RESEARCH ARTICLE Improvement in detecting cytomegalovirus drug resistance mutations in solid organ transplant recipients with suspected resistance using next generation sequencing Rubén López-Aladid1☯, Alba Guiu1☯, Maria Mar Mosquera1, Francisco López-Medrano2, Frederic Cofán3, Laura Linares4, Julián Torre-Cisneros5, Elisa Vidal5, Asunción Moreno4, Jose Marı́a Aguado2, Elisa Cordero6, Cecilia Martin-Gandul6, Jordi Carratalá7, a1111111111 Nuria Sabé7, Jordi Niubó8, Carlos Cervera ID9, Alicia Capón1, Anna Cervilla1, a1111111111 Marta Santos ID1, Marta Bodro4, Patricia Muñoz10, Maria Carmen Fariñas11, a1111111111 Andrés Antón12, Maitane Aranzamendi13, Miguel Montejo14, Pilar Pérez-Romero15, a1111111111 Oscar Len16, Maria Ángeles Marcos ID1* a1111111111 1 Department of Clinical Microbiology, Hospital Clinic, Universidad de Barcelona, Institute for Global Health, (ISGlobal), Barcelona, Spain, 2 Unit of Infectious Diseases, Instituto de Investigación Hospital 12 Octubre (i + 12) University Hospital 12 de Octubre, Universidad Complutense, Madrid, Spain, 3 Department of Nephrology and Renal Transplant, Hospital Clinic, Universidad de Barcelona, Barcelona, Spain, 4 Department of Infectious Diseases, Hospital Clinic, Institut d’Investigacions Biomediques August Pi I OPEN ACCESS Sunyer (IDIBAPS), Universidad de Barcelona, Barcelona, Spain, 5 Clinical Unit of Infectious Diseases, Hospital Universitario Reina Sofia-IMIBIC-UCO, Córdoba, Spain, 6 Department of Infectious Diseases, Citation: López-Aladid R, Guiu A, Mosquera MM, Instituto de Biomedicina de Sevilla (IBIS), University Hospital Virgen del Rocı́o, Sevilla, Spain, 7 Department López-Medrano F, Cofán F, Linares L, et al. (2019) of Infectious Diseases, Bellvitge University Hospital, IDIBELL, Barcelona, Spain, 8 Department of Clinical Improvement in detecting cytomegalovirus drug Microbiology, Bellvitge University Hospital, IDIBELL, Barcelona, Spain, 9 Department of Medicine, Division of resistance mutations in solid organ transplant Infectious Diseases, University of Alberta, Edmonton, Canada, 10 Department of Clinical Microbiology and recipients with suspected resistance using next Infectious Diseases, Hospital General Universitario Gregorio Marañón, Instituto de Investigación Sanitario Gregorio Marañón, Madrid, Spain, 11 Unit of Infectious Diseases, University Hospital Marqués de Valdecilla, generation sequencing. PLoS ONE 14(7): Universidad de Cantabria, Santander, Spain, 12 Microbiology Service, Hospital Vall d’Hebron, Barcelona, e0219701. https://doi.org/10.1371/journal. Spain, 13 Microbiology Service, Hospital Universitario de Cruces, Bilbao, Spain, 14 Department of Infectious pone.0219701 Diseases, Hospital Universitario de Cruces, Bilbao, Spain, 15 National Center for Microbiology, Instituto de Editor: Stanislaw Stepkowski, University of Toledo, Salud Carlos III, Majadahonda, Madrid, Spain, 16 Department of Infectious Diseases, Hospital Universitari Vall d’Hebrón, Uniiversidad Autónoma de Barcelona, Barcelona, Spain UNITED STATES Received: February 20, 2019 ☯ These authors contributed equally to this work. * mmarcos@clinic.ub.es Accepted: May 7, 2019 Published: July 18, 2019 Copyright: © 2019 López-Aladid et al. This is an Abstract open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and Objetives reproduction in any medium, provided the original author and source are credited. The aim of this study was to identify CMV drug resistance mutations (DRM) in solid organ transplant (SOT) recipients with suspected resistance comparing next-generation sequenc- Data Availability Statement: All relevant data are within the manuscript and its Supporting ing (NGS) with Sanger sequencing and assessing risk factors and the clinical impact of Information files. resistance. Funding: M.A.M. was supported in part by: Agency for Health Technology Assessment and Research Methods Supported by Ministerio de Economı́a y Using Sanger sequencing as the reference method, we prospectively assessed the ability of Competitividad, Instituto de Salud Carlos III (PS12/ 02131 and PI17/02150); Agència de Gestió d´Ajuts NGS to detect CMV DRM in the UL97 and UL54 genes in a nationwide observational study Universitaris I de Recerca, Generalitat de from September 2013 to August 2016. PLOS ONE | https://doi.org/10.1371/journal.pone.0219701 July 18, 2019 1 / 13 Cytomegalovirus resistance in solid organ transplant recipients Catalunya, 2017 SGR 794; and Fundació Marató Results TV3 project code 201824. Among 44 patients recruited, 14 DRM were detected by Sanger in 12 patients (27%) and 20 Competing interests: The authors have declared DRM were detected by NGS, in 16 (36%). NGS confirmed all the DRM detected