Surveillance of CMV Terminase Gene Mutations in Allogeneic Hematopoietic Stem Cell Transplant Recipients Receiving Letermovir Prophylaxis

Surveillance of CMV Terminase Gene Mutations in Allogeneic Hematopoietic Stem Cell Transplant Recipients Receiving Letermovir Prophylaxis | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 9 October 2025 V1 Latest version Share on Surveillance of CMV Terminase Gene Mutations in Allogeneic Hematopoietic Stem Cell Transplant Recipients Receiving Letermovir Prophylaxis Authors : Amandine Caillault 0009-0007-7262-5287 , Alienor XHAARD 0000-0002-4449-989X , séverine mercier-delarue , Linda Feghoul , Julien Robert , Nathalie Schnepf , David Michonneau , … Show All … , Marie Robin , Flore Sicre de Fontbrune , Eleonore Kaphan , Mathilde Ruggiu-Goiran , Pedro Henrique de Lima Prata , Florian Chevillon , Matthieu Jestin , Nathalie Dhedin , Gerard Socie , Régis Peffault de Latour 0000-0001-6222-4753 , Maud Salmona 0000-0001-7985-6132 , and Jerome LeGoff 0000-0001-7111-1865 [email protected] Show Fewer Authors Info & Affiliations https://doi.org/10.22541/au.176004557.70870678/v1 204 views 132 downloads Contents Abstract Keywords Abstract Introduction Material and methods Discussion Bibliography Figure legend Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Background : Letermovir (LMV), a CMV-specific terminase inhibitor, is recommended for cytomegalovirus (CMV) prophylaxis in CMV-seropositive allogeneic hematopoietic stem cell transplant (alloHSCT) recipients. The association between low-level CMV DNAemia during prophylaxis and the emergence of mutations in CMV terminase complex genes remains unclear. Methods : We retrospectively analyzed 127 alloHSCT recipients at Saint-Louis Hospital, Paris, receiving LMV prophylaxis (108 primary, 24 secondary, 5 both). Weekly CMV DNAemia monitoring was performed, and samples with viral loads ≥3 log IU/mL underwent whole-genome next-generation sequencing to detect mutations in terminase complex genes. Results : During LMV prophylaxis, CMV DNAemia was detected in 31.5% of primary and 62.5% of secondary prophylaxis patients; however, viral loads ≥3 log IU/mL remained low in both groups (8.3%). The frequency of polymorphisms in terminase complex genes increased 2.5-fold during prophylaxis compared with pre-prophylaxis, and then decreased 3.0-fold after prophylaxis withdrawal. Confirmed LMV resistance was found in two patients, representing 1.6% of all patients and 18.2% of those with CMV DNAemia ≥ 3 log IU/mL. Two additional patients harbored mutations at resistance-associated positions in UL56 and UL89, totaling 36% of patients with viral loads ≥3 log IU/mL displaying mutations at known resistance sites. Clinically significant CMV infections were successfully managed with preemptive therapies. Conclusions : This study confirms the efficacy of LMV prophylaxis and demonstrates that LMV exerts selective pressure on the terminase complex, increasing the risk of resistance when CMV DNA levels exceed 3 log IU/mL, underscoring the importance of timely consideration of alternative antiviral therapies when CMV replication persists. Surveillance of CMV Terminase Gene Mutations in Allogeneic Hematopoietic Stem Cell Transplant Recipients Receiving Letermovir Prophylaxis Amandine Caillault 1 , Alienor Xhaard 2 , Severine Mercier-Delarue 1 , Linda Feghoul 1 , Julien Robert 1 , Nathalie Schnepf 1 , David Michonneau 2 , Marie Robin 2 , Flore Sicre de Fontbrune 2 , Eleonore Kaphan 2 , Mathilde Ruggiu-Goiran 2 , Pedro Henrique de Lima Prata 2 , Florian Chevillon 3 , Matthieu Jestin 3 , Nathalie Dhedin 3 , Gerard Socie 2 , Régis Peffault de Latour 2 , Maud Salmona 1,3 , Jérôme Le Goff 1,3 1. Virology Department, AP-HP, Hôpital Saint Louis, F-75010 Paris, France 2. Service Hématologie Greffe, Hôpital Saint-Louis, Université Paris Cité, Paris, France 3. Haematology for adolescents and young adults, AP-HP, Hôpital Saint Louis, F-75010 Paris, France 3. Université Paris Cité, INSERM UMR 1342, Biology and Pathogenesis of Viral infections, Saint Louis Research Institute, F-75010 Paris, France Keywords Cytomegalovirus, letermovir, antiviral resistance, whole genome sequencing Corresponding author: Jérôme Le Goff E-mail address: [email protected] Postal address: Laboratoire de Virologie, AP-HP, Hôpital Saint-Louis , 1 avenue Claude Vellefaux, 75010, Paris, France Telephone number: +33142499493 Abstract Background : Letermovir (LMV), a CMV-specific terminase inhibitor, is recommended for cytomegalovirus (CMV) prophylaxis in CMV-seropositive allogeneic hematopoietic stem cell transplant (alloHSCT) recipients. The association between low-level CMV DNAemia during prophylaxis and the emergence of mutations in CMV terminase complex genes remains unclear. Methods : We retrospectively analyzed 127 alloHSCT recipients at Saint-Louis Hospital, Paris, receiving LMV prophylaxis (108 primary, 24 secondary, 5 both). Weekly CMV DNAemia monitoring was performed, and samples with viral loads ≥3 log IU/mL underwent whole-genome next-generation sequencing to detect mutations in terminase complex genes. Results : During LMV prophylaxis, CMV DNAemia was detected in 31.5% of primary and 62.5% of secondary prophylaxis patients; however, viral loads ≥3 log IU/mL remained low in both groups (8.3%). The frequency of polymorphisms in terminase complex genes increased 2.5-fold during prophylaxis compared with