Ganciclovir Therapy Research Articles

Half-dose ganciclovir preemptive treatment of cytomegalovirus infection after pediatric allogeneic hematopoietic stem cell transplantation.

Ganciclovir (GCV) has been widely used as preemptive therapy after hematopoietic stem cell transplantation (HSCT), although bone marrow suppression is a known accompaniment, with secondary infection or bleeding as potential complications. Our aim was to evaluate clinical outcomes in pediatric patients with low cytomegalovirus (CMV) antigenemia levels using half the dosage of GCV generally given preemptively. Patients received half doses of intravenous GCV (5 mg/kg once daily, 6 days/week) at CMV antigenemia levels <10/200,000 cells. At higher levels of CMV antigenemia, conventional doses of GCV (5 mg/kg every 12 h) were administered. A total of 130 patients were evaluated, detecting CMV antigenemia in 87 (66.9%). Of these patients, 74 (85.1%) were treated preemptively with half-dose GCV, which proved effective as sole therapy in 51 (68.9%). CMV retinitis developed in 4 patients, 2 of whom initially were given half-dose GCV. All infections resolved successfully, with no CMV-related deaths. CMV seropositivity in recipients was the only significant risk factor for positive CMV antigenemia (hazard ratio [HR] = 10.05, P = 0.046). Compared with half-dose GCV administration, conventional GCV dosing resulted in a higher rate of severe neutropenia, defined as absolute neutrophil count <0.5 × 10(9) /L (HR = 4.30, P = 0.015). Half-dose GCV therapy at CMV antigenemia levels <10/200,000 cells is an effective and safe means of preemptively treating pediatric CMV infection after HSCT.

Assessment of the Human Cytomegalovirus UL97 Gene for Identification of Resistance to Ganciclovir in Iranian Immunosuppressed Patients.

BackgroundHuman cytomegalovirus (HCMV) infections are a major cause of morbidity and mortality among immunocompromised patients. Prolonged antiviral therapy is a cause of mutation and drug resistance in the HCMV genome.ObjectivesThe aim of this study was to identify resistance to ganciclovir (GCV) in Iranian immunosuppressed patients at two different stages of the disease: early (before GCV is initiated) and late (after six months of GCV therapy).Patients and MethodsIn this study, 87 specimens from Iranian patients were amplified using nested PCR amplification of the UL97 gene. Sequence analyses of products were performed for identifying the mutated codons.ResultsThe present study show that the most frequent GCV-resistant mutations occurred in codons A594V (26.43%), H520Q (18.39%), and M460V (13.79%), consequently occurring at a low frequency in the L595S (2.29%), E596G (1.14%), and Del 594 (1.14%) codons, and with intermediate frequency in the C592G (10.34%), M460I (9.19%), and C603W (6.89%) codons. We describe for the first time a new GCV-resistance mutation, the deletion of codon 594, in the UL97 gene of Iranian HCMV patients after GCV therapy, following renal transplantation.ConclusionsThe findings of the present study can be utilized to detect GCV resistance patterns among Iranian immunocompromised patients and to treat HCMV infections accordingly.

Aqueous humor immune factors and cytomegalovirus (CMV) levels in CMV retinitis through treatment – The CRIGSS study

PurposeThis study aims to perform comprehensive longitudinal immune factor analysis of aqueous humor in relation to the aqueous CMV viral load and systemic CD4 counts during treatment of patients with co-infection of HIV and CMVR. MethodsAqueous humor samples were collected from 17 HIV-positive patients with CMVR scheduled to undergo weekly intravitreal ganciclovir therapy as part of the prospective CMV Retinitis Intravitreal Ganciclovir Singapore Study (CRIGSS) over the course of 1year. Full data across all the 4 time points was obtained and analyzed for CMV DNA viral load, 41 cytokine and chemokine factors using real-time PCR with the FlexMAP 3D (Luminex®) platform and assessed using the Milliplex Human Cytokine® kit. ResultsThe following immune factors (Spearman correlation coefficient r value in parenthesis, p<0.05) showed strong correlation with CMV DNA load in the aqueous - MCP-1 (0.80, IFN-g (0.83), IP-10 (0.82), IL-8 (0.81), fractalkine (0.73), RANTES (0.68) - while the following showed moderate correlation - PDGF-AA (0.58), Flt-3L (0.59) and G-CSF (0.53). Only PDGF-AA revealed a statistically significant negative correlation with serum CD4 levels (r=−0.74). ConclusionImmune factors that correlate with intraocular CMV DNA load are identified. They are indicative of a Th1 and monocyte-macrophage mediated response, and exhibit a decreasing trend longitudinally through the course of treatment. These factors may be an important new consideration in individualizing the treatment of patients with CMVR.

Ganciclovir and Foscarnet Therapy of Cytomegalovirus-Associated Meningoencephalitis in a Hemodialysis Patient With Liver Transplantation: Case Report

Cytomegalovirus (CMV) infection in patients with liver transplantation (LT) remains a highly prevalent complication with a significant increase in morbidity and mortality. However, CMV-associated meningoencephalitis is rarely diagnosed, and treatment is very difficult. The aim of the present report is to review the experience of successful treatment with combined ganciclovir and foscarnet of CMV-associated meningoencephalitis refractory to ganciclovir alone in a hemodialysis (HD) patient after LT. A 54-year-old woman with end-stage renal disease on HD developed a seizure with loss of consciousness. She had received a liver transplant 4 months before. Blood CMV polymerase chain reaction was positive, and cerebrospinal fluid (CSF) analysis was compatible with viral meningitis. Brain magnetic resonance imaging (MRI) showed extensive dural thickening with enhancement and a round ring-like enhancement in the left centrum semiovale. She was diagnosed with CMV-associated meningoencephalitis. At that time, ganciclovir was started intravenously. After that, there were no improvements in mental state, CSF analysis, or brain MRI. Intravenous foscarnet at reduced dose was added to ganciclovir therapy. With combined ganciclovir and foscarnet, there was a slight improvement in her mental state and brain MRI.

