofins Viracor: Employee Clayton Thomas, B.S., Eurofins Viracor: Employee Jamie Nutt, n/a, Eurofins Viracor: Employee Pam Morris, M.S., PMP, Eurofins Viracor: Employee Zachary Kockler, PhD, Eurofins Viracor: Employee Steve Kleiboeker, PhD, Eurofins: employee|Eurofins: Stocks/Bonds (Public Company)

Torque Teno Virus (TTV) has emerged as a promising marker reflecting the net state and trajectory of immunosuppression (IS). We analyzed longitudinal TTV data from 252 kidney transplant recipients in a multi-center observational study (average 7.8 visits/patient over 2 years). Patient-specific TTV trajectories, computed as slopes over defined time windows, captured the direction of immune response dynamics. A past 1-year logTTV slope < 0.0066, combined with a low current logTTV (<4.3) and a relatively high historical logTTV average (>5.7), was associated with a 13.88-fold increase in the odds of subclinical AR (95% CI: 5.49-37.42) relative to patients whose slope exceeded 0.0066. In contrast, a past 1-year logTTV slope > 0.076 conferred a 12.15-fold rise in the odds of infection over TX (95% CI: 2.99-81.42). Decision trees incorporating TTV trajectories achieved AUCs of 0.67 for both subclinical AR and infection versus TX-outperforming models using a single TTV measurement. We identified optimal two-sided logTTV thresholds-(4.5,7.8)-stratifying patients into Under-IS, Even-IS, and Over-IS states, where Under-IS status increases subclinical AR odds over TX by 2.39-fold (95% CI: 1.53-3.83), while Over-IS status increases infection odds over subclinical AR by 2.5-fold (95% CI: 1.03-6.42). These findings provide a framework for personalized IS management.

Abstract Background Fungal and acid-fast bacteria (AFB) infections can be a serious complication for immunosuppressed patients. Identification of the pathogen involved in invasive fungal/AFB infections (IFIs) can allow for reduced time to initiation of the appropriate targeted therapy through elimination of ineffective therapies. Current fungal/AFB diagnostics suffer from low sensitivity/specificity, invasiveness (e.g., biopsy or antigen), have long turnaround times (e.g., culture), and/or require an a priori hypothesis regarding the causative pathogen (e.g., PCR based methods). To overcome shortcomings in fungal/AFB diagnostics, an unbiased next-generation sequencing (NGS) approach can be utilized to characterize pathogen cfDNA present in a patient blood sample. Methods Sequencing of IFIs through hybrid capture has the potential to detect a broad range of pathogens yet can still specifically target the most clinically relevant. Compared to standard whole-genome sequencing methods, hybrid capture followed by NGS can be more specific and sensitive due to an enrichment step using probes specific to cfDNA targets of interest. This additional enrichment step can result in more sequencing reads being dedicated to cfDNA derived from pathogens of interest rather than to cfDNA derived from non-relevant organisms. Here is described a hybrid capture assay that uses >30,000 probes to detect >600 Fungal and select AFB species in plasma. Results Cutoffs for target coverage and kmers/million were established through ROC analysis of 170 contrived plasma samples resulting in an analytical specificity of ∼96% and a sensitivity of ∼1,200 molecules/mL. 98% of the 40 contrived samples used for accuracy were correctly identified while 100% intra- and 93%-inter assay precision was achieved. In silico validation demonstrated the assay’s ability to detect closely related species unable to be tested at the bench. Several species have been detected in the initial clinical samples received thus far, however, all have been below the reportable cutoff values. Conclusion The analytical validation described here demonstrates the analytical performance of hybrid capture in human plasma. Disclosures Curtis Bacon, PhD, Eurofins Viracor: employee Ellis Bixler, n/a, Eurofins Viracor: Employee Scott Cowden, n/a, Eurofins Viracor: Employee Jamie Nutt, n/a, Eurofins Viracor: Employee Steve Kleiboeker, PhD, Eurofins: employee|Eurofins: Stocks/Bonds (Public Company)