pre-prophylaxis, and then decreased 3.0-fold after prophylaxis withdrawal. Confirmed LMV resistance was found in two patients, representing 1.6% of all patients and 18.2% of those with CMV DNAemia ≥ 3 log IU/mL. Two additional patients harbored mutations at resistance-associated positions in UL56 and UL89, totaling 36% of patients with viral loads ≥3 log IU/mL displaying mutations at known resistance sites. Clinically significant CMV infections were successfully managed with preemptive therapies. Conclusions : This study confirms the efficacy of LMV prophylaxis and demonstrates that LMV exerts selective pressure on the terminase complex, increasing the risk of resistance when CMV DNA levels exceed 3 log IU/mL, underscoring the importance of timely consideration of alternative antiviral therapies when CMV replication persists. Introduction Cytomegalovirus (CMV) causes significant morbidity in allogeneic hematopoietic stem cell transplantation (alloHSCT) recipients. The spectrum of CMV infection is broad, ranging from asymptomatic viremia to fatal disease affecting gut, liver, lungs or brain. CMV infection also has indirect effects such as immunosuppression or graft failure, possibly facilitating concomitant infectious complications. 1 Letermovir (LMV) is a recently developed CMV-specific antiviral drug that inhibits the CMV terminase complex. 2 Its use as prophylaxis has demonstrated a significant reduction in the incidence of clinically significant CMV infection. LMV is now indicated as primary prophylaxis of CMV infection in CMV seropositive alloHSCT recipients. 2–4 Treatment is initiated within the first 28 days after alloHSCT and continued until day 100 to 200, depending on risk factors and immunosuppression levels. 5 LMV can also be used as secondary prophylaxis after effective control of CMV viremia. 2–4,6 Resistance to LMV is relatively infrequent, around 1.1% and 3.8% in primary and secondary prophylaxes, respectively. 3,7,8 The most frequent and impactful mutation occurs in the C325 locus of UL56. Other mutations have been described, conferring either low or high levels of resistance. 9,10 As LMV enzymatic target acts after CMV DNA replication, CMV DNA can be detected during LMV prophylaxis, causing short-term increase in viral load, usually at low levels, named “blips”. 11,12 The potential for such low-level reactivation to select specific mutations conferring resistance to LMV and anticipating treatment failure, remains to be investigated. 6,8,9 LMV resistance mutations can be researched using a variety of techniques. Until recently, Sanger was considered as the gold standard. However, it does not detect minor sub-populations (< 20%). On the contrary, whole genome sequencing or next-generation sequencing (NGS) allows to detect minor sub-populations of variants present at a frequency of 1-3%. 13–15 This greater sensitivity enables early identification of resistance mutations. In addition, NGS allows to study the abundance, dynamics and diversity of viruses. 13–15 In this single-center retrospective study, we used NGS to investigate CMV terminase complex genes mutations in alloHSCT recipients receiving primary or secondary LMV prophylaxis. The objectives were to determine whether the detection of CMV DNA during LMV prophylaxis was associated with the emergence of mutations in the terminase complex genes, and to evaluate their potential association with treatment failure. Material and methods Study population The study was conducted in the Hematology-Transplantation Adolescents and Young Adults Hematology units of Saint-Louis Hospital, Paris, France, including all CMV-seropositive alloHSCT recipients transplanted between January 1, 2020 and December 31, 2021, who received LMV as primary and/or secondary prophylaxis. Patients were classified into two groups based on the type of prophylaxis. The primary prophylaxis (PP) group included patients receiving LMV as PP started within 28 days after alloHSCT and continued for at least 100 days. The secondary prophylaxis (SP) group included patients receiving LMV as SP, initiated after CMV reactivation was controlled with curative treatment. Patients receiving both primary and secondary prophylaxes were included into both groups as per the respective episodes of prophylaxis. CMV load was monitored 1–2 times per week from the day of alloHSCT until at least day +100 or death, whichever occurred first. Samples with CMV load ≥ 3 log IU/mL were selected for whole-genome sequencing to detect resistance mutations. For patients with multiple positive samples, only those collected at least one week apart were analyzed while others were considered redundant. Clinically significant CMV infection (CS-CMVi) was defined as either the occurrence of CMV disease or the initiation of anti-CMV preemptive therapy based on prespecified CMV DNAemia thresholds. 3,16 Antiviral resistance was defined as the identification of a viral genetic mutation known to confer reduced sensitivity to one or more anti-viral drug. 16,17 CMV load determination The CMV viral load (VL) was measured at the virology laboratory of Saint Louis Hospital for all samples by quantitative real-time PCR in whole blood (WB) using the Abbott RealTime CMV assay on the m2000 RealTime platform (Abbott Molecular Diagnostics, Rungis, France). The lower limit of quantification was 62 IU/mL (1.79 log 10 IU/mL). 