Cytomegalovirus Viral Load Research Articles

Drug Resistant CMV Infection in Lung Transplantation Patients: Incidence, Characteristics and Clinical Outcomes

Purpose Cytomegalovirus (CMV) infection and development of CMV drug resistance can cause significant morbidity and mortality in lung transplantation (LTX) patients. We investigated the incidence of CMV drug resistance in adult LTX patients and characterized this patient group and its outcomes. Methods We analyzed a single center retrospective cohort of LTX patients who had undergone CMV UL97 and UL54 genotyping for clinically suspected drug resistance. Of the 852 patients who received LTX between January 2012 and May 2018, 27 patients were genotyped, and 11 were confirmed to have drug resistance. Case-matched controls (3 control patients for each resistant patient) were identified by matching for CMV serostatus, development of CMV disease or significant CMV viremia (over 3000 IU/ml), and transplantation date. Results The cumulative incidence of drug resistant CMV was 1.29% (11/852). Altogether 27 patients were tested for drug resistance and 40.7% (11/27) of strains were resistant, 29.6% (8/27) sensitive, and 29.6% (8/27) inconclusive. The median follow-up time was 630 days (range 100-1950 days). Most patients (7/10) who developed resistant CMV infection were seronegative recipients who received a lung from a CMV seropositive donor. Comparison of characteristics and clinical outcomes between resistant CMV patients and control patients is provided in Table 1. No differences in immunosuppression, acute rejection, or pre-transplant sensitization were seen between groups. The peak CMV viral load and duration of CMV viremia were significantly higher in the resistant group. The resistant group had a significantly increased overall mortality after onset of viremia compared to the controls. Conclusion We conclude that drug resistant CMV infection is rare but patients who develop it have decreased overall survival. Risk factors for developing resistant CMV infection were peak CMV viral load and duration of CMV viremia.

Evaluation of four commercial extraction-quantification systems to monitor EBV or CMV viral load in whole blood

BackgroundMeasurement of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) viral loads is commonly used to monitor posttransplant patients. Two new systems (eMAG/eSTREAM and Versant/kPCR) have been recently commercialized. ObjectivesTo evaluate the performance of four systems to quantify CMV and EBV in whole blood. Study designThree extraction and real-time PCR amplification systems: m2000SP/RT (M2000), eMAG/eSTREAM (EMAG), and Versant/kPCR (KPCR) were compared with our routine system Qiasymphony/RGQ (QS/RGQ). The 4 systems were tested using 3 dilutions in triplicate according to the WHO international standard (WHO-IS) for intra-assay reproducibility; 56 whole blood samples (24 patients, 4 follow-ups) for CMV and 45 samples (27 patients, 3 follow-ups) for EBV. ResultsFor CMV, the mean of the WHO-IS (expected value: 4.7 Log IU/ml) was: QS/RGQ=4.84, M2000=4.61, EMAG=4.33, and KPCR=4.79. One patient (10 samples) presented a major underquantification by QS/RGQ. Of the 46 remaining samples, 41 were quantified with QS/RGQ, 43 with M2000, 33 with EMAG and 24 with KPCR. For EBV, the mean of the WHO-IS was: QS/RGQ=4.70, M2000=4.61, EMAG=4.62, and KPCR=4.57. Among the 45 samples, 43 were quantified with QS/RGQ, 39 with M2000, 40 with EMAG and 32 with KPCR. ConclusionThe results obtained with the WHO-IS were very good. The results of patients' samples were well correlated with the announced sensitivity of each system. The elevated threshold of the KPCR CMV assay may be problematic for the follow-up of highly immunocompromised patients who require early introduction of treatment.

Relationship of Ganciclovir Therapeutic Drug Monitoring with Clinical Efficacy and Patient Safety.

The clinical utility of ganciclovir therapeutic drug monitoring (TDM) is unknown. We retrospectively analyzed adult patients treated for cytomegalovirus (CMV) infection with ganciclovir with TDM between 2005 and 2015. The primary outcome was an association between ganciclovir TDM and clinical efficacy endpoints within 30 days, defined by viral load and symptomatology. Secondary outcomes included safety endpoints, evaluated within 7 days of the last administered dose of ganciclovir. Of 175 patients evaluated, 82 patients with CMV infection were included in our analysis with a median (interquartile range) baseline CMV viral load of 5,500 (3,000 to 15,200) copies/ml. The majority achieved undetectable or reduced CMV viral load below the lower limit of quantification (74.4%) with improvement in symptomatology (70.7%) at 30 days. Among patients with detectable CMV viremia at 30 days, the viral load had declined to a median of 1,000 (1,000 to 3,090) copies/ml. We did not observe significant associations between the efficacy outcomes and ganciclovir trough (P = 0.20 and P = 0.20, respectively) or peak concentrations (P = 0.14 and P = 0.14, respectively). Similarly, there was no significant association between ganciclovir trough or peak concentrations and safety endpoints, including leukopenia (P = 0.48 and P = 0.69), neutropenia (P = 0.59 and P = 0.69), thrombocytopenia (P = 0.29 and P = 0.37), anemia (P = 0.51 and P = 0.35), nephrotoxicity (P = 0.41 and P = 0.57), and neurotoxicity (P = 0.22 and P = 0.48). We did not observe any associations between ganciclovir TDM and clinical efficacy or safety endpoints. Routine ganciclovir TDM may be of limited value. Future studies may be warranted to identify specific populations with unpredictable pharmacokinetic and pharmacodynamics profiles in whom ganciclovir TDM may be of benefit.