Abstract Background Predicting respiratory viral infection (RVI) outcomes may lead to earlier intervention and improved prognosis in immunocompromised hosts. A microarray and RNA-Seq results meta-analysis identified a conserved gene expression host response to RVI of 396 genes called the Meta-Virus Signature (MVS). While the MVS score positively correlates to RVI severity, a 42-gene subset called the Severe-or-Mild (SoM) score distinguishes severity groups with higher accuracy (PMID 33765435). This assay has not been validated for use in immunocompromised patients with RVIs. We aim to determine if SoM scores are associated with the severity of RVIs in hematopoietic cell transplant (HCT) recipients.Table 1:Baseline characteristics and clinical outcomes following respiratory infection (n=50) Abbreviations: Allo=allogenic, BiPAP= bilevel positive airway pressure, BMI= Body mass index, CKD= chronic kidney disease, COPD= chronic obstructive pulmonary disease, CPAP=continuous positive airway pressure, ESRD= end-stage renal disease, GVHD= graft-versus-host disease, HCT= hematopoietic stem cell transplantation, HFNC= high-flow nasal cannula, HM=hematologic malignancy, ISI= immunodeficiency scoring index, IQR= interquartile range, LRI= lower respiratory tract infection, MRD= matched related donor, MUD= matched unrelated donor, PIV= parainfluenza virus, RSV= respiratory syncytial virus, RVI= respiratory virus infection, SD=standard deviation, SoM= Severe-to-mild, URI= upper respiratory tract infection. Methods We prospectively enrolled 40 HCT recipients with different RVIs (respiratory syncytial virus, influenza, parainfluenza, and SARS-CoV-2; 10 for each cohort) and 10 post-transplant patients with respiratory symptoms but negative viral respiratory panel (control cohort). Patients were followed for 30 days. Whole blood was collected in HemaSure-OMICS tubes on Day 0 and Day 5. Expression of the SoM score genes was measured by RT-PCR on the Fluidigm Biomark. Data was normalized to the Delta Ct method using the geometric mean of YWHAZ and UBC Ct values. The SoM score was calculated by taking the sum of the geometric means of previously defined modules 3 and 4 divided by the sum of the geometric means of modules 1 and 2. Infection severity was defined based on 30-day maximal oxygen requirements.Figure 1. Median Severe-or-Mild scoring according to 30-day maximal oxygen requirements (among patients with RVI, n=40).Abbreviations: BiPAP= bilevel positive airway pressure, CPAP= continuous positive airway pressure, IQR= intra quartile range, RVI= respiratory virus infection, SoM= Severe-or-Mild.*Mild infection is defined as no oxygen requirement, moderate infection is defined as the maximal O2 requirement of a nasal cannula or face mask, and severe infection as the need for high flow nasal cannula, BiPAP, CPAP, or mechanical ventilation. Results Among the 50 patients enrolled, 33 (66%) had a mild infection, 13 (26%) were moderate, and 4 (8%) experienced severe infections leading to mortality (Table 1). We observed a trend between higher SoM scores and RVI severity, with median SoM scores of 0.694, 0.860, and 1.550 in patients with mild, moderate, and severe infections, respectively (p=0.370) (Figures 1,2). The association between an elevated SoM score and severe infections was stronger for HCT recipients infected with parainfluenza (Figure 2).Figure 2. Median Severe-or-Mild scoring and severity of respiratory infection (n=50).Abbreviations: BiPAP= bilevel positive airway pressure, CPAP= continuous positive airway pressure, PIV= parainfluenza virus, RSV= respiratory syncytial virus, SoM= Severe-or-Mild.*Mild infection is defined as no oxygen requirement, moderate infection is defined as the maximal O2 requirement of a nasal cannula or face mask, and severe infection as the need for high flow nasal cannula, BiPAP, CPAP, or mechanical ventilation. Conclusion SOM score was elevated in HCT recipients with severe RVIs, although not statistically significant. Larger prospective cohort studies will be needed to identify clinically relevant applications of gene expression analysis to predict the severity of infection. Disclosures Ronnie LaCombe, PhD, Eurofins Viracor: Employee Genevieve S. Hanna, B.S. Biology, Eurofins Viracor: Employee Isabelle Ulloa, n/a, Eurofins-Viracor: Employment Rohita Sinha, PhD, Eurofins Viracor: I am a salaried employee of Eurofins Viracor Pam Morris, M.S., PMP, Eurofins Viracor: Employee Karen Howard-Shipley, M.S., PMP, Eurofins Viracor: Employment Steve Kleiboeker, PhD, Eurofins: employee|Eurofins: Stocks/Bonds (Public Company) Fareed Khawaja, MBBS, Eurofins Viracor: Grant/Research Support|Symbio: Grant/Research Support Roy F. Chemaly, MD/MPH, AiCuris: Advisor/Consultant|AiCuris: Grant/Research Support|Ansun Pharmaceuticals: Advisor/Consultant|Ansun Pharmaceuticals: Grant/Research Support|Astellas: Advisor/Consultant|Eurofins-Viracor: Grant/Research Support|InflaRX: Advisor/Consultant|Janssen: Advisor/Consultant|Karius: Advisor/Consultant|Karius: Grant/Research Support|Merck/MSD: Advisor/Consultant|Merck/MSD: Grant/Research Support|Moderna: Advisor/Consultant|Oxford Immunotec: Advisor/Consultant|Oxford Immunotec: Grant/Research Support|Roche/Genentech: Advisor/Consultant|Roche/Genentech: Grant/Research Support|Shinogi: Advisor/Consultant|Takeda: Advisor/Consultant|Takeda: Grant/Research Support|Tether: Advisor/Consultant

