Structure, receptor recognition, and antigenicity of the human coronavirus CCoV-HuPn-2018 spike glycoprotein.

The isolation of CCoV-HuPn-2018 from a child respiratory swab indicates that more coronaviruses are spilling over to humans than previously appreciated. We determined the structures of the CCoV-HuPn-2018 spike glycoprotein trimer in two distinct conformational states and showed that its domain 0 recognizes sialosides. We identified that the CCoV-HuPn-2018 spike binds canine, feline, and porcine aminopeptidase N (APN) orthologs, which serve as entry receptors, and determined the structure of the receptor-binding B domain in complex with canine APN. The introduction of an oligosaccharide at position N739 of human APN renders cells susceptible to CCoV-HuPn-2018 spike-mediated entry, suggesting that single-nucleotide polymorphisms might account for viral detection in some individuals. Human polyclonal plasma antibodies elicited by HCoV-229E infection and a porcine coronavirus monoclonal antibody inhibit CCoV-HuPn-2018 spike-mediated entry, underscoring the cross-neutralizing activity among ɑ-coronaviruses. These data pave the way for vaccine and therapeutic development targeting this zoonotic pathogen representing the eighth human-infecting coronavirus.

Convergent clonal selection of donor- and recipient-derived CMV-specific T cells in hematopoietic stem cell transplant patients.

Competition between antigen-specific T cells for peptide:MHC complexes shapes the ensuing T cell response. Mouse model studies provided compelling evidence that competition is a highly effective mechanism controlling the activation of naïve T cells. However, assessing the effect of T cell competition in the context of a human infection requires defined pathogen kinetics and trackable naïve and memory T cell populations of defined specificity. A unique cohort of nonmyeloablative hematopoietic stem cell transplant patients allowed us to assess T cell competition in response to cytomegalovirus (CMV) reactivation, which was documented with detailed virology data. In our cohort, hematopoietic stem cell transplant donors and recipients were CMV seronegative and positive, respectively, thus providing genetically distinct memory and naïve T cell populations. We used single-cell transcriptomics to track donor versus recipient-derived T cell clones over the course of 90 d. We found that donor-derived T cell clones proliferated and expanded substantially following CMV reactivation. However, for immunodominant CMV epitopes, recipient-derived memory T cells remained the overall dominant population. This dominance was maintained despite more robust clonal expansion of donor-derived T cells in response to CMV reactivation. Interestingly, the donor-derived T cells that were recruited into these immunodominant memory populations shared strikingly similar TCR properties with the recipient-derived memory T cells. This selective recruitment of identical and nearly identical clones from the naïve into the immunodominant memory T cell pool suggests that competition is in place but does not interfere with rejuvenating a memory T cell population. Instead, it results in selection of convergent clones to the memory T cell pool.

Serial Quantitation of Plasma Microbial Cell-Free DNA Before and After Diagnosis of Pulmonary Invasive Mold Infections After Hematopoietic Cell Transplant.

BACKGROUND: Plasma microbial cell-free DNA sequencing (mcfDNA-Seq) is a noninvasive test for microbial diagnosis of invasive mold infection (IMI). The utility of mcfDNA-Seq for predicting IMI onset and the clinical implications of mcfDNA concentrations are unknown. METHODS: We retrospectively tested plasma from hematopoietic cell transplant (HCT) recipients with pulmonary IMI and ≥1 mold identified by mcfDNA-Seq in plasma collected within 14 days of clinical diagnosis. Samples collected from up to 4 weeks before and 4 weeks after IMI diagnosis were evaluated using mcfDNA-Seq. RESULTS: Thirty-five HCT recipients with 39 IMIs (16 Aspergillus and 23 non-Aspergillus infections) were included. Pathogenic molds were detected in 38%, 26%, 11%, and 0% of samples collected during the first, second, third, and fourth week before clinical diagnosis, respectively. In non-Aspergillus infections, median mcfDNA concentrations in samples collected within 3 days of clinical diagnosis were higher in infections with versus without extrapulmonary spread (4.3 vs 3.3 log10 molecules per microliter [mpm], P = .02), and all patients (8/8) with mcfDNA concentrations >4.0 log10 mpm died within 42 days after clinical diagnosis. CONCLUSIONS: Plasma mcfDNA-Seq can identify pathogenic molds up to 3 weeks before clinical diagnosis of pulmonary IMI. Plasma mcfDNA concentrations may correlate with extrapulmonary spread and mortality in non-Aspergillus IMI.