by Sanger. that no competing interests exist. The additional six mutations detected by NGS were present in <20% of the sequenced pop- ulation, being located in the UL97 gene and conferring high-level resistance to ganciclovir. The presence of DRM by NGS was associated with lung transplantation (p = 0.050), the administration of prophylaxis (p = 0.039), a higher mean time between transplantation and suspicion of resistance (p = 0.038) and longer antiviral treatment duration before suspicion (p = 0.024). However, the latter was the only factor independently associated with the pres- ence of DRM by NGS in the multivariate analysis (OR 2.24, 95% CI 1.03 to 4.87). Conclusions NGS showed a higher yield than Sanger sequencing for detecting CMV resistance muta- tions in SOT recipients. The presence of DRM detected by NGS was independently associ- ated with longer antiviral treatment. Introduction Cytomegalovirus (CMV) infection is one of the most common complications in solid organ transplant (SOT) patients. Currently, prophylaxis and preemptive treatments have decreased the incidence of disease and death by CMV. However, mutations associated with resistance to antivirals have become an important problem with a high associated morbidity and mortality [1, 2]. In the transplant setting, the incidence of drug resistance mutations (DRM) is 5–12% depending on the group of patients studied but may be higher than 20% in patients with sus- pected DRM [3, 4]. Diagnostic laboratory testing should be performed whenever there is a criterion of suspi- cion of resistance since suboptimal response to treatment may have other causes, and inade- quate empiric changes in therapy may have adverse effects [5, 6]. Genotypic testing by Sanger sequencing of the protein kinase gene (UL97) and DNA polymerase gene (UL54) is the usual method for detecting drug resistance and is the basis of the selection of alternative therapy [7]. However, Sanger DNA sequencing is unable to detect and quantify subpopulations of viral mutants of less than 20–30%, which may be crucial for selecting the most adequate therapy [8]. In the last years next generation sequencing (NGS) technologies have provided better understanding of the complexity of variant populations, increasing the detection of subpopula- tions of minority variants which have a prevalence of 1–3%. Nevertheless, to date, only a few studies have evaluated CMV DRM using NGS and most of these studies were carried out in a reduced number of patients [9– 12]. The present study was conducted within the context of the Group for the Study of Infection in Transplantation (GESITRA), constituting one of its major lines of clinical research. The nationwide network of Spanish hospitals participating in GESITRA has allowed the inclusion of a wide variety of SOT recipients. The aim of this study was to identify CMV drug resistance mutations in solid organ trans- plant recipients with suspected resistance comparing NGS with Sanger sequencing and to assessing risk factors and the clinical impact of resistance detected by NGS. PLOS ONE | https://doi.org/10.1371/journal.pone.0219701 July 18, 2019 2 / 13 Cytomegalovirus resistance in solid organ transplant recipients Materials and methods Settings and study population We conducted a prospective observational study in nine hospitals included in the Spanish Net- work for Research in Infectious Diseases (REIPI). Adult SOT patients with suspected DRM were included in the study from September 2013 to August 2016. Resistance was suspected when there was persistent or increasing CMV viral load despite � 2 weeks of appropriate anti- viral treatment, or failure to have a significant improvement in clinical symptoms despite 2 weeks of full-dose antiviral therapy.[2,5,6] The coordinating centre was the Hospital Clinic of Barcelona (Spain), which performed the genotypic resistance testing as well as the data analysis. Patient treatment and follow-up were conducted as per the protocol of each centre. All patients were initially treated with standard- dose ganciclovir/valganciclovir (GCV/VGCV) (adjusted to renal function). Monitoring of CMV viral load was based on local quantitative PCR-based assays. On the suspicion of resistance, plasma samples were collected and frozen at -80˚C in the respective hospital and sent in batches to the Microbiology Laboratory of the Hospital Clinic every 4 months. Thus, genotypic testing was not carried out in real time and all patients received empiric treatment on the presence of suspected drug resistance. Data at patient inclusion and during follow-up were registered in a clinical database at each participating hospital. We reviewed medical histories in terms