Background. Kidney transplant recipients require potent immunosuppression and are predisposed to opportunistic infections, many of which have a viral etiology. Currently, viral assays detect and quantify single pathogens using PCR or qPCR. An unbiased sequencing method with comparable accuracy would allow simultaneous monitoring of multiple viral pathogens and nonpathogenic Anelloviridae. The quantification of donor-derived cell-free DNA (dd-cfDNA) is an established method for the detection of allograft rejection, and a single workflow combining ddcfDNA quantification and viral detection represents an opportunity to improve patient monitoring and management. Methods. Whole genome sequencing of cell-free DNA was performed using 1,980 plasma samples from 256 subjects enrolled in a multi-center study. Non-human sequences underwent reference-assisted assembly and taxonomic annotation of the viral DNA pathogens. Results. Of the 1,980 samples tested, 1,453 (73.4%) had >= 1 viral detection(s), either a known viral pathogen or torque teno virus (TTV), with positivity rates generally declining 12-18 months post-transplant. Concordance of metagenomic NGS (mNGS) viral detection with qPCR detection was 97.7% (94.1% sensitivity, 98.2% specificity), and a linear relationship was demonstrated between mNGS viral quantitation and qPCR results. BK virus, cytomegalovirus, and Epstein-Barr virus were detected by sequencing up to 60 days prior to independently established clinical diagnoses. Conclusions. Whole-genome sequencing allows simultaneous quantification of dd-cfDNA as well as sensitive and early detection of viral infection through secondary analysis of the same sequencing results. In combination with dd-cfDNA, mNGS viral detection may provide additional pathogen surveillance results and serve as a useful biomarker for both over- and underimmunosuppression.

IntroductionTransplant Associated Thrombotic Microangiopathy (TA-TMA) is a complication in hematopoietic stem cell transplant (HSCT) patients due to overactivation of the complement cascade. TA-TMA can result in injury to multiple organs including the kidneys and requires rapid initiation of treatment. Markers used to diagnose and monitor treatment of TA-TMA include a high-risk injury marker sC5b-9, the soluble form of terminal complement complex (TCC), and CH50 that measures potential complement activity. Eculizumab is a monoclonal antibody drug that inhibits cleavage of C5 in the complement cascade and has been used to treat TA-TMA. An assay to measure sC5b-9 was developed and validated, and an FDA approved assay for CH50 was verified, to aid in early diagnosis and treatment of TA-TMA. These assays are performed rapidly and results provided the same day. An assay to measure free eculizumab concentration in the blood is being developed to assist in monitoring patient treatment.MethodsThe sC5b-9 ELISA assay measures the soluble TCC in EDTA plasma using a monoclonal antibody specific for the complex. The CH50 total complement assay is a liposomal turbidimetric assay that measures CH50 in serum. The Eculizumab ELISA measures levels in serum or plasma and is specific for the free form of eculizumab, not bound to endogenous C5. The validations/verifications of the assays are performed to meet requirements for certification by the College of American Pathologists and the Clinical and Laboratory Standards Institute.ResultsThe sC5b-9 lower limit of quantification (LLOQ) was 81ng/mL, the reference range was ≤ 256 ng/mL correlating with the published normal of ≤ 244 ng/mL and the CVs for precision were ≤ 19%. The CH50 LLOQ was 13 U/mL, the reference range was 36-95 U/mL and the CVs for precision were ≤ 5%. Preliminary testing of the eculizumab ELISA indicated an LLOQ of 28 µg/mL and precision CVs ≤ 15%.Clinical samples previously analyzed for sC5b-9 were analyzed for eculizumab (Figure 1) to determine correlation of the results. Three samples with sC5b-9 levels > 256 ng/mL (blue line) had eculizumab levels that were below the recommended trough level of 100 mg/mL (yellow line). These results indicate excessive complement activation and eculizumab treatment had not controlled the activation. Patients with low levels of sC5b-9 either had no detectable eculizumab or had detectable eculizumab with low sC5b-9. These results suggest the patients did not require treatment or were being treated and the complement activation was controlled.ConclusionsAssays measuring sC5b-9 and CH50 with same day turnaround time have been validated or verified so that timely results can be provided to physicians with patients at risk of organ damage due to TA-TMA. An assay to measure Eculizumab levels within 24 hours is currently under development.

BK polyomavirus (BKPyV) is associated with disease in transplant patients. BKPyV genomic diversity is thought to contribute to functional differences in virulence factors. Here, we present the near full-length BKPyV genome sequence from a patient who underwent hematopoietic cell transplant and was infected with subtype III, a rare subtype.