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 of the long-acting mAb sotrovimab targeting severe acute respiratory syndrome coronavirus 2 in hematopoietic cell transplant (HCT) recipients. METHODS: All participants received an intravenous infusion of sotrovimab within 1 week prior to initiating the pretransplant preparative regimen. The serum concentration of sotrovimab was measured longitudinally for up to 24 weeks posttransplant. 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 graft-versus-host disease (GVHD) posttransplant. CONCLUSIONS: These data show that sotrovimab exposure may be reduced in HCT recipients, possibly related to increased gastrointestinal 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.

Variants of Concern Are Overrepresented Among Postvaccination Breakthrough Infections of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in Washington State.

Across 20 vaccine breakthrough cases detected at our institution, all 20 (100%) infections were due to variants of concern (VOCs) and had a median Ct of 20.2 (IQR, 17.1-23.3). When compared with 5174 contemporaneous samples sequenced in our laboratory, VOCs were significantly enriched among breakthrough infections (P < .05).

Evaluation and utilization of preassembled frozen commercial fast real-time qPCR master mixes for detection of cytomegalovirus and BK virus.

Quantitative DNA detection of cytomegalovirus (CMV) and BK virus (BKV) is critical in the management of transplant patients. Quantitative laboratory-developed procedures for CMV and BKV have been described in which much of the processing is automated, resulting in rapid, reproducible, and high-throughput testing of transplant patients. To increase the efficiency of such assays, the performance and stability of four commercial preassembled frozen fast qPCR master mixes (Roche FastStart Universal Probe Master Mix with Rox, Bio-Rad SsoFast Probes Supermix with Rox, Life Technologies TaqMan FastAdvanced Master Mix, and Life Technologies Fast Universal PCR Master Mix), in combination with in-house designed primers and probes, was evaluated using controls and standards from standard CMV and BK assays. A subsequent parallel evaluation using patient samples was performed comparing the performance of freshly prepared assay mixes versus aliquoted frozen master mixes made with two of the fast qPCR mixes (Life Technologies TaqMan FastAdvanced Master Mix, and Bio-Rad SsoFast Probes Supermix with Rox), chosen based on their performance and compatibility with existing PCR cycling conditions. The data demonstrate that the frozen master mixes retain excellent performance over a period of at least 10 weeks. During the parallel testing using clinical specimens, no difference in quantitative results was observed between the preassembled frozen master mixes and freshly prepared master mixes. Preassembled fast real-time qPCR frozen master mixes perform well and represent an additional strategy laboratories can implement to reduce assay preparation times, and to minimize technical errors and effort necessary to perform clinical PCR.

Evaluation of the Tasso+ blood self-collection device for quantitation of plasma cytomegalovirus (CMV) DNAemia in adult solid organ transplant recipients (SOTr).

Quantitative monitoring of cytomegalovirus (CMV) DNAemia in venous blood is standard in solid organ transplant recipients (SOTr) but is limited by the need for phlebotomy facilities and personnel. The aim of the study was to evaluate the Tasso+ capillary blood (CB) self-collection device for quantitation of plasma CMV DNAemia. Thirty adult SOTr with suspected CMV DNAemia were enrolled to have a supervised Tasso+ CB sample collection within 24 h of a venous sample. CMV DNA was quantitated in paired samples by using the Abbott M2000 Real-Time qPCR instrument. The participants were provided with a study-specific survey that measured patient acceptability of the Tasso+ device compared with venipuncture. A Tasso + CB sample was successfully collected in 28/30 (93%) patients, and 44 paired samples were analyzed. Concordance for detection of CMV DNAemia above the limit of detection (LOD) was 91% (42/44), and the Tasso + CB sample was estimated to be 95% sensitive at a viral load (VL) of 308 IU/mL. Among samples with a quantifiable DNAemia result with both methods (N = 31), there was excellent correlation between methods (Spearman R2 = 0.99). The difference in CMV VL between venous and Tasso+ CB samples was not dependent on time (P > 0.1). Of 12 who completed the survey, 11 (92%) expressed a preference for Tasso+ CB collection over venipuncture. Collection of CB with the Tasso+ device is feasible, patient-acceptable, and yields generally comparable CMV DNAemia load to standard venous samples, but with lower sensitivity. Future studies to optimize and evaluate this methodology for patient self-collected samples are warranted. IMPORTANCE: We evaluate an FDA-cleared blood self-collection device (Tasso+) and demonstrate that it is patient-acceptable and yields a liquid blood sample with quantitative CMV DNAemia results comparable to those of standard venipuncture samples. This opens up possibilities for self-blood collection to monitor for CMV and potentially other viruses in transplant and other at-risk populations.