of viral response, rejection and mortality at 3 months after suspicion of resistance. The DRM detected by Sanger sequencing and NGS were also included in the database. CMV infection and disease and acute cellular rejection were defined as described previously [5, 6]. The study was endorsed by Group for the study of Infection in Transplantation (GESITRA) and approved by the Ethical Committee of Hospital Clı́nic (Comité Ético de Investigación del Hospital Clı́nic de Barcelona, CEIC), approval number HCB/2012/7598) as the reference com- mittee for the all participating centres according to legal regulations in Spain. Microbiological studies In the coordinating laboratory, all samples were kept at -80˚C until processing. DNA extrac- tion was performed in 500 μL of each plasma sample using the QIAsymphony system (Qiagen, Hilden, Germany). CMV viral load was confirmed by CMV Real Time PCR (Nanogen Advanced Diagnostics, Italy) according to the manufacturer’s instructions. Subsequently, all samples were analysed by Sanger sequencing based on PCR amplification of the CMV UL97 protein kinase gene (codons 400–670) in a single fragment and the UL54 DNA polymerase gene (codons 300–1000) in four fragments [3]. Afterwards, antiviral resistance NGS, using Ion Torrent PGM (Ion Personal Genome Machine, Ion Torrent Life Technologies, South San Francisco, CA) was performed by flanking both the UL97 and UL54 gene. Primer and library design for NGS To create DNA library, UL97 and UL54 primers and PCR conditions previously validated and described by Sahoo MK et al were used [10]. After establishing the whole technique, pilot tests were performed in Ion Torrent PGM in order to adjust the reproducibility of the technique. The test of reproducibility was carried out in a total of 6 control samples which included: 2 samples without suspicion of resistance, 2 samples from QCMD with known DRMs, and 2 clinical samples with known DRMs tested by PLOS ONE | https://doi.org/10.1371/journal.pone.0219701 July 18, 2019 3 / 13 Cytomegalovirus resistance in solid organ transplant recipients Sanger. The results were the same in two sequencing experiments. Each run had a pool of the 6 samples in 3 different libraries with the same DNA extract. [13] The limit of detection to avoid false positive results was defined as 5% according to Sahoo et al [10] Sequencing and bioinformatic analyses Forty-eight samples were sequenced in the Ion PGMTM 35 System for NGS. Quality assess- ment of the FASTQ files was performed using FastQC–version 0.11.3 [14]. The resulting reads were aligned to the human herpesvirus 5 strain Merlin reference genome (GenBank: AY446894.2) using the BWA-MEM algorithm from BWA version 0.7.12 with default parame- ters [15]. Alignments were sorted and indexed using Sambamba version 0.5.1 [16]. Duplicate reads in sorted BAM files were identified and removed using Picard tools [17]. GATK base quality score recalibration, insertion and delection (INDEL) realignment, and duplicate removal were applied [18]. The discovery of single nucleotide polymorphisms (SNP) and INDEL and genotyping were simultaneously performed across 12 samples to optimise this method using standard filtering and variant quality score recalibration according to the GATK Best Practices recommendations [19]. Genetic variant annotation and prediction of the effects of variants on genes (such as amino acid changes) were done using snpEff [20]. To do this a snpEff database was created using the information of the reference genome provided by Gen- Bank. The resulting variant call format (VCF) files were post-processed with in-house R [21] scripts using Bioconductor packages [22] to identify mutations by gene (i.e. UL54 and UL97) in order to find the relative position of each mutation to the first position of the corresponding gene and to filter mutations by missing calls by depth at a sample level (DP> = 5), by allele depth (AD> = 5), and by poor quality calls (in which the percentile of minimum quality accepted was 5%). An average 247382 raw sequences per sample were obtained in the samples sequenced in this study. After pipeline passing quality filters 142.078 reads were obtained per sample.The mean coverage among the different amplicons was up to 2000X Statistical analysis A database was developed for the registry of the patients enrolled in Open Clinica 3.1 [GNU- Lesser General Public License (GNU LGPL)]. Categorical variables are expressed as number and percentage of patients, and the median (interquartile range [IQR]) is used for continuous variables with a non-normal distribution and the mean (standard deviation [SD]) for those with a normal distribution. Categorical variables were