Recovery of CMV-Specific T Cells Following Alternative Donor Allogeneic Transplant with Post-Transplant Cyclophosphamide

Background: Post-transplant cyclophosphamide (PTCy) is increasingly used for GVHD prophylaxis after allogeneic hematopoietic stem cell transplantation (HCT) using alternative donors. However, immune reconstitution can be delayed posing an increased risk for CMV reactivation. We evaluated the outcomes of patients who received HCT-apheresis products comparing the impact of PTCy on lymphocyte recovery, CMV reactivation and CMV-specific CD8+ T cell recovery following haplo-identical (HAPLO), matched unrelated donor (MUD), and mismatched unrelated donor (mMUD) grafts vs. with conventional matched related donor (MRD) graft recipients.Methods: We examined 26 patients (median age, 49 years; range, 20-72 years) with advanced hematologic malignancies; n=5 (HAPLO); 6 (MRD); 15 (MUD). All patients received myeloablative conditioning regimens that was either busulfan- or total body irradiation (TBI)-based. PTCy (50 mg/kg/day) was administered on days +3 and +4 following HAPLO and on day +3 following MUD/mMUD transplant. Peripheral blood lymphocyte reconstitution and frequency of circulating CMV-directed CD8+ T cells was assessed (day ± 10 days) on post-transplant days +30, +60, and +90. Circulating anti-CMV T cell frequency was assessed using a phycoerythrin-tagged MHC dextramer against HLA-specific CMV pp65, IE-1, or pp50 peptides (Immudex; Copenhagen, DK) in combination with Tru-Count¨ tubes and fluorescent-labeled monoclonal antibodies against CD3, CD8, CD4, CD16/56, and CD19 (BD Biosciences; San Jose, CA). Anti-CMV CD8+ T cell immunity was defined as a CMV-dextramer (CMV/DEX) positive count of ≥7cells/ml. CMV reactivation was defined as a serologic titer of >500IU/mL. All patients with CMV reactivation received ganciclovir therapy until CMV titer became negative.Results: Day +30 total T cell recovery was significantly faster in MRD than CY-treated recipients (p=0.015) due principally to more robust CD8+ T cell recovery. CD4 T cell recovery remained below normal range in all groups through day +100. NK cells recovered to normal numbers at day +28 in all groups. Neither PTCy nor donor source significantly impacted the percentage of patients that recovered anti-CMV CD8+ T cells at each time interval (p = 0.8232). Excluding donors (D) and recipients (R) that were both negative, CMV/DEX+ T cells recovery was >7/mL in 4/5 MRD, 7/14 MUD, and 3/5 HAPLO by day +100. Among MRD recipients either D+ or R+ (n=5), 2 patients showed CMV reactivation within 40 days of transplant that was associated with <7 CMV/DEX+ T cells on day +30. Subsequent high (>90/mL) CMV/DEX T cell response in one patient shortened the duration of viremia to 10 days (vs. 16 days with poor responder) and 3 patients showed no CMV reactivation and a high CMV/DEX+ T cell response by day +60. For MUD CMV D+ and/or R+ recipients (n=14), 3 showed CMV reactivation within 50 days of transplant. All 3 patients had suboptimal CMV/DEX T cell response on day +30. Robust CMV/DEX+ T cell response on day +60 predicted shorter duration of viremia (20 days vs. average of 32 days). For HAPLO CMV D+ and/or R+ (n=5) recipients, 4 experienced CMV reactivation within 50 days of transplant. All patients had a <7 CMV/DEX+ T cells/mL +30. Robust CMV/DEX+ T cell response by day +60 was associated with shorter duration of viremia (range 7-21 days), while one patient with <7/mL CMV/DEX+ T cells had continued CMV viremia for 36 days.Conclusion: In this preliminary analysis, neither PTCy nor donor source significantly impacted the percentage of patients that recovered anti-CMV CD8+ T cells at each time interval. A weak CMV/DEX+ response (<7 cells/mL) on day +30 was consistent with increased risk of CMV reactivation (viremia) in all groups. A CMV/DEX+ T cell count ≥7 cells/mL was not immediately protective against CMV reactivation, but higher counts were associated with a shortened duration of viremia while on antiviral therapy. Conversely, subnormal counts were associated with a longer duration of viremia. This interim analysis suggests that CMV/DEX+ T cell enumeration is a useful biologic correlate for determining clinical response to antiviral therapy, and that donor-derived CMV specific T cell immunity is not further compromised with following PTCy in alternative donor HCT. DisclosuresNo relevant conflicts of interest to declare.