Letermovir for Secondary Cytomegalovirus (CMV) Prophylaxis in a Pediatric Stem Cell Transplant Patient

Patients who undergo stem cell transplants are at a significant risk of infections. Cytomegalovirus (CMV) is an organism of particular concern in this population. Current treatments options for CMV include ganciclovir, valganciclovir, foscarnet, and cidofovir. These treatment options have adverse effects such as myelosuppression or nephrotoxicity, making them less than ideal as a treatment options. Increasing rates of drug resistance is another concern. The need for alternative treatment options is paramount. Many patients require secondary prophylaxis. Letermovir is a newly approved CMV DNA terminase complex inhibitor indicated for prophylaxis of CMV infection and disease in adults. Its role in secondary prophylaxis has not been clearly defined. We report the case of an 11-year old male who had undergone a 4/6 HLA-matched cord blood transplant for Fanconi Anemia. The patient developed CMV viremia that later progressed to CMV colitis and pneumonitis. He was initially treated with ganciclovir then was found to have UL97 mutation. He was then switched to foscarnet for induction therapy, to which the patient showed a good response. The patient was subsequently transitioned to cidofovir for maintenance therapy where CMV viremia recurred. Foscarnet was re-initiated with good response then transitioned to cidofovir maintenance therapy. Unfortunately, CMV viral load began to increase prompting a change to high-dose ganciclovir. Due to concerns for myelosuppression with using high-dose ganciclovir he was switched back to foscarnet. While on foscarnet the patient had nephrotoxicity and electrolyte disturbances and the decision was made to re-initiate high-dose ganciclovir. When the patient's CMV viral load was undetectable the patient was transitioned to letermovir 480 mg daily with successful suppression of CMV viral load. Letermovir has been well tolerated and the patient has experienced no adverse effects associated with it. The patient has not required further therapy for CMV viremia since starting letermovir. Letermovir prophylaxis was stopped after four months of therapy. The patient's CMV viral load began to rise after stopping for one month with the peak at 381 copies/mL. After restarting letermovir the CMV viral load became undetectable and has remained so. Letermovir could be useful in secondary prophylaxis in pediatric patients with ganciclovir-resistant CMV who are at risk of recurrences. Further investigation is warranted for the use of letermovir in the pediatric population.

Efficacy and Pharmacoeconomic Impact of Letermovir for CMV Prophylaxis in Allogeneic Hematopoietic Cell Transplant Recipients

Cytomegalovirus (CMV) seropositivity and early CMV reactivation after hematopoietic-cell transplantation (HCT) remain associated with increased mortality. Those at high risk include CMV seropositive recipient/donor pairs (CMV R+ or D+) and those on escalated immunosuppression (prednisone ≥ 20mg/kg) for graft versus host disease (GVHD). Historically, our institutional CMV reactivation rate (RR) was 63% in our high risk cohort, similar to national average. Prophylaxis with letermovir, a new CMV-antiviral therapy, significantly reduced the incidence of CMV reactivation and viremia in a prospective trial (Marty FM, et al. NEJM 2017). The purpose of this study was to assess CMV reactivation, defined as CMV viral load > 500 IU/mL, with letermovir prophylaxis and its pharmacoeconomic impact in an outpatient transplant program. This was a retrospective review of adult HCT patients receiving letermovir from December 2017-August 2018 at Vanderbilt University Medical Center. Use of letermovir was standardized at a program level. Therapy was used in high risk patients, undergoing HCT with matched-unrelated donor (MUD), cord blood donor or haploidentical donor transplants with no active CMV reactivation prior to initiation and no anti-CMV treatment post-HCT. Primary endpoint compared CMV RR to historical controls utilizing a two-sided t-test. Secondary outcomes evaluated logistics and cost impact. 30 patients met entry criteria (26 CMV R+, 21 D+), RR was significantly lower than our historical control (20% vs 63%; p = 0.0003). Median time to reactivation was 38 days post-HCT (29-58). 4 of the 6 patients with reactivation received induction therapy with ganciclovir (50%), valganciclovir (25%), or letermovir (25%, peak viral load 740 IU/mL) for ∼40 days (31-52). All were CMV R-/D+ with AML and received HCT from MUD (75%) or haploidentical donors (25%). The remaining 2 patients (CMV R+/D+) had low level reactivation (137-500 IU/mL) and continued letermovir through day 100 without additional CMV treatment. Patients started letermovir by day 14 post-HCT (5-65) with a median 83 days (7-158) of prophylaxis. 3 patients received letermovir as secondary prophylaxis due to prior CMV reactivation or escalated immunosuppression for GVHD. Of those patients, reactivation occurred in 1 patient (AML MUD transplant; CMV D-/R+) who had 2 reactivations while on foscarnet therapy. No letermovir resistance was observed. Letermovir copay was between $10 and $360/month. 76% of prescriptions required prior authorization, 19% monetary grant, 18% patient assistance. Majority had private insurance (49%) and Medicare/Medicaid (36%) with a median of 3 days for approval and 5 days from prescription submission to patient medication pick-up. Primary prophylaxis with letermovir is associated with significantly lower RR compared to historical controls and is financially feasible with dedicated pharmacy services.

Impact of Post-Transplant High-Dose Cyclophosphamide and Donor Sources on CMV Reactivation.

Introduction Cytomegalovirus (CMV) reactivation remains a common cause of morbidity and mortality following allogeneic hematopoietic cell transplant (HCT) despite attempts at prophylaxis. Following transplantation, surveillance of the peripheral blood CMV viral load with early aggressive treatment is commonly employed and has been an effective strategy at preventing overt infection. While a matched related donor (MRD) is often the preferred transplant type, the use of alternative donors including matched unrelated donors (MUD), mismatched unrelated donors (MMUD) and haploidentical (HI) transplants, has greatly increased over the past several years. The data regarding CMV infection among these alternative donor types is scarce. Objectives To better characterize the extent and risk factors for CMV reactivation among patients who received an allogeneic HCT for hematologic malignancies, particularly among HI transplant recipients and those who received post-transplant high-dose cyclophosphamide (PTCy) for graft-versus-host disease (GVHD) prophylaxis. Methods A retrospective analysis was performed on 88 adults with hematologic malignancies who received an allogeneic HCT at our institution from 2012-2017. Only cases with CMV seropositive donors and/or recipients were included. We compared donor types/sources, conditioning regimens, GVHD prophylaxis regimens, and assessed for CMV reactivation utilizing the peripheral blood polymerase chain reaction (PCR) values that are monitored in post-transplant clinic visits. Results Nineteen (22%) patients received MRD transplants, while the remainder received alternative donor sources, including 17 (19%) HI transplants. Overall, the rate of CMV reactivation was 27% with a significant difference noted between donor sources (p= Conclusion HI HCT transplant recipients are at high risk for CMV reactivation. The use of myeloablative conditioning regimens or PTCy did not affect the rate of CMV reactivation.