compared using the X2 test or the Fisher exact test. Continuous variables were compared using the t test or the non-parametric Mann-Whitney test. Logistic regression analyses were performed to identify variables associated with the presence of mutations. Var- iables showing a significant result in the univariate analyses (p<0.05) were included in the multivariate model (forward stepwise procedure). The Hosmer-Lemeshow goodness-of-fit test was performed to assess the overall fit of the models [23]. The area under the receiver operating characteristic (ROC) curve of the multivariate model to predict the presence of mutations was calculated. Internal validation of the prediction model was conducted using ordinary nonparametric bootstrapping with 1,000 bootstrap samples and bias-corrected, accelerated 95% CIs [24] (S1 Text; S1 Table). The level of significance was set at 0.05 (2-tailed). All analyses were performed using IBM SPSS Statistics 22.0 (Armonk, New York, USA). PLOS ONE | https://doi.org/10.1371/journal.pone.0219701 July 18, 2019 4 / 13 Cytomegalovirus resistance in solid organ transplant recipients Table 1. Comparison of the clinical characteristics of the study population at baseline. Variables Overall Without Mutations With Mutations P value (n = 44) (n = 28) (n = 16) Age, mean (SD), years 52.5 (12.3) 54.1 (11.8) 49.8 (12.9) 0.26 Male sex, n (%) 32 (72.7) 22 (78.6) 10 (62.5) 0.30 Type of transplant, n (%) 0.24 Kidney 19 (43.2) 13 (46.4) 6 (37.5) 0.57 Liver 9 (20.5) 6 (21.4) 3 (18.8) >0�99 Heart 8 (18.2) 5 (17.9) 3 (18.8) >0.99 Lung 5 (11.4) 1 (3.6) 4 (25.0) 0.050 Kidney-pancreas 3 (6.8) 3 (10.7) 0 (0) 0.29 CMV serostatus, n (%)a 0.21 D+/R- 26 (61.9) 14 (53.8) 12 (75.0) 0.17 D+/R+ 13 (31.0) 10 (38.5) 3 (18.8) 0.30 D-/R+ 2 (4.8) 2 (7.7) 0 (0) 0.52 D-/R- 1 (2.4) 0 (0) 1 (6.3) 0.38 Induction therapy, n (%) Basiliximab 21 (47.7) 16 (57.1) 5 (31.3) 0.098 Antithymocyte globulin 10 (22.7) 7 (25.0) 3 (18.8) 0.72 Initial maintenance therapy, n (%)b Mycophenolate mofetil 36 (81.8) 23 (82.1) 13 (81.3) >0.99 Tacrolimus 35 (79.5) 24 (85.7) 11 (68.8) 0.25 Steroids 34 (77.3) 21 (75.0) 13 (81.3) 0.72 Cyclosporine 6 (13.6) 2 (7.1) 4 (25.0) 0.17 mTOR inhibitors 5 (11.4) 2 (7.1) 3 (18.8) 0.34 CMV prophylaxis, n (%) 24 (54.5) 12 (42.9) 12 (75.0) 0.039 �3 monthsc 18 (75.0) 10 (83.3) 8 (66.7) 0.64 �6 monthsc 6 (25.0) 2 (16.7) 4 (33.3) Abbreviations: D-, negative donor; D+, positive donor; R-, negative recipient; R+, positive recipient; mTOR, mammalian target of rapamycin; n, number of cases; SD, standard deviation a CMV serostatus not specified in 2 patients b May have had more than 1 initial maintenance c Percentages calculated for patients with CMV prophylaxis https://doi.org/10.1371/journal.pone.0219701.t001 Results During the study period, 48 adults who had undergone SOT and in whom CMV antiviral resistance was suspected were included in the study. Four samples with a low CMV viral load (< 1.000 copies/ml) were excluded from the analysis because neither Sanger sequencing nor NGS could be carried out. Therefore, 44 patients were finally included. The baseline character- istics of the study population are shown in Table 1. Based on sequence analysis, 12 out of 44 (27%) patients showed CMV DRM by Sanger sequencing, while 16 patients (36%) showed mutations by NGS (Table 2). All the 16 patients had mutations in the UL97 gene and in addition, two patients had one mutation each in the UL54 gene (D413N and P522A). The NGS identified six low abundance resistance mutations that had not been reported by Sanger sequencing, since they were presented in <20% of the viral population. Patients 8 and 16 showed two mutations in UL97 (C592G + M460V and L595W + A594V respectively); in both cases. In these two patients, M460V and A594V, PLOS ONE | https://doi.org/10.1371/journal.pone.0219701 July 18, 2019 5 / 13 Cytomegalovirus resistance in solid organ transplant recipients Table 2. Detection of cytomegalovirus drug resistance mutations in the UL97 and UL54 genes by Sanger and next-generation sequencing. PATIENT SANGER UL97 NGS (Coverage %) UL97 GCVa SANGER UL54 NGS Coverage % UL54 GCV/FOS/CDVa RATIO RATIO 1 L595S (15%) 9.2 2 M460V (19%) 8.3 3 A594V A594V (20%) 8.3 4 M460V M460V (29%) 8.3 5 M460V (19%) 8.3 6 L595S (14%) 9.2 7 L595S L595S (20%) 9.2 8 C592G C592G+M460V (26%/14%) 2.9/8.3 9 H520Q H520Q (30%) 10 10 M460V M460V (31%) 8.3 P522A P522A (22%) 3/1/4.1 11 L595S L595S (20%) 9.2 12 M460V M460V (26%) 8.3 13 M460I M460I (31%) 5 D413A D413A (24%) 6.5/0.8/11 14 A594P A594P (31%) 3 15 L595S L595S (23%) 9.2 16 L595W L595W+A594V (21%/7%) 5.1/8.3 a IC50 of mutant /IC50 of wild type. If IC50 >5 the resistance was considered of high grade while an IC50 <3 was