CMV-DNA Copies Research Articles

Cytomegalovirus detected by qPCR in iris and ciliary body of immunocompetent corneal donors

BackgroundCytomegalovirus (CMV) can cause a wide panel of ocular infections. The involvement of CMV as a cause of anterior uveitis in the immunocompetent patient is recent and remains poorly understood. ObjectiveTo investigate the presence of CMV in anterior uveal tissues of immunocompetent corneal donors. Study DesignWe collected aqueous humor, iris, and ciliary body from both eyes of 25 donors died at the Limoges University Hospital between January 2020 and July 2021. CMV serology was determined for all patients from post-mortem blood sample. Ocular tissues were split in 2 fragments for qPCR and 2 for histological analysis. CMV genomes copies were quantified by Multiplex qPCR after DNA extraction. Results16 of 25 patients (64%) displayed positive CMV serology, with a median age of 67 years. Viremia was positive in 3 of 16 (19%) CMV-positive patients. No CMV DNA copies were found from the aqueous humor samples. CMV DNA was detected in iris and ciliary body of 28 of 32 eyes of seropositive donors, and 5 of 18 eyes of seronegative donors. The median viral copy number [IQR] was 2.41 × 102 [8.91 × 101 - 1.01 × 103] copies/1 × 106 cells in the CMV-positive group and 0.00 [0.00 - 3.54 × 102] copies/1 × 106 cells in the CMV-negative group (p<0.001). Histology and immunohistochemistry did not reveal any CMV lesions from any sample. ConclusionCMV DNA was found in iris and ciliary body of immunocompetent seropositive patients, but also, although less frequently, from seronegative donors. These results highlight mechanisms of infection, latency and reactivation of CMV in ocular tissues.

Effect of Plasmacytoid Dendritic Cell Dose in Grafts on CMV Infection after Allogeneic Hematopoietic Stem Cell Transplantation

To investigate the correlation between plasmacytoid dendritic cell (pDC) dose in grafts and the occurrence of cytomegalovirus (CMV) infection after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The clinical data of 80 children who received allo-HSCT in Children's Hospital of Soochow University from August 20, 2020 to June 11, 2021 were retrospectively analyzed. Proportions of DC subsets and T-cell subsets in grafts were detected by flow cytometry in order to calculate infused cell dose of each cell. Weekly monitoring of CMV-DNA copies in peripheral blood for each child were performed after transplantation. The last follow-up date was December 31, 2021. All the children gained hematopoietic reconstitution. CMV infection was observed in 51 children (63.8%±5.4%) within the first 100 days after transplantation, including 2 cases developing CMV disease. Univariate analysis indicated that infused doses of DC and pDC were significantly associated with CMV infection within 100 days after allo-HSCT (P <0.05). Multivariate analysis indicated that a high dose infusion of pDC was an independent protective factor for CMV infection within 100 days after allo-HSCT (P <0.05). By the end of follow-up, 7 children died of transplantation-related complications, including 2 deaths from CMV disease, 2 deaths from extensive chronic graft-versus-host disease, and 3 deaths from capillary leak syndrome. The overall survival rate was 91.2%. The pDC in grafts may be associated with early infection of CMV after allo-HSCT, while a high infused pDC dose may serve as a protective factor for CMV infection after transplantation.

Efficacy of Mesenchymal Stromal Cells for Refractory Late-Onset Hemorrhagic Cystitis after Allogeneic Hematopoietic Stem Cell Transplantation

INTRODUCTION Nowadays, the treatment of refractory late-onset hemorrhagic cystitis (LOHC) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) still faces many difficulties and challenges. Mesenchymal stromal cells (MSCs) possess the capacity to modulate immune or inflammation response, support hematopoiesis and repair tissue. We explore the efficacy of mesenchymal stromal cells for refractory LOHC treatment after allo-HSCT. METHORDS Twenty-four patients with refractory LOHC from July 2008 to June 2021 received conventional therapy combined with MSCs obtained from bone marrow (BM) of third-party donors. MSCs were given intravenously at a dose of 1×106 cells/kg once weekly until the symptoms improving or LOHC not improving after continuous infusion of four times. The number of BK viruria (BKV)-DNA, JC viruria (JCV)-DNA, cytomegalovirus (CMV)-DNA copies in the urine were detected by real-time quantitative PCR before and at 8 weeks after the MSCs infusion. RESULTS The median number of MSC infusions for each patient was 3 (range, 2-8). Twenty patients achieved complete response, one partial response after treatment. The overall response rate was 87.5%. With a median follow-up of 397.5 days (range 39-937 days) post-transplantations, 17 patients survived and 7 died. The causes of death included aGVHD (n=1), infections (n=5) and TMA (n=1), respectively. Compared with that before MSCs infusion, the number of BKV-DNA and CMV-DNA copies were significantly lower at 8 weeks after MSCs treatment (12.105×108/mL vs 0.004×108/ml, P=0.041; 3.131×104/mL vs 0.000×104/ml，P=0.008). CONCLUSIONS The results indicate that MSCs derived from BM of a third-party donor are effective to treat refractory LOHC after allo-HSCT. MSCs might improve LOHC by regulating immune and anti-viral activity.