Rapid regression of cystoid macular edema associated with cytomegalovirus retinitis in adult acute myeloid leukemia by intravitreal methotrexate combined with oral valganciclovir: A case report with comparison of binocular outcome

Cytomegalovirus (CMV) retinitis is a late complication of organ and hematopoietic stem cell transplant, the risk of which depends on the degree of immunosuppression. With the institution of preemptive ganciclovir therapy early after transplant, most patients survive episodes of life-threatening CMV infection during the early months (usually the first 3 months) after transplant and hence late onset of CMV disease, such as CMV retinitis, is being recognized more frequently. Direct involvement of the macula or optic head remains the leading cause of visual loss in patients with CMV retinitis, but there are few studies investigating the management of this condition.Herein, we present the case of 28-year-old man who had acute myeloid leukemia and developed CMV retinitis with bilateral cystoid macular edema and optic swelling in the right eye 6 months after bone marrow transplant. He received treatment with intravitreal methotrexate in the right eye in combination with oral valganciclovir. Visual acuity improved 1 month after four weekly injections of intravitreal methotrexate 400 μg/0.1 mL. Resolved disc swelling and regression of macular edema were also observed. By comparing binocular outcome, we present our findings and discuss the possible efficacy and safety of this treatment with respect to regression of anatomical damage and improvement in visual acuity.

The Incidence of Venous Thromboembolism (VTE) in 892 Allogeneic Hematopoietic Cell Transplant (allo-HCT) Recipients ( A single institution study comparison of VTE incidence with sirolimus versus non-sirolimus-based GVHD prophylaxis)

s / Biol Blood Marrow Transplant 21 (2015) S266eS321 S297 multivariate analysis using the LASSO approach to logistic regression analysis. Results: Of 134 allo-HSCTs, 29 (21.6%) patients experienced CMV viremia. Among these patients, median age was 51 years (range 27-67), with 48 episodes of viremia. Nine (31%) viremic patients developed CMV disease. CMV disease occurred at a median of 124 days post HSCT (range 61-322). Patients with CMV disease had a median of 2 viremic episodes before disease (range 1-4). Disease occurred at a median of 33 days from the start of the last viremic episode, and 75 days from the start of the first episode of viremia. On univariate analysis, factors associated with progression to CMV disease were: steroid-refractory acute GVHD (60 vs. 20%, p1⁄40.028); number of episodes of viremia >1x103 copies/mL (mean 2.4 vs 1.1, p1⁄40.016); longer duration of viremia (mean 38 vs. 22 days, p1⁄40.01); higher peak viral load (mean 4.49x105 vs. 8.31x103 copies/mL, p 1x103 copies/mL, a longer duration of viremia, and to have failed first-line pre-emptive ganciclovir therapy. This study, while limited, suggests that these risk factors may be predictive of CMV disease. Larger, prospective studies are needed to confirm these risk factors, some of which may be amenable to more aggressive anti-viral therapy.

Risk factors for ganciclovir-induced thrombocytopenia and leukopenia.

Ganciclovir is a nucleoside guanosine analogue that exhibits therapeutic activity against human cytomegalovirus infection, and is primarily excreted via glomerular filtration and active tubular secretion. The adverse effects induced by ganciclovir therapy are generally of a hematological nature and include thrombocytopenia and leukopenia. Low marrow cellularity and elevated serum creatinine have been identified as risk factors for ganciclovir-induced neutropenia. However, the risk factors for thrombocytopenia have yet to be determined. Therefore, this study investigated patients administered ganciclovir to determine the risk factors for thrombocytopenia and leukopenia. Thrombocytopenia occurred in 41 of these patients (30.6%). Multivariate logistic regression analysis identified three independent risk factors for thrombocytopenia: cancer chemotherapy (odds ratio (OR)=3.1), creatinine clearance (<20 mL/min) (OR=12.8), and the ganciclovir dose (≥12 mg/kg/d) (OR=15.1). Leukopenia occurred in 36 patients (28.6%), and white blood cell count (<6000 cells/mm(3)) (OR=3.7) and the ganciclovir dose (≥12 mg/kg/d) (OR=7.8) were identified as risk factors. These results demonstrated that several factors influenced the occurrence of ganciclovir-induced thrombocytopenia and leukopenia, and suggest that special attention should be paid to patients receiving cancer chemotherapy with a low creatinine clearance (<20 mL/min) and high dose (≥12 mg/kg/d) in order to avoid ganciclovir-induced thrombocytopenia.

Full Donor Myeloid Engraftment with Minimal Toxicity in Dyskeratosis Congenita Patients Undergoing Allogeneic Bone Marrow Transplantation without Radiation or Alkylating Agents

