Cytomegalovirus In Peripheral Blood Research Articles

Cytomegalovirus antigen-specific T cell immune responses in patients with autoimmune diseases under different cytomegalovirus infection status.

Cytomegalovirus antigen-specific T cell immune responses in patients with autoimmune diseases under different cytomegalovirus infection status.

924. Cytomegalovirus (CMV) Retinitis during Maintenance Chemotherapy for Acute Lymphoblastic Leukemia

Abstract Background Acute leukemia patients are at risk for cytomegalovirus (CMV) retinitis following hematopoietic stem cell transplantation, though the disease can also occur in non-transplant adult leukemia patients. Emerging data suggest a shift to pediatric-inspired chemotherapy regimens in adults with acute lymphoblastic leukemia (ALL) can lead to increasing cytopenias and impaired functional immunity, placing these patients at risk for this opportunistic infection. Here we describe a case of CMV retinitis in an ALL patient following a lower-intensity regimen during maintenance chemotherapy. Methods Chart review. Results A 55-year-old male with ALL presented to his optometrist with complaints of visual changes including “fogginess” and “floaters”. The patient had completed 8 cycles of dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab (EPOCH-R) and achieved complete remission. He had been on maintenance chemotherapy with 6-mercaptopurine, vincristine, methotrexate, and prednisone (POMP) for 2 months at the time of symptom onset. He was referred to his local ophthalmologist who had concerns for bilateral, zone 1 CMV retinitis based on fundoscopic exam (Figure 1). Vitreous aspiration was performed and CMV DNA PCR returned positive at 1.6 million IUs/ml. Peripheral blood CMV DNA PCR was also positive at 1133 IU/ml. He was started on combination therapy with intravitreal ganciclovir injections and oral valganciclovir 900 mg twice daily (Figure 2). The patient received 14 intravitreal injections with resultant stability of his eye exam, though he remained on induction valganciclovir for 5 months due to persistent blood CMV DNAemia. Letermovir was added to help suppress his peripheral CMV DNAemia and he attained partial vision recovery. Figure 1. Fundoscopic images Conclusion CMV retinitis is an uncommon and highly morbid infection that can occur during maintenance chemotherapy in adult non-transplant ALL patients. Early identification of the disease is imperative as delay can result in blindness or further systemic CMV disease. Treatment is challenging, involving systemic and intravitreal antiviral therapy, serial ophthalmologic exams, serum CMV monitoring, and close coordination with the treating hematologist. Disclosures Michael Boeckh, MD PhD, AlloVir (Consultant)Ansun Biopharma (Grant/Research Support)Astellas (Grant/Research Support)EvrysBio (Consultant, Other Financial or Material Support, Options to acquire equity, but have not exercised them)Gilead Sciences (Consultant, Grant/Research Support)GlaxoSmithKline (Consultant)Helocyte (Consultant, Other Financial or Material Support, Options to acquire equity, but have not exercised them)Janssen (Grant/Research Support)Kyorin (Consultant)Merck (Consultant, Grant/Research Support)Moderna (Consultant)Symbio (Consultant)Takeda (formerly known as Shire) (Consultant, Grant/Research Support)VirBio (Consultant, Grant/Research Support) Ryan Cassaday, MD, Amgen (Grant/Research Support, Advisor or Review Panel member)Kite/Gilead (Grant/Research Support, Advisor or Review Panel member)Merck (Grant/Research Support)Pfizer (Grant/Research Support, Advisor or Review Panel member)Seagen (Other Financial or Material Support, Spouse is employee and hold stock)Vanda Pharmaceuticals (Grant/Research Support)

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.

T-cell immune response against cytomegalovirus in peripheral blood and colonic mucosa from ulcerative colitis and Crohn's disease patients.

T-cell immune response against cytomegalovirus in peripheral blood and colonic mucosa from ulcerative colitis and Crohn's disease patients.

Report of a Myocarditis Outbreak among Pediatric Patients: Human Herpesvirus 7 as a Causative Agent?

The etiology of myocarditis in children has not yet been completely elucidated. Medical records of eight pediatric patients diagnosed with acute myocarditis within a 41-day period in a small-town hospital were retrospectively analyzed. We examined antibody titers of adenovirus, Epstein-Barr virus, herpes simplex virus, respiratory syncytial virus, varicella-zoster virus and cytomegalovirus in peripheral blood. We used polymerase chain reaction (PCR) amplification to detect genetic sequences from Human herpesvirus (HHV) 7, HHV 6, enterovirus, measles or parvovirus in peripheral blood. The causative agent was HHV 7 in four patients. HHV 7 sequences were detected through PCR in one patient with rapid deterioration. Of four patients with HHV 7, two presented with dilated cardiomyopathy. To our knowledge, this is the first report to suggest HHV 7 as a causative agent for acute myocarditis. We believe HHV 7 should be considered as a possible etiologic pathogen for patients with suspected myocarditis.