Analysis of risk factors and prognosis of cytomegalovirus infection post umbilical cord blood stem cell transplantation in children with primary immunodeficiency diseases

Objective: To investigate the risk factors and outcomes of cytomegalovirus (CMV) infection post umbilical cord blood stem cell transplantation (UCBT) in children with primary immunodeficiency diseases (PID). Methods: Clinical data of 143 PID children who received UCBT in the Children's Hospital of Fudan University from January 2015 to June 2020 were collected retrospectively. CMV-DNA in the plasma was surveilled once or twice a week within 100 days post-UCBT. According to the CMV-DNA test results, children were divided into the CMV-infected group and the CMV-uninfected group. The incidence and risk factors of CMV infection were analyzed. At 1-month post-UCBT, the absolute lymphocyte count, ratio of lymphocyte subsets and immunoglobulin levels were compared between those whose CMV infection developed 1-month later post-UCBT and those not. Mann-Whitney U test and chi-squared test were used for comparision between groups. Kaplan-Meier survival analysis was used to analyze the impact of CMV infection on survival. Results: Among 143 patients, there were 113 males and 30 females, with a age of 14 (8, 27) months at UCBT. Chronic granulomatosis disease (n=49), very-early-onset inflammatory bowel disease (n=43) and severe combined immunodefiency (n=29) were the three main kinds of PID. The rate of CMV infection was 21.7% (31/143), and the time of infection occurring was 44 (31, 49) days post-UCBT. The incidence of recurrent CMV infection was 4.2% (6/143) and refractory CMV infection was 4.9% (7/143).There was no significant difference in the first time CMV-DNA copy and peak CMV-DNA copy during treatment between the recurrent CMV infection group and the non-recurrent CMV infection group (32.8 (18.3, 63.1)×106 vs. 22.5 (13.2, 31.9)×106 copies/L, Z=-0.95, P=0.340;35.2 (20.2, 54.6)×106 vs. 28.4 (24.1, 53.5)×106copies/L, Z=-0.10, P=0.920), so were those between the refractory CMV infection group and non-refractory CMV infection group (21.8 (13.1, 32.2)×106 vs. 25.9 (14.2, 12.2)×106copies/L, Z=-1.04, P=0.299; 47.7 (27.9, 77.6)×106 vs. 27.7 (19.7,51.8)×106copies/L, Z=-1.49, P=0.137). The CMV-infected group accepted more reduced-intensity conditioning (RIC) regimen than the CMV-uninfected group (45.2% (14/31) vs. 25.0% (28/112), χ2=4.76, P<0.05). The rate of CMV-seropositive recipients and Ⅱ-Ⅳ acute graft versus host diseases (aGVHD) are significantly higher in the CMV-infected group than the CMV-uninfected group (100% (31/31) vs. 78.6% (88/112), 64.5% (20/31) vs. 26.8% (30/112), χ2=7.98,15.20, both P<0.05). The follow-up time was 31.6 (13.2, 45.9) months, CMV infection had no effect on overall survival (OS) rate (χ2=0.02, P=0.843). There was significant difference in the survival rate among three groups of refractory CMV infection, non-refractory CMV infection and the CMV-uninfected (4/7 vs.95.8% (23/24) vs. 86.6% (97/112), χ2=5.91, P=0.037), while there was no significant difference in the survival rate among three groups of recurrent CMV infection, non-recurrent CMV infection and the CMV-uninfected (5/6 vs. 88.0% (22/25) vs. 86.6% (97/112), χ2=0.43, P=0.896). Children who developed CMV infection after 30 days post-UCBT had lower absolute count and rate of CD4+ T cells and immunoglobulin G (IgG) level than those in the CMV-uninfected group (124.1 (81.5, 167.6) ×106 vs. 175.5 (108.3, 257.2) ×106/L, 0.240 (0.164, 0.404) vs. 0.376 (0.222, 0.469), 9.3 (6.2, 14.7) vs. 13.6 (10.7, 16.4) g/L, Z=-2.48, -2.12,-2.47, all P<0.05), but have higher rate of CD8+T cells than those in CMV-uninfected group (0.418 (0.281, 0.624) vs. 0.249 (0.154, 0.434), Z=-2.56, P=0.010). Conclusions: RIC regimen, grade Ⅱ-Ⅳ aGVHD and CMV-seropositive recipients are the main risk factors associated with CMV infection in PID patients post-UCBT. Survival rate of children with refractory CMV infection after UCBT is reduced. Immune reconstitution in children after UCBT should be regularly monitored, and frequency of CMV-DNA monitoring should be increased for children with delayed immune reconstitution.