The effect of gastrointestinal graft-versus-host disease and diarrhea on the pharmacokinetic profile of sotrovimab in hematopoietic stem cell transplant recipients.

BACKGROUND: Monoclonal antibodies (mAbs) are utilized broadly to treat cancer and infectious diseases, and mAb exposure (serum concentration over time) is one predictor of overall treatment efficacy. Herein, we present findings from a clinical trial evaluating the pharmacokinetics (PK) of the long-acting mAb sotrovimab targeting SARS-CoV-2 in hematopoietic cell transplant (HCT) recipients. METHODS: All participants received an intravenous infusion of sotrovimab within one week prior to initiating the pre-transplant preparative regimen. The serum concentration of sotrovimab was measured longitudinally for up to 24 weeks post-transplant. RESULTS: Compared to non-HCT participants, we found that mAb clearance was 10% and 26% higher in autologous and allogeneic HCT recipients, respectively. Overall sotrovimab exposure was approximately 15% lower in HCT recipients compared to non-HCT recipients. Exposure was significantly reduced in HCT recipients who developed diarrhea and lower gastrointestinal (GI) graft-versus-host disease (GVHD) post-transplant. CONCLUSIONS: These data show that sotrovimab exposure may be reduced in HCT recipients, possibly related to increased GI clearance in patients with GVHD. This phenomenon has implications for dose selection and duration of efficacy with sotrovimab and potentially other mAbs in this vulnerable patient population. Thus, mAb dose regimens developed in non-HCT populations may have to be optimized when applied to HCT populations.

Cytomegalovirus (CMV) Reactivation and CMV-Specific Cell-Mediated Immunity After Chimeric Antigen Receptor T-Cell Therapy.

BACKGROUND: The epidemiology of cytomegalovirus (CMV) after chimeric antigen receptor-modified T-cell immunotherapy (CARTx) is poorly understood owing to a lack of routine surveillance. METHODS: We prospectively enrolled 72 adult CMV-seropositive CD19-, CD20-, or BCMA-targeted CARTx recipients and tested plasma samples for CMV before and weekly up to 12 weeks after CARTx. We assessed CMV-specific cell-mediated immunity (CMV-CMI) before and 2 and 4 weeks after CARTx, using an interferon γ release assay to quantify T-cell responses to IE-1 and pp65. We tested pre-CARTx samples to calculate a risk score for cytopenias and infection (CAR-HEMATOTOX). We used Cox regression to evaluate CMV risk factors and evaluated the predictive performance of CMV-CMI for CMV reactivation in receiver operator characteristic curves. RESULTS: CMV was detected in 1 patient (1.4%) before and in 18 (25%) after CARTx, for a cumulative incidence of 27% (95% confidence interval, 16.8-38.2). The median CMV viral load (interquartile range) was 127 (interquartile range, 61-276) IU/mL, with no end-organ disease observed; 5 patients received preemptive therapy based on clinical results. CMV-CMI values reached a nadir 2 weeks after infusion and recovered to baseline levels by week 4. In adjusted models, BCMA-CARTx (vs CD19/CD20) and corticosteroid use for >3 days were significantly associated with CMV reactivation, and possible associations were detected for lower week 2 CMV-CMI and more prior antitumor regimens. The cumulative incidence of CMV reactivation almost doubled when stratified by BCMA-CARTx target and use of corticosteroids for >3 days (46% and 49%, respectively). CONCLUSIONS: CMV testing could be considered between 2 and 6 weeks in high-risk CARTx recipients.

A human coronavirus evolves antigenically to escape antibody immunity.