Cytomegalovirus (CMV) Pentameric Complex (PC) Neutralizing Antibodies (nAb) from a Randomized, Controlled Trial of CMV-Specific Intravenous Immunoglobulin (IVIG) for the Prevention of Primary CMV Infection after Hematopoietic Cell Transplantation (HCT)

Prevention of CMV disease after HCT remains a challenge. In addition to antivirals, immune-based strategies such as active and passive immunization may have potential roles preventing infection. CMV entry into epithelial and endothelial cells requires distinct viral proteins including UL128, UL130, UL131A, gB and gH/gL that combine to form a PC. A nAb assay was developed by Gerna et al. that measures nAb activity against PC, although its clinical application in HCT patients has not been described. In 1991, Bowden et al. conducted a study where CMV IVIG was administered as prophylaxis to D+/R- patients early after allogenic HCT. Although it showed reduced CMV infection in CMV IVIG recipients, CMV disease was not reduced thus its use was never adopted. This impact on infection suggests the possibility that CMV IVIG inhibits infection via neutralization of an unidentified entry pathway that may include PC. From the original cohort, we tested 538 serum samples from 61 patients using CMV DNA polymerase chain reaction (PCR) to compare clinically significant viral load (≥ 4 log10 IU/ml) or histopathologically detected CMV between groups. We also used a CMV nAb assay to evaluate anti-PC neutralizing capacity. The assay uses retinal pigmented epithelial (ARPE-19) cells exposed to serial dilutions of serum combined with a laboratory CMV strain, BADrUL131-Y4, that contains a green fluorescent protein (GFP) cassette. After one hour incubation, cells are washed and incubated for one week. Fluorescence is measured and we construct curves that plot viral neutralization against serum dilution and from these we extrapolate an IC-50; that is the dilution where 50% virus neutralization is achieved. After excluding three patients infected prior to day 21 post-transplant, there were 27 CMV IVIG and 31 control patients. Neither cumulative incidence of CMV infection (Figure 1, p=0.55), nor IC-50 values (Figure 2, p=0.19) differed by study arm, though observed median IC-50 values were higher in the CMV IVIG arm (5.8) than in the control arm (4.8). We found no association between IC-50 and risk of CMV infection (HR=1.8, 95% CI 0.6-5.3, p=0.31). Using modern CMV detection methods, our study suggests CMV IVIG does not result in decreased CMV viral load. Although the PC nAb composition of the CMV IVIG used is unknown, it did not appear protective against CMV infection. We successfully evaluated CMV PC entry-specific neutralizing activity in the HCT setting using a nAb assay, however evaluation among larger cohorts is needed to determine its clinical utility.

Determination of Optimal Viral Kinetic Markers for Predicting Antiviral Treatment Effect for the Prevention of Cytomegalovirus (CMV) Disease after Hematopoietic Cell Transplant (HCT) Using Machine Learning and a Novel Non-Parametric Estimation Method

The United States Food and Drug Administration recently issued guidance that CMV DNAemia (quantitative CMV viral load as measured by PCR) could be used in a composite endpoint along with tissue-invasive CMV disease as the primary endpoint in allogeneic HCT CMV prophylaxis trials. However, the specific time points after antiviral treatment initiation at which to measure viral load and the viral load thresholds that most accurately predict clinical CMV disease have not been identified. In order to estimate the treatment effect of an antiviral agent on CMV clinical outcomes, placebo-controlled, randomized trial data with accompanying viral load measurements are needed. Unfortunately, in the modern era of PCR and early preemptive treatment, ethically, placebo-controlled, randomized trials that allow for the development of CMV disease after HCT can no longer be performed. To overcome this limitation, we performed CMV DNA PCR testing on frozen plasma samples collected during the first 100 days post-HCT in the only placebo-controlled, double-blind RCT of ganciclovir for the early treatment of CMV infection after HCT (Goodrich et al. NEJM 1991). We used three analytic methods (Cox proportional hazards models, machine learning, and a novel non-parametric method) to determine which of 14 viral load markers, measured in the first four weeks of treatment, are the most useful surrogates of antiviral treatment for the prevention of CMV disease. All methods identified peak viral load and percentage of positive samples by week 4 as highly associated with or predictive of tissue-invasive CMV disease by week 8 post-randomization. Cox proportional hazards models (Figure 1) yielded significant associations for peak viral load measured by week 4 (HR 1.4, 95% CI 1.1-1.9) and percentage of positive samples by week 4 (HR 1.6, CI 1.1-2.3). The machine-learning ensemble algorithm SuperLearner implemented in R (Figure 2) demonstrated cross-validated area under the receiver-operator curves of at least 75% for peak viral load (75%, CI 62-87%) and percentage of positive plasma samples (77%, CI 63-90%) measured by week 4. Using a novel statistical technique that allows direct estimation of biomarker surrogacy (Parast et al. Stat Med 2017), we estimated that the percentage of ganciclovir treatment effect explained by peak viral load and percentage of positive samples measured by week 4 was greater than 85%—peak viral load 89%, CI 56-100%; percent positive samples 91%, CI 60-100% (Figure 3). Peak viral load and percentage of positive samples by week 4 are highly predictive of CMV disease and the treatment effect of ganciclovir. Our analysis suggests that these may be the best surrogate markers of ganciclovir treatment and introduces novel methods for identifying optimal candidate biomarkers as surrogates for clinical endpoints.