A novel ganciclovir desensitization protocol for a pediatric patient: A case report

A nine year-old girl, having high temperature and stomach ache symptoms, was diagnosed with primary haematophagocytic lymphohistiocytosis. During the treatment, she had neutropenic fever, dyspnea and bronchospasm. Laboratory tests revealed a 7,189,566 cytomegalovirus (CMV)-DNA copies/mL. Ganciclovir therapy was started for CMV pneumonia. At the second infusion of ganciclovir, she had dyspnea, bronchospasm, tachycardia and urticaria. Adrenaline, antihistamines and corticosteroids were administered for anaphylaxis. Treatment was replaced with valganciclovir but there was no clinical or laboratory regression. Because valganciclovir was ineffective and foscarnet could not be provided, we decided to apply desensitization with ganciclovir without performing skin tests because the reaction was anaphylactic. Total dose was 350mg, for the patient's weight of 35kg, according to 10mg/kg daily dose. A (1/1000 dilution), B (1/100 dilution), C (1/10 dilution) and D (no dilution) solutions were prepared. Desensitization began with a 1/10,000e of the therapeutic dose, followed by 14 administrations of increasing doses every 30 minutes and lasted 6.5 hours. There was no reaction during desensitization. Patient was subsequently treated with ganciclovir every day without any problem for 21 days. After the treatment, clinical symptoms resolved and CMV-DNA copies were found<31IU/mL. In conclusion, we present a rare case of ganciclovir hypersensitivity (allergic or non-allergic?) and a novel protocol of ganciclovir desensitization.

Citomegalovirusna reaktivacija kod pacijenata u procesu alogene transplantacije matičnh ćelija hematopoeze

Introduction: Opportunistic CMV reactivation is the most common viral complication after allogenic hematopoietic stem cell transplantation (allo-HSCT). Aim: The aim of our study is to evaluate the frequency of CMV reactivation in relation to the serostatus od the donor and the recipient, and the correlation with the day of leukocyte (Le) and thrombocyte (Tr) engraftment. We compared the frequency of CMV reactivation in myeloablative conditioning (MAC) versus reduced intensity conditioning (RIC), as well as in match related donor (MRD) versus match unrelated donor (MUD) allo-HSCT. We analyzed whether CMV reactivation affected the overall survival (OS) after allo-HSCT. Materials and methods: In a retrospective cohort study, we inspected 42 patients over the age of 18 years, who were treated at the Clinic for Hematology of the Clinical Center of Serbia, from December 2017 to November 2019. Results: Most CMV reactivations were noticed if the recipient (R) was seropositive, and the donor (D) was seronegative (R+/D= 60.0%). The number of CMV DNA copies corelated with the day of leukocyte engraftment of (p = 0.031), but not of thrombocyte engraftment (p = 0.598). The frequency of reactivation in patients treated with RIC was 25.0%, and it was 63.5%, if they were treated with MAC. The intensity of the conditioning regimen corelated with the number of CMV DNA copies (p = 0.025%). There was no correlation found between the type of transplantation (MRD or MUD) and CMV reactivation (p = 0.515). OS after allo-HSCT was 36.39 months (95% CI 26,0 - 46,78). The mean OS in patients with CMV reactivation was 7.39 months (95% CI 5,72 - 9,06), but we did not prove that CMV reactivation had an impact on OS (p = 0.527). Conclusion: CMV reactivation was most common in the R+/Dgroup. CMV reactivation did not affect OS after allo-HSCT in our group of patients.

Hyperbaric Oxygen Therapy for Late-Onset Hemorrhagic Cystitis after Allogeneic Hematopoietic Stem Cell Transplantation and Effective Factors Prediction