Dyskeratosis congenita (DC) is an inherited bone marrow failure (BMF) syndrome caused by defects in telomere biology genes. BMF occurs in approximately 80% of patients by 30 years of age and, in addition to pulmonary failure and malignancies, remains a major contributor to mortality. Hematopoietic cell transplantation (HCT) cures BMF in DC patients but is associated with a high incidence of graft failure and transplant-related mortality. Radiation and/or alkylating agents (e.g. cyclophosphamide, busulfan, melphalan) used in conditioning may have a role in the poor outcomes in DC patients following HCT. Recently, reduced-intensity conditioning approaches have improved short-term outcomes, but concerns remain about the long-term effects of DNA damaging agents in these regimens, given the multisystem disease and lifelong predisposition to malignancy conferred by telomere dysfunction.Radiation and alkylating agents are mainstays of allogeneic HCT preparative regimens: their highly effective myeloablative and immunosuppressive properties provide a niche for donor hematopoietic progenitors and decrease graft rejection. Based on a rationale that presumes (1) niche availability in DC patients with BMF, and (2) an intrinsic replicative defect in DC hematopoietic cells, we hypothesized that engraftment would be feasible in DC patients using a regimen of immunosuppressive therapy alone.Here, we report that in a single-institution prospective study, 4 consecutive patients with DC have undergone successful HCT using a radiation- and alkylator-free preparative regimen. Patients received alemtuzumab and fludarabine, followed by bone marrow transplantation (BMT) from unrelated donors. Cyclosporine A and mycophenolate mofetil were used for graft versus host disease (GVHD) prophylaxis. The primary outcome measures were neutrophil engraftment at day +30 and overall survival at day +100. Secondary outcome measures included acute toxicities and rates of graft failure, infections, and GVHD.The first two patients were 18 month-old twins with TINF2 mutations and transfusion-dependent BMF who received a BMT from a fully HLA-matched unrelated donor. The third patient was an 18 year-old female with CTC1 mutations and transfusion-dependent BMF, who underwent a B-mismatched unrelated donor BMT. The fourth patient, a 22 year-old male with clinical DC and short telomeres, had significant pulmonary disease (diffusing capacity of lung for carbon monoxide 28% of predicted) and BMF, and underwent a fully HLA-matched unrelated donor BMT.All 4 patients engrafted neutrophils by day +30, and are alive and well with follow-up ranging from 6 months (1 patient) to 2 years (3 patients). Platelet transfusion-independence occured between day +4 to +18, and red cell transfusion-independence occured between day 0 to +90. No patients required transfusions thereafter. All patients showed full donor myeloid chimerism by day +60. 3 of 4 patients showed full donor lymphoid chimerism by year 2; the fourth patient has high and increasing mixed donor lymphoid chimerism (60%) at day +180. There were no significant, unexpected toxicities, bacterial or fungal bloodstream or tissue infections. All patients were monitored weekly by serum PCR for CMV, EBV, adenovirus and HHV6 until day +100; only one patient showed CMV reactivation which was controlled with pre-emptive ganciclovir therapy. There was no acute GVHD. Only one patient developed chronic GVHD with limited skin involvement controlled with topical steroids.These results provide promising early data for engraftment and a favorable short-term toxicity profile using a radiation- and alkylator-free conditioning regimen in HCT for BMF due to DC. This approach could spare DC patients the acceleration of non-hematologic complications and malignancies, which has been attributed to HCT, and improve long-term survival. The limited toxicity of the alemtuzumab/fludarabine regimen may also allow patients with severe disease-related co-morbidities such as pulmonary dysfunction to safely undergo HCT. To our knowledge, this is the first prospective HCT study to achieve full donor myeloid engraftment in a series of patients following conditioning without radiation or alkylating agents. DisclosuresOff Label Use: alemtuzumab - conditioning for bone marrow transplantation fludarabine - conditioning for bone marrow transplantation cyclosporine A - graft versus host disease prophylaxis in bone marrow transplantation mycophenolate mofetil - graft versus host disease prophylaxis in bone marrow transplantation.

Long-term outcomes of cytomegalovirus reactivation in patients with moderate to severe ulcerative colitis: a multicenter study.

Background/AimsCytomegalovirus (CMV) reactivations are frequently observed in patients with active ulcerative colitis (UC), and ganciclovir therapy is effective in patients with steroid-refractory UC. This study aimed to determine the long-term outcomes of CMV reactivation and the long-term therapeutic efficacy of ganciclovir treatment.MethodsThis retrospective multicenter study included a cohort of 72 patients with moderate-to-severe UC who were evaluated for CMV reactivation at the time of their initial UC flare. Colectomy, disease relapse, and the recurrence rate of CMV reactivation were investigated.ResultsThe mean duration of follow-up for the 72 patients was 43.16±19.78 months (range, 1 to 67 months). The cumulative colectomy (log-rank, p=0.025) and disease flare-up rates (log-rank, p=0.048) were significantly higher in the CMV-positive group. Of the 11 patients who were successfully treated with ganciclovir in the initial treatment, three patients (27.3%) experienced CMV reactivation, and six patients (54.5%) experienced poor outcomes, such as the need for colectomy or a steroid-dependent state.ConclusionsThe patients who had CMV-reactivated UC showed poor outcomes at the long-term follow-up, and the long-term efficacy of ganciclovir therapy was marginal. Careful assessment is necessary for patients who exhibit evidence of CMV reactivation.