There is intense interest in antibody immunity to coronaviruses. However, it is unknown if coronaviruses evolve to escape such immunity, and if so, how rapidly. Here we address this question by characterizing the historical evolution of human coronavirus 229E. We identify human sera from the 1980s and 1990s that have neutralizing titers against contemporaneous 229E that are comparable to the anti-SARS-CoV-2 titers induced by SARS-CoV-2 infection or vaccination. We test these sera against 229E strains isolated after sera collection, and find that neutralizing titers are lower against these "future" viruses. In some cases, sera that neutralize contemporaneous 229E viral strains with titers >1:100 do not detectably neutralize strains isolated 8-17 years later. The decreased neutralization of "future" viruses is due to antigenic evolution of the viral spike, especially in the receptor-binding domain. If these results extrapolate to other coronaviruses, then it may be advisable to periodically update SARS-CoV-2 vaccines.

Cytomegalovirus-specific T-cell reconstitution following letermovir prophylaxis after hematopoietic cell transplantation.

Decreased cytomegalovirus (CMV)-specific immunity after hematopoietic cell transplantation (HCT) is associated with late CMV reactivation and increased mortality. Whether letermovir prophylaxis-associated reduction in viral exposure influences CMV-specific immune reconstitution is unknown. In a prospective cohort of allogeneic HCT recipients who received letermovir, we compared polyfunctional CMV-specific T-cell responses to those of controls who received PCR-guided preemptive therapy before the introduction of letermovir. Thirteen-color flow cytometry was used to assess T-cell responses at 3 months after HCT following stimulation with CMV immediate early-1 (IE-1) antigen and phosphoprotein 65 (pp65) antigens. Polyfunctionality was characterized by combinatorial polyfunctionality analysis of antigen-specific T-cell subsets. Use of letermovir and reduction of viral exposure were assessed for their association with CMV-specific T-cell immunity. Polyfunctional T-cell responses to IE-1 and pp65 were decreased in letermovir recipients and remained diminished after adjustment for donor CMV serostatus, absolute lymphocyte count, and steroid use. Among letermovir recipients, greater peak CMV DNAemia and increased viral shedding were associated with stronger CD8+ responses to pp65, whereas the CMV shedding rate was associated with greater CD4+ responses to IE-1. In summary, our study provided initial evidence that letermovir may delay CMV-specific cellular reconstitution, possibly related to decreased CMV antigen exposure. Evaluating T-cell polyfunctionality may identify patients at risk for late CMV infection after HCT.

Donor-Derived CD4+ T Cells and Human Herpesvirus 6B Detection After Allogeneic Hematopoietic Cell Transplantation.

We sought to determine whether donor-derived human herpesvirus (HHV) 6B-specific CD4+ T-cell abundance is correlated with HHV-6B detection after allogeneic hematopoietic cell transplantation. We identified 33 patients who received HLA-matched, non-T-cell-depleted, myeloablative allogeneic hematopoietic cell transplantation and underwent weekly plasma polymerase chain reaction testing for HHV-6B for 100 days thereafter. We tested donor peripheral blood mononuclear cells for HHV-6B-specific CD4+ T cells. Patients with HHV-6B detection above the median peak viral load (200 copies/mL) received approximately 10-fold fewer donor-derived total or HHV-6B-specific CD4+ T cells than those with peak HHV-6B detection at ≤200 copies/mL or with no HHV-6B detection. These data suggest the importance of donor-derived immunity for controlling HHV-6B reactivation.

Liquid Biopsy for Invasive Mold Infections in Hematopoietic Cell Transplant Recipients With Pneumonia Through Next-Generation Sequencing of Microbial Cell-Free DNA in Plasma.