Background: Late-onset hemorrhagic cystitis (LO-HC) is one of the common complications and major causes of morbidity in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). The classic treatment for HC includes hydration, alkalization, bladder irrigation, immunosuppressant reduction, and platelet transfusion. When the HC was considered to be associated with aGVHD, more immunosuppressive agents such as glucocorticoids will be added in. However, too intensive immunosuppressive measures leave the patients susceptible to infection and, in turn, increases the accidence of non-relapse mortality (NRM). It is important to explore novel, less-toxic, more effective, and cost-effective strategies for LO-HC. Hyperbaric oxygen therapy (HBOT) can stimulate fibroblast proliferation, angiogenesis, and wound healing. We deduce that HBOT might benefit patients with LO-HC after allo-HSCT. Methods: In this study, we retrospectively analyzed 238 patient underwent allo-HSCT in our center. From 2018, patients with LO-HC have been given HBOT in addition to conventional treatment in our center. The data in HBOT and the control group were collected and compared. Patients' characteristics were compared using the x2 test in case of discrete variables or the Mann-Whitney test in case of continuous variables. Logistic regression examined factors might be used as effective predictors for HBOT. Sensitivity and specificity were estimated based on four parameters cut-off point for additional glucocorticoid necessity. Results: A total of 238 patients who underwent allo-HSCT from 2014 to 2020 were enrolled in this study. Among them, 55 (23.11%) developed LO-HC (36 received regular treatment, and 19 received HBOT in addition to the regular therapy). The clinical &amp; transplantation parameters of the patients with LO-HC were listed in table 1. There is no significant difference in the age, gender, primary disease distribution, conditioning regimen, donor, HLA compatibility, stem cell sources, the occurrence of aGVHD between two groups. There is also no significant difference in the initial day of LO-HC post-HSCT and duration, peak red cells count in the urine, and whether additional glucocorticoid for HC control had been added between the two groups. It is worth noting that compared with the HBOT group, we found higher HC grade distribution, higher CMV-DNA copies, and decreased levels of SOD2 within the 1st week of the initial therapy for LO-HC in the control group. The comparison of the demographic and medical characteristics of subjects with LO-HC were listed in table 2. According to the results of logistic regression analysis, patients who developed LO-HC with higher HC grade (p = 0.001) and decreased SOD2 level (p = 0.002) might benefit more from HBOT, indicating that they might be used as effective predictors for HBOT (Figure 1A). Only higher HC grade (p = 0.011) could predict whether or not additional glucocorticoid would be necessary for LO-HC control (Figure 1B). The predictive power of additional glucocorticoid during the LO-HC treatment process for 4 different titers (HC grade and duration, CMV, and SOD2 level) was evaluated by ROC analysis. According to the area under the curve (AUC), the best diagnostic capabilities of four parameters were shown in Figure 2A, 2B, 2C, and 2D. None of our patients experienced severe adverse events of HBOT, such as gas emboli, pulmonary edema, and seizures. Only one patient with AML discontinued after 1 session due to the reversible ear barotrauma which was suspected with HOBT. Conclusions: In summary, HBOT was effective and well-tolerated in patients with LO-HC post-allo-HSCT. Patients who received HBOT exhibited with short disease duration and mild LO-HC grade. Higher HC grade and decreased SOD2 levels might be used as effective predictors for HBOT. Higher HC grade might be used as a predictor to determine whether or not additional glucocorticoid would be necessary for HC control. In the future, HBOT might be not only used as an adjunctive care application for LO-HC, but also might be as the first-line treatment therapy for this population, regardless of the causes of LO-HC. Based on our preliminary result, we plan to enroll more eligible patients to establish the definitive efficacy and safety of HBOT in patients with LO-HC after allo-HSCT (NCT04502628). Disclosures No relevant conflicts of interest to declare.

Efficacy, safety, and pharmacokinetics of oral valganciclovir in patients with congenital cytomegalovirus infection

ObjectivesValganciclovir (VGCV) has been shown to improve sensorineural hearing loss (SNHL) and neurological outcomes in patients with neonatal symptomatic congenital cytomegalovirus (cCMV) infection. However, reports on the pharmacokinetics, efficacy and safety of oral VGCV are limited. The aim of this study is to evaluate the pharmacokinetics of VGCV for use in the treatment of cCMV. MethodsThis was a single-center, retrospective observational study conducted at Saitama Children's Medical Center in Japan between 2012 and 2017. CMV DNA copy number, maximum plasma VGCV concentration (Cmax), and adverse events (ADEs) during treatment were evaluated. ResultsA total of 26 patients with cCMV who received VGCV were included in this study. The median age at VGCV initiation was 9.5 months (range 0–46). Twenty-one patients (81%) had SNHL at baseline. Of these, five patients (19%) presented with improved SNHL, and none experienced worsened SNHL during treatment. The mean VGCV Cmax was 3.5 μg/mL (range 2–5.3), with no significant variation among individual values, and the values were maintained during treatment. Furthermore, there were no correlations between the Cmax values and age, sex, SNHL improvement or ADEs. Neutropenia (<1000/mm3) was observed in six patients (23%); however, no serious ADEs occurred. ConclusionsVGCV prevented the progression of SNHL without serious ADEs due to its stable pharmacokinetics. This study provides safety and tolerability of VGCV for the treatment of cCMV patients.

Clinical characteristics of cytomegalovirus gastritis: A retrospective study from a tertiary medical center.