Cytomegalovirus, inflammatory bowel disease, and anti-TNFα.

Anti-TNFα agents emerged in inflammatory bowel disease (IBD) as an effective option in situations that, otherwise, would be refractory to medical therapy. Cytomegalovirus infection may present with a high spectrum of manifestations and lead to high morbidity and mortality. However, its clinical significance in IBD course remains unknown and data on its association with anti-TNFα are limited. This study aims to evaluate cytomegalovirus (CMV) infection/disease in patients with IBD treated with anti-TNFα; if possible, possible risk factors associated with CMV infection/disease in IBD patients under anti-TNFα as well as the influence of CMV infection/disease in IBD course would be determined. During three consecutive years, all IBD patients starting infliximab in our department were included. Cytomegalovirus status before anti-TNFα was evaluated. Data regarding IBD, therapeutic and IBD course after infliximab, were recorded. CMV analysis was performed with polymerase chain reaction (PCR)-cytomegalovirus in peripheral blood and colonoscopy with biopsies (histopathology/immunohistochemistry). We included 29 patients: female-83%; Crohn's disease-51.8%, ulcerative colitis-44.8%, non-classified colitis-3.4%; 23 cytomegalovirus seropositive. Median follow-up: 19months (3-36). During follow-up, 14 patients were under combination therapy with azathioprine and 5 did at least 1cycle of corticosteroids. Twenty-one patients responded to infliximab. We registered 8 exacerbations of IBD. Four patients discontinued infliximab: none had CMV infection. We documented 1 case of intestinal cytomegalovirus infection-detected in biopsies performed per protocol in an asymptomatic UC patient, who responded to valganciclovir without infliximab discontinuation. Infliximab, with/without immunosuppression, does not confer an increased risk of (re)activation of cytomegalovirus. Cytomegalovirus was not responsible neither for significant morbidity nor mortality in IBD.

Immunological Prediction of Cytomegalovirus (CMV) Replication Risk in Solid Organ Transplantation Recipients: Approaches for Regulating the Targeted Anti-CMV Prevention Strategies.

The current cytomegalovirus (CMV) prevention strategies in solid organ transplantation (SOT) recipients have contributed towards overcoming the detrimental effects caused by CMV lytic infection, and improving the long-term success rate of graft survival. Although the quantification of CMV in peripheral blood is the standard method, and an excellent end-point for diagnosing CMV replication and modulating the anti-CMV prevention strategies in SOT recipients, a novel biomarker mimicking the CMV control mechanism is required. CMV-specific immune monitoring can be employed as a basic tool predicting CMV infection or disease after SOT, since uncontrolled CMV replication mostly originates from the impairment of immune responses against CMV under immunosuppressive conditions in SOT recipients. Several studies conducted during the past few decades have indicated the possibility of measuring the CMV-specific cell-mediated immune response in clinical situations. Among several analytical assays, the most advancing standardized tool is the QuantiFERON®-CMV assay. The T-Track® CMV kit that uses the standardized enzyme-linked immunospot assay is also widely employed. In addition to these assays, immunophenotyping and intracellular cytokine analysis using flow cytometry (with fluorescence-labeled monoclonal antibodies or peptide-major histocompatibility complex multimers) needs to be adequately standardized and validated for potential clinical applications.

Monitoring of trough plasma ganciclovir levels and peripheral blood cytomegalovirus (CMV)-specific CD8+ T cells to predict CMV DNAemia clearance in preemptively treated allogeneic stem cell transplant recipients.

It is uncertain whether monitoring plasma ganciclovir (GCV) levels is useful in predicting cytomegalovirus (CMV) DNAemia clearance in preemptively treated allogeneic stem cell transplant recipients. In this observational study, including 13 episodes of CMV DNAemia treated with intravenous (i.v.) GCV or oral valganciclovir, we showed that monitoring trough plasma GCV levels does not reliably predict response to therapy. Rather, immunological monitoring (pp65 and immediate-early [IE]-1-specific gamma interferon [IFN-γ]-producing CD8+ T cells) appeared to perform better for this purpose.