BACKGROUND: Noninvasive diagnostic options are limited for invasive mold infections (IMIs). We evaluated the performance of a plasma microbial cell-free DNA sequencing (mcfDNA-Seq) test for diagnosing pulmonary IMI after hematopoietic cell transplant (HCT). METHODS: We retrospectively assessed the diagnostic performance of plasma mcfDNA-Seq next-generation sequencing in 114 HCT recipients with pneumonia after HCT who had stored plasma obtained within 14 days of diagnosis of proven/probable Aspergillus IMI (n = 51), proven/probable non-Aspergillus IMI (n = 24), possible IMI (n = 20), and non-IMI controls (n = 19). Sequences were aligned to a database including >400 fungi. Organisms above a fixed significance threshold were reported. RESULTS: Among 75 patients with proven/probable pulmonary IMI, mcfDNA-Seq detected ≥1 pathogenic mold in 38 patients (sensitivity, 51% [95% confidence interval {CI}, 39%-62%]). When restricted to samples obtained within 3 days of diagnosis, sensitivity increased to 61%. McfDNA-Seq had higher sensitivity for proven/probable non-Aspergillus IMI (sensitivity, 79% [95% CI, 56%-93%]) compared with Aspergillus IMI (sensitivity, 31% [95% CI, 19%-46%]). McfDNA-Seq also identified non-Aspergillus molds in an additional 7 patients in the Aspergillus subgroup and Aspergillus in 1 patient with possible IMI. Among 19 non-IMI pneumonia controls, mcfDNA-Seq was negative in all samples, suggesting a high specificity (95% CI, 82%-100%) and up to 100% positive predictive value (PPV) with estimated negative predictive values (NPVs) of 81%-99%. The mcfDNA-Seq assay was complementary to serum galactomannan index testing; in combination, they were positive in 84% of individuals with proven/probable pulmonary IMI. CONCLUSIONS: Noninvasive mcfDNA-Seq had moderate sensitivity and high specificity, NPV, and PPV for pulmonary IMI after HCT, particularly for non-Aspergillus species.

Comprehensive viromewide antibody responses by systematic epitope scanning after hematopoietic cell transplantation.

Further insight into humoral viral immunity after hematopoietic cell transplantation (HCT) could have potential impact on donor selection or monitoring of patients. Currently, estimation of humoral immune recovery is inferred from lymphocyte counts or immunoglobulin levels and does not address vulnerability to specific viral infections. We interrogated the viral antibody repertoire before and after HCT using a novel serosurvey (VirScan) that detects immunoglobulin G responses to 206 viruses. We performed VirScan on cryopreserved serum from pre-HCT and 30, 100, and 365 days after myeloablative HCT from 37 donor-recipient pairs. We applied ecologic metrics (α- and ß-diversity) and evaluated predictors of metrics and changes over time. Donor age and donor/recipient cytomegalovirus (CMV) serostatus and receipt systemic glucocorticoids were most strongly associated with VirScan metrics at day 100. Other clinical characteristics, including pre-HCT treatment and conditioning, did not affect antiviral repertoire metrics. The recipient repertoire was most similar (pairwise ß-diversity) to that of donor at day 100, but more similar to pre-HCT self by day 365. Gain or loss of epitopes to common viruses over the year post-HCT differed by donor and recipient pre-HCT serostatus, with highest gains in naive donors to seropositive recipients for several human herpesviruses and adenoviruses. We used VirScan to highlight contributions of donor and recipient to antiviral humoral immunity and evaluate longitudinal changes. This work builds a foundation to test whether such systematic profiling could serve as a biomarker of immune reconstitution, predict clinical events after HCT, or help refine selection of optimal donors.

Cytomegalovirus Humoral Response Against Epithelial Cell Entry-Mediated Infection in the Primary Infection Setting After Hematopoietic Cell Transplantation.

BACKGROUND: The influence of humoral immunity on the prevention of primary cytomegalovirus (CMV) infection after hematopoietic cell transplantation (HCT) is poorly understood. METHODS: To determine whether neutralizing antibodies (nAbs) against CMV pentameric complex (PC)-mediated epithelial cell entry decrease CMV infection after HCT, samples were analyzed from a randomized controlled trial of CMV intravenous immunoglobulin (IVIG) prophylaxis. Weekly serum from 61 CMV donor-positive/recipient-negative (D+/R-) HCT patients (33 control, 28 CMV IVIG) was tested using a PC-entry nAb assay and quantitative CMV polymerase chain reaction (PCR). RESULTS: There was a trend toward higher weekly PC-entry nAb titers (P = .07) and decreased CMV infection by PCR at viral load cutoffs of ≥1000 and ≥10 000 IU/mL in the CMV IVIG arm. High nAb titers were not significantly protective against CMV infection later after HCT in both study arms. Among CMV-infected patients, each log2 increase in nAb titer was associated with an average 0.2 log10 decrease in concurrent CMV viral load after infection (P = .001; adjusted for study arm). CONCLUSIONS: This study provides initial support that CMV IVIG prophylaxis moderately enhances PC-entry nAB activity in D+/R- HCT recipients.