PO-0104 Different Clinical Spectrum Of Cytomegalovirus Hepatitis In Infants

Background and aim Cytomegalovirus (CMV) is, a member of the herpes viridae family, found widely in nature and the most common congenital infection in newborns. The average incidence of CMV infection in newborn infants is 1%. Irreversible signs of central nervous system involvement (microcephaly, deafness, mental-motor retardation) develops in 5–10% congenitally infected infants. Signs of perinatal infections, hepatosplenomegaly, pneumonia, hepatitis, are seen, but in this period neurological sequelae are rare. In this study, CMV-infected patients who were admitted to our clinic within 3 months were examined. Methods Between the date of December 2013 and February 2014, in total five CMV infection (min 45 days, max 2 years–4 months old) have been detected at Uludag University, Faculty of Medicine, Department of Paediatric Gastroenterology. Three of those patients admitted with jaundice and other two patients were detected during the pancytopenia aetiology and vomiting aetiology investigation. In our series, which consist of developed CMV hepatitis cases due to different etiological reasons, retrospective examination is conducted with clinical and laboratory findings. Results Biliary atresia was detected in three patients. One is by intraoperative cholangiography and other two, as evidenced by histopathology. In the fourth case, CMV infection was detected simultaneously with vitamin B 12 deficiency during pancytopenia investigation. In the fifth patient, performed liver transplantation due to neonatal hepatitis, CMV infection was detected during investigating for vomiting and elevated aminotransferase levels in outpatient control. In one of the cases of biliary atresia with microcephaly, deafness and mental-motor retardation was considered due to congenital CMV infection. Other cases were evaluated as perinatally infection. Five patients were given ganciclovir therapy. Duration of the therapy was; six weeks for patient with congenital CMV infection and two weeks for the other four patients. Conclusion Biliary atresia were seen in all our cases with cholestasis and there is importance of seeing it with CMV hepatitis which, in our conclusion, requires comprehensive studies. In addition, seeing these patient in such a short period of time was found interesting in epidemiological perspective.

Cytomegaloviral Infection in Solid Organ Transplant Recipients: Preliminary Report of One Transplant Center Experience

BackgroundDespite advances in immunosuppressive therapy and post-transplantation care, antiviral prevention, and therapy, cytomegalovirus (CMV) infection remains the most common viral infection after solid organ transplantation (SOT). MethodsThis study included 2,375 patients under the care of our transplant center during the 1-year period from June 2012 to June 2013. There were 351 patients (14.78%) suspected and tested for CMV infection with the use of viral DNA amplification test. ResultsSymptoms that triggered diagnostics were graft dysfunction in 24 (55.8%), diarrhea in 18 (41.9%), fever in 15 (34.9%), leukopenia in 14 (32.6%), abdominal pain in 13 (30.2%), nausea in 7 (16.3%), cough in 6 (14%), and shivers in 2 (4.7%). Positive test results were obtained in 43 patients (12.3% of patients tested and 1.8% of the entire cohort). The group consisted of 17 women (39.5%) and 26 men (60.5%), 26 kidney (60.5%) and 17 liver (39.5%) transplant recipients, aged 49.3 years (SD 14.9). The initial viral load was median 8,093 (range: 4,232–219,180) copies/mL. The mean ganciclovir (GCV) treatment duration was 19.05 (SD 8.1) days. GCV doses ranged from 100 to 1,000 mg/d, mean 370.6 (SD 254.2) mg/d. Clinical resistance to treatment was diagnosed in 5 patients (11.6%). We found a positive correlation of GCV treatment duration with natural logarithm of initial CMV viremia (r = 0.56; P = .0002) and of time in months to CMV infection with mean cyclosporine level (r = −0.74; P = .04) and GCV dose (r = −0.34; P = .03). The duration of GCV therapy was positively influenced by CMV load and tacrolimus administration and negatively by patient's age and male sex. ConclusionsAppearance of any symptoms occurring after transplantation, even nonspecific, should lead to diagnostics for CMV infection. The duration of treatment depends on the severity of the infection expressed by CMV viremia. Clinical resistance to GCV is not frequent, but it is an important transplantologic problem.

Clinical Outcomes in Cytomegalovirus-Positive Posner-Schlossman Syndrome Patients Treated With Topical Ganciclovir Therapy

To evaluate the clinical characteristics and therapeutic outcomes of cytomegalovirus (CMV)-positive Posner-Schlossman syndrome patients undergoing topical ganciclovir treatment. Retrospective, comparative, and interventional case series. One eye of each of 126 consecutive Posner-Schlossman syndrome patients was investigated using aqueous polymerase chain reaction (PCR) between January 2006 and June 2013. The initial presentations and follow-up data of the CMV-positive patients (68 eyes) and CMV-negative patients (58 eyes) were compared. Severe endothelial cell loss (P < .001) and a higher number of eyes requiring glaucoma filtering surgery (P = .017) were observed in CMV-positive Posner-Schlossman syndrome patients. All CMV-infected eyes treated with continual topical 2% ganciclovir exhibited an undetectable CMV level at the following taps. During follow-up, the average number of antiglaucomatous agents decreased, and a similar frequency of intraocular pressure (IOP) spikes was observed in both groups (P = .358). Patients with CMV-positive eyes with a disease duration over 5 years were likely to require glaucoma surgery (P = .024, log-rank test). All patients receiving surgery exhibited CMV-negative PCR during the IOP attack, but experienced severe peripheral anterior synechiae and pigment clogging. Both groups exhibited a similar endothelial cell decrease (P = .243) and probability of progressive endothelial cell loss (P = .219, log-rank test). Ganciclovir treatment was effective for clearing the viral load, assisting the IOP control, and preserving the corneal endothelium of CMV-positive Posner-Schlossman syndrome patients. Early diagnosis and proper treatment could decrease the risk of advanced glaucoma and avoid glaucoma surgery in long-lasting cases.