18 Whole CMV genome sequencing CMV whole genome sequencing was carried out by CMV probe capture (Agilent technologies, Santa Clara, California, USA) as previously described. 19 The libraries were normalized and pooled in two separate runs (52 and 45 samples by runs) before sequencing on Illumina’s NextSeq500 using a NextSeq 550 System Mid-output-kit cartridge (San Diego, California, USA). Raw sequencing reads were preprocessed to remove adapter sequences and low-quality bases using Trimmomatic v0.36. (SLIDINGWINDOW:4:25). 20 Cleaned reads were then aligned to the CMV reference genome (accession number FJ616285.1) using BWA v0.7.15, 21 followed by marking duplicates with Picard v2.8.1 22 and realignment with GATK v3.8 23 to refine alignment accuracy. Variant calling was performed on the generated BAM files using 4 different variant callers, including VarScan2 v2.4.3, VarDict v1.8.2, BCFtools v1.5, and Pindel v0.2.5 24–27 and validated using Clintools checkvariants v1.1. 28 For each sample, a detailed individual HTML report was generated by a python script using the Jinja2 package. This report included information on nucleotide changes, their positions within the target genes, corresponding amino acid changes with their effects, variant allele frequencies, sequencing coverage depths, and the percentage of reads supporting each nucleotide change. Variants associated with antiviral resistance were flagged, and regions of the genome that were not covered by sequencing were identified. Only CMV genes associated with the activity of CMV DNA terminase complex inhibitors are reported in this study, including UL51, UL56, UL89, and UL104. To analyze the sequences of each sample, it was necessary to establish a threshold of interpretability. Despite the absence of precise recommendations, intra-host diversity analysis is usually set at 50-100x global CMV genome coverage, compared with CMV reference sequence (FJ616285 TOWNE). 19,29,30 We did, however, have access to data from samples sequenced below 50x CMV coverage, enabling us to analyze them as required. This was particularly useful for intra-patient comparisons, when researching for the appearance of mutations over time, especially following LMV prophylaxis. All variants with a frequency of at least 5% are reported. The pipeline is accessible on the following GitHub repository: https://github.com/jrobert35/CMVar.git Data collection Clinical data were retrospectively collected from clinical records. Virological data and associated information were collected prospectively, and blood tubes prospectively frozen (-80°C). Viral genome analysis was then carried out retrospectively. Ethics This study involved a retrospective analysis of anonymized data collected in the context of routine clinical care. All patients gave written informed consent for data collection in accordance with good clinical practice protocols and the principles of the Helsinki Declaration. This study was approved by the ethical comity with the number IRB00003888. Statistical analysis Statistical comparisons of primary and secondary prophylaxes were carried out using the student test (age, median duration of LMV prophylaxis, median CMV load) and X² tests (sex, proportion of donor/recipient serostatus, proportion of patients receiving LMV prophylaxis for less or more than 100 days). Continuous variables are expressed as median [interquartile range (IQR)]. These tests were carried out using https://biostatgv.sentiweb.fr/ tools and GraphPad Prism (V8). Patient characteristics Primary prophylaxis group included 108 patients and secondary prophylaxis group 24 patients (among which 5 had previously received primary prophylaxis). Patient characteristics are reported in Table 1 . The CMV serological donor/recipient status was not significantly different between both groups, with a predominance of seropositive donor /seropositive recipient (D+/R+) in both groups. The average duration of prophylaxis was not significantly different between both groups. Most patients received prophylaxis longer than 100 days. The number of patients and samples analyzed, and their results are presented in Figures 1 and 2 . There was no case of CMV disease. In the PP group, before the start of LMV, 13/80 patients (16.3%) had at least one positive CMV load, including 3 (3.8%) with at least one VL ≥ 3 log IU/mL (median time from alloHSCT to CMV detection: 5 days [0; 48]; median time from CMV detection to LMV initiation: 6 days [0; 73]). During LMV PP, 34/108 patients (31.5%) had at least one positive CMV load (median time from LMV initiation to CMV detection: 9.5 days [0; 206]), including 9 patients (8.3%) with at least one VL ≥ 3 log IU/mL. Among the 61 patients for whom CMV monitoring was continued after PP withdrawal, 24 (39.3%) had at least one positive CMV detection, including 15 (24.6%) with at least one VL ≥ 3 log IU/mL (average follow-up time: 86.5 days [2; 335]; median time from LMV withdrawal to CMV detection: 43 days [4; 296]). In the SP group, before the start of LMV (median time from CMV detection to LMV initiation: 14.5 days [2;158]), 14/24 patients (58.3%) had available samples with at least one CMV load ≥ 3 log IU/mL. During LMV SP, 15/24 patients (62.5%) had at least one positive CMV load, including 2 patients (8.3%) with at least one VL ≥ 3 log IU/mL (median time from LMV initiation to CMV detection: 5 days [1; 147]). Among the 15 patients for whom CMV monitoring was continued