Features of Cytomegalovirus DNAemia Blips in Allogeneic Hematopoietic Stem Cell Transplant Recipients: Implications for Optimization of Preemptive Antiviral Therapy Strategies

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Features of Cytomegalovirus DNAemia Blips in Allogeneic Hematopoietic Stem Cell Transplant Recipients: Implications for Optimization of Preemptive Antiviral Therapy Strategies ´ Dixie Huntley , Alberto Talaya , Estela Gimenez , ´ Ariadna Mart´ınez , Juan Carlos Hernandez-Boluda , Rafael Hernani , Ignacio Torres , Juan Alberola , Eliseo Albert , ˜ Jose´ Luis Pinana , Carlos Solano , David Navarro PII: DOI: Reference:

S1083-8791(20)30047-1 https://doi.org/10.1016/j.bbmt.2020.01.015 YBBMT 55908

To appear in:

Biology of Blood and Marrow Transplantation

Received date: Accepted date:

18 November 2019 22 January 2020

´ Please cite this article as: Dixie Huntley , Alberto Talaya , Estela Gimenez , Ariadna Mart´ınez , ´ Juan Carlos Hernandez-Boluda , Rafael Hernani , Ignacio Torres , Juan Alberola , Eliseo Albert , ˜ Jose´ Luis Pinana , Carlos Solano , David Navarro , Features of Cytomegalovirus DNAemia Blips in Allogeneic Hematopoietic Stem Cell Transplant Recipients: Implications for Optimization of Preemptive Antiviral Therapy Strategies, Biology of Blood and Marrow Transplantation (2020), doi: https://doi.org/10.1016/j.bbmt.2020.01.015

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HIGHLIGHTS  CMV DNAemia blips occur frequently in allo-HSCT recipients.  CMV DNAemia recurrences develop frequently following blips  Initial CMV DNA load does not reliably predict the course of CMV DNAemia episodes.  Receipt of an allograft from a CMV seropositive donor was associated with the occurrence of blips.

1

Features of Cytomegalovirus DNAemia Blips in Allogeneic Hematopoietic Stem Cell Transplant Recipients: Implications for Optimization of Preemptive Antiviral Therapy Strategies Dixie Huntley,1Alberto Talaya,1 Estela Giménez,1Ariadna Martínez,2 Juan Carlos Hernández-Boluda,2,3 Rafael Hernani,2 Ignacio Torres,1 Juan Alberola,4 Eliseo Albert,1 José Luis Piñana,2† Carlos Solano,2,3 and David Navarro1,4 1

Microbiology Service, Hospital Clínico Universitario, INCLIVA Research Institute,

Valencia, Spain 2

Hematology Service, Hospital Clínico Universitario, INCLIVA Research Institute,

Valencia, Spain. 3

Department of Medicine, School of Medicine, University of Valencia, Valencia, Spain.

4

Department of Microbiology, School of Medicine, University of Valencia, Valencia,

Spain. †

Present address: Hematology Department, Hospital Universitari i Politècnic La Fe.

*Correspondence:

David

Navarro,

Microbiology

Service,

Hospital

Clínico

Universitario, and Department of Microbiology, School of Medicine, Av. Blasco Ibáñez 17, 46010 Valencia, Spain. Phone: +34 963864657; Fax: +3963864173; E-mail: [email protected]. Running head: CMV DNAemia blips in allo-HSCT recipients. Financial disclosure: This work was supported by a Fondo de Investigaciones Sanitarias (FIS) grant from Carlos III Health Institute (15/0060). The authors report no conflicts of interest. 2