REPEATED INTRANASAL APPLICATION OF NEURAL STEM CELL-MEDIATED ENZYM/PRODRUG THERAPY USING A NOVEL HSV-THYMIDINE KINASE VARIANT IMPROVES THERAPEUTIC EFFICIENCY IN AN INTRACRANIAL GLIOBLASTOMA MODEL

BACKGROUND: Neural stem cells (NSCs) have an inherent brain tumor tropism that can be exploited for targeted delivery of therapeutic genes to invasive gliomas. We have recently introduced the concept of intranasal application of NSCs as a noninvasive and direct alternative delivery method for glioma-targeting NSCs. Here, we demonstrate that the repeated non-invasive intranasal administration of tumor-targeting NSC is able to deliver a novel suicide gene (TK.007) to intracerebrally growing human glioblastoma xenografts. METHODS: Murine NSC were genetically modified to stably express a novel, codon-optimized HSVtk(A168H) mutant (TK.007). The biological activity of the NSC-mediated TK.007/ganciclovir (GCV) system was assessed in cell survival and bystander assays using various human glioma cell lines. Therapeutic effects of intratumoral and intranasal NSC-TK007 applications alone and the sequential combinations of both was tested using an intracranial U87 human glioblastoma model in nude mice. All animals received 50 mg/kg GCV i.p. for five consecutive days. In all experiments two control groups received either NaCl instead of GCV or NSC containing the control vector instead of NSC-TK.007. Therapeutic efficiency was determined by assessment of tumor size by 7T MR-imaging and by establishment of Kaplan-Meier survival curves. RESULTS: Cell survival and bystander assays confirmed the GCV catalytic activity of NSC-expressed TK007 in a GCV dose dependent manner and a significant bystander effect at 12.5% of cells. Tumor targeted migration was demonstrated by intravital imaging of luciferase-expressing NSC. In glioma-bearing mice a single intratumoral application of NSC-TK007 followed by systemic prodrug application of GCV lead to a significant tumor growth inhibition of 72% versus the control groups at day 22 and translated in a prolonged survival time. Mice recieving a cyclic intranasal application of NSC-TK.007 alone displayed a signifcant longer survival than the controls. This effect was significantly enhanced when cyclic intranasal NSC-TK.007 therapy was preceded by a single intratumoral application of NSC-TK007 and led to a significant tumor growth inhibition of >95% and improved survival time. CONCLUSIONS: Our data demonstrates that the NSC-mediated TK.007/GCV therapy, which is based on the clinically most widely used suicide system to date, is safe, nontoxic, and effective in glioblastoma-bearing mice. Most importantly, our findings establish that the intranasal application of therapeutically-modified NSC is a safe and non-invasive application method which allows a chronic treatment during the disease course. SECONDARY CATEGORY: Clinical Neuro-Oncology.

Cytomegalovirus infection in immunocompetent wheezy infants: the diagnostic value of CMV PCR in bronchoalveolar lavage fluid.

Cytomegalovirus (CMV) pneumonitis in immunocompetent hosts is uncommon but is being recognized more frequently, particularly when presenting as severe viral pneumonia. The objective of this study was to examine lower respiratory tract CMV infection in immunocompetent wheezy infants, based on polymerase chain reaction (PCR) in bronchoalveolar lavage (BAL) fluid, to compare CMV PCR results in BAL and in blood samples and to evaluate the benefits of antiviral ganciclovir therapy in these patients. Retrospective review of the records of patients referred to our tertiary care hospital between January 2000 and July 2010 who had unexplained persistent wheezing and underwent fibreoptic flexible bronchoscopy (FFB). Fibreoptic flexible bronchoscopy was applied to 102 infants with persistent wheezing and diffuse interstitial infiltration on radiological investigations; so CMV PCR in BAL fluid was performed. CMV PCR in BAL fluid was positive in 51 patients. Retrospectively, we had access to the files of 25 of these patients. The mean CMV PCR in BAL fluid was 334840copies/mL. Only eight patients had CMV PCR positivity in their blood samples (mean: 2026·3copies/mL). There was not a relationship between BAL and blood CMV PCR values based on Spearman's correlation analysis (r=-0·008). Fourteen patients had severe respiratory symptoms and received ganciclovir therapy. Twelve of them fully recovered. Bronchoalveolar lavage fluid CMV PCR was superior to blood CMV PCR in diagnosing lower respiratory tract infections caused by CMV in immunocompetent infants. Ganciclovir therapy may be effective in selected immunocompetent wheezy infants with CMV PCR positivity in BAL fluid.

Risk factors of Ganciclovir-related neutropenia after allogeneic stem cell transplantation: a retrospective monocentre study on 547 patients

Cytomegalovirus (CMV) infection is a serious complication that may occur in the weeks or months following bone marrow transplantation. However, both Ganciclovir and the CMV infection itself can cause marrow toxicity, notably neutropenia, that may consequently expose these immunosuppressed patients to life-threatening bacterial and/or fungal infections. The aim of this retrospective study was to identify factors associated with the occurrence of grade III–IV neutropenia among patients receiving pre-emptive Ganciclovir therapy after allogeneic stem cell transplantation at our Institution. We identified 547 consecutive patients transplanted from January 2005 to June 2011 at our Institution. In all, 190 patients (35%) presented with CMV reactivation of whom 30 patients (5%) were excluded from the analysis because they already had neutropenia at the time of reactivation. Finally, 160 (29%) patients were analysed. According to multivariate analysis, at the time of treatment initiation, the risk factors significantly associated with a grade III–IV Ganciclovir-related neutropenia included a high viral load (hazard ratio (HR) = 2.68, 95% CI 1.25–5.737, p 0.01); an absolute neutrophil count >3000 was a protective factor (HR = 0.26, 95% CI 0.125–0.545, p <0001) whereas serum creatinine >2 mg/dL was associated with higher Ganciclovir-related neutropenia (HR = 2.4, 95% CI 1.11–5.17, p 0.002). This large analysis revealed three risk factors for Ganciclovir-related neutropenia among patients with CMV reactivation after allogeneic stem cell transplantation; prompt identification of patients at risk when antiviral therapy is started may allow clinicians to adopt adequate preventive measures, so reducing the morbidity and mortality associated with CMV reactivation.