Prophylactic Pretransplant Ganciclovir to Reduce Cytomegalovirus Infection after Hematopoietic Stem Cell Transplantation

Introduction Cytomegalovirus (CMV) reactivation remains a serious complication after allogeneic hematopoietic stem cell transplantation (HCT) occurring in 35-55% of HCT recipients. CMV reactivation can lead to adverse outcomes including end-organ damage and graft failure. Objective To reduce the incidence of CMV reactivation and disease by using prophylactic ganciclovir pretransplant. Methods We conducted a retrospective analysis of all recipient or donor CMV seropositive patients who received their first allogeneic HCT between 2012 and 2017. Ganciclovir was administered pretransplant (5 mg/kg twice daily IV from day −8 to day −2). Patients received valacyclovir or acyclovir starting day 0 until one-year posttransplant. Patients were monitored weekly with serum CMV PCR through Day 100 posttransplant. CMV reactivation was defined as a CMV viral load of more than 135 IU/ml, and was treated with preemptive therapy. All patients received GVHD prophylaxis with calcineurin inhibitor. Results Seventy-nine consecutive patients were treated. The median age was 58 years (range 20 to 72). The most common diagnoses were acute myeloid leukemia (49%) and acute lymphocytic leukemia (14%). Graft sources were matched related donor (30%), matched unrelated donor (35%), haploidentical (5%) and cord blood (29%). 43 patients (55%) received myeloablative conditioning. 36 patients (45%) received fludarabine based reduced intensity/nonmyeloablative conditioning. 24 patients (30%) had CMV reactivation with median time of reactivation of 47 days posttransplant (range 27 to 229). 22 out of the 24 (88%) patients required treatment for CMV reactivation with a median treatment duration of 37 days (range 14 to 315) and were treated with ganciclovir or foscarnet. The cumulative incidence of CMV reactivation at day 100 posttransplant was 27% with a 95% CI (18%-37%) depicted in Figure 1. There were no patients with biopsy proven CMV disease or deaths attributed to CMV reactivation or disease. The median time-to neutrophil recovery (≥500 K/ul) was 18 days and the median time to platelet recovery (≥20,000 K/ul) was 19 days. The incidence of acute GVHD Grades II-IV was 25 %. The incidence of significant kidney injury (Creatinine >2.5 mg/dL) was 2.4%. Conclusions The incidence of CMV reactivation, in this high-risk patient population, by day 100 of 27% with pretransplant ganciclovir may be improved compared to historical controls of 35-55%. The use of pretransplant ganciclovir was associated with no CMV disease and was safe, with low incidence of kidney damage. These data suggest that pretransplant ganciclovir with preemptive therapy for viral reactivation should be considered regardless of graft source. Future prospective randomized trials are needed to evaluate strategies for CMV prophylactic regimens.

Cytomegalovirus viral load parameters associated with earlier initiation of pre-emptive therapy after solid organ transplantation.

BackgroundHuman cytomegalovirus (HCMV) can be managed by monitoring HCMV DNA in the blood and giving valganciclovir when viral load exceeds a defined value. We hypothesised that such pre-emptive therapy should occur earlier than the standard 3000 genomes/ml (2520 IU/ml) when a seropositive donor transmitted virus to a seronegative recipient (D+R-) following solid organ transplantation (SOT).MethodsOur local protocol was changed so that D+R- SOT patients commenced valganciclovir once the viral load exceeded 200 genomes/ml; 168 IU/ml (new protocol). The decision point remained at 3000 genomes/ml (old protocol) for the other two patient subgroups (D+R+, D-R+). Virological outcomes were assessed three years later, when 74 D+R- patients treated under the old protocol could be compared with 67 treated afterwards. The primary outcomes were changes in peak viral load, duration of viraemia and duration of treatment in the D+R- group. The secondary outcome was the proportion of D+R- patients who developed subsequent viraemia episodes.FindingsIn the D+R- patients, the median values of peak viral load (30,774 to 11,135 genomes/ml, p<0.0215) were significantly reduced on the new protocol compared to the old, but the duration of viraemia and duration of treatment were not. Early treatment increased subsequent episodes of viraemia from 33/58 (57%) to 36/49 (73%) of patients (p< 0.0743) with a significant increase (p = 0.0072) in those episodes that required treatment (16/58; 27% versus 26/49; 53%). Median peak viral load increased significantly (2,103 to 3,934 genomes/ml, p<0.0249) in the D+R+ but not in the D-R+ patient subgroups. There was no change in duration of viraemia or duration of treatment for any patient subgroup.InterpretationPre-emptive therapy initiated at the first sign of viraemia post-transplant significantly reduced the peak viral load but increased later episodes of viraemia, consistent with the hypothesis of reduced antigenic stimulation of the immune system.

Correlation of Cytomegalovirus (CMV) Disease Severity and Mortality With CMV Viral Burden in CMV-Seropositive Donor and CMV-Seronegative Solid Organ Transplant Recipients.