Rapid detection of human cytomegalovirus UL97 and UL54 mutations directly from patient samples.

Human cytomegalovirus (CMV) is a significant contributor to morbidity and mortality in immunocompromised patients, particularly in the transplant setting. The availability of anti-CMV drugs has improved treatment, but drug resistance is an emerging problem. Here, we describe an improved, rapid, sequencing-based assay for the two genes in CMV where drug resistance occurs, the UL97 and UL54 genes. This assay is performed in 96-well format with a single master mix and provides clinical results within 2 days. It sequences codons 440 to 645 in the UL97 gene and codons 255 to 1028 in the UL54 gene with a limit of detection of 240 IU/ml. With this assay, we tested 43 specimens that had previously been tested for UL97 drug resistance and identified 3 with UL54 mutations. One of these patients had no concurrent UL97 mutation, pointing toward the need for an assay that facilitates dual UL97/UL54 gene testing for complete resistance profiling.

Cytomegalovirus breakthrough and resistance during letermovir prophylaxis.

Letermovir is a relatively new antiviral for prophylaxis against cytomegalovirus (CMV) after allogeneic hematopoietic cell transplantation (HCT). CMV-seropositive HCT recipients who received letermovir prophylaxis from 2018 to 2020 at our center were evaluated for letermovir resistance and breakthrough CMV reactivation. Two-hundred twenty-six letermovir recipients were identified and 7/15 (47%) with CMV DNAemia ≥200 IU/mL were successfully genotyped for UL56 resistance. A single C325Y resistance mutation was identified in an umbilical cord blood recipient. Ninety-five (42%), 43 (19%), and 15 (7%) patients had breakthrough CMV at any level, ≥150 IU/mL, and ≥500 IU/mL, respectively. Risk factors for breakthrough CMV reactivation at each viral threshold were examined. Cumulative steroid exposure was the strongest risk factor for CMV at all evaluated viral thresholds. Graft-versus-host disease prophylaxis with post-transplantation cyclophosphamide (aHR 2.34, 95% CI 1.28-4.28, p = 0.001) or calcineurin inhibitors plus mycophenolate (aHR 2.24, 95% CI 1.30-3.86, p = 0.004) were also associated with an increased risk of CMV reactivation at any level. De novo letermovir resistance is rare and can be successfully treated using other antivirals. Letermovir effectively prevents clinically significant CMV, however, subclinical CMV reactivation occurs frequently at our center.

Clinical Performance Characteristics of the Swift Normalase Amplicon Panel for Sensitive Recovery of Severe Acute Respiratory Syndrome Coronavirus 2 Genomes.

Amplicon-based sequencing methods are central in characterizing the diversity, transmission, and evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but need to be rigorously assessed for clinical utility. Herein, we validated the Swift Biosciences' SARS-CoV-2 Swift Normalase Amplicon Panels using remnant clinical specimens. High-quality genomes meeting our established library and sequence quality criteria were recovered from positive specimens, with 95% limit of detection of 40.08 SARS-CoV-2 copies/PCR. Breadth of genome recovery was evaluated across a range of CT values (11.3 to 36.7; median, 21.6). Of 428 positive samples, 413 (96.5%) generated genomes with <10% unknown bases, with a mean genome coverage of 13,545× ± SD 8382×. No genomes were recovered from PCR-negative specimens (n = 30) or from specimens positive for non-SARS-CoV-2 respiratory viruses (n = 20). Compared with whole-genome shotgun metagenomic sequencing (n = 14) or Sanger sequencing for the spike gene (n = 11), pairwise identity between consensus sequences was 100% in all cases, with highly concordant allele frequencies (R2 = 0.99) between Swift and shotgun libraries. When samples from different clades were mixed at varying ratios, expected variants were detected even in 1:99 mixtures. When deployed as a clinical test, 268 tests were performed in the first 23 weeks, with a median turnaround time of 11 days, ordered primarily for outbreak investigations and infection control.

Rapid detection directly from patient serum samples of human cytomegalovirus UL97 mutations conferring ganciclovir resistance.

Ganciclovir-resistant cytomegalovirus can cause disease and death in transplant recipients. We describe here a rapid PCR- and sequencing-based assay for ganciclovir resistance that can be performed in 1 to 2 working days directly from patient specimens, without the need for amplification of the virus by cell culture. An evaluation of 120 sequential samples submitted for clinical testing revealed a variety of silent and amino acid mutations.