after SP withdrawal, 6 (40%) had at least one positive CMV detection, including 3 (20%) with at least one VL ≥ 3 log IU/mL (average follow-up time: 133 days [1; 602]; median time from LMV withdrawal to CMV detection: 30 days [13; 301]). During LMV treatment, the rate of patients with at least one positive CMV load was significantly higher during SP than during PP (62.5% vs 31.5%, p= 0.009). However, the rate of patients with at least one VL ≥ 3 log IU/mL was similar between both groups (8.3% vs 8.3%). There was no significant difference in the median CMV load between both groups (2.28 vs. 2.21 log IU/mL for PP and SP groups, respectively, p= 0.69), even when considering only samples with VL ≥ 3 log IU/mL (3.14 vs. 3.29 log IU/mL, respectively, p= 0.67). The range of CMV load did not differ significantly between both groups ([1.79–5.38] vs. [1.80–3.79] log IU/mL, respectively, p= 0.23) ( Table 2 and Figure 3 ). No PP patient with a positive CMV load before the start of LMV received any pre-emptive treatment. Among the nine PP patients who had at least one CMV load ≥ 3 log IU/mL during LMV prophylaxis, four patients with a CS-CMVi (# 4, 5, 6, 32) received preemptive anti-CMV treatment (3 received valganciclovir, and 1 successively received foscarnet, ganciclovir and valganciclovir). All patients cleared CMV within a median of 23 days [11-35] and three patients (# 4, 5, 32) subsequently received LMV secondary prophylaxis. In another three patients (# 3, 8 and 99), LMV was continued and the CMV load decreased spontaneously. In the last two patients (# 1 and 12), LMV prophylaxis was discontinued without preemptive treatment in the context of palliative care for steroid-resistant graft-versus-host disease (GVHD). The two SP patients (# 2 and 34) with at least one VL ≥ 3 log IU/mL during LMV prophylaxis were considered with CS-CMVi and received valganciclovir or foscarnet treatments, allowing successful CMV clearance within 8 days [7-13]. LMV was subsequently reintroduced in one patient, and no further CMV replication was observed. The other patient experienced positive CMV VL 13 days after LMV withdrawal: VL remained below 3 log IU/mL, without any need for pre-emptive treatment. Patients with LMV PP or SP who experienced CMV reactivation received preemptive treatment if their CMV load rose above 4 log IU/mL, or if it persisted for more than 10 days with a viral load ≥ 3 log IU/mL. In contrast, patients who did not receive any preemptive treatment had CMV loads that remained below 3 log IU/mL, or stayed within the range of 3 to 3.5 log for less than 10 days. In total, after excluding the 2 patients in palliative situation, 3 out of 9 patients (33%) had a spontaneous viral clearance while letermovir was continued. CMV Gene Polymorphisms and Resistance Mutations Only samples with a CMV load ≥ 3 Log IU/mL were analyzed for polymorphisms and resistance mutations. A total of 97 samples from 34 different patients were sequenced, including 51 from the PP group and 46 from the SP group ( Figure 1 and Table S2 ). According to the genome interpretability rules (see methods), we obtained 58 interpretable samples (59.7%), corresponding to 22 patients ( Table S2 ). The analysis of viremia across all samples revealed that uninterpretable samples had lower CMV loads ( Figure S1) , median CMV load 3.32 log IU/mL in uninterpretable samples versus 4.10 log IU/mL in interpretable ones (p< 0.0001)). To accurately identify mutations potentially selected during LMV treatment in the terminase complex genes, we considered both previously reported polymorphisms and mutations observed among patients with CMV replication before LMV was started. We then assessed whether additional, previously unreported mutations emerged during LMV prophylaxis and persisted after treatment discontinuation. Before LMV was started, 42 samples (PP, n= 4; SP, n= 38) corresponding to 15 patients were sequenced, with 29 interpretable sequences (PP, n= 1; SP, n= 28) corresponding to 10 patients ( Figure 1 and Table S2). In total, polymorphisms were observed at 16 positions out of 151 amino acids in UL51, 61 out of 850 in UL56, 61 out of 674 in UL89, and 48 out of 687 in UL104, corresponding to mean polymorphism frequencies per sample of 0.37%, 0.25%, 0.25%, and 0.22%, respectively. We identified 147 polymorphism mutations that had not been previously described (11 in UL51, 49 in UL56, 46 in UL89 and 41 in UL104), including 20 observed in at least two different samples or with a variant frequency ≥ 50% within the sample (Supplementary results, Table S1 and Figure S2 ). No known resistance mutations were detected. During LMV, interpretable sequences were obtained for four out of seven PP patients (patients # 1, 3, 4 and 5) and one out of two SP patients (patient # 2) ( Figure 1 and Table S2 ). The mean frequency of polymorphisms per sample increased significantly compared with pre-prophylaxis levels, with ratios (during vs. before treatment) of 3.6 for UL56 (p< 0.0001), 2.5 for UL89 (p= 0.0001), and 1.8 for UL104 (0.0463). No significant change was observed for UL51 (ratio of 2.1, p = 0.09). The overall fold increase across the four genes was 2.5. These results suggest that LMV treatment exerted selective pressure on the terminase complex ( Table S3 ). Known resistance mutations were identified in two patients (patient # 1 with PP and patient # 2 with SP) in the UL56 gene ( Figure 4, A and B ), confirming LMV resistance in 0.9% of patients during PP and 4.2% during SP (1.6% among all patients), and overall, in 18.2% in those with VL ≥ 3 log IU/mL. Additional mutations were also detected at positions previously associated with LMV resistance (365 and 368 in UL56; 350 in UL89) but involving different amino acid changes in 3 patients (patients # 2, 3, and 4). In patient # 5, two deletions leading to a frameshift were detected in UL56 and UL89, at positions not known to be associated with resistance. Thus, during LMV treatment, 36.4% (4/11) of patients with VL ≥ 3 log IU/mL experienced CMV strains with mutations in UL56 or UL89 at positions associated with resistance to LMV ( Figure 4 ). In those patients, after LMV discontinuation, some polymorphism mutations were not consistently detected in consecutive samples, and all mutations known to confer resistance or at positions associated with resistance disappeared (patients # 1, 2, 3 and 4, Figure 4 ). The mean frequency of polymorphisms per sample decreased by 3-fold in UL51 (p=0.03), 3.5-fold in UL56 (p<0.0001), 3.2-fold in UL89 (p<0.0001), and 2.6-fold in UL104 (p= 0,005) compared with levels observed during prophylaxis ( Table S3 ), with an overall fold decrease across the four genes of 3.0. Discussion In this single-center observational study, we conducted a longitudinal analysis of CMV detection in blood and CMV genetic evolution in 127 alloHSCT recipients receiving letermovir as primary and/or secondary prophylaxis. CMV detection in blood was frequent during prophylaxis, with a significantly higher rate in secondary prophylaxis compared with primary prophylaxis (62.5% vs. 31.5%; p=0.009). However, most detections remained at low levels, with only 8.3% of patients in both groups presenting at least one CMV load ≥ 3 log IU/mL, and 33% of them showing spontaneous CMV clearance. Our results corroborate other studies of LMV use in routine practice in alloHSCT, with CMV detection in plasma or whole blood ranging from 11.1% to 28.5% during primary prophylaxis, and from 5% to 39% during secondary prophylaxis. 7,8,16,33–35 Our findings also confirm that CS-CMVi remains infrequent, in line with pivotal trials and real-world studies. 2,35 In our study, CMV resistance to letermovir was confirmed in 0.9% of patients during primary prophylaxis and 4.2% during secondary prophylaxis, all related to mutations in the UL56 gene. Our results are consistent with previous reports, with mutation rates of 1.1 to 2% in primary prophylaxis and 3.8 to 5% in secondary prophylaxis. 3,7,8 When focusing on patients with CMV loads ≥ 3 log IU/mL, resistance rates in our population increased to 18.2%. In addition, our extensive sequencing analysis of samples before and during prophylaxis provided evidence of selective pressure exerted by LMV on three CMV terminase complex genes (UL56, UL89, and UL104), with a significant 1.8- to 3.6-fold increase in polymorphism rates during LMV treatment. Although the small number of cases warrants caution in interpreting these frequencies, these results highlight the risk of CMV resistance when CMV load increases. 3,7,8 Using a next-generation sequencing approach with CMV whole-genome sequencing in order to detect all mutations in the genes involved in the activity of the CMV terminase complex, we were able to extend the catalog of polymorphisms with new substitutions. While previous studies have described the emergence of specific resistance mutations, few have systematically quantified the broader genetic diversification of the CMV terminase complex under drug pressure. Nägele et al . (2025) 15 and Komatsu et al . (2019) 36 provided initial evidence that letermovir can drive the selection of minor variants in UL56. Our observations show a high diversity and reversibility of polymorphisms across the UL56 region and other genes of the terminase complex. We found 3 mutations already described for resistance (R369M, R369T and V231L), 7,9,31 and observed 2 mutations in UL56 and UL89, at positions previously associated with resistance (but with another amino acid substitution). 31,32,37,38 Mutations arising in other genes were not known to confer resistance. A role in compensating for deleterious mutations in UL56 would deserve further study using phenotyping assays. Of note, many of the mutations in UL51, UL89, and UL104 were not consistently detected over time, suggesting that they may have raised from DNA replication but were not associated with virus production. This phenomenon might reflect a selective landscape imposed by letermovir, which, by targeting the terminase complex, allows for the transient expansion of viral subpopulations harboring diverse mutations. Importantly, the decline in polymorphism and the disappearance of resistant mutations after drug discontinuation supports the hypothesis that many of these variants carry a fitness cost in the absence of selection, as previously suggested for other antiviral agents. It also suggests that such reversion may allow to use again LMV after CMV clearance. In our study, actually, LMV was successfully reintroduced as secondary prophylaxis in five patients. However, further in vitro analysis is required to confirm the impact of these mutations. In conclusion, although confirmed resistance to letermovir is rare, persistent low-level CMV replication may indicate ongoing viral adaptation. Letermovir prophylaxis is associated with a reversible increase in CMV genetic polymorphism, underscoring the importance of regular viral load monitoring and timely consideration of alternative antiviral therapies when CMV replication increases. Bibliography 1. Cho SY, Lee DG, Kim HJ. Cytomegalovirus Infections after Hematopoietic Stem Cell Transplantation: Current Status and Future Immunotherapy. Int J Mol Sci . 