BackgroundThe rate of cytomegalovirus (CMV) viral load increase and peak viral loads are associated with CMV disease in kidney and liver transplant recipients, but relationships to disease severity or mortality have not been shown.MethodsUsing stored serial serum specimens from renal (n = 59) and liver (n = 35) transplant recipients (D+R-; CMV-seropositive donors, CMV-seronegative recipients) from 2 prospective, randomized, controlled, interventional prophylaxis trials of CMV immune globulin (CMVIG), CMV viral load was measured using the COBAS quantitative polymerase chain reaction assay and the World Health Organization CMV standard. Patients with severe CMV-associated disease were classified according to trial definitions. Pairwise comparisons of mean viral load among deceased, surviving diseased, and nondiseased patients were analyzed by 2-way analysis of variance. To determine if viral load could predict mortality, receiver operating characteristic (ROC) curves were constructed using area under the curve (AUC) of the viral load and peak viral concentration (Vmax).ResultsViral load (mean log10 [AUC], peak viral load [Vmax]) for patients with severe CMV disease was significantly higher compared with nondiseased patients (P < .001). Similarly, higher viral burden was significantly associated with mortality (P < .001). Viral load AUC and Vmax AUROCs for predicting mortality were 0.796 and 0.824, respectively, for renal patients, and 0.769 and 0.807, respectively, for liver patients.ConclusionsUsing specimens from studies preceding the antiviral prophylaxis era, CMV viral load was associated with severe CMV disease and death, supporting CMV viral load quantification as a proxy for CMV disease severity and disease-associated mortality end points in solid organ transplantation.

Monitoring of cytomegalovirus infection in non-transplant pediatric acute lymphoblastic leukemia patients during chemotherapy.

Cytomegalovirus (CMV) infection is a significant cause of morbidity and mortality in the posttransplant setting; however, it is increasingly recognized in pediatric leukemia during chemotherapy. This study assessed the prevalence and associated factors of CMV infection in pediatric non-transplant leukemia patients.This was a cross-sectional study of 50 pediatric acute lymphoblastic leukemia (ALL) patients receiving chemotherapy at Ramathibodi Hospital from December 2015 to December 2016. CMV viral load quantified by DNA polymerase chain reaction (PCR) was monitored in different phases of chemotherapy: enrolment, post-induction, post-consolidation, post-intensification, and maintenance.One hundred forty one blood tests were evaluated from 50 patients. Overall prevalence of CMV DNAemia (≥20 copies/mL) and high-level CMV DNAemia (≥1000 copies/mL) was 52% (26 of 50) and 16.0% (8 of 50), respectively. All patients with high-level CMV DNAemia were in the maintenance phase of chemotherapy. One patient had CMV retinitis, while the rest had no end-organ CMV diseases. Increased lymphocyte count was significantly associated with protection from high-level CMV DNAemia (odds ratio 0.997, P = .02). Receiver operating characteristic curve identified a cut-off value of 798 cells/mm3 of absolute lymphocyte count (ALC) as a discriminator for the presence of high-level CMV DNAemia (area under the curve 0.756, 95% CI 0.645–0.867, P = .001) with 88.9% sensitivity and 50.4% specificity.CMV infection predominantly occurred during maintenance chemotherapy. Low ALC was significantly associated with high-level CMV DNAemia. CMV infection surveillance by quantitative CMV DNA PCR during maintenance chemotherapy in patients with ALC <800 cells/mm3 may be considered.

Congenital Cytomegalovirus Infection Presenting as Pneumonia with Respiratory Distress and Thrombocytopenia

Human Cytomegalovirus (CMV) is a member of Herpes viridae family, that affects most of the human population at some stage of live and is the most common congenital infection causing sensorineural hearing loss and neurodevelopmental delay in newborn. CMV infection may be acquired in a newborn congenitally or after delivery, but except for the congenital infection, other mode of infection rarely result in significant symptoms or sequel in them. Clinical findings of congenital CMV infection include IUGR, hydrops, generalized petechiae, purpura, thrombocytopenia, jaundice, hepatosplenomegaly, pneumonitis, microcephaly, periventricular calcifications, seizures, chorioretinitis, sensorineural hearing loss, bone abnormalities, abnormal dentition, and hypocalcified enamel. Here we present a case of congenital CMV infection who presented with Pneumonia with Respiratory distress and thrombocytopenia. A single, live, term, female child delivered at home developed low grade fever, cough and increased oral secretion at 28 day of life and was initially diagnosed as Pneumonia with respiratory distress with thrombocytopenia. Mother and child, both were investigated for TORCH infection which came out to be positive for CMV infection (child’s serum CMV Ab IgM 94 U/ml). Her CMV Viral Load Real Time PCR tested positive with 56380 copies/ml.

A Step Forward Towards Cytomegalovirus Free Hematopoietic Stem Cell Transplantation