ABSTRACT Cytomegalovirus (CMV) DNAemia occurs frequently in allogeneic hematopoietic stem cell transplant recipients (allo-HSCT). There is limited information about the incidence, features and clinical impact of CMV DNAemia blips (episodes defined by an isolated positive PCR result) in this setting. In this retrospective study, 225 consecutive adult patients undergoing any modality of allo-HSCT at our center between May 2012 and July 2019 were included. Plasma CMV DNA load was monitored using a highly sensitive real-time PCR assay. In all, 187 out of 225 patients had CMV DNAemia through day 365 after allo-HSCT (total number of episodes, n=379). Eighty-three out of the 187 patients had one or more blips (n=104). Blips occurred as a first episode of CMV DNAemia as opposed to prolonged CMV DNAemia (≥2 consecutive positive PCR results) in 47 patients; in 20 of these patients, blips represented the only documented episode throughout the study period, and in 27 patients, blips preceded a prolonged CMV DNAemia episode. In the remaining 36 patients blips developed as recurrences. Blips presenting as initial episodes occurred more frequently (P=<0.001) in patients receiving an allograft from a CMV seropositive donor. The cumulative incidence of recurrent CMV DNAemia following initial blips, self-resolving prolonged CMV DNAemia episodes or CMV DNAemia episodes treated preemptively with antivirals was not significantly different (P=0.34). ROC curve analysis indicated that a CMV DNA load cut-off of 48 IU/ml yielded the highest combined sensitivity (66%) and specificity (70.2%) for predicting a prolonged CMV DNAemia episode. The practical implications of our data in optimization of preemptive antiviral therapy strategies are discussed. Keywords: CMV, CMV DNAemia, blips, allo-HSCT, recurrences, CMV DNA load. 3

INTRODUCTION Cytomegalovirus (CMV) infection continues to be a clinically relevant problem in allogeneic hematopoietic stem cell transplant recipients (allo-HSCT) [1]. Preemptive antiviral therapy (PET) triggered upon detection of CMV DNA in the blood compartment has long been the first-choice strategy for prevention of CMV disease in this setting [1]. How this strategy will be positioned in the management of CMV infection in the new era of letermovir prophylaxis [2,3] remains to be established. Neither is there consensus on the CMV DNA load threshold that should prompt initiation of PET, nor on whether this cut-off should be adapted according to the patient’s risk for CMV end-organ disease [1,4,5]. Some authors advocate starting PET at low CMV DNA loads, even at any level, often yielded by the first PCR positive blood sample, on the grounds that this strategy appears to be associated with shorter CMV DNAemia episodes and courses of antiviral therapy [6], and that low-magnitude CMV DNAemia might be associated with increased overall (OM) and non-relapse mortality (NRM) [7]. Other authors support delaying initiation of PET until higher CMV DNA levels in blood are reached (i.e. 1,500 IU/ml) to avoid overtreatment and consequent drug-related toxicity, given that some CMV DNAemia episodes resolve spontaneously [8,9]. In this context, a trend towards increased risk of OM and NRM was seen in patients with one or more episodes of CMV DNAemia requiring PET [10], but not in those experiencing one or more self-resolving episodes. In a recent metaanalysis, use of PET was reported to increase the strength of association between CMV DNAemia and OM and NRM [11]. CMV DNAemia blips (episodes in which an isolated positive PCR result is documented) have been reported to occur with variable frequency in the allo-HSCT setting, in particular in patients allografted with CMV4

seropositive donors [12,13]. Hill et al., [12] also found that blips, as opposed to prolonged episodes of DNAemia, are temporally associated with lower incidence of end-organ disease. Here we further characterized the virological features of blips, and assessed whether they might have an impact on clinical outcomes. Deeper insight on this topic may help to improve current PET strategies. PATIENTS AND METHODS Study population In this single center observational study, we retrospectively reviewed CMV DNA PCR results and clinical charts of 225 consecutive adult patients who underwent any modality of allo-HSCT at the Hospital Clínico Universitario of Valencia (HCUV) between May 2012 and July 2019. Demographic and relevant clinical characteristics of the patients are shown in Table 1. Regarding baseline transplant factors known to be associated with the occurrence of CMV DNAemia in allo-HSCTs [1], our cohort comprised 43% of patients receiving an HLA-mismtached allograft, of which 73% were haploidentical, 3.1% of patients undergoing umbilical cord blood transplantation, 28.9% of patients receiving a myeloablative conditioning and 12.1% of patients treated with anti-thymocyte globulin (ATG). The study period comprised the first year after alloHSCT. This study was approved by the Hospital Clínico, Fundación INCLIVA Ethics Committee. Informed consent was obtained from participants. CMV DNA monitoring and management of CMV infection Plasma CMV DNA load was monitored using the CMV RealTime CMV PCR (Abbott Molecular, Des Plaines, IL, USA) [14], that has a limit of detection (LOD) and quantification of 31.4 IU/ml (95% CI) [15]. The lower limit of linearity is 50 IU/ml, according to the manufacturer. This assay may detect, but not quantify, CMV DNA 5