Inflammatory Cytokine Profile in Individuals with Inherited Chromosomally Integrated Human Herpesvirus 6.

Acute graft-versus-host-disease (aGVHD) is a major complication following hematopoietic cell transplantations (HCTs). We have shown that HCT recipients in whom either the donor or patient had inherited chromosomally integrated human herpesvirus 6 (iciHHV-6) have a higher incidence of developing more severe aGVHD. Previous studies established that increased proinflammatory cytokines are associated with increased risk for aGVHD and nonrelapse mortality post-HCT. We hypothesized that HCT recipients with donor or recipient iciHHV-6 (iciHHV-6pos HCT cases) will have higher cytokine levels compared with HCT recipients without iciHHV-6 (iciHHV-6neg HCT controls). We identified 64 iciHHV-6pos HCT cases with plasma from days 7, 14, and/or 21 post-HCT and before aGVHD onset in patients who developed aGVHD. We identified 64 iciHHV-6neg HCT controls matched for aGVHD risk factors. We also identified 28 donors with iciHHV-6 and 56 matched donors without iciHHV-6. We measured plasma cytokine concentrations for IL-6, suppression of tumorigenicity 2, T cell immunoglobulin and mucin-domain containing 3, TNFα, soluble TNF receptor 1 (TNFRp55), and C-reactive protein (CRP). We used Mann-Whitney tests and repeated-measures models to compare cytokine levels. iciHHV-6pos HCT cases had higher CRP levels on day 7 and day 21 and higher TNFRp55 levels on day 14 and day 21 compared with iciHHV-6neg HCT controls. These findings were recapitulated in a repeated-measures model. The differences were most evident among patients who subsequently developed aGVHD grades 2 to 4. Additionally, iciHHV-6pos HCT cases had earlier-onset aGVHD (median, 20 versus 27 days post-HCT; P = .02). There were no differences in cytokine levels among healthy donors with or without iciHHV-6. This study demonstrates that HCT recipients with iciHHV-6 have higher proinflammatory cytokines that may be associated with increased risk for aGVHD.

Effect of Preemptive Therapy vs Antiviral Prophylaxis on Cytomegalovirus Disease in Seronegative Liver Transplant Recipients With Seropositive Donors: A Randomized Clinical Trial.

Importance: Despite the use of a cytomegalovirus (CMV) prevention strategy of antiviral prophylaxis for high-risk CMV-seronegative liver transplant recipients with seropositive donors, high rates of delayed-onset postprophylaxis CMV disease occur. An alternate approach, preemptive therapy (initiation of antiviral therapy for early asymptomatic CMV viremia detected by surveillance testing), has not previously been directly compared with antiviral prophylaxis in these patients. Objective: To compare preemptive therapy with antiviral prophylaxis in CMV-seronegative liver transplant recipients with seropositive donors for the prevention of CMV disease. Design, Setting, and Participants: Randomized clinical trial of preemptive therapy vs antiviral prophylaxis in 205 CMV-seronegative liver transplant recipients with seropositive donors aged older than 18 years. The trial was conducted at 6 academic transplant centers in the United States between October 2012 and June 2017, with last follow-up in June 2018. Interventions: Patients were randomized 1:1 to receive either preemptive therapy (valganciclovir, 900 mg, twice daily until 2 consecutive negative tests a week apart) for viremia detected by weekly plasma CMV polymerase chain reaction for 100 days (n = 100) or valganciclovir, 900 mg, daily for 100 days as antiviral prophylaxis (n = 105). Main Outcomes and Measures: The primary outcome was incidence of CMV disease by 12 months, defined as CMV syndrome (CMV viremia and clinical or laboratory findings) or end-organ disease. Secondary outcomes included acute allograft rejection, opportunistic infections, graft and patient survival, and neutropenia. Results: Among 205 patients who were randomized (mean age, 55 years; 62 women [30%]), all 205 (100%) completed the trial. The incidence of CMV disease was significantly lower with preemptive therapy than antiviral prophylaxis (9% [9/100] vs 19% [20/105]; difference, 10% [95% CI, 0.5% to 19.6%]; P = .04]). The incidence of allograft rejection (28% vs 25%; difference, 3% [95% CI, -9% to 15%]), opportunistic infections (25% vs 27%; difference, 2% [95% CI, -14% to 10%]), graft loss (2% vs 2%; difference, <1% [95% CI, -4% to 4%]), and neutropenia (13% vs 10%; difference, 3% [95% CI, -5% to 12%]) did not differ significantly for the preemptive therapy vs antiviral prophylaxis group, respectively. All-cause mortality at last follow-up was 15% in the preemptive therapy vs 19% in the antiviral prophylaxis group (difference, 4% [95% CI, -14% to 6%]; P = .46). Conclusions and Relevance: Among CMV-seronegative liver transplant recipients with seropositive donors, the use of preemptive therapy, compared with antiviral prophylaxis, resulted in a lower incidence of CMV disease over 12 months. Further research is needed to replicate these findings and assess long-term outcomes. Trial Registration: ClinicalTrials.gov Identifier: NCT01552369.