Cytomegalovirus (CMV) gastritis is a rare opportunistic infection with diverse clinical manifestations. Our study aimed to investigate the clinical features of Chinese patients with CMV gastritis.Six inpatients diagnosed with CMV gastritis were retrospectively enrolled, based on the finding of inclusion bodies in routine hematoxylin and eosin staining or positive anti-CMV monoclonal antibodies under immunohistochemistry in the gastric biopsy. Data, including demographics, diagnostic measurements, and medications, were collected.Abdominal pain was the most frequently reported symptom, occurring in 4 patients. Five patients were immunocompromised with associated underlying diseases, and 3 patients had decreased leukocyte differentiation antigen 4 positive (CD4+) T lymphocyte counts. Only 3 patients had either positive cytomegalovirus (CMV)-immunoglobulin (Ig) M or increased copies of CMV-DNA peripherally. All patients had gastric lesions in the antrum of the stomach, including ulcers or erosions observed by gastroscopy. All patients received ganciclovir by intravenous injection (IV) as the first line anti-CMV therapy, and attained complete (4) or partial remission (2) during the follow-up.CMV gastritis should be taken into consideration in patients with immunocompromised status who have abdominal pain, nausea, or vomiting. Gastroscopy and necessary biopsy are the major diagnostic methods for CMV gastritis. Early diagnosis leads to a better prognosis for these patients.

The Efficacy and Safety of Cidofovir in Salvage Therapy for CMV Infection in the Patients with Haploid Hematopoietic Stem Cell Transplantation

Cidofovir (CDV) is a nucleotide analogue with broad antiviral activity and has been approved for the treatment of cytomegalovirus (CMV) retinitis in patients with acquired immunodeficiency syndrome. Data on the use of CDV for anti-CMV treatment in the patients with allogeneic hematopoietic stem cell transplantation (allo-HSCT) are limited. To evaluate the efficacy and safety of CDV in salvage treatment of CMV infection after allo-HSCT, we prospectively enrolled 18 patients with CMV infection after allo-HSCT in this study, of whom failed or intolerant to preemptive antiviral therapy. Dose of CDV as 5 mg/kg per week was used until CMV-DNA negative twice with quantification polymerase chain reaction (qPCR) detection or disease progression. All these 18 patients received haploid HSCT and suffered from a median of II (I~IV) ° acute graft versus host disease (aGVHD) before or at the same time of CMV infection. The patients showed as CMV DNAemia (n=7), CMV syndrome (n=5) and CMV disease (n=6) at a median time of 37 (range, 26 to 49) days post transplantation. Before CDV, all the patients received ganciclovir (n=9) or/and foscarnet (n=10) as pre-empty treatment for CMV infection, and 15 cases failed while 3 untolerated to the side effect. After a median of 2 (1-4) course of CDV therapy, 14 (77.8%) patients acquired clinical cure, 1 improved, and 3 (16.7%) failed, presenting a median response time of 2 courses. 11 (61.1%) patients had a 1-log decrease in CMV-DNA copy number after 1 cycle of CDV treatment, and 10 (55.6%) patients obtained virus clearance after 1 to 2 courses of treatment. With a median follow-up of 5 (1 to 12) months, 14 patients remained continuously CMV-DNA negative. During CDV treatment, 4 (22.2%) patients developed mild reversible renal damage and 5 (27.8%) experienced I~II° gastrointestinal reactions. Taking together, it could be effective and safe to use cidofovir in salvage treatment for CMV infection in the patients with allo-HSCT, including haplo-HSCT. Disclosures No relevant conflicts of interest to declare.

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.

Predictors of mortality in autoimmune disease patients with concurrent cytomegalovirus infections detected by quantitative real-time PCR.

ObjectiveCytomegaloviruses (CMV) can have a significant impact on the prognosis of immunocompromised patients. Unlike in the transplantation and AIDS fields, only a few studies on CMV infections have been published in the field of autoimmunity. In this study, we examined the clinical outcomes of CMV infections in patients with autoimmune diseases at a single tertiary medical institution.MethodsA retrospective study was performed to identify the mortality risk factors associated with CMV infections in patients with autoimmune diseases. We reviewed the medical records of patients with autoimmune diseases who were diagnosed with CMV infections using real-time quantitative polymerase chain reaction between December 2005 and March 2016. Clinical and laboratory parameters as well as treatment outcomes were analyzed.ResultsSeventy-three CMV infected patients were separated into survivors and non-survivors. Non-survivors had significantly higher median CMV-DNA copy numbers than survivors (95,500 vs 6,700 copies/mL, p = 0.005) and demonstrated significantly more frequent incidents of CMV pneumonitis (69.2 vs 36.2%, p = 0.007). After adjusting for multiple confounding covariates, the log CMV-DNA copies/mL (hazard ratio, 1.48; 95% confidence interval, 1.14–1.92; p = 0.003) and the presence of concurrent infections (hazard ratio, 22.00; 95% confidence interval, 2.75–175.97, p = 0.004) were identified as independent mortality risk factors. Furthermore, patients with high CMV copy numbers (> 60,000 copies/mL) had higher in-hospital mortality than those with low CMV copy numbers (p < 0.05).ConclusionsCMV-DNA copy numbers and concurrent infections are predictors of in-hospital mortality in CMV-infected patients with autoimmune diseases. Therefore, serial measurements of CMV-DNA copy numbers and close observation for signs of other infections are recommended for patients with autoimmune diseases who have concurrent CMV infection.