considered as low-grade resistance. GCV: ganciclovir, FOS: foscarnet, CDV:cidofovir https://doi.org/10.1371/journal.pone.0219701.t002 respectively, were only detected by NGS. These six mutations were reproducibly detected at similar levels in two independent sequencing experiments. All the DRM identified by Sanger were also identified by NGS. The lowest depth coverage obtained related to a resistance muta- tion was 7% of the viral subpopulations. The detection of mutations by NGS allowed the differentiation and comparison of the patients with and without mutations (Tables 1 and 3). The presence of mutations was more frequent in lung transplant recipients (p = 0.050) and in patients receiving prophylaxis (42.9% vs. 75%, p = 0.039), regardless of its duration. The median time between transplantation and suspicion of resistance was significantly longer in the group with DRM (211.5 days [87.5; 285.0] vs 108.5 days [43.5; 201.5] p = 0.038) as was the median treatment duration before sus- picion of resistance (41.5 days [18.0; 75.5] vs 16.0 days [14.5; 22.5] (p = 0.024). At the time of suspicion of resistance, 75% of patients without mutations were receiving therapy for asymp- tomatic CMV replication and 50% of patients with DRM were receiving treatment for CMV disease. CMV disease was diagnosed in 54.5% of patients. However, biopsy confirmation was achieved in only 3 patients. All the patients were receiving standard GCV/VGCV doses at the time of suspicion of resistance and in the absence of DRM study results the treatment was empirically modified accordingly. In patients with a DRM, mTORi was added or changed empirically (50%, p = 0.009). No statistically significant differences were observed between the patients with and without mutations in relation to outcome at 3 months (Table 3). Table 4 shows the clinical and virological data of the SOT recipients with known CMV DRM. Of the variables associated with the presence of resistance mutations in the univariate logistic regression analyses (S2 Text. S2 Table), treatment duration before suspicion of resis- tance was the only factor independently associated with the presence of mutations in the mul- tivariate analysis (log-transformed scale, OR 2.24, 95% CI 1.03 to 4.87). The area under the ROC curve was 0.71 (95% CI 0.53 to 0.88) for the final model of mutations (S1 Fig). PLOS ONE | https://doi.org/10.1371/journal.pone.0219701 July 18, 2019 6 / 13 Cytomegalovirus resistance in solid organ transplant recipients Table 3. Data at the onset and beyond the suspicion of resistance. Variables Overall Without Mutations With Mutations P value (n = 44) (n = 28) (n = 16) Time interval between transplantation and suspicion, median (IQR), days 116.5 (60.0; 232.5) 108.5 (43.5; 201.5) 211.5 (87.5; 285.0) 0.038 Days of therapy before suspicion, median (IQR) 20.0 (15.0; 48.0) 16.0 (14.5; 22.5) 41.5 (18.0;75.5) 0.024 Treatment at the time of suspicion, n (%) Preemptive treatment 28 (63.5) 21 (75.0) 7 (43.8) 0.038 Disease treatment 15 (34.1) 7 (25.0) 8 (50.0) 0.092 Prophylaxis 1 (2.3) 0 (0) 1 (6.3) 0.36 CMV viral load at the time of suspicion, median (IQR), copies/ml 11,304 (3,521; 64,105) 7,088 (2,415; 47,555) 36,236 (9,609; 65,247) 0.17 Symptoms at the time of suspicion, n (%)a 24 (54.5) 14 (50) 10 (62) 0.53 Viral syndromeb 13 (29.5) 10 (35.7) 3 (18.8) 0.31 Gastrointestinal diseasec 8 (18.2) 3 (10.7) 5 (31.3) 0.12 Pneumonitis 4 (9.1) 2 (7.1) 2 (12.5) 0.61 Retinitis 2 (4.5) 2 (7.1) 0 (0) 0.53 Hepatitis 1 (2.3) 1 (3.6) 0 (0) >0.99 Disseminated (>1 end organ) 1 (2.3) 0 (0) 1 (6.3) 0.36 Therapeutic strategy on suspicion, n (%)d Increase GCV/VGC dose 15 (34.1) 10 (35.7) 5 (31.3) 0.764 Continue with standard GCV/VGCe dose 18 (40.9) 13 (46.4) 5 (31.3) 0.325 Decreased immunosuppression 12 (27.3) 6 (21.4) 6 37.5) 0.303 Switch or add mTOR inhibitors 11 (25.0) 3 (10.7) 8 (50.0) 0.009 Switch or add foscanet 8 (18.2) 3 (10.7) 5 (31.3) 0.12 Outcome (at 3 months) Viral response Total suppression of CMV replication 25 (56.8) 18 (64.3) 7 (43.8) 0.186 Incomplete suppression 19 (43.2) 10 (35.7) 9 (56.3) 0.156 Final outcomef Graft rejection 5 (11.9) 3 (11.1) 2 (13.3) >0.99 Death 5 (11.9) 3 (11.1) 2 (13.3) >0.99 Abbreviations: IQR, interquartile range; mTOR, mammalian target of rapamycin; n, number of case a May have had more than 1 symptom at the time of suspicion b Viral syndrome: fever >38˚C(for at least 2 days in a 4 day period) associated with leucopenia, thrombocytopenia or increase in transaminases c Gastrointestinal disease include: colitis, gastritis d May have had more than 1 therapeutic strategy on suspicion e Continue with standard prophylactic or treatment dose of GCV/VGC f Final outcome not specified in 