Multiplex polymerase chain reaction for six herpesviruses after hematopoietic stem cell transplantation

Herpesviruses cause life-threatening diseases after hematopoietic stem cell transplantation (HSCT). It is necessary that viral diseases are identified early and safely diagnosed. The purpose of this study was to evaluate the efficacy of multiplex polymerase chain reaction (PCR) for qualitative detection of the six herpesviruses simultaneously: herpes simplex virus type 1 and type 2, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus (EBV) and human herpesvirus 6B. Multiplex PCR was applied to patients with various clinical manifestations including central nervous system, cutaneous and mucosal complications after allogeneic HSCT, and the data were retrospectively analyzed. Patients positive for cytomegalovirus in peripheral blood by multiplex PCR might need pre-emptive treatment, but a positive result for EBV had no specific correlation with EBV-associated post-transplant lymphoproliferative disease, and positive result for human herpesvirus 6B failed to show any clinical significance. The multiplex PCR was safe and helpful to diagnose viral diseases of local regions, for example, the central nervous system, skin and mucosa. It may be worthwhile to survey the six herpesviruses with multiplex PCR after HSCT.

Cytomegalovirus infection and immunosuppressant treatment in allogeneic hematopoietic stem cell transplantation recipients

To explore the correlation between peripheral blood cytomegalovirus (CMV) DNA level and cyclosporine A (CsA) plasma concentration among allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients who received immunosuppressant treatment, and to evaluate the potential clinical value. A total of 32 allo-HSCT patients were enrolled and their data were analyzed retrospectively. Ganciclovir was used to prevent CMV infection before the transplantation. Routine fluorescence PCR was admitted to test the blood CMV DNA level. The patients were divided into 2 groups: a CMV DNA positive group and a CMV DNA negative group. Enzyme multiplied immunoassay technique was adopted regularly to monitor the blood CsA concentration. The correlation between CMV DNA level and CsA concentration was analyzed. The CMV infection rate in patients who received allo-HSCT was 53.13%. The blood CsA concentration in the CMV DNA positive group was significantly higher than that in the CMV DNA negative group (P<0.05). Through the ROC curve, the area under the curve on Day 1, 7, and 14 had statistical significance compared with 0.5, and the corresponding blood CsA concentration was 203.15, 215.55, and 302.65 ng/mL, respectively. Immunosuppressive drug concentration can affect the dynamic changes of CMV DNA. High blood CsA concentration may be one of the reasons for CMV infection. Monitoring the blood CsA concentration may provide guidance for clinical treatment.

Persistent pyrexia post-liver transplantation: A unique case of pericardial tuberculosis

A 60-year-old Caucasian woman was admitted into the hospital with a two-week history of dry cough, fever and breathlessness. There were no other complaints. She had an orthotopic liver transplant (OLT) three months previously for end-stage primary biliary cirrhosis (PBC) and was on azathioprine 125 mg and tacrolimus 9 mg per day. On examination, she was pale, tachycardic and temperature was 38.7 °C. There was medium to coarse crepitations in the left lung base. Initial investigations were: white cell count (WCC) 8.5 (NR 4–11×10/L), neutrophils 4.5 (NR: 2–7.5×10/L), lymphocytes 1.5 (NR 1.5– 4×10/L), haemoglobin (Hb) 7.1 (NR: 11–16 g/dL), platelets 375 (NR: 150–400×10/L), C-reactive protein (CRP) was 182 (NR: b10 mg/L), AST 86 (NR: 5–43 U/L), ALP 965 (NR: 70– 330 U/L), bilirubin 12 (NR:1–22 μmol/L) and creatinine 133 (NR: 50–111 μmol/L). Chest radiograph (CXR) showed clear lung fields but increased cardio-thoracic ratio (C: T=0.6). Blood, urine, sputum and stool cultureswere negative. Shewas started on intravenous tazocin (piperacillin–tazobactam) 4.5 g t.d.s. for a possible lower respiratory tract infection. Despite completing antibiotics, she remained unwell with swinging pyrexia (37.5 to 39 °C). Abdominal ultrasound scan was normal but a trans-thoracic echocardiogram confirmed loculated pericardial effusion. Pericardiocentesis provided symptomatic relief; pericardial fluid was sent for microscopy, culture and cytology. All were negative, including a Ziehl– Neilsen (ZN) stain. Peripheral blood cytomegalovirus (CMV) polymerase chain reaction (PCR) showed high viral loads at 1.7×10 copies/mL.