Prospective Assessment of Cytomegalovirus Immunity in High-Risk Donor-Seropositive/Recipient-Seronegative Liver Transplant Recipients Receiving Either Preemptive Therapy or Antiviral Prophylaxis.

The differential impact of preemptive therapy (PET) and antiviral prophylaxis (AP) on development of cytomegalovirus (CMV)-specific neutralizing antibody (nAb) and T-cell responses have not previously been directly compared in high-risk donor-seropositive/recipient-seronegative (D+R-) organ transplant recipients. We prospectively assessed T-cell and nAb responses 3 months after transplantation in cohorts of high-risk D+R- liver transplant recipients who received either PET (n = 15) or AP (n = 25) and a control group of CMV-seropositive transplant recipients (R+) (AP; n = 24). CMV phosphoprotein 65 (pp65)- and immediate early protein 1-specific multifunctional T-cell responses were determined by means of intracellular cytokine staining and nAbs against BADrUL131-Y4 CMV in adult retinal pigment epithelial cell line-19 human epithelial cells; nAbs were detected in 8 of 12 (67%) in the PET group, none of 17 in the AP group, and 20 of 22 (91%) in the R+ group. Multifunctional CD8 and CD4 T-cell responses to pp65 were generally similar between PET and R+ groups, and lower for the AP group; multifunctional CD4 responses were similar across all groups. Among D+R- liver transplant recipients, PET was associated with the development of greater nAb and multifunctional CD8 T-cell responses compared with AP, providing a potential mechanism to explain the relative protection against late-onset disease with PET. Future studies are needed to define specific immune parameters predictive of late-onset CMV disease with AP.

The cumulative burden of double-stranded DNA virus detection after allogeneic HCT is associated with increased mortality.

Strategies to prevent active infection with certain double-stranded DNA (dsDNA) viruses after allogeneic hematopoietic cell transplantation (HCT) are limited by incomplete understanding of their epidemiology and clinical impact. We retrospectively tested weekly plasma samples from allogeneic HCT recipients at our center from 2007 to 2014. We used quantitative PCR to test for cytomegalovirus, BK polyomavirus, human herpesvirus 6B, HHV-6A, adenovirus, and Epstein-Barr virus between days 0 and 100 post-HCT. We evaluated risk factors for detection of multiple viruses and association of viruses with mortality through day 365 post-HCT with Cox models. Among 404 allogeneic HCT recipients, including 125 cord blood, 125 HLA-mismatched, and 154 HLA-matched HCTs, detection of multiple viruses was common through day 100: 90% had ≥1, 62% had ≥2, 28% had ≥3, and 5% had 4 or 5 viruses. Risk factors for detection of multiple viruses included cord blood or HLA-mismatched HCT, myeloablative conditioning, and acute graft-versus-host disease (P values < .01). Absolute lymphocyte count of <200 cells/mm3 was associated with greater virus exposure on the basis of the maximum cumulative viral load area under the curve (AUC) (P = .054). The maximum cumulative viral load AUC was the best predictor of early (days 0-100) and late (days 101-365) overall mortality (adjusted hazard ratio [aHR] = 1.36, 95% confidence interval [CI] [1.25, 1.49], and aHR = 1.04, 95% CI [1.0, 1.08], respectively) after accounting for immune reconstitution and graft-versus-host disease. In conclusion, detection of multiple dsDNA viruses was frequent after allogeneic HCT and had a dose-dependent association with increased mortality. These data suggest opportunities to improve outcomes with better antiviral strategies.