2 patients https://doi.org/10.1371/journal.pone.0219701.t003 Discussion Drug-resistant CMV infection is an important emerging problem in SOT recipients. However, it may be underdiagnosed since mutation analysis is not routinely performed and the current gold-standard Sanger sequencing method lacks sufficient sensitivity [1]. In the present study, a NGS was compared with Sanger sequencing to identify mutations associated with resistance in SOT patients with suspicion of resistance. Among the 44 patients recruited, 14 DRM were detected by Sanger in 12 patients (27%) and 20 DRM were detected with NGS in 16 patients (36%), respectively. NGS showed a higher sensitivity for the detection of mutations present in <20% of the population sequenced which were not found by Sanger sequencing. Univariate analysis of the clinical data revealed that the presence of DRM detected by NGS was associated PLOS ONE | https://doi.org/10.1371/journal.pone.0219701 July 18, 2019 7 / 13 Cytomegalovirus resistance in solid organ transplant recipients Table 4. Clinical and virological data in solid organ transplant recipients with cytomegalovirus resistance mutations. Type of Induction Prophylaxis Treatment on Symptomatic on UL97/UL54 CMV Therapeutic Virological and/ Transplant therapy (months) suspicion suspicion of DRM (Days viral b adaptation on or clinical (CMV of resistancea resistance post-TX) (copies/ suspicion of outcome c serostatus) (Days) ml) resistance 1 Kidney D+/R- ATG 3M Disease VGC Suspected colitis L595S (91) 4.180 GCV/VGC IS. 900mg/12h (16) increased mTORi 2 Kidney D+/R+ Basiliximab No Preemptive No M460V (68) 55.253 GCV/VGC CS.Total treatment VGC increased resolution 450mg/12h (15) 3 Liver D+/R- No No Preemptive No A594V 63) 9.300 GCV/VGC IS. Cell-mediated treatment VGC increased ISP rejection 450mg/12h(15) decreased 4 Liver D+/R- No 3M Disease VGC Viral syndromed, M460V (203) 3.849 ISP decreased CS.Total 450mg/12h(15) Suspected colitis Continue with resolution GCV/VGCV 5 Kidney D+/R+ Basiliximab 1M Disease VGC Suspected gastritis M460V (235) 11.234 ISP decreased GCV/ CS. Total 450mg/12h(84) VGC increased resolution mTORi 6 Kidney D+/R+ ATG No Disease VGC Viral syndrome d L595S (58) 114.167 GCV/VGC CS.Total 450mg/12h(67) Suspected colitis increased resolution 7 Heart D+/R- No 3M Disease VGC Viral syndrome d L595S (350) 154.486 GCV/VGC IS 100mg/12h (20) increased 8 Kidney D+/R- ATG 3M Disease VGC Suspected colitis C592G 11.605 GCV/VGC IS 450mg/12h(61) +M460V increased. mTORi (102) 9 Lung D+/R- No 12M Prophylaxis VGC Confirmed H520Q (210) 33.772 ISP Decreased. FOS. CS.Total 900mg/24h(38) pneumonitis mTORi resolution 10 Lung D+/R- No 12M Preemptive No M460V/ 68.494 ISP Decreased .FOS. IS treatment VGC P522A (335) mTORi 900mg/12h(96) 11 Lung D+/R- No 6M Preemptive No L595S (210) 38.700 ISP Decreased .FOS. CS.Total treatment VGC mTORi resolution 900mg/12h(26) 12 Liver D+/R- No No Disease VGC Confirmed M460V (340) 9.919 Continue with IS. Death 450mg/12h (45) pneumonitis and GCV/VGCV. retinitis mTORi 13 Lung D+/R- Basiliximab 12M Preemptive No M460I/ 62.000 GCV/VGC IS. Cell-mediated treatment VGC D413N (400) increased. rejection 450mg/12h(231) Leflunomide 14 Heart D+/R- Basiliximab 1M Preemptive Viral syndromed A594P (331) 59.879 Continue with IS treatment VGC GCV/VGCV. 450mg/12h(113) Maribavir 15 Kidney D+/R- No 1M Disease VGC Viral syndromec L595S (112) 721.000 Switch to FOS IS. Death 450mg/12h(28) Suspected colitis 16 Heart D+/R- Basiliximab 2M Preemptive Viral syndromed L595W 4.120 GCV/VGC CS.Total treatment VGC +A594V (84) increased. FOS. resolution 900mg/12h(51) mTORi CMV, cytomegalovirus; D, donor; R, recipient; TX, transplantation; GCV, ganciclovir; VGC, valganciclovir; FOS, foscarnet, CDV, cidofovir; ATG, antithymocyte globulin; ISP, immunosuppression; IS, incomplete suppression; CS, total supression of CMV replication a Drug/doses b CMV viral load on suspicion of resistance c Virological and clinical outcomes at 3 months d Presence of fever >38˚C (for at least 2 days in a 4 day period) associated with leucopenia, thrombocytopenia or an increase in transaminases https://doi.org/10.1371/journal.pone.0219701.t004 PLOS ONE | https://doi.org/10.1371/journal.pone.0219701 July 18, 2019 8 / 13 Cytomegalovirus resistance in solid organ transplant recipients with lung transplantation (p = 0.050), prophylactic treatment (p = 0.039), a higher mean time between transplantation and suspicion of resistance (p = 0.038) and longer treatment duration with GCV or VGCV before suspicion (p = 0.024). However, the latter was the only factor inde- pendently associated with the presence of resistance mutations in the multivariate analysis. Several aspects