Introduction: Cytomegalovirus (CMV) reactivation remains one of the most serious complications after allogeneic hematopoietic stem cell transplantation (HSCT) occurring in up to 30-50% of HSCT recipients. CMV reactivation can lead to adverse outcomes including end-organ damage and graft failure. Prevention of CMV infection may improve outcomes of HSCT recipients; however, CMV reactivation can still occur in HSCT recipients despite receiving prophylactic acyclovir. In this study, we used prophylactic ganciclovir pre-transplant to reduce the incidence of CMV reactivation and CMV disease.Methods: To reduce the incidence of CMV reactivation and disease, ganciclovir was administered before transplantation (5 mg/kg twice daily intravenously from day −8 to day −2) for all donor and recipient seropositive allogeneic HSCT. This was followed by high dose valacyclovir or acyclovir starting day 0 until one year post-transplant. Patients were monitored weekly with serum CMV PCR through Day 100 post transplantation. Preemptive therapy was started for an elevated CMV viral load. CMV reactivation was defined as a CMV viral load of more than 135 IU/ml. Over the course of the analysis period, two different PCR methods were used for CMV viral load; since 2015 Roche Cobas AmpliPrep/Cobas TaqMan CMV test was implicated. CMV disease was defined as detection of CMV by one of the following diagnostic tests including: culture, immunohistochemistry staining, or histopathology examination accompanied by documentation of disease signs and symptoms of the affected organ. Statistical analysis was performed using SAS version 9.4. Logistic regression models were explored to assess the association of age, graft source, and disease type on CMV reactivation. We performed a retrospective analysis of all recipient or donor CMV seropositive patients who received their first allogeneic HSCT at the University of Virginia between 2012 and 2017.Results: Seventy-nine consecutive patients were treated. The median age was 58 years (range 20 to 72). The most common diagnoses were acute myeloid leukemia (n=39, 49%) and acute lymphocytic leukemia (n=11, 14% ). Graft sources were matched related donor, (n=24, 30%), matched unrelated donor, (n=28, 35%), haploidentical (n=4, 5%) and cord blood (n=23, 29%). 43 patients (55%) received myeloablative (CyTBI, BuCy and FluBu) conditioning. 36 patients (45%) received reduced intensity/nonmyeloablative conditioning (Flu/Cy/TBI+/- ATG, Flu/Cy/ATG ,Flu Mel, Flu Bu and Cy ATG). All patients received calcineurin inhibitor based prophylactic immunosuppressive therapy for GVHD prevention.24 patients (30%) had CMV reactivation with median time of reactivation of 47 days post-transplant (range 27 to 229). 22 out of the 24 (88%) patients required treatment for CMV reactivation with a median treatment duration of 37 days (range 14 to 315). Patients were treated with either ganciclovir or foscarnet, as clinically indicated. The cumulative incidence of CMV reactivation at day 100 post-transplant was 27% with a 95% CI (18%-37%). The median highest viral load was 2130 copies/ml (range 151 to 3,250,000 copies/ml). There were no patients with biopsy proven CMV disease. There were no deaths attributed to CMV reactivation or disease.The median time-to neutrophil recovery (ANC > 500 k/uL) was 18 days and the median time to platelet count greater than 20,000 k/ul unsupported was 19 days. The incidence of acute GVHD (Grades II-IV) was 25 %. The incidence of significant acute kidney injury defined by serum creatinine of more than 2.5 mg/dL was 2.4 %. 1 year overall survival estimate was 54% with 95% CI (42-65%). In multivariate analysis, patients who received cord blood transplants, were approximately 4 times more likely to have a CMV reactivation (Odds Ratio 3.9 with a 95% CI(1.4-10.9)), p= 0.01, than those who did not.Conclusions:The incidence of CMV reactivation by day 100 of 27% with pre-transplant ganciclovir may be improved compared to historical controls of 30-50%.The use of pre-transplant ganciclovir was associated with no CMV disease, in this single center study.The use of pre-transplant ganciclovir is safe, with low incidence of kidney damage.These data suggest that pre-transplant ganciclovir with Preemptive therapy for viral reactivation should be considered regardless of graft source. Future prospective randomized trials are needed to evaluate strategies for CMV prophylactic regimens. DisclosuresNo relevant conflicts of interest to declare.

Protective Effects of Higher Cytomegalovirus Virus Load in Patients with Acute Myeloid Leukemia after Umbilical Cord Blood Transplantation

Objective: The aim is to explore the potential relationship between the virus load of Cytomegalovirus (CMV)infection and disease relapse in acute myeloid leukemia (AML)patients after Umbilical cord blood transplantation (UCBT), The mechanism of interaction in which lymphocyte subsets may be involved is also analyzed.Design and method: All 126 patients with acute myeloid leukemia receiving a single UCB graft at the institution between Oct 2010 to Dec 2017 were enrolled in this retrospective study and engraft failure and death within 100 days excluded. All patients received common transplant processing: intensive myeloablative conditioning without ATG, Cyclosporine (CSA) and mycophenolate mofetil (MMF) for GVHD prophylaxis. All patients had negative plasma CMV DNA prior to the transplantation and examined twice a week for at least 100 days after transplantation. CMV were measured by Quantitative real-time PCR using TaqMan (ABI)-based method. Patients were divided into three groups according to the CMV viral load within 100 days: CMV-negative groups (no relevant special treatment), <1,000/mL CMV DNA copy group (reduced immunosuppressor and close follow up) and >= 1,000 / mL CMV DNA copy group (reduced immunosuppressor, antiviral treatment and close follow up), the first group was merged into the second group due to the 92% incidence of CMV infection after UCBT .To compare the two regimens which aimed to focus on disease relapse ,non-relapse mortality (NRM),overall survivor (OS),disease free survival(DFS) ,GVHD-free /relapse-free survivor (GRFS)and graft versus host disease (GVHD) after transplantation for two years. Also, retrospectively compared the lymphocyte ratio recovery at different time points after transplantation .Results: Patients characteristic had no significant differences between high CMV virus load (>=10^3/mL) group and low CMV virus load(<10^3/mL) group. There were 63 males and 63 females with a median age of 13 years(range,1-52) and a median weight of 40 Kg (range,11-87) .The median follow-up time among survivors was 736 days (range, 100-2184).Univariate and multivariate analysis showed, high CMV load group has lower two-year cumulative incidence of relapse [(2.9% vs. 17.9%, P=0.017); adjusted HR 0.19, P=0.045], higher cumulative incidence of DFS [(88.3% vs. 63.2%, P=0.003), adjusted HR0.28, P=0.005],higher cumulative incidence of OS[(88.0% vs. 72.3%, P=0.037),adjusted HR0.35,P=0.027].The immune reconstruction data of the two groups showed that the median value of CD8+ T cells in the high CMV load group at 1, 4, 6, 9, 12, 15, 18 and 24-36 months after transplantation was higher than the low CMV load Group (30.3% vs. 13.9%, 46.8% vs. 27.6%, 46.5% vs. 27.9%, 37.0% vs. 31.5%, 37.7% vs. 20.9%, 42.3% vs. 22.8%, 30.4% vs. 23.5%, 29.1% Vs.22.1%; P=0.032, P=0.014, P=0.01, P=0.621, P=0.005, P=0.008, P=0.088, P=0.371).Conclusion: Higher CMV load within 100 days after UCBT presents lower relapse,improved OS and DFS in AML patients. At the same time, the proportion of CD8+ T cells increased significantly in patients with high CMV load after 1, 4, and 6 months after transplantation. The above data indicates that CMV virus infection may stimulates the activation of CD8+ T cells further induces a GVL effect.keywords:Cytomegalovirus,acute myeloid leukemia,cord blood transplantation,virus load, relapse. DisclosuresNo relevant conflicts of interest to declare.