Evaluation of a Probe-Based PCR-ELISA System for Simultaneous Semi Quantitative Detection and Genotyping of Human Cytomegalovirus (HCMV) Infection in Clinical Specimens.

Background:Human cytomegalovirus (HCMV) is a common opportunistic pathogen that causes serious complications in immunosuppressed patients and infected newborns. In this study, PCR-ELISA was optimized for semi-quantitative detection of infection in clinical specimens and simultaneous genotyping of glycoprotein B for 4 major genotypes, due to its significance.Method:During DIG-labeling PCR, a pair of primers amplifies a fragment of variable region of the glycoprotein B encoding sequence. Under optimized conditions, labeled Target amplicons hybridize to biotinated specific probes and are detected in an ELISA system.Results:PCR-ELISA system showed specific performance with detection limit of approximately 100 copies of CMV DNA. The linear correlation was observed between the PCR-ELISA results (OD) and logarithmic scale of CMV (r=0.979). Repeatability of PCR-ELISA detection system for intra-assay and inter-assay was evaluated for negative and positive samples. In optimized conditions of hybridization, differentiation between genotypes of glycoprotein B was feasible using genotype-specific probes in PCR-ELISA genotyping system.In comparison with sequencing method, genotyping system was confirmed with kappa index of 1.Conclusion:PCR-ELISA is proposed as an applicable and reliable technique for semi-quantitative diagnosis and typing of the infection. This technique is flexible to apply in a variety of molecular fields.

Screening for seemingly healthy newborns with congenital cytomegalovirus infection by quantitative real-time polymerase chain reaction using newborn urine: an observational study

ObjectiveApproximately 8–10% of newborns with asymptomatic congenital cytomegalovirus (cCMV) infection develop sensorineural hearing loss (SNHL). However, the relationship between CMV load, SNHL and central nervous system (CNS) damage in cCMV...

Low total IgM values and high cytomegalovirus loads in the blood of newborns with symptomatic congenital cytomegalovirus infection.

Neurological outcomes differ considerably between symptomatic and asymptomatic infants with congenital cytomegalovirus (CMV) infection. Our objective was to characterize laboratory markers in symptomatic newborns in comparison with asymptomatic newborns with congenital CMV infection. Ten newborns with symptomatic and 13 newborns with asymptomatic congenital CMV infection were included in this 3-year prospective cohort study. Total immunoglobulin M (IgM), CMV-IgM, CMV antigenemia, and CMV-DNA in blood and urine were measured and their positive rates and quantitative values compared between the symptomatic and asymptomatic groups. Fifty percent of newborns in the symptomatic group were positive based on total IgM; this was significantly lower than in the asymptomatic group (100%). Quantitative total IgM values were significantly lower, and there were significantly more copies of CMV-DNA in the blood of symptomatic newborns than in asymptomatic newborns (median values for total IgM: 14 vs. 43 mg/dL and blood CMV-DNA: 3.2×102 vs. 3.5×101 copies/106 white blood cells). CMV-IgM, CMV antigenemia, and urine CMV-DNA did not differ significantly between groups. Low total IgM values and high blood CMV loads were associated with the presence of symptoms in newborns with congenital CMV infection.

Preemptive treatment of Cytomegalovirus infection in kidney transplant recipients with letermovir: results of a Phase 2a study

Cytomegalovirus (CMV) infection remains a significant cause of morbidity and mortality in transplant recipients. Letermovir (AIC246), is a novel anti-HCMV drug in development, acting via a novel mechanism of action. In this proof-of-concept trial with first administration of letermovir to patients, 27 transplant recipients with active CMV replication were randomly assigned to a 14-day oral treatment regimen of either letermovir 40 mg twice a day, letermovir 80 mg once a day, or local standard of care (SOC) in a multicenter, open-label trial. Efficacy, safety, and limited pharmacokinetic parameters were assessed. All groups had a statistically significant decrease in CMV-DNA copy number from baseline (40 mg BID: P = 0.031; 80 mg QD: P = 0.018; SOC: P = 0.001), and comparison of viral load reduction between treatment groups showed no statistically significant differences. Viral clearance was achieved for 6 of 12 patients (50%) in the letermovir groups versus two of seven SOC patients (28.6%). Letermovir treatment was generally well tolerated, no patient developed CMV disease during the trial. Both letermovir treatment regimens resulted in equally high trough level plasma concentrations. The efficacy, safety, and pharmacokinetics observed in these viremic transplant recipients indicate that letermovir is a promising new anti-CMV drug.