Kinetics of Double-Stranded DNA Viremia After Allogeneic Hematopoietic Cell Transplantation.

Background: Improved understanding of double-stranded DNA (dsDNA) virus kinetics after hematopoietic cell transplantation (HCT) would facilitate development of therapeutic strategies. Methods: We tested weekly plasma samples from 404 patients through day 100 after allogeneic HCT for cytomegalovirus (CMV), human herpesvirus (HHV) 6A and 6B, BK polyomavirus (BKV), adenovirus (AdV), and Epstein-Barr virus (EBV) using quantitative polymerase chain reaction. Episodes lasting ≤1 week were defined as blips and >1 week as persistent. We described virus-specific kinetics, analyzed the association of virus area under the curve (AUC) with overall mortality, and identified risk factors for persistent episodes. Results: We identified 428 episodes of CMV, 292 of BKV, 224 of HHV-6B, 46 of AdV, and 53 of EBV. CMV and BKV had the highest proportions of persistent episodes (68% and 80%, respectively). Detection and kinetics varied by virus. HHV-6B episodes reached maximum levels fastest and had the shortest intervals between detection and end-organ disease. End-organ disease occurred within 14 days of viremia in 68% of cases, generally during persistent episodes. For all viruses, higher viral load AUC increased risk for overall mortality through day 365, persistent episodes had higher viral load than blips, and higher first positive viral load significantly increased risk for persistent episodes. First viral load >2 log10 copies/mL (range, 2.04-3.06 per virus) had high specificity for persistent episodes. Conclusions: Persistent high viral load dsDNA viremia episodes after allogeneic HCT predict mortality. Virus-specific kinetics can guide timing and thresholds for early intervention in studies of novel agents.

Prolonged Shedding of Human Coronavirus in Hematopoietic Cell Transplant Recipients: Risk Factors and Viral Genome Evolution.

Background: Recent data suggest that human coronavirus (HCoV) pneumonia is associated with significant mortality in hematopoietic cell transplant (HCT) recipients. Investigation of risk factors for prolonged shedding and intrahost genome evolution may provide critical information for development of novel therapeutics. Methods: We retrospectively reviewed HCT recipients with HCoV detected in nasal samples by polymerase chain reaction (PCR). HCoV strains were identified using strain-specific PCR. Shedding duration was defined as time between first positive and first negative sample. Logistic regression analyses were performed to evaluate factors for prolonged shedding (≥21 days). Metagenomic next-generation sequencing (mNGS) was conducted when ≥4 samples with cycle threshold values of <28 were available. Results: Seventeen of 44 patients had prolonged shedding. Among 31 available samples, 35% were OC43, 32% were NL63, 19% were HKU1, and 13% were 229E; median shedding duration was similar between strains (P = .79). Bivariable logistic regression analyses suggested that high viral load, receipt of high-dose steroids, and myeloablative conditioning were associated with prolonged shedding. mNGS among 5 subjects showed single-nucleotide polymorphisms from OC43 and NL63 starting 1 month following onset of shedding. Conclusions: High viral load, high-dose steroids, and myeloablative conditioning were associated with prolonged shedding of HCoV in HCT recipients. Genome changes were consistent with the expected molecular clock of HCoV.