differentiate the present stydy from other similar studies in the field reported to date, and these aspects can be summarized as follows:NGS was addressed to the UL97 and UL54 genes, a relatively high number of patients, undergoing different solid organ transplanted were studied, and associated risk factors were also evaluated. The current gold standard for the genotypic detection of CMV drug resistance is Sanger sequencing of the UL97 and UL54 genes. However, this technique cannot detect viral variants that represent < 20% of the viral population [9, 10]. This lack of sensitivity can lead to contin- ued administration of an antiviral to which resistance has developed and the subsequent expan- sion of initially minority populations of resistant variants leading to the development of treatment failure, increased morbidity and shortened graft survival. On the other hand, modifi- cations in therapy following the identification of GCV resistance is associated with more rapid viral clearance, further emphasising the importance of timely detection of resistance [7, 11, 12]. Recent studies have shown that NGS methods can provide new insights into viral diversity and in the detection of low-abundance variants by the analysis of thousands of amplicons in a single experiment [10, 25]. However, so far, only a few studies have focused on CMV-resistant mutants. Most studies have included few patients and only some cover the UL97 gene [11, 12]. An exception to this is the study by Sahoo et al which evaluated DRM in both of the genes involved using NGS in a considerable number of specimens [10]. However, this study did not include an analysis of clinical factors. In our study, NGS identified six low abundance resistant mutations that had not been reported by Sanger sequencing, thus suggesting that NGS is more sensitive than Sanger since the use of high throughput sequencing allowed the detection of viral subpopulations with a prevalence of 7%. All six mutations were located in the UL97 gene and showed high-level resistance to GCV [26, 27]. In addition, two of these six DRM identified were present as mixed mutations in two patients which may have important implications in patient management. Therefore, studies with prospective monitoring of the abundance of such muta- tions over time and an assessment of their association with virologic failure are necessary. UL54 DNA polymerase mutations are typically selected after prolonged GCV exposure and were revealed in 2 lung transplant patients who had received a longer prophylaxis and preemptive treatment [28, 29]. However, compared to Sanger sequencing NGS did not improve the detec- tion sensitivity of DRM in the UL54 gene in concordance with the results of Sahoo et al [10]. In the present study longer treatment duration with antivirals before suspicion of resistance was identified as a risk factor associated with DRM (p = 0.24)[1, 2], and is considered a major factor in the selection and emergence of resistance. Lung transplantation showed the highest incidence of DRM. It is important to note that 4 out of 5 lung transplant patients in our study presented DRM and all had received lengthy preemptive treatment and antiviral prophylaxis [29, 30]. Moreover, the presence of DRM was more frequent among transplant recipients who had received prophylaxis (p = 0.039). Nonetheless, other factors may have also contributed to this finding. The interdependence of host, virus and antiviral therapy is likely associated with the development of DRM [31]. In fact, all the patients included in this study had one or more known risk factors predisposing to DRM [1, 2]. Therefore, according to current consensus guidelines genotypic testing confirmation should be done irrespective of any exclusion criteria whenever antiviral resistance is suspected [5, 6], since resistance might otherwise be underesti- mated similar to what has been described in previous reports [32]. In the present study, DRM were found in unusual situations, such as during prophylaxis in one patient and in 3 patients with CMV D+/R+ serostatus. PLOS ONE | https://doi.org/10.1371/journal.pone.0219701 July 18, 2019 9 / 13 Cytomegalovirus resistance in solid organ transplant recipients Previous studies have reported antiviral resistance to be associated with increased attribut- able morbidity and mortality [2]. This was not found in the present study, likely because patients were only clinically followed during the first three months. Moreover, clinicians were unaware of the presence of DRM in real time, and therefore, treatment was empirically modi- fied. Thus, we were unable to evaluate the outcome of the patients according to Sanger or NGS sequencing