CMV Infection Alone Does Not Affect The Survival In Patients After Umbilical Cord Blood Transplantation: Can We Put Off The Time Of Preemptive Therapy?

ObjectiveAnalysis and discuss the time of cytomegalovirus (CMV) preemptive therapy after umbilical cord blood transplantation (UCBT). Analysis the risk factors of CMV infection and the effect on survival of CMV infection after UCBT. MethodFrom April 2000 to March 2013, 176 cases of patients undergo UCBT who were detected plasma CMV-DNA twice a week routinely. Preemptive antiviral therapy was initiated with ganciclovir or foscarnet for positive PCR of 103 DNA copies or more per ml. Results125 of 176 patients developed CMV infection (71%) at a median of 32d (range 19 to 98) post-transplant and only 4 patients developed CMV disease (2.3%). The 5-year overall (OS) was 59.4% in the patients with CMV infection and 64.9% in patients without CMV infection (P=0.193). The study identified that younger age, high-risk disease, infused little CD34 positive cells, developed III-IV degree acute graft-versus-host disease (GVHD), high-dose methylprednisolone, and treated with basiliximab and other strong immunosuppressive agent as significant risk factors for developing CMV infection. It had nothing to do with the gender of patients, transplantation of UCB units, the conditioning regime, the day of neutrophil recovery and chronic GVHD. Preemptive therapy when the CMV-DNA copies are greater than 103 does not increase the incidence of CMV disease, and the survival has not been impacted. ConclusionPreemptive therapy can be initiated when CMV-DNA copies are greater than 103 which can achieve the same effects in UCBT. Disclosures:No relevant conflicts of interest to declare.

Comparison of two quantitative real‐time CMV‐PCR tests calibrated against the 1st WHO international standard for viral load monitoring of renal transplant patients

Cytomegalovirus (CMV) replication in organ transplant recipients is commonly diagnosed by quantitative PCR methods. However, there has been a poor inter-laboratory correlation of viral load values due to the lack of an international reference standard. In a recent study, the COBAS® AmpliPrep/COBAS® TaqMan® (CAP/CTM) CMV test calibrated to the 1st WHO CMV standard, showed good reproducibility in CMV load values across multiple laboratories. Fifty-seven follow-up plasma specimens from 10 kidney transplant recipients with CMV replication were examined using the new quantitative CAP/CTM CMV test and the "in-house" quantitative CMV real-time PCR method, also calibrated against the 1st WHO CMV standard for their clinical applicability for monitoring CMV load in renal transplant patients. By CAP/CTM CMV test 49/57 specimens were CMV-DNA positive compared to 44/57 by the "in-house" PCR test. The "in-house" PCR and CAP/CTM CMV test correlated well in monitoring individual kidney transplant patients. Conversion of the CMV-DNA copies to IUs made the results of the "in-house" PCR and CAP/CTM CMV test less uniform in analysis of the patient samples. In specimens of one patient, significant underquantification of CMV load with "in-house" PCR emerged during follow-up due to a point mutation in the "in-house" PCR primer sequence. The CAP/CTM CMV test was found suitable for diagnosing and monitoring CMV replication in renal transplant patients. Multicenter studies are needed to provide more information of the commutability of the 1st WHO CMV standard and to define the clinical thresholds.

A Study on the Timeline of Onset of Opportunistic Cytomegalovirus: Infection in Renal Transplant Recipients and Comparison of Various Methods of Its Detection

Infectious complications are still a significant cause of morbidity and death in recipients of organ transplant. Among them Cytomegalovirus is the single most important agent affecting these patients, with at least two-thirds of these patients developing CMV infection 1~4 months after transplantation. This study was aimed to bring out the best and effective methodology or combination to detect CMV disease in post renal transplant Indian patients. We compared Quantitative CMV–DNA Real time PCR with pp65 Antigenemia, serology (anti-CMV IgM/IgG antibody assay) and Anti-CMV IgG avidity. We tried to evaluate the time line of onset of Cytomegalovirus disease post renal transplant. Fifty consecutive renal transplant recipients with clinical suspicion of CMV disease were studied prospectively. CMV Disease was defined clinically based on some criteria. These patients were subjected to a detailed viral diagnostic workup. The clinical response to treatment was considered as a strong clinical evidence of CMV disease in retrospect. The time course of onset of CMV disease post-transplant in majority of patients was found to be between 8 to 14 weeks. Five tests were done in all 50 patients and 20 healthy subjects (control group).Out of the 50 patients who formed our study population, 32 (64%) were found to have CMV disease. All the diseased cases were picked by the Real-Time PCR giving a sensitivity of 100% and specificity of 88.8%. The sensitivity and specificity of pp-65 antigenemia was found out to be 53% and 100%. 32% of patients were found positive for CMV IgM antibody showing a sensitivity 47.05%. Out of the 32 positive patients, 9 had low avidity showing recent infection, 11 patients had high avidity showing long term infection and 12 patients had avidity in the indeterminate range. The Quantitative Real Time PCR stood out as a sensitive test and detected all CMV infections which were considered to be clinically significant and went on to receive treatment whereas pp-65 antigenemia was found to be a more specific and confirmatory test. CMV DNA copy numbers of >500 detected by the Real-time PCR correlate 100% with the presence of CMV disease. We found CMV-IgM antibody assay was not sensitive enough for diagnosis and monitoring of active CMV disease since there is a prolonged presence of IgM antibodies after recent CMV infection. Anti-CMV IgG antibody avidity was found to have a marginal role in diagnosis of this disease.