loads below the LOD/LOQ, with a lower probability (<95%). CMV DNA load monitoring was performed on a weekly basis from start of conditioning regimen through day +100. From day +100 until day +365 patients at risk for recurrent episodes of CMV DNAemia [1] were also monitored once a week [8]. The remaining patients were monitored at each planned visit and if testing positive they were monitored on a weekly basis until resolution of CMV DNAemia. Preemptive antiviral therapy with (val)ganciclovir or foscarnet at conventional doses was initiated when the plasma CMV DNA load reached levels of ≥1,500 IU/ml [8], or when the CMV doubling time (dt) was ≤2.0 days (9), whichever occurred first (this latter strategy since May 2014). Definitions In this study, CMV DNAemia, either initial (first episode) or recurrent, refers to the presence of CMV DNA in one or more consecutive plasma specimens. Blips are defined by the presence of CMV DNA at any level in a single plasma specimen, preceded and succeeded by a negative (undetectable) PCR specimen, usually drawn 7 days apart. An episode of prolonged CMV DNAemia is defined by the presence of CMV DNA in 2 or more consecutive specimens (usually drawn 7 days apart). Selfresolving episodes of CMV DNAemia were those that cleared without requiring PET. Detectable but not quantifiable CMV DNA loads were arbitrarily ascribed a nominal value of 15.2 IU/ml (midpoint between 0 and the LOD of the assay). Recurrent episodes of CMV DNAemia (either blips or prolonged CMV DNAemia episodes) were ones that developed at least 14 days after clearance of the previous one. CMV disease was defined following recently established criteria [16]. Acute graft-versus-host disease (aGvHD) was diagnosed and graded as previously reported [17].

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Statistical analysis Cumulative incidence of events of interest was estimated by the cumulative incidence method using the R statistical software (http://www.r-project.org/) with the survival and cmprsk packages. The Fine-Gray test was used for comparisons. The cumulative incidence of any type of CMV DNAemia episode was calculated considering death as a competitive event. The odds ratio (OR) of the initial episode being a blip or any other type of CMV DNAemia (self-resolving prolonged CMV DNAemia or treated CMV DNAemia) was determined by logistic regression analysis. For multivariate analyses, only variables with parameter estimates with a P value ≤ 0.10 in the univariate analyses were included; two-sided P values <0.05 were deemed to be significant. Differences between medians were compared using the Mann-Whitney U-test and the KruskalWallis test, when appropriate. The latter statistical analyses were performed using SPSS version 20.0 (SPSS, Chicago, IL, USA). Receiver operating characteristic (ROC) curves were generated and the area under a curve (AUC) calculated with the GraphPad Prism v8.0. statistical software package to determine the sensitivity and specificity of first positive CMV DNA threshold for predicting the virological course of the CMV DNAemia episode (blip vs. prolonged CMV DNAemia) with the highest combined sensitivity and specificity.

7

RESULTS Incidence and features of CMV DNAemia blips in allo-HSCT recipients Relevant data on the incidence and virological outcomes of CMV DNAemia episodes throughout the study period are summarized in Figure 1. In all, 187 out of 225 patients had one (n=95) or more (n=92) episodes of CMV DNAemia through day 365 after alloHSCT (cumulative incidence, 83.1%; 95% CI, 80.4-85.4%). The total number of episodes experienced by these patients was 379. Eighty-three out of the 187 patients had one (n=68) or more (n=15) blips, with an overall cumulative incidence of 36.4% (95% CI, 36.8-45.2%). In total, 104 blips were documented out of 379 episodes of CMV DNAemia (27.4%). The time elapsed between the plasma specimen testing positive by PCR and the subsequent negative one was a median of 7 days (range, 4-8 days). Blips occurred as a first episode of CMV DNAemia in 47 patients (overall cumulative incidence, 20.8%; 95% CI, 12.4-30.8%), at a median of 23 days after allo-HSCT (range, 2-202 days), and represented the only documented episode within the study period in 20 patients (overall cumulative incidence, 8.89%; 95% CI, 2.3-21%). In the remaining 27 patients, blips preceded a prolonged CMV DNAemia episode that occurred at a median of 35 days following allo-HSCT (range, 10-176 days). Finally, 36 patients had CMV DNAemia blips that developed as recurrences (in total 57 episodes). The median CMV DNA load in blips was similar (P=0.92) across comparison groups (Table 2). No blip was treated preemptively with antivirals. When only considering initial episodes of CMV DNAemia, the median CMV DNA load in blips was significantly lower (P=<0.001) than in the first positive PCR specimen in prolonged self-resolving or treated CMV DNAemia episodes (Figure 2). ROC curve 8