Congenital cytomegalovirus infection in Canada: Active surveillance for cases diagnosed by paediatricians

To determine the rate of diagnosis; demographic, clinical and laboratory characteristics; and management of congenital cytomegalovirus (cCMV) cases identified by paediatricians in routine clinical practice in Canada. National active monthly surveillance of all clinically practicing general and subspecialty paediatricians in Canada was performed for cCMV from March 1, 2005 to February 28, 2008, through the Canadian Paediatric Surveillance Program. Forty-nine cases of cCMV infection were reported (4.5 per 100,000 births): 40.8% were born before 36 weeks' gestation; 55.1% had a birth weight <2500 g; and 28.6% and 30.6% were below the third percentile for weight and head circumference, respectively. The median maternal age was 23 years, and 18.4% were <20 years of age. Eight mothers (16.3%) were Aboriginal (non-Aboriginal birth prevalence 3.9 per 100,000; Aboriginal birth prevalence 15.8 per 100,000; P<0.005). The most common laboratory abnormality was thrombocytopenia (53.1%). Thirty-three (67.3%) infants exhibited neurological manifestations; ganciclovir therapy was administered to 12 (36.4%) and was not administered to 21 (63.6%) of the infants with neurological manifestations. Only a minority of the expected number of symptomatic cCMV-infected infants were reported in the present study. The majority of these severely affected infants, including those with neurological disease, are not being treated with ganciclovir. The present description of current diagnosis and practice highlights the need for more complete case identification in the population as well as the need for increased awareness of the optimal therapy for symptomatic cCMV.

Impact Of CMV Reactivation On Relapse In Patients With Acute Leukemia Who Received Allogeneic Stem Cell Transplantation In First Remission: Possible GVL Effect In CMV Seropositive Population

In western countries, several studies have reported that CMV seronegative acute leukemia (AL) patients might have a beneficial effect from CMV seropositive donor for reduction of relapse. These observations resulted from subclinical CMV viremia with delayed initiation of antiviral therapy which showed a stimulatory effect on natural-killer cells and cytotoxic T-cells that may enhance graft-versus-leukemia (GVL) effect. We tried to evaluate the relationship between CMV reactivation and relapse of AL in Korean population, in which CMV serostatus is mostly positive for both donors and recipients.Three hundred eighty-nine AL patients with AML (n=197), ALL (n=192) who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) in first remission between 2007 and 2011 were retrospectively analyzed. Most of the donors (95.1%) and recipients (98.7%) were CMV seropositive. After engraftment, we serially measured CMV RQ-PCR once a week for surveillance of CMV reactivation and generally initiated preemptive ganciclovir (GCV) therapy when the titer reaches over 15,000copies/mL. Below 15,000, patients without GVHD were observed with tapering immunosuppressive agents. To evaluate the anti-leukemic activity of CMV viremia, patients with early death (<150 days) were excluded and subgroup analysis was performed according to the occurrence of chronic GVHD. OS, EFS and relapse incidence was compared between the 3 groups; undetected viremia, GCV-treated viremia with higher-level and untreated viremia with lower-level of CMV RQ-PCR.In the entire group, CMV viremia was an adverse factor for OS (p<0.001) and EFS (p<0.001), and patients with acute-GVHD significantly showed more CMV viremia (91.5% vs. 83.6%, p=0.017). After excluding early death (n=51), 195 patients without chronic-GVHD were finally analyzed. Untreated lower-CMV group (n=93) showed significantly superior OS (p<0.001) and EFS (p<0.001) with lower relapse rate (p=0.003) compared to the treated higher-CMV (n=72) and CMV-undetected group (n=30). Patients with other than untreated lower-CMV viremia and adverse-risk karyotype were independent adverse-risk factor for OS, EFS and relapse by multivariate analyses in subgroup without chronic GVHD. In chronic-GVHD subgroup (n=143), CMV-undetected group showed superior OS (p=0.006) and EFS (p=0.010), but the relapse rate was not significantly different (p=0.238). The results were similar in subgroup analyses for AML and ALL.Our data showed the possible GVL effect associated with CMV reactivation measured by RQ-PCR. This finding should be evaluated by larger validated studies to disclose the role of CMV reactivation in AL in the CMV seropositive population. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Prophylactic Infusion Of Multi-Virus Specific T Cells For Management Of Viral Reactivation and Infection In Patients Post Allogeneic Hematopoietic Stem Cell Transplantation (HSCT)