CMV and EBV Reactivation Post HSCT Impairs Survival of Patients If They Do Not Produce IgM Antibodies Againts These Viruses

Abstract 4552It is known that CMV infection/reactivation influences the survival of patients post HSCT. Risk factors are associated with both (i) the CMV serostatus of donors and recipients and (ii) the immunological potential of the recipients post transplantation.In this study 142 patients - 69 males and 73 females suffering from hematological malignancies, SAA and inborn errors at the age from 0.6 to 64 (median 40) - were followed for the presence of CMV and EBV DNA copies and the presence of CMV and EBV IgM antibodies in blood post HSCT (61 sibling and 81 matched unrelated donors) with 625 days of median observation time. In addition the number of lymphocytes in blood and the proportions and numbers of CD4, CD8, CD20 positive cells in blood were analyzed in association with the CMV and EBV findings.CMV and EBV DNA copies were determined with the use of qPCR at 1-week intervals until 30 day post HSCT, then monthly until one year post HSCT and then at routine follow-up examinations (usually in 3 months intervals), and always when clinically suggested. Values exceeding 50 copies/10^5 cells were recognized as positive and clinically significant. At the same time the presence of IgM and IgG antibodies specific for CMV and EBV were examined with the use of ELISA. The first positive results for CMV DNA copies were seen at median time of 48 days post-transplant and IgM antibodies if present were detected in a majority of cases within 30 days after DNA copies finding. EBV copies were detected at the similar time post transplantation (median day of positive results: 48 days) and IgM antibodies if present were usually seen within 2 months after DNA copies detection. Notably, in 9 patients EBV reactivations were associated by the appearance of IgM CMV but not EBV antibodies. Therefore the presence of IgM antibodies against either CMV or EBV was recognized as a sign of the ability of a given patient to respond immunologically to CMV or EBV viruses.For the sake of this study the patients group was divided into subgroups as follow: (1) CMV and/or EBV copies present but IgM antibodies absent, (2) CMV and/or EBV DNA copies as well as CMV and/or EBV antibodies present, (3) neither CMV or EBV copies nor IgM antibodies present, (4) CMV and EBV DNA copies absent but IgM CMV and/or EBV antibodies present.Patients lacking DNA copies or having both copies and IgM antibodies against CMV and/or EBV had significantly better survival than those having DNA copies but lacking IgM antibodies any time during observation period (2 yrs survival 70% vs 41%, p<0.001). To dissect the influence of the immune response against these two viruses similar evaluation was performed for CMV and EBV separately. It became apparent that the worst survival (Kaplan-Meier overall survival curve) had pts with CMV copies but lacking IgM antibodies (2 yrs survival 73% vs 40%, p=0.001), with respect to EBV similar tendency was observed but less pronounced (2 yrs survival 73% vs 44%, p=0.108).To understand the immunological background to this observation lymphocytosis and blood lymphocyte subsets were analyzed at 30 day post HSCT. It became apparent that pts having CMV and/or EBV copies but lacking IgM antibodies had significantly lower number of lymphocytes and their (CD4+ cells subset) in blood as compared to patients having IgM antibodies against CMV and/or EBV irrespective whether they had CMV and/or EBV DNA copies with median values as follow: 650/ul (128/ul) vs 370/ul (60/ul), p=0.020 (p=0.033) or lacked CMV and EBV DNA copies, LYM (CD4+) median values: 750/ul (161/ul) vs 370/ul (60/ul), p=0.047 (p=0.060).CMV and EBV infection/reactivation affects the survival of patients post HSCT if they are unable to generate IgM antibodies against these viruses. Inability to mount humoral response to CMV and EBV was associated with an impairment of the immune system potential (low number of lymphocytes and CD4+ cells in blood).Supported by the grants N R13 0082 06 from the Polish Ministry of Science & Higher Education. Disclosures:No relevant conflicts of interest to declare.