analysis (Figure 3) indicated that a CMV DNA load cut-off of 48 IU/ml gave the highest combined sensitivity (66%) and specificity (70.2%) for predicting a prolonged CMV DNAemia episode (ROC area under a curve-AUC-, 0.76; 95% CI, 0.68-0.85). In addition, the median first CMV DNA load was comparable in prolonged selfresolving and treated CMV DNAemia episodes (P=0.46). Recurrent CMV DNAemia following blips We next looked at the incidence of recurrent CMV DNAemia following initial episodes presenting either as blips, prolonged self-resolving CMV DNAemia episodes or CMV DNAemia episodes that required PET. The data are depicted in Figure 4. The cumulative incidence of recurrent CMV DNAemia was no different across comparison groups (P=0.34). Occurrence of CMV end-organ disease There were two cases of CMV end-organ disease (esophagitis and colitis) in the cohort, and these occurred within CMV DNAemia episodes under PET. Factors associated with occurrence of blips We next investigated whether blips occurring as initial episodes of CMV DNAemia were associated with any demographic or clinical characteristics of patients, including sex, age, type of allograft, source of stem cells, conditioning regimen, aGvHD prophylaxis and donor/recipient CMV serostatus. As shown in Table 3, blips occurred more frequently (P=<0.001) in patients receiving an allograft from a CMV seropositive donor, irrespective of the receptor CMV serostatus. Univariate regression analysis of risk factors for blips as initial episodes of CMV DNAemia is shown in Table 4. Of the two variables with P values ≤0.1, namely the conditioning regimen and donor CMV 9

seropositivity, the latter was found to be independently associated with the development of blip episodes in multivariate models (OR, 2.82; 95% C.I., 1.21-6.54; P=0.015), while the former was not (OR, 2.12; 95% C.I.,0.90 – 4.98; P=0.084). DISCUSSION The term blip was first coined in the human immunodeficiency virus (HIV) infection setting to describe low-level, intermittent episodes of viremia occurring in patients who had achieved virological suppression following antiretroviral therapy (ART) [18]. Although initially under debate, the importance of blips in treatment outcomes for HIVinfected patients on ART is now recognized and considered in clinical decision-making [19]. In the HIV setting, blips occur while patients are on ART, and could reflect lack of adherence to therapy or impaired immunological control of HIV infection [18,19]. In contrast, in the allo-HSCT setting, blips commonly develop as first episodes of CMV DNAemia in the absence of anti-CMV treatment [12,13]. Very little is known about the incidence and virological course of CMV DNAemia blips in the allo-HSCT setting [12,13], or more importantly about their effect on clinical outcomes. Here, taking advantage of a conservative strategy for PET (CMV DNA threshold of 1,500 IU/ml in most patients) and the use of a highly-sensitive real-time PCR assay for CMV infection monitoring [8-11,14], we investigated the natural history of blips in this clinical setting. This knowledge may contribute towards optimizing current PET strategies. Our first finding was that blips occur frequently in allo-HSCT recipients; in fact, 25% of patients developed blips as a first episode of CMV DNAemia. Moreover, 10% of patients only experienced blips throughout the first year after transplantation. In all, 27.4% of all CMV DNAemia episodes developing within the study period were blips. These numbers are close to those reported by Hill and colleagues (32%) [12] and higher 10