2019;20(11):2666. doi:10.3390/ijms201126662. Chemaly RF, Ullmann AJ, Stoelben S, et al. Letermovir for Cytomegalovirus Prophylaxis in Hematopoietic-Cell Transplantation. N Engl J Med . 2014;370(19):1781-1789. doi:10.1056/NEJMoa13095333. Marty FM, Ljungman P, Chemaly RF, et al. Letermovir Prophylaxis for Cytomegalovirus in Hematopoietic-Cell Transplantation. N Engl J Med . 2017;377(25):2433-2444. doi:10.1056/NEJMoa17066404. Ljungman P, Alain S, Chemaly RF, et al. Recommendations from the 10th European Conference on Infections in Leukaemia for the management of cytomegalovirus in patients after allogeneic haematopoietic cell transplantation and other T-cell-engaging therapies. Lancet Infect Dis . 2025;25(8):e451-e462. doi:10.1016/S1473-3099(25)00069-65. Russo D, Schmitt M, Pilorge S, et al. Efficacy and safety of extended duration letermovir prophylaxis in recipients of haematopoietic stem-cell transplantation at risk of cytomegalovirus infection: a multicentre, randomised, double-blind, placebo-controlled, phase 3 trial. Lancet Haematol . 2024;11(2):e127-e135. doi:10.1016/S2352-3026(23)00344-76. Clinical Review Report: Letermovir (Prevymis): (Merck Canada Inc.): Indication: For the Prophylaxis of Cytomegalovirus (CMV) Infection in Adult CMV-Seropositive Recipients (R+) of an Allogeneic Hematopoietic Stem Cell Transplant . Canadian Agency for Drugs and Technologies in Health; 2018. Accessed May 27, 2024. http://www.ncbi.nlm.nih.gov/books/NBK539334/7. Douglas CM, Barnard R, Holder D, et al. Letermovir Resistance Analysis in a Clinical Trial of Cytomegalovirus Prophylaxis for Hematopoietic Stem Cell Transplant Recipients. J Infect Dis . 2020;221(7):1117-1126. doi:10.1093/infdis/jiz5778. Robin C, Thiebaut A, Alain S, et al. Letermovir for Secondary Prophylaxis of Cytomegalovirus Infection and Disease after Allogeneic Hematopoietic Cell Transplantation: Results from the French Compassionate Program. Biol Blood Marrow Transplant . 2020;26(5):978-984. doi:10.1016/j.bbmt.2020.01.0279. Chou S. Advances in the genotypic diagnosis of cytomegalovirus antiviral drug resistance. Antiviral Res . 2020;176:104711. doi:10.1016/j.antiviral.2020.10471110. Shigle TL, Handy VW, Chemaly RF. Letermovir and its role in the prevention of cytomegalovirus infection in seropositive patients receiving an allogeneic hematopoietic cell transplant. Ther Adv Hematol . 2020;11:2040620720937150. doi:10.1177/204062072093715011. Royston L, Royston E, Masouridi‐Levrat S, Chalandon Y, Van Delden C, Neofytos D. Predictors of breakthrough clinically significant cytomegalovirus infection during letermovir prophylaxis in high‐risk hematopoietic cell transplant recipients. Immun Inflamm Dis . 2021;9(3):771-776. doi:10.1002/iid3.43112. Cassaniti I, Colombo AA, Bernasconi P, et al. Positive HCMV DNAemia in stem cell recipients undergoing letermovir prophylaxis is expression of abortive infection. Am J Transplant Off J Am Soc Transplant Am Soc Transpl Surg . 2021;21(4):1622-1628. doi:10.1111/ajt.1645013. Garrigue I, Boucher S, Couzi L, et al. Whole blood real-time quantitative PCR for cytomegalovirus infection follow-up in transplant recipients. J Clin Virol . 2006;36(1):72-75. doi:10.1016/j.jcv.2006.01.00214. Lodding IP, Jørgensen M, Bennedbæk M, et al. Development and Dynamics of Cytomegalovirus UL97 Ganciclovir Resistance Mutations in Transplant Recipients Detected by Next-Generation Sequencing. Open Forum Infect Dis . 2021;8(10):ofab462. doi:10.1093/ofid/ofab46215. Nägele K, Bättig V, Gosert R, et al. Revealing the Unseen: Next-Generation Sequencing for Early Detection of Drug-Resistant Cytomegalovirus Variants Upon Letermovir Prophylaxis Failure. J Infect Dis . 2025;231(1):e177-e182. doi:10.1093/infdis/jiae41416. Ljungman P, Chemaly RF, Khawaya F, et al. Consensus Definitions of Cytomegalovirus (CMV) Infection and Disease in Transplant Patients Including Resistant and Refractory CMV for Use in Clinical Trials: 2024 Update From the Transplant Associated Virus Infections Forum. Clin Infect Dis Off Publ Infect Dis Soc Am . 2024;79(3):787-794. doi:10.1093/cid/ciae32117. Chemaly RF, Chou S, Einsele H, et al. Definitions of Resistant and Refractory Cytomegalovirus Infection and Disease in Transplant Recipients for Use in Clinical Trials. Clin Infect Dis Off Publ Infect Dis Soc Am . 2019;68(8):1420-1426. doi:10.1093/cid/ciy69618. Helary M, Schnepf N, Mahjoub N, et al. Prospective comparison of cytomegalovirus quantification in whole blood and plasma samples among hematopoietic stem cell transplant and kidney transplant recipients. J Clin Virol Off Publ Pan Am Soc Clin Virol . 2024;174:105690. doi:10.1016/j.jcv.2024.10569019. Houldcroft CJ, Bryant JM, Depledge DP, et al. Detection of Low Frequency Multi-Drug Resistance and Novel Putative Maribavir Resistance in Immunocompromised Pediatric Patients with Cytomegalovirus. Front Microbiol . 2016;7:1317. doi:10.3389/fmicb.2016.0131720. Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics . 2014;30(15):2114-2120. doi:10.1093/bioinformatics/btu17021. Li H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. Published online May 26, 2013. doi:10.48550/arXiv.1303.399722. Picard Tools - By Broad Institute. Accessed April 7, 2025. http://broadinstitute.github.io/picard/23. Auwera G van der. Genomics in the Cloud: Using Docker, GATK, and WDL in Terra . First edition. O’Reilly Media; 2020.24. Koboldt DC, Chen K, Wylie T, et al. VarScan: variant detection in massively parallel sequencing of individual and pooled samples. Bioinformatics . 2009;25(17):2283-2285. doi:10.1093/bioinformatics/btp37325. Lai Z, Markovets A, Ahdesmaki M, et al. VarDict: a novel and versatile variant caller for next-generation sequencing in cancer research. Nucleic Acids Res . 2016;44(11):e108. doi:10.1093/nar/gkw22726. Li H. A statistical framework for SNP calling, mutation discovery, association mapping and population genetical parameter estimation from sequencing data. Bioinformatics . 2011;27(21):2987-2993. doi:10.1093/bioinformatics/btr50927. Ye K, Schulz MH, Long Q, Apweiler R, Ning Z. Pindel: a pattern growth approach to detect break points of large deletions and medium sized insertions from paired-end short reads. Bioinformatics . 2009;25(21):2865-2871. doi:10.1093/bioinformatics/btp39428. bioinfo-pf-curie/clintools. Published online April 30, 2018. Accessed April 7, 2025. https://github.com/bioinfo-pf-curie/clintools29. Cudini J, Roy S, Houldcroft CJ, et al. Human cytomegalovirus haplotype reconstruction reveals high diversity due to superinfection and evidence of within-host recombination. Proc Natl Acad Sci U S A . 2019;116(12):5693-5698. doi:10.1073/pnas.181813011630. Goya S, Ruis C, Neher RA, et al. Standardized Phylogenetic Classification of Human Respiratory Syncytial Virus below the Subgroup Level - Volume 30, Number 8—August 2024 - Emerging Infectious Diseases journal - CDC. doi:10.3201/eid3008.24020931. Codex_V5. Accessed April 9, 2025. https://www.unilim.fr/cnr-herpesvirus/outils/codexmv/database/antiviral/54/32. Pilorgé L, Burrel S, Aït-Arkoub Z, Agut H, Boutolleau D. Human cytomegalovirus (CMV) susceptibility to currently approved antiviral drugs does not impact on CMV terminase complex polymorphism. Antiviral Res . 2014;111:8-12. doi:10.1016/j.antiviral.2014.08.01433. Ljungman P, Boeckh M, Hirsch HH, et al. Definitions of Cytomegalovirus Infection and Disease in Transplant Patients for Use in Clinical Trials. Clin Infect Dis Off Publ Infect Dis Soc Am . 2017;64(1):87-91. doi:10.1093/cid/ciw66834. Desai N, Pasic I, Law AD, et al. Letermovir as secondary prophylaxis of cytomegalovirus infection after allogeneic hematopoietic cell transplantation: A single center experience. Eur J Haematol . 2024;113(4):477-484. doi:10.1111/ejh.1425835. Sassine J, Khawaja F, Shigle TL, et al. Refractory and Resistant Cytomegalovirus After Hematopoietic Cell Transplant in the Letermovir Primary Prophylaxis Era. Clin Infect Dis Off Publ Infect Dis Soc Am . 2021;73(8):1346-1354. doi:10.1093/cid/ciab29836. Komatsu TE, Hodowanec AC, Colberg-Poley AM, et al. In-depth genomic analyses identified novel letermovir resistance-associated substitutions in the cytomegalovirus UL56 and UL89 gene products. Antiviral Res . 2019;169:104549. doi:10.1016/j.antiviral.2019.10454937. Champier G, Couvreux A, Hantz S, et al. Putative functional domains of human cytomegalovirus pUL56 involved in dimerization and benzimidazole D-ribonucleoside activity. Antivir Ther . 2008;13(5):643-654.38. Champier G, Hantz S, Couvreux A, et al. New functional domains of human cytomegalovirus pUL89 predicted by sequence analysis and three-dimensional modelling of the catalytic site DEXDc. Antivir Ther . 2019;12(2):217-232. N = 108 24 / Men (%) 53% 46% 0.67 Median age (years) [min-max] 45.5 [14 ; 71] 41 [16 ; 68] 0.24 Donor-recipient CMV serological status D+/R+ 71 (66%) 20 (83%) 0.15 N (%) D-/R+ 37 (34%) 4 (17%) Median duration of letermovir prophylaxis (days) [min-max] 136 [12 ; 524] 113 [12 ; 518] 0.97 Prophylaxis ≤ 100 days – N (%) 22 (20%) 10 (42%) 0.052 Prophylaxis > 100 days – N (%) 86 (80%) 14 (58%) Hematological disease AML 32 (29.6%) 10 (41.7%) 0.92 MDS 27 (25%) 5 (20.8%) SCD 12 (11.1%) 2 (8.3%) ALL 9 (8.3%) 3 (12.5%) NHL 9 (8.3%) 2 (8.3%) AA 8 (7.4%) 0 (0%) MPN 6 (5.6%) 2 (8.3%) CML 3 (2.8%) 0 (0%) PID 2 (1.9%) 0 (0%) Donor type Haplo-identical 32 (29.6%) 5 (20.8%) 0.11 MRD 27 (25.0%) 8 (33.3%) MUD 27 (25.0%) 10 (41.7%) MMUD 22 (20.4%) 1 (4.2%) Stem cell source PBSC 86 (79.6%) 21 (87.5%) 0.87 BM 19 (15.6%) 3 (12.5%) CB 3 (2.8%) 0 (0%) Conditioning RIC 74 (68.5%) 13 (54.2%) 0.37 MAC 33 (30.6%) 11 (45.8%) Sequential 1 (0.9%) 0 (0.0%) ATG 37 (34.3%) 7 (29.2%) 0.66 Alemtuzumab 13 (12.0%) 1 (5.6%) N = 108 24 / Men (%) 53% 46% 0.67 Median age (years) [min-max] 45.5 [14 ; 71] 41 [16 ; 68] 0.24 Donor-recipient CMV serological status D+/R+ 71 (66%) 20 (83%) 0.15 N (%) D-/R+ 37 (34%) 4 (17%) Median duration of letermovir prophylaxis (days) [min-max] 136 [12 ; 524] 113 [12 ; 518] 0.97 Prophylaxis ≤ 100 days – N (%) 22 (20%) 10 (42%) 0.052 Prophylaxis > 100 days – N (%) 86 (80%) 14 (58%) Hematological disease AML 32 (29.6%) 10 (41.7%) 0.92 MDS 27 (25%) 5 (20.8%) SCD 12 (11.1%) 2 (8.3%) ALL 9 (8.3%) 3 (12.5%) NHL 9 (8.3%) 2 (8.3%) MPN 6 (5.6%) 2 (8.3%) AA (6.5%) 0 (0%) CML 3 (2.8%) 0 (0%) PID 2 (1.9%) 0 (0%) Aplasia 1 (0.93%) 0 (0%) Donor type Haplo-identical 32 (29.6%) 5 (20.8%) 0.11 MRD 27 (25.0%) 8 (33.3%) MUD 27 (25.0%) 10 (41.7%) MMUD 22 (20.4%) 