Cytomegalovirus (CMV) DNA Quantitation in Bronchoalveolar Lavage Fluid From Hematopoietic Stem Cell Transplant Recipients With CMV Pneumonia.

Background: Quantitative cytomegalovirus (CMV) DNA-specific polymerase chain reaction (PCR) analysis is widely used as a surveillance method for hematopoietic stem cell transplant (HCT) recipients. However, no CMV DNA threshold exists in bronchoalveolar lavage (BAL) to differentiate pneumonia from pulmonary shedding. Methods: We tested archived BAL fluid samples from 132 HCT recipients with CMV pneumonia and 139 controls (100 patients with non-CMV pneumonia, 18 with idiopathic pneumonia syndrome [IPS], and 21 who were asymptomatic) by quantitative CMV and ß-globin DNA-specific PCR. Results: Patients with CMV pneumonia had higher median viral loads (3.9 log10 IU/mL; interquartile range [IQR], 2.6-6.0 log10 IU/mL) than controls (0 log10 IU/mL [IQR, 0-1.6 log10 IU/mL] for patients with non-CMV pneumonia, 0 log10 IU/mL [IQR, 0-1.6 log10 IU/mL] for patients with IPS, and 1.63 log10 IU/mL [IQR, 0-2.5 log10 IU/mL] for patients who were asymptomatic; P < .001 for all comparisons to patients with CMV pneumonia). Receiver operating characteristic curve analyses and predictive models identified a cutoff CMV DNA level of 500 IU/mL to differentiate between CMV pneumonia and pulmonary shedding, using current CMV pneumonia prevalence figures. However, different levels may be appropriate in settings of very high or low CMV pneumonia prevalence. The presence of pulmonary copathogens, radiographic presentation, or pulmonary hemorrhage did not alter predictive values. Conclusion: CMV DNA load in BAL can be used to differentiate CMV pneumonia from pulmonary shedding.

Valganciclovir for the prevention of complications of late cytomegalovirus infection after allogeneic hematopoietic cell transplantation: a randomized trial.

BACKGROUND: Optimal prevention of late cytomegalovirus (CMV) disease is poorly defined. OBJECTIVE: To compare valganciclovir prophylaxis with polymerase chain reaction-guided preemptive therapy. DESIGN: Randomized, double-blind trial. (ClinicalTrials.gov: NCT00016068). SETTING: Multicenter trial. PATIENTS: 184 recipients of hematopoietic cell transplantation (HCT) who were at high risk for late CMV disease (95 patients received valganciclovir and 89 received placebo). INTERVENTION: 6 months of valganciclovir (900 mg/d) or placebo. Patients with polymerase chain reaction positivity at 1000 copies/mL or greater or a 5-fold increase over baseline were treated with ganciclovir or valganciclovir (5 mg/kg or 900 mg twice daily, respectively). MEASUREMENTS: The composite primary end point was death, CMV disease, or other invasive infections by 270 days after HCT. Secondary end points were CMV disease, CMV DNAemia, death, other infections, resource utilization, ganciclovir resistance, quality of life, immune reconstitution, and safety. RESULTS: The primary composite outcome occurred in 20% of valganciclovir recipients versus 21% of placebo-preemptive therapy recipients (treatment difference, -0.01 [95% CI, -0.13 to 0.10]; P = 0.86). There was no difference in the primary end point or its components 640 days after HCT. The incidence of a CMV DNAemia level of 1000 copies/mL or greater or a 5-fold increase over baseline was reduced in the valganciclovir group (11% vs. 36%; P < 0.001). Neutropenia was not significantly different at the absolute neutrophil count of less than 0.5 × 109 cells/L (P = 0.57); however, more patients received hematopoietic growth factors in the valganciclovir group (25.3% vs. 12.4%; P = 0.026). No significant differences were seen in other secondary outcomes. LIMITATION: Some high-risk patients were not included. CONCLUSION: Valganciclovir prophylaxis was not superior in reducing the composite end point of CMV disease, invasive bacterial or fungal disease, or death when compared with polymerase chain reaction-guided preemptive therapy. Both strategies performed similarly with regard to most clinical outcomes. PRIMARY FUNDING SOURCE: Roche Laboratories.