than the ones published by Lodding et al. (16%) [13]. This apparent discrepancy is likely related to differences in the limits of detection of the PCR assays used to quantify the plasma CMV DNA load across centers (lower in Hill’s study and ours), and perhaps to differences in the clinical characteristics of patients in the cohorts (i.e. type of alloHSCT, use of T-cell depleting agents, such as ATG, or the type of conditioning regimen). Interestingly, blips were found to develop with comparable frequency across different allo-HSCT modalities (HLA-matched vs. HLA-mismatched allografts, or related vs. unrelated transplants), suggesting that the virological course of initial CMV DNAemia episodes might not be anticipated on clinical grounds. However, occurrence of blips was independently associated with receipt of an allograft from a CMVseropositive donor. In contrast, neither sex, age, type of allograft, source of stem cells, conditioning regimen, nor aGvHD prophylaxis regimen were found to be correlated. The above observation needs to be validated in cohorts enriched in patients with characteristics underrepresented in our series (i.e. ATG use in the conditioning regimen or umbilical cord blood allografts), known to increase the risk of CMV DNAemia [1]. In our series, overall, the magnitude of the CMV DNA load in blips was comparable regardless of whether they developed as isolated episodes of CMV DNAemia or occurred either preceding or succeeding an episode of prolonged CMV DNAemia. However, when only considering initial episodes of CMV DNAemia, the median CMV DNA load in blips was significantly lower than in the first PCR-positive specimen in episodes of prolonged CMV DNAemia (combining treated and self-resolving episodes). Despite this, there was a high degree of overlap in CMV DNA load values across comparison groups. The best CMV DNA load cut-off discriminating between blips and prolonged CMV DNAemia episodes, 48 IU/ml, returned a sensitivity of 66% and 11

specificity of 70.2% for predicting a prolonged CMV DNAemia episode. In a previous study Hill et al. [12] reported that magnitude of first positive CMV DNA load was associated with risk of persistent CMV DNAemia (duration longer than 4 weeks). In that study [12] the authors proposed two CMV DNA load cut-offs (2.0 log10 and 1.75 log10 copies/ml) to discriminate between blips (CMV DNA loads below the cut-off) and persistent episodes (CMV DNA loads above the threshold), whose respective false positive rates of 32% and 57% indicated the probability that an episode with a CMV DNA load above the threshold would be a blip. In light of the above, and considering that inter-assay variations of CMV real-time PCR assays are highest at CMV DNA values close to the LOD [1], in our view a reliable threshold for predicting the virological outcome of initial episodes of CMV DNAemia is unlikely to be established. Finally, the impact of blips on mortality could not be addressed in the present study due to the low number of patients developing only this type of CMV DNAemia episode (n=20); although speculative, mortality associated with appearance of isolated blips could well be comparable to mortality in self-resolving episodes of CMV DNAemia, which was also found to show similar rates in the absence of CMV DNAemia [20]. Limitations of the current study include its retrospective nature, the lack of systematic CMV testing beyond day 100 after allo-HSCT which may have resulted in an underestimation of the real incidence of blips and prolonged episodes, and the relative heterogeneity of the cohort. The clinical importance of low-level CMV DNAemia remains a debated issue [21]. Starting PET at low CMV DNA levels, frequently yielded by the first PCR-positive sample, is common practice at some centers, particularly for initial CMV DNAemia episodes developing in high-risk patients, such as those undergoing HLA-mismatched allo-HSCT or receiving high doses of corticosteroids for severe aGvHD [1,4,6,7]. The 12

rationale behind this approach stems from two facts: first, this strategy seems to be associated with shorter episodes of CMV DNAemia, with lower AUCs, and courses of antiviral therapy [6], and second, there appears to be a “dose-dependent” relationship between CMV DNAemia AUCs and the risk of OM and NRM [12,22]. However, the lower the CMV DNA threshold is set the greater the number of patients receive PET, leading to overtreatment. In this context, our observations argue against initiating PET at very low CMV DNA thresholds, since blips, which by definition resolve spontaneously and display low AUCs, occur quite frequently in this clinical setting (27%), and the virological course of an episode of CMV DNAemia cannot be accurately predicted either on clinical grounds or by the magnitude of the initial CMV DNA load. Further studies are nevertheless warranted to determine whether our conclusions can be extrapolated to other centers using CMV PCR assays with different analytical performances or to patient cohorts differing from ours in baseline or post-transplant clinical characteristics. ACKNOWLEDGEMENTS Estela Giménez holds a Juan Rodés research contract from the Carlos III Health Institute (Ref.JR18/00053). Eliseo Albert holds a Río Hortega research contract from the Carlos III Health Institute (Ref.CM18/00221). AUTHOR CONTRIBUTIONS J.L.P, J.C. H-B, RH, A.M. and C.S. attended the patients and collected the clinical data. D.H., A.T., E.G., I.T., J.A., and E.A. analyzed the data. D.N. analyzed the data and wrote the manuscript.

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REFERENCES 1.

Pérez-Romero P, Blanco P, Giménez E, Solano C, Navarro D. An update on the management and prevention of cytomegalovirus infection following allogeneic hematopoietic stem cell transplantation. Future Virology. 2015; 10: 113-134.