1 (4.2%) Stem cell source PBSC 86 (79.6%) 21 (87.5%) 0.87 BM 19 (15.6%) 3 (12.5%) CB 3 (2.8%) 0 (0%) Conditioning RIC 74 (68.5%) 13 (54.2%) 0.37 MAC 33 (30.6%) 11 (45.8%) Sequential 1 (0.9%) 0 (0.0%) ATG 37 (34.3%) 7 (29.2%) 0.66 Alemtuzumab 13 (12.0%) 1 (5.6%) Abbreviations: AA: aplastic anemia, ALL: acute lymphoblastic leukemia, AML: acute myeloblastic leukemia, ATG: anti-thymoglobulin, BM: bone marrow, CB: cord blood, CML: chronic myelomonocytic leukemia, MAC: Myeloablative Conditioning, MDS: myelodysplastic syndrome, MMUD: mismatched unrelated donor, MPN: myeloproliferative neoplasms, MRD: matched related donor, MUD: matched unrelated donor, NHL: non-Hodgkin lymphoma, PBSC: peripheral blood stem cells, PID: primary immune deficiency, RIC: Reduced-Intensity Conditioning, SCD: sickle cell disease Table 2. CMV DNA loads in patients with primary or secondary letermovir prophylaxis. Median CMV DNA loads (samples > 1,8 log IU/mL) [min-max] During prophylaxis 2.28 [1.79 - 5.38] 2.21 [1.80 - 3.79] 0.69 After prophylaxis withdrawal 3.24 [1.80 - 6.01] 2.31 [1.80 - 3.42] 0.006 Median CMV DNA loads (samples with VL ≥ 3 log IU/mL) [min-max] During prophylaxis 3.14 [3.02 - 5.38] 3.29 [3.18 - 3.79] 0.56 After prophylaxis withdrawal 3.74 [3.03 - 6.01] 3.40 [3.28 - 3.42] 0.17 Figure legend Figure 1 Flowchart of the study Numbers of patients and total samples for each group * Five patients received both primary and secondary prophylaxes, and were then included in both groups **One sample of patient #1 during PP is not included in the group as the first sequencing of UL56 was performed only using the Sanger technique at the CMV National Reference Center. Other samples were tested using NGS. CMV detection during primary and secondary prophylaxis Among all patients, the pie chart depicts the proportions of patients (A) with (i) no detectable CMV, (ii) at least one CMV DNA value < 3 log IU/mL, or (iii) at least one CMV DNA value ≥ 3 log IU/mL. Among patients with positive CMV detection, the pie chart depicts (B) the proportions of samples with (i) no detectable CMV, (ii) CMV DNA (left) and secondary (right) prophylaxis. Figure 3 CMV DNA loads in patients with at least one positive CMV detection Box plots display the median and interquartile range of CMV DNA loads during primary or secondary letermovir prophylaxis. Panel (A) includes all positive CMV DNA values, panel (B) includes only values ≥ 3 log IU/mL. Figure 4 CMV viremia, antiviral treatments and mutations in the CMV terminase complex genes Evolution of CMV DNA loads and mutations in CMV terminase complex genes over time after alloHSCT in patients who had CMV positive detection during primary (patients #1, 3, 4, 5) and secondary (patient 2) letermovir prophylaxis. CMV loads are represented by dark circles. Periods with antiviral treatment are outlined in grey (GCV=ganciclovir, LMV=letermovir, FCV=foscarnet). Circled numbers correspond to samples with interpretable CMV sequencing. Polymorphism and resistance mutations detected are reported with their proportion compared to wild amino acid for each gene of the CMV terminase complex. Amino acid substitutions previously reported to confer letermovir resistance are formatted in bold. Mutations located at known resistance-associated positions unreported yet and frameshifts (fs) and are underlined. *In patient # 1, the first detection of the mutation R369M in UL56 was found using a Sanger sequencing technique and could not be retested using the NGS technique. Information & Authors Information Version history V1 Version 1 09 October 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords cytomegalovirus genetics infection mutation resistance virus classification Authors Affiliations Amandine Caillault 0009-0007-7262-5287 Hopital Saint-Louis View all articles by this author Alienor XHAARD 0000-0002-4449-989X Hopital Saint-Louis View all articles by this author séverine mercier-delarue Hopital Saint-Louis View all articles by this author Linda Feghoul Hopital Saint-Louis View all articles by this author Julien Robert Hopital Saint-Louis View all articles by this author Nathalie Schnepf Hopital Saint-Louis View all articles by this author David Michonneau Hopital Saint-Louis View all articles by this author Marie Robin Hopital Saint-Louis View all articles by this author Flore Sicre de Fontbrune Hopital Saint-Louis View all articles by this author Eleonore Kaphan Hopital Saint-Louis View all articles by this author Mathilde Ruggiu-Goiran Hopital Saint-Louis View all articles by this author Pedro Henrique de Lima Prata Hopital Saint-Louis View all articles by this author Florian Chevillon Hopital Saint-Louis View all articles by this author Matthieu Jestin Hopital Saint-Louis View all articles by this author Nathalie Dhedin Hopital Saint-Louis View all articles by this author Gerard Socie Hopital Saint-Louis View all articles by this author Régis Peffault de Latour 0000-0001-6222-4753 Hopital Saint-Louis View all articles by this author Maud Salmona 0000-0001-7985-6132 Hopital Saint-Louis View all articles by this author Jerome LeGoff 0000-0001-7111-1865 [email protected] Hopital Saint-Louis View all articles by this author Metrics & Citations Metrics Article Usage 204 views 132 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Amandine Caillault, Alienor XHAARD, séverine mercier-delarue, et al. 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