Cytomegalovirus reactivation exacerbated thrombocytopenia and haemolysis in a patient with systemic lupus erythematosus

To the editor, Immunosuppressive therapy, including high-dose corticosteroids and other immunosuppressive agents, has markedly improved systemic lupus erythematosus (SLE) patient survival. However, these patients are deemed at increased risk for opportunistic infection because of impaired immunity due to immunosuppressive therapy. We encountered a patient with diVuse lupus nephritis complicated with severe thrombocytopenia and haemolytic anaemia, whose haematological manifestations might have been related to cytomegalovirus (CMV) reactivation. A 23-year-old Japanese man was hospitalized because of urinary abnormalities. Two months prior to admission, proteinuria and haematuria had been detected on a routine health examination. On admission, physical Wndings were unremarkable; however, laboratory testing revealed active nephritic urinary sediments with marked urinary protein excretion (2.6 g per day), low serum complement [C3 (24 mg/dl) and C4 (6 mg/dl)] values, and high titre of antidouble-stranded DNA (ds-DNA) antibody (63.1 IU/ml; reference range < 20). Renal biopsy revealed diVuse mesangial proliferation and capillary wall thickening on light microscopy, “full-house” pattern on immunoXuorescence microscopy, and mesangial/subendothelial electron-dense deposits on electron microscopy. During hospitalization, constitutional manifestations (i.e., fever and general myalgia), arthritis, and lymphopenia developed; thus, we diagnosed him to have SLE associated with diVuse lupus nephritis [International Society of Nephrology (ISN), Class IV]. Intravenous methyl prednisolone (0.5 g, three consecutive days) once every 2 weeks and oral corticosteroid (prednisolone, 55 mg per day) was started. This treatment improved his immunological abnormalities (i.e., hypocomplementaemia and elevated anti-ds-DNA antibody levels); however, his renal function deteriorated and serum creatinine levels were elevated to around 3.0 mg/dl with nephrotic-range proteinuria. On the 60th hospital day, gross haematuria, systemic petechia, melena, and epistaxis suddenly developed. He was afebrile, and physical examination was unremarkable except the bleeding tendency; however, laboratory data revealed marked thrombocytopenia (9,000/ l). Peripheral blood smear showed no morphological abnormalities. Coagulation screening results and serum transaminases were normal, but a haptoglobin level was undetectable. Bone marrow aspiration test showed a normal level and morphology of megakaryocytes, an active erythroid system, and no signs of haemophagocytosis. Abdominal computed tomography revealed no remarkable abnormalities. Platelet-associated IgG (157 ng/10 cells; reference range 5.0–25.0 ng/10 cells) was positive, but direct Coombs’ test, anti-granulocyte antibody, anti-cardiolipin-IgM/IgG antibodies, anti2 glycoprotein I antibody, cryoglobulin, and plasma activity of von Willebrand factorcleaving protease (69.5%) showed negative or normal results. Evaluation for bacterial infection, including blood and urine cultures, yielded negative results. Anti-parvovirus B 19 IgM antibody and Epstein–Barr (EB) virus plasma viremia were also negative, but peripheral blood CMV antigenaemia assay showed an equivocal result (5/34,000 peripheral blood leukocytes) [1]. Platelets transfusion for the marked bleeding symptoms was initiated. Intravenous methyl prednisolone (1.0 g per day, three consecutive days) followed by oral corticosteroid (prednisolone, 55 mg per T. Sugimoto (&) · M. Aoyama · N. Takeda · M. Sakaguchi · Y. Nishio · T. Uzu · A. Kashiwagi Department of Internal Medicine, Shiga University of Medical Science, Seta, Otsu, Shiga 520-2192, Japan e-mail: toshiro@belle.shiga-med.ac.jp

Recovery from CMV esophagitis after allogeneic bone marrow transplantation using non-myeloablative conditioning: The role of immunosuppression

Cytomegalovirus (CMV) positive recipients of CMV negative bone marrow bear a significantly higher risk of developing CMV disease compared to all other constellations. Here, we report a case of severe CMV induced esophagitis after allogeneic bone marrow transplantation for paroxysmal nocturnal hemoglobinuria. The patient developed the first symptoms between day 10 and 20 after dose reduced conditioning and HLA-matched unrelated stem cell transplantation. Esophageal tissue biopsies as well as peripheral blood proved positive for CMV DNA by PCR. Treatment with acyclovir, ganciclovir, foscarnet, cidofovir, and immunoglobulines resulted in elimination of CMV in peripheral blood but not in clinical improvement. Only tapering of cyclosporine at day +120 eventually led to the development of CMV-specific T-cells and resolution of esophagitis.