2.

Chemaly RF, Ullmann AJ, Stoelben S, et prophylaxis in hematopoietic-cell

al. Letermovir for cytomegalovirus

transplantation. New Engl J Med. 2014;

370: 1781-1789. 3.

Marty FM, Ljungman P, Chemaly RF, et al. Letermovir prophylaxis for cytomegalovirus in hematopoietic-cell

transplantation. New Engl J Med.

2017; 377: 2433-2444. 4.

Boeckh M, Ljungman P. How we treat cytomegalovirus in hematopoietic cell transplant recipients. Blood. 2009;113:5711-5719.

5.

Ljungman P, de la Camara R, Robin C, et

al. Guidelines for the management

of cytomegalovirus infection in patients with haematological malignancies and after stem cell transplantation from the 2017 European Conference on Infections in Leukaemia (ECIL 7). Lancet Infect Dis. 2019;19:e260-e27. 6.

Tan SK, Waggoner JJ, Pinsky BA. Cytomegalovirus load at treatment initiation is predictive of time to resolution of viremia and duration of therapy in hematopoietic cell transplant recipients. J Clin Virol. 2015;69:179-83

7.

Green ML, Leisenring W, Xie H, et al. CMV viral load and mortality after hematopoietic cell transplantation: a cohort study in the era of preemptive therapy. Lancet Haematol. 2016; 3: e119.

14

8.

Solano C, Muñoz-Cobo B, Giménez E, et al. Pre-emptive antiviral therapy for active CMV infection in adult allo-SCT patients guided by plasma CMV DNAemia quantitation using a real-time PCR assay: clinical experience at a single center. Bone Marrow Transplant. 2013;48:1010-1012.

9.

Solano C, Giménez E, Piñana JL, et al. Preemptive antiviral therapy for CMV infection in allogeneic stem cell transplant recipients guided by the viral doubling time in the blood. Bone Marrow Transplant. 2016;51:718-721.

10.

Solano C, Giménez E, Piñana JL, et al. Impact of cytomegalovirus DNAemia on overall and non-relapse mortality in allogeneic stem cell transplant recipients. Transpl Infect Dis 2017;19(4).

11.

Giménez E, Torres I, Albert E, et al. Cytomegalovirus (CMV) infection and risk of mortality in allogeneic hematopoietic stem cell transplantation (Allo-HSCT): A systematic review, meta-analysis, and meta-regression analysis. Am J Transplant. 2019;19:2479-2494.

12.

Hill JA, Mayer BT, Xie H, et al. Kinetics of Double-Stranded DNA Viremia After Allogeneic Hematopoietic Cell Transplantation. Clin Infect Dis. 2018;66:368-375.

13.

Lodding IP, Mocroft A, da Cunha Bang C, et al. Impact of CMV PCR Blips in Recipients of Solid Organ and Hematopoietic Stem Cell Transplantation. Transplant Direct. 2018; 4:e355

14.

Clari MÁ, Bravo D, Costa E, et al. Comparison of the new Abbott Real Time CMV assay and the Abbott CMV

PCR Kit for the quantitation of plasma

cytomegalovirus DNAemia. Diagn Microbiol Infect Dis. 2013;75:207-209.

15

15.

Tremblay MA, Rodrigue MA, Deschênes L, et al. Cytomegalovirus quantification in plasma with Abbott RealTime CMV and Roche Cobas Amplicor CMV assays. J Virol Methods. 2015;225:1-3.

16.

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 2017;64:87-91.

17.

Glucksberg H, Storb R, Fefer A, et al. Clinical manifestations of graft-versushost disease in human recipients of marrow

from

HL-A-matched

sibling

donors. Transplantation. 1974;18:295-304. 18

Havlir DV, Bassett R, Levitan D, et al. Prevalence and predictive value of intermittent viremia with combination HIV therapy. JAMA. 2001;286:171-179.

19

Poveda E, Crespo M. Hot News: Impact of Low-level Viremia on Treatment Outcomes During ART - Is it Time to Revise the Definition of Virological Failure? AIDS Rev. 2018;20:71-72.

20

Talaya A, Giménez E, Piñana JL, et al. Spontaneously-resolving episodes of cytomegalovirus DNAemia in allogeneic hematopoietic stem cell transplant recipients: Virological features and clinical

outcomes.