Aim Adoptive antiviral immunotherapy has recently gained popularity as a prophylactic and therapeutic strategy for viral infections post allogeneic stem cell transplant (HSCT). We report the outcomes of 10 patients who received multi-virus specific T cells (CTL) prophylactically post HSCT, and compare the outcomes of these patients to a contemporaneous cohort control. Methods Donor derived CMV, EBV, adenoviral and VZV specific T cells were generated in a 21-day culture, which involved stimulation of peripheral blood mononuclear cells by antigen-pulsed monocyte derived dendritic cells (DC) on days 0 and 7 and culture with 20-50units/ml of IL2. DC were transfected with an adenoviral vector encoding the CMV immune-dominant antigen pp65 or epitopes of EBV antigens EBNA1, LMP1 and LMP2. A commercial VZV vaccine (Varivax®, Merck &amp; Co) was used to stimulate VZV specific T cells. Patients undergoing matched sibling HSCT at Westmead Hospital from Feb 2011 to Jun 2012 were recruited. Patients with no active acute graft versus host disease were given 2x107/m2 multi-virus CTLs from day 35 onwards. After CTL infusion, patients were monitored for 12 months. Clinical outcome measures included adverse events related to CTL infusion, incidence of acute and chronic graft versus host disease and the incidence of CMV, EBV, adenoviral and VZV reactivations and infection. Patients were treated for viral reactivation according to standard institutional guidelines. A contemporaneous cohort of matched sibling transplant recipients treated during the same period at Westmead Hospital was used for comparison. A landmark analysis was performed on both groups using day 35 as a landmark to focus on outcome events related to CTL infusion. Results 10 patients who underwent matched sibling donor HSCT from Feb 2011 to Jun 2012 were given multi-virus specific T cells. There were no adverse events in any of the patients within 24 hours of T cell infusion. Three patients developed grade II-IV acute graft versus host disease (aGVHD) after transplant, 1 prior to CTL infusion, and 2 post CTL infusion. 7 patients developed chronic graft versus host disease. 8 patients reactivated CMV at any time post-transplant, 6 prior to CTL infusion of whom 2 required ganciclovir therapy; 5 patients developed low level CMV reactivation (median peak titre 600 copies/ml) post CTL infusion. 1 patient received ganciclovir for CMV PCR positivity on colonic tissue. No CMV disease developed and no evidence of EBV, VZV or adenoviral reactivation was noted during 12 months follow up. Using the day 35 landmark to exclude patients with grade II-IV acute GVHD, CMV reactivation and premature death in both CTL and control cohorts, 9 patients in the study cohort and 21 patients in the control cohort were available for comparison. 22% in the study cohort and 19% in the control cohort developed grade II-IV acute GVHD. 67% in the CTL cohort and 57% in the control cohort developed chronic GVHD. 7 of 9 patients in the study cohort and 9 of 21 patients in the control cohort had a CMV reactivation post transplant. The median peak CMV copy number was lower in the study cohort (600 vs 2840 copies/ml). 29% and 67% of patients received ganciclovir in the CTL and control cohort respectively. There was no clinically documented CMV disease in either cohort. 1 EBV, 1 adenoviral and 1 VZV reactivation were identified in the control cohort. No reactivations were identified in the study cohort. Conclusion Multi-virus T cell therapy appears safe, with no evidence of increased GVHD, or excess of adverse events observed as a result of CTL administration. Patients receiving T cells in this study had a lower median peak CMV copy number during reactivation episodes and fewer required anti-CMV pharmacotherapy. These results confirm out previous data on the efficacy of prophylactically administered CMV specific T cells given post HSCT but a larger study is required to determine their efficacy in preventing EBV, adenovirus and varicella zoster virus disease. Disclosures: No relevant conflicts of interest to declare.

Combined RNA interference of adenine nucleotide translocase-2 and ganciclovir therapy in hepatocellular carcinoma

The purpose of this study was to investigate the anticancer effects of combined RNA interference (RNAi) of the adenine nucleotide translocase-2 (ANT2) gene and ganciclovir (GCV) therapy for treatment of hepatocellular carcinoma cells (Huh 7) in an animal model. The Huh 7/NTG stable cell line was established by transfection of a vector with the human sodium iodide symporter (hNIS), HSV1-sr39 thymidine kinase (tk), and enhanced green florescent protein (EGFP) fusion gene into Huh 7 cells. mRNA expressions of these genes were evaluated by RT-PCR analysis. The functions of hNIS and HSV1-sr39tk were verified with (125)I uptake and (3)H-penciclovir (PCV) uptake tests. EGFP and hNIS expression was confirmed with confocal microscopy after immunocytochemical staining. We treated the tumor cells with ANT2 shRNA or GCV or both ANT2 shRNA and GCV and treated the in vivo mouse model with a Huh 7/NTG tumor xenograft. The therapeutic effects of the in vivo study were assessed with caliper measurements and gamma camera imaging using (99m)Tc-pertechnetate. Huh 7/NTG cells showed a cell number-dependent increase in (125)I uptake and a 24-fold higher (3)H-PCV uptake compared to parent Huh 7 cells. Huh 7/NTG cells transfected with ANT2 shRNA had lower ANT2 mRNA expression and more impaired proliferation activity than cells transfected with scramble shRNA. Proliferation of Huh 7/NTG cells was also inhibited by GCV treatment. Combined GCV and ANT2 shRNA therapy further inhibited cell proliferation in the in vitro study. The combined therapy with GCV and ANT2 shRNA showed a further decrease in tumor growth in the mouse model. Our results suggest that the combined RNA interference with ANT2 and GCV therapy inhibited hepatocellular carcinoma cell proliferation more than single GCV therapy or ANT2 shRNA therapy in vitro and in vivo. Therefore it could be applied treating incurable hepatocellular carcinoma.