Quantitation of peripheral blood cytomegalovirus DNA for monitoring recurrent cytomegalovirus retinitis in pediatric solid organ transplant recipients.

Cytomegalovirus (CMV) infection is a major concern following solid organ transplantation, especially in the pediatric population who remain at high risk of primary infection. CMV disease leads not only to increased patient and graft morbidity, but also to increased health care costs. This study describes the usefulness of a quantitative CMV polymerase chain reaction (PCR) technique for monitoring peripheral blood CMV DNA in pediatric recipients of kidney and liver allografts who had recurrent CMV retinitis. The incidence of CMV disease in 28 pediatric transplant recipients was 28.6%, one-half of whom developed retinitis. Two of these patients had recurrent retinitis on cessation of anti-viral treatment. A peripheral blood CMV DNA copy number of > or =500/microg of DNA was associated with recrudescence of the retinitis in these patients. We conclude that the measurement of peripheral blood CMV DNA by PCR is a useful tool for the surveillance of disease resolution and recurrence. This is particularly important in patients with CMV retinitis, who may remain asymptomatic for a period of time, despite recurrences.

Significance of leukocyte concentration in the performance of the quantitative cytomegalovirus (CMV) antigenemia assay

Background: The quantitative cytomegalovirus-antigenemia (CMV-Ag) assay is an important technology in the regimen of tests utilized in the care and management of acquired immunodeficiency syndrome (AIDS) and other immunocompromised patient groups. Performance of this assay is contingent upon the appropriate processing of the polymorphonuclear leukocyte (PMNL) compartment of the peripheral blood. However, a cell input standard in the performance of the CMV-Ag assay, has not been established. Interpretive differences between laboratories utilizing the CMV-Ag assay may reflect this lack of test uniformity. Objectives: To determine the effect of different PMNL concentrations on the quantitation of CMV in peripheral blood. The leukocyte concentration resulting in optimal rates of viral detection, will be compared with the shell vial assay-indirect immunofluorescent assay (SVA-IFA), and conventional tube culture (TC-CPE). Study design: A total of 74 freshly collected blood specimens were tested by the CMV-Ag assay, using cytospin preparations consisting of 2×10 5, 4×10 5 and in preliminary experiments, 8×10 5 PMNLs/slide. Data obtained from these studies were compared to SVA-IFA and TC-CPE. Viral load was monitored among 11 symptomatic patients through sequential testing of these patients at the start of ganciclovir (GCV), foscarnet (PFA), or combination drug therapy. Results: Among 74 blood specimens tested by the CMV-Ag assay, cytospin preparations consisting of 4×10 5 compared with 2×10 5 PMNLs/slide, affected a mean positive cell increase of 215% ( P=0.03). PMNL slide preparations consisting 8×10 5 cells produced background levels which prevented accurate reading of slides. The CMV-Ag assay was more sensitive than the SVA-IFA, but equivalent to TC-CPE. Among 11 patients started on drug therapy, viral load was markedly reduced in 8 within 2–3 weeks; three patients (2 deceased within 3 weeks after receiving therapy), showed no decrease in viral load. One patient was identified as harboring a PFA resistant strain. Conclusions: A PMNL concentration of 4×10 5 cells facilitated the reading of CMV-Ag assay slide preparations. The modified CMV-Ag assay furthermore, is applicable in the monitoring of viral load for the tracking of susceptible or resistant CMV strains.

Cell culture-PCR technique for detection of infectious cytomegalovirus in peripheral blood

We determined the efficacy of PCR for the direct detection of infectious cytomegalovirus (CMV) in specimen-inoculated MRC-5 tube cultures (cell culture-PCR [CC-PCR]). Parallel testing was performed by a shell vial assay-indirect immunofluorescence assay (SVA-IFA) and isolation (conventional MRC tube cultures [TC-CPE]. The sensitivity, specificity, and positive and negative predictive values for CC-PCR, SVA-IFA, and TC-CPE were 81, 99, 94, and 94%, 86, 100, 100, and 96%, and 77, 100, 100, and 93%, respectively. Future application of CC-PCR may be directed toward its use as a screening tool for cytomegalovirus genotypic variants.

Persistence of cytomegalovirus in peripheral blood from blood donors.

Persistence of cytomegalovirus in peripheral blood from blood donors.