J

Med

Virol.

2019;91:1128-1135. 21.

Meesing A, Germer JJ, Yao JD, Gartner ML, Digmann BJ, Razonable RR. Differences in Duration and Degree of Cytomegalovirus DNAemia Observed with Two Standardized Quantitative Nucleic Acid Tests and Implications for Clinical Care. J Infect Dis. 2019 Aug 31. pii: jiz452.

16

22.

Giménez E, Solano C, Vinuesa V,

et al. Cytomegalovirus DNAemia Burden

and Mortality Following Allogeneic Hematopoietic Stem Cell Transplantation: An Area Under a Curve-Based Investigational Approach. Clin Infect Dis. 2018;67:805-807.

FIGURE LEGENDS Figure 1.

Chart depicting the incidence of different types of CMV DNAemia episodes

throughout

the

study

period.

Allo-HSCT,

allogeneic

hematopoietic stem cell transplant recipients. PET refers to preemptive antiviral therapy.

Figure 2.

CMV DNA loads detected in first PCR-positive plasma specimens according to CMV DNAemia episode type. 17

Figure 3.

Receiver operating characteristics (ROC) analysis to assess the sensitivity and specificity of first positive CMV DNA load thresholds as predictors of blips or prolonged CMV DNAemia episodes. The arrow points to the cut-off value giving the highest combined sensitivity.

18

Figure 4.

Cumulative incidence of recurrent CMV DNAemia following blips, prolonged self-resolving episodes of CMV DNAemia and CMV DNAemia episodes treated preemptively with antivirals.

19

Table 1. Patient demographic and clinical characteristics Parameter

no. of patients (%)

Sex Male

137 (60.9)

Female

88 (39.1)

Underlying disease Acute leukemia

87 (38.7)

Chronic leukemia

25 (11.1)

Lymphoma

77 (34.2)

Myelodysplastic syndrome / Myelofibrosis

28 (12.48)

Multiple Myeloma

8 (3.6)

HLA-matching Matched

128 (56.9)

Haploidentical

71 (31.6)

Mismatched

26 (11.6)

Donor type Related

138 (61.3)

Unrelated

87 (38.7)

Stem cell source

20

Peripheral blood

210 (93.3)

Umbilical cord blood

7 (3.1)

Bone marrow

8 (3.6)

Conditioning regimen Reduced intensity

160 (71.1)

Myeloablative

65 (28.9)

Graft vs. Host Disease prophylaxis Regimen with Cyclophosphamide

96 (42.7)

Regimen with Cyclosporin A

69 (30.7)

Regimen with Sirolimus

60 (26.7)

ATG use prophylaxis Yes

28 (12.4)

No

197 (87.6)

CMV serostatus D+/R+

128 (56.9)

D+/R-

18 (8.0)

D-/R+

61 (27.1)

D-/R-

18 (8.0)

Acute Graft vs. Host Disease

21

Grade 0-I

153 (68.0)

Grade II-IV

72 (32.0)

ATG, anti-thymocyte globulin; CMV, Citomegalovirus; HLA, Human Leukocyte Antigen; D, Donor; R, Recipient.

Table 2. Virological features of CMV DNAemia blips in allogeneic hematopoietic stem cell transplant recipients Parameter Type of CMV DNAemia blip Isolated Blip Blip followed Blip followed Blip occurring by a prolonged by an episode after an initial self-resolving of prolonged episode of episode of CMV prolonged CMV DNAemia CMV DNAemia requiring PET DNAemia Median CMV 33.4 (15.233.4 (15.242.5 (15.233.4 (15.2DNA load in 463.6) 132.2) 389.1) 1,267.6) IU/ml (range) CMV, Cytomegalovirus; PET, preemptive antiviral therapy.