Early CMV Replication-Associated Anti-Leukemia Effect Is Related to the Reconstitution of Specific Subsets of Natural Killer Cells after Haploidentical Stem Cell Transplantation in Acute Leukemia

Background: Cytomegalovirus (CMV) infection is a major infectious complication after allogeneic hematopoietic cell transplantation. Recently, it has been reproducibly demonstrated that CMV reactivation is associated with decreased relapse rate in patients with acute myeloid leukemia (AML). The aim of this study is to evaluate the impact of early CMV reactivation on the incidence of disease relapse in relation to the reconstitution of subsets of natural killer (NK) cells after haploidentical stem cell transplantation (haploSCT) in acute leukemia.Methods: Clinical data from 47 adult patients diagnosed with acute leukemia who underwent their first haploSCT between September 2009 and December 2017 was retrospectively analyzed. All patients were unable to find a suitable HLA-matched donor in their families or donor registries. Patient blood samples were collected prior to conditioning therapy and following haploSCT at day 30 and day 90. Peripheral blood mononuclear cells obtained from 28 patients abundant cells at every time points were analyzed for flow cytometric immunophenotyping. Expression of specific receptors (NKG2A, NKG2C, NKG2D, DNAM1 and NKp46) on the NK cells (CD56brightCD16- or CD56dim/-CD16+ cells) were serially quantified by multiparametric flow cytometry using appropriate monoclonal antibodies.Results: Median age was 38 years (range, 21-62 years), and 28 (54%) patients were male. Thirty-six (69%) patients received a transplant in their first complete remission (CR) status. Median follow-up duration was 54 months (range, 6.6-83.3 months), and all patients were CMV seropositive before receiving a transplant. Early CMV replication occurred at a median of 23 days after haploSCT in 40 of 47 patients (85%). Among these patients, 14 had more than two episodes of CMV replication throughout the follow-up period. The median peak viral load during CMV replication was 54,000 copies/mL. In univariate analysis, early CMV replication (P < 0.001), older donor age (P = 0.018), high dose of infused T cells (P = 0.022) and chronic graft-versus-host disease (GVHD, P = 0.001) were significantly associated with lower 3-year cumulative incidence of relapse after haploSCT. Early CMV replication was correlated with higher leukemia-free survival (LFS, P < 0.001). In multivariate analysis, early CMV replication (hazard ratio [HR], 0.24; 95% confidence interval [CI], 0.060 to 0.930, P = 0.039) and chronic GHVD (HR, 0.25; CI, 0.089 to 0.695; P = 0.008) were identified as independent factors for higher LFS rate. CMV reactivation was associated with higher overall survival (OS) rates and increased nonrelapse mortality, although there was no statistical significance. The viral load at the initiation of CMV-specific treatment was significantly associated with OS rates. Patients with CMV viral load higher than 45,000 copies/mL had lower OS rate compared to those with lower CMV load (34.5% versus 89.5%, P = 0.022). Longitudinal analysis of NK cell reconstitution after haploSCT showed that the CMV infection was associated with the increased expansion of CD56brightCD16dim/- NK cell, particularly in DNAM1+ NK cell subset. The rate of CD56brightCD16dim/-DNAM1+ NK cells increment was significantly higher in the patients with CMV infection compared with patients without CMV infection (P = 0.022). Importantly, we observed that the cumulative relapse rate was significantly decreased in patients with an increased CD56brightCD16dim/- DNAM1+ NK cells compared to patients with low CD56brightCD16dim/- DNAM1+ NK cells (20.4% versus 63.6% , P = 0.016).Conclusion: Early CMV replication was identified as an independent prognostic factor for LFS in acute leukemia in the haploSCT setting. Increased reconstitution of CD56brightCD16dim/- DNAM1+ NK cells was associated with CMV infection-related reduction in the relapse rate. Further studies are required to elucidate the anti-leukemia effects of these NK cell subsets associated with CMV infection in haploSCT. DisclosuresKim:Novartis Korea: Honoraria.

Comparison of intravitreal ganciclovir monotherapy and combination with foscarnet as initial therapy for cytomegalovirus retinitis.

To compare the effectiveness between multiple intravitreal injections of ganciclovir alone and combined with foscarnet as initial treatment for patients with newly-onset cytomegalovirus retinitis (CMVR). The retrospective study observed 37 patients (58 eyes) who suffered from CMVR onset between 2013 and 2015. Among them, 35 eyes underwent 4 weekly intravitreal injections of 3.0 mg ganciclovir, and 23 eyes underwent 4 weekly injections of 3.0 mg ganciclovir combined with 2.4 mg foscarnet. Visual acuity, intraocular pressure and viral load of cytomegalovirus (CMV) in aqueous humor measured by real-time polymerase chain reaction were compared before and after each injection. CMV-DNA copies in aqueous humor decreased remarkably in both groups. The average of CMV-DNA copies in patients' aqueous decreased from 38.3×104 copies/mL at baseline to 2.2×104 copies/mL after the 4th injection in patients who were treated with ganciclovir monotherapy, and decreased from 76.9×104 copies/mL to 11.3×104 copies/mL after 4 continuous injections of ganciclovir combined with foscarnet. No significant difference was found in reduction of viral load, change of visual acuities or intraocular pressures between monotherapy or combined therapy. Results of this study show that the initial effectiveness of treating CMVR after 4 weekly intravitreal injections is not significantly different from ganciclovir alone or combined with foscarnet. Continuous injection of ganciclovir alone is sufficient in treating immunosuppressive patients with newly-onset CMVR.