results. This should be addressed in future studies. In our cohort, 64% of the patients suspected of having resistance to antivirals did not pres- ent DRM. Therefore, as suggested previously other factors might contribute to treatment fail- ure [33]. Emphasis should be given to prioritizing the optimization of host factors rather than switching antiviral medications, thereby adjusting immunosuppressive therapy whenever pos- sible. Additionally, blood GCV levels were not available and were not performed during the clinical care of the patients studied, and thus, sub-therapeutic GCV levels may be a possible cause of non-response to treatment in some patients. On the other hand, a recent study of serial specimens received for genotypic testing showed that the first specimen tested negative for DRM in more than half of the patients, although about two-thirds eventually became posi- tive on repeated testing [4]. It could therefore be suggested that patients without DRM but with suspected criteria of resistance should be closely monitored and new samples should be studied for viral mutations. Our study has limitations regarding the modest sample size and the lack of balance among groups although it does reflect real life in a nation-wide cohort study. Our results should be confirmed in larger and better balanced cohorts. Another limitation is that the analysis of GCV resistance of the mutations was performed only when there was suspicion; Nonetheless, these patients represented the group with the highest risk for antiviral resistant CMV. In summary, the development of DRM may be a direct effect of a longer duration of antiviral treatment, and thus, the implementation of effective CMV prevention strategies that minimise drug exposure could lead to lower resistance rates. Although the clinical risk factors for drug resistance have been relatively well defined, genotypic testing should be performed in all patients with suspected criteria of resistance. Despite the lack of standardized protocols and the complexity of the bioinformatics analysis for the use of NGS in routine clinical practice, the results of this study suggests that NGS technology improves the genotypic diagnosis of DRM, fundamentally in low-abundance variants and mixed populations allowing early detection of the emergence of CMV-resistant strains and subsequent targeted adjustment of therapy. Supporting information S1 Text. Methods of statistical analysis. (DOC) S2 Text. Results of statistical analysis. (DOC) S3 Text. References of statistical analysis. (DOC) S1 Table. Internal validation of prediction model for mutations using nonparametric bootstrap technique. (DOC) S2 Table. Significant univariate and multivariable logistic regression analyses for muta- tions. (DOC) PLOS ONE | https://doi.org/10.1371/journal.pone.0219701 July 18, 2019 10 / 13 Cytomegalovirus resistance in solid organ transplant recipients S1 Fig. Receiver operating characteristic curve analysis of significant variables derived from the logistic regression model for the ability to predict mutations. (DOC) Acknowledgments We would like to acknowledge GESITRA-SEIMC, Group for Study of Infection in Transplan- tation of the Spanish Society of Infectious Diseases and Clinical Microbiology and REIPI, the Spanish Network for Research in Infectious Diseases. We thank Albert Gabarrus (Statistician in Applied Research in Respiratory Diseases, IDI- BAPS, UB, CIBERES) for their statistical analysis. Thanks to Institute for Research in Biomedi- cine of Barcelona, specially at Jose Luis Mosquera and Camille Stephan to contribute in bioinformatic analysis. We thank Donna Pringle, an English-Spanish translator/reviewer based in Barcelona (Spain) for revision of the manuscript. Author Contributions Conceptualization: Francisco López-Medrano, Frederic Cofán, Julián Torre-Cisneros, Asun- ción Moreno, Jose Marı́a Aguado, Carlos Cervera, Maria Ángeles Marcos. Funding acquisition: Maria Ángeles Marcos. Investigation: Rubén López-Aladid, Alba Guiu, Maria Ángeles Marcos. Methodology: Rubén López-Aladid, Alba Guiu, Maria Mar Mosquera, Elisa Cordero, Jordi Niubó, Anna Cervilla, Marta Santos, Andrés Antón, Maria Ángeles Marcos. Project administration: Maria Ángeles Marcos. Resources: Francisco López-Medrano, Frederic Cofán, Laura Linares, Julián Torre-Cisneros, Elisa Vidal, Jose Marı́a Aguado, Elisa Cordero, Cecilia Martin-Gandul, Jordi Carratalá, Nuria Sabé, Jordi Niubó, Alicia Capón, Marta Bodro, Patricia Muñoz, Maria Carmen Far- iñas, Maitane Aranzamendi, Miguel Montejo, Pilar Pérez-Romero, Oscar Len. Supervision: Maria Ángeles Marcos. Validation: Maria Mar Mosquera. 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