22

Table 3. Patient demographic and clinical characteristics by initial CMV DNAemia episode type Type of CMV DNAemia episode. no. of patients (%) Prolonged

Parameter Blip (n = 47)

Prolonged

CMV

self-resolving

DNAemia

CMV

episode

DNAemia (n

subjected to

= 71)

PET

P value

(n = 69) Sex Male

26 (23.2)

43 (38.4)

43 (38.4) 0.744

Female

21 (28.0)

28 (37.3)

26 (34.7)

Acute leukemia

14 (18.9)

29 (39.2)

31 (41.9)

Chronic leukemia

6 (27.3)

10 (45.5)

6 (27.3)

Lymphoma

16 (26.2)

22 (36.1)

23 (37.7)

Underlying disease

0.670 Myelodysplastic syndrome /

8 (34.8)

7 (30.4)

8 (34.8)

3 (42.9)

3 (42.9)

1 (14.3)

Myelofibrosis Multiple Myeloma HLA-matching

23

Matched

28 (26.2)

42 (39.3)

37 (34.6)

Haploidentical

14 (23.3)

20 (33.3)

26 (43.3)

Mismatched

5 (25.0)

9 (45.0)

6 (30.0)

32 (28.1)

41 (36.0)

41 (36.0)

0.762

Donor type Related

0.502 Unrelated

15 (20.5)

30 (41.1)

28 (38.4)

Peripheral blood

46 (26.1)

69 (39.2)

61 (34.7)

Umbilical cord blood

0 (0.0)

2 (40.0)

3 (60.0)

Bone marrow

1 (16.7)

0 (0.0)

5 (83.3)

39 (28.5)

50 (36.5)

48 (35.0)

Stem cell source

0.080

Conditioning regimen Reduced intensity

0.219 Myeloablative

8 (16.0)

21 (42.0)

21 (42.0)

Graft vs. Host Disease prophylaxis Regimen with

17 (21.8)

32 (41.0) 29 (37.2)

Cyclophosphamide 0.846 Regimen with Cyclosporin A

15 (26.8%)

21 (37.5)

20 (35.7)

Regimen with Sirolimus

15 (28.3%)

21 (39.6)

17 (32.1)

6 (27.3)

10 (45.5)

6 (27.3)

ATG use Yes

24

0.595

No

41 (24.8)

61 (37.0)

63 (38.2)

D+/R+

34 (28.8)

42 (35.6)

42 (35.6)

D+/R-

5 (50.0)

1 (10.0)

4 (40.0)

D/R CMV serostatus

0.042 D-/R+

6 (10.9)

27 (49.1)

22 (40.0)

D-/R-

2 (50.0)

1 (25.0)

1 (25.0)

D+

39 (30.5)

43 (33.6)

46 (35.9)

D-

8 (13.6)

28 (47.7)

23 (39.0)

30 (24.2)

53 (42.7)

41 (33.1)

<0.001

Acute Graft vs. Host Disease Grades 0-I

0.149 Grades II-IV

17 (27.0)

18 (28.6)

28 (44.4)

ATG, anti-thymocyte globulin; CMV, Cytomegalovirus; D, Donor ; HLA, Human Leukocyte Antigen; PET, preemptive antiviral therapy; R, Recipient.

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Table 4. Risk factors for the occurrence of blips as the initial episode of CMV DNAemia in allogeneic hematopoietic stem cell transplant recipients Univariant logistic regression Parameter

OR

95% C.I.

P value

1.28

0.65 – 2.50

0.46

1

-

-

Chronic leukemia

1.60

0.53 – 4.84

0.40

Lymphoma

1.52

0.67 – 3.44

0.31

2.28

0.81 – 6.44

0.11

3.21

0.64 – 16.01

0.15

1

-

-

Haploidentical

0.85

0.41 – 1.79

0.68

Mismatched

0.94

0.31 – 2.82

0.91

Donor type (related vs. unrelated)

1.50

0.75 – 3.03

0.24

Stem cell source (peripheral blood vs. no peripheral blood)

3.53

0.44 – 28.40

0.23

Conditioning regimen (reduce intensity vs. myeloablative)

2.08

0.90– 4.85

0.08

Sex (female vs. male) Underlying disease Acute leukemia

Myelodysplastic syndrome Myelofibrosis

/

Multiple Myeloma HLA-matching Matched

26

Acute Graft vs. Host Disease prophylaxis Regimen with Cyclophosphamide

1

-

-

Regimen with Cyclosporin A

1.31

0.59 – 2.91

0.50

Regimen with Sirolimus

1.41

0.63 – 3.16

0.39

ATG use (no vs. yes)

1.13

0.41 – 3.09

0.80

Donor CMV serostatus (D+ vs. D-)

2.79

1.21 – 6.43

0.01

ATG, anti-thymocyte globulin; C.I., confidence interval; CMV, Cytomegalovirus; D, donor; HLA, Human Leukocyte Antigen; OR, Odds ratio; R, recipient.

27