A Rare Case of Cytomegalovirus Colitis in an Immunocompetent Patient Mimicking Clostridium difficile Colitis

Cytomegalovirus (CMV) colitis usually occurs in immunocompromised patients and can cause significant morbidity and mortality. We report a 68 year old immunocompetent male presenting with abdominal pain and diarrhea who was found to have CMV colitis. 68 year old african american male with a history of end stage renal disease on hemodialysis presented with a one month history of watery diarrhea, diffuse abdominal pain, and weakness. Patient reported a 15-lb weight loss. No blood or mucus seen in the stool. He denied nausea or vomiting, dietary changes, international travel, sick contacts, or recent antibiotic use. Physical examination revealed tachycardia, dry mucous membranes, and a normal abdominal exam. Laboratory data was only remarkable for electrolyte abnormalities: sodium 150 mg/dL, potassium 2.1 mg/dL, and phosphorus 1.1 mg/dL. The patient received intravenous fluids and his electrolytes were corrected. CBC, TSH, urinary 5-HIAA, and celiac panel were normal. Fecal leukocytes, clostridium difficile toxin PCR, rotavirus ELISA, bacterial cultures, giardia, and cryptosporidium antigen were negative. An abdominal computed tomography was concerning for an infectious or inflammatory colitis in the descending and sigmoid colon. ESR, CRP, and fecal calprotectin were elevated. Patient underwent a colonoscopy with biopsies, which revealed diffuse viability with ulcers, exudates and pseudomembranes concerning for clostridium difficile (CD) colitis. Patient was started on oral vancomycin. Despite treatment, his diarrhea did not improve. Later, colonic biopsies surprisingly revealed cytopathic evidence consistent with CMV colitis ( Figure 1 & 2). CMV viral load was elevated at 2592 copies/mL. Human immunodeficiency virus serology was negative. Vancomycin was stopped, and the patient was started on ganciclovir with resolution of diarrhea.1554_A Figure 1. Left image: the arrows indicate CMV “owl's eye inclusions on H & E ( 40x) Right image: corresponding CMV infected cells light up brown with IHC (immunohistochemistry) stain. (40x)1554_B Figure 2. Left Image: the arrows indicate CMV “owl's eye inclusions on H & E ( 20 x) Right Image: corresponding CMV infected cells light up brown with IHC (immunohistochemistry) stain. ( 20 x)CMV colitis rarely occurs in immunocompetent patients making it a diagnostic challenge. Furthermore, it can mimic CD colitis because of some similar overlapping endoscopic findings. Recognizing that CMV colitis may occur in immunocompetent patients can prevent serious morbidity and mortality, especially in those with advanced age and multiple medical comorbidities.

NKG2C Natural Killer Cells in Bronchoalveolar Lavage Are Associated With Cytomegalovirus Viremia and Poor Outcomes in Lung Allograft Recipients.

Cytomegalovirus (CMV) infection is a risk factor for chronic lung allograft dysfunction (CLAD), which limits survival in lung allograft recipients. Natural killer (NK) cells that express the NKG2C receptor mediate CMV-specific immune responses. We hypothesized that NKG2C NK cells responding to CMV in the lung allograft would reduce CMV-related inflammation and would improve CLAD-free survival. We prospectively followed 130 subjects who underwent lung transplantation from 2012 to 2016. Bronchoalveolar lavage (BAL) NK cells were immunophenotyped for NKG2C, maturation, and proliferation markers. CMV viral load, serologies, serial spirometry, and mortality were recorded from medical records. Natural killer cell subset association with CMV endpoints were made using generalized estimating equation-adjusted linear models. BAL NKG2C NK cell association with CLAD-free survival was assessed by Cox proportional hazards modeling. NKG2C NK cells were more mature and proliferative than NKG2C NK cells and represented a median of 7.8% of BAL NK cells. The NKG2C NK cell proportion increased prior to the first detection of viremia and was nearly tripled in subjects with high level viremia (>1000 copies/mL) compared with no detected viremia. Subjects with increased BAL NKG2C NK cells, relative to the median, had a significantly increased risk for CLAD or death (hazard ratio, 4.2; 95% confidence interval, 1.2-13.3). The BAL NKG2C NK cell proportion may be a relevant biomarker for assessing risk of CMV viremia and quantifying potential CMV-related graft injury that can lead to CLAD or death.

CMV on surfaces in homes with young children: results of PCR and viral culture testing

BackgroundCaring for young children is a known risk factor for cytomegalovirus (CMV) infection mainly through exposure to their saliva and urine. In a previous study, 36 CMV-seropositive children 2 mo. to 4 years old were categorized as CMV shedders (n = 23) or non-shedders (n = 13) based on detection of CMV DNA in their saliva and urine. The current study evaluated the presence of CMV on surfaces in homes of the children.MethodsStudy staff made 4 visits to homes of the 36 enrolled children over 100 days. Saliva was collected by swabbing the mouth and urine was collected on filter paper inserted into diapers. In addition, five surface specimens were collected: three in contact with children’s saliva (spoon, child’s cheek, washcloth) and two in contact with children’s urine (diaper changing table, mother’s hand). Samples were tested by PCR and viral culture to quantify the presence of CMV DNA and viable virus.ResultsA total of 654 surface samples from 36 homes were tested; 136 were CMV DNA positive, 122 of which (90%) were in homes of the children shedding CMV (p < 0.001). Saliva–associated samples were more often CMV positive with higher viral loads than urine-associated samples. The higher the CMV viral load of the child in the home, the more home surfaces that were PCR positive (p = 0.01) and viral culture positive (p = 0.05).ConclusionsThe main source for CMV on surfaces in homes was saliva from the child in the home. Higher CMV viral loads shed by children correlated with more viable virus on surfaces which could potentially contribute to viral transmission.