CMV monitoring using blood cells and plasma: a comparison of apples with oranges?

Quantitative cytomegalovirus (CMV) monitoring is still far from being standardized between transplant centers. In the present study, we compared assays for quantitative CMV monitoring using blood cells and plasma. Four hundred and thirty-five consecutive samples from 29 patients with active CMV infection after allogeneic T-cell-depleted hemopoietic stem cell transplantation were tested in parallel using pp65 antigenemia and quantitative CMV polymerase chain reaction (PCR) in blood cells and plasma (COBAS AMPLICOR CMV MONITOR). Although only 142 (53.1%) of 253 positive samples were concordantly identified by all three assays, the number of positive samples detected by each assay was not different and the quantitative values were correlated, provided that nucleic acid (NA) in plasma was isolated by COBAS AmpliPrep and not by the manual protocol. Six (18%) of 34 episodes with active CMV infection were not detected using CMV PCR in plasma; whereas in times of white blood cell aplasia or blast crisis of leukemia, samples with active CMV infection in plasma could not be detected using blood cells. We conclude that CMV monitoring in whole blood could be favorable compared with assays using plasma or blood cells alone. Automated NA isolation could become an attractive tool for a more sensitive and better standardized molecular diagnostics.     

Evaluation of the AMPLICOR CMV, COBAS AMPLICOR CMV monitor and antigenemia assay for cytomegalovirus disease

The AMPLICOR CMV (qualitative DNA assay by PCR), COBAS AMPLICOR CMV Monitor (quantitative DNA assay by PCR), and antigenemia assay were tested for their ability to diagnose cytomegalovirus (CMV) infection in 115 immunocompromised patients. The AMPLICOR qualitative assay and the antigenemia assay were positive for all nine patients with a clinical diagnosis of CMV disease. The AMPLICOR quantitative assay was negative for one of the nine patients. In 106 patients without CMV disease, the AMPLICOR qualitative test was positive in 22, the quantitative test was positive in 23, and the antigenemia test was positive in 55 patients. The AMPLICOR qualitative and quantitative assays had specificities of 79% and 78% in patients without CMV disease, while that of the antigenemia assay was 48%. Diagnostic efficiencies were 79% for the AMPLICOR qualitative assay, 69% for the AMPLICOR quantitative assay, and 48% for the antigenemia assay. All three tests yielded positive results before, or at the same time as, the onset of CMV disease in most cases, which suggests they can be used to predict disease before the onset of symptoms. During antiviral treatment, test results tended to decrease quantitatively and finally became negative; negative results were followed by remission of symptoms. This suggests that the AMPLICOR quantitative assay and the antigenemia assay could be useful for monitoring therapeutic efficacy. The AMPLICOR qualitative and quantitative assays, as well as the antigenemia assay were considered effective for all of the following: diagnosing CMV disease, predicting the onset of disease, and evaluating the effectiveness of antiviral chemotherapy. The antigenemia assay was at times difficult to perform in the case of severely neutropenic patients, whereas the AMPLICOR assays could be used in such cases.     

A short-term preemptive treatment for cytomegalovirus infection in seropositive patients after liver transplantation

Background/purpose

Cytomegalovirus (CMV) infection remains a challenge following liver transplantation. Preemptive treatment is an effective strategy for CMV infection. However, how long preemptive treatment should be applied is not defined.

Methods

Clinical records of preemptive treatment for CMV infection in patients who underwent liver transplantation were collected. CMV antigenemia (pp65) was monitored weekly during hospital stay and subsequently on follow up whenever indicated clinically. Antiviral treatment was administered based on positive antigenemia (>1 positive cell per 500,000 leukocytes) and discontinued when antigenemia became negative.

Results

CMV infection was diagnosed in 58 (43.9%) of 132 liver transplantation patients. All 58 patients were seropositive for CMV before transplantation. CMV infection was first diagnosed at a median time of 20?days (interquartile range [IQR] 15.3?C26) after transplantation. Twelve (20.7%) patients developed repeated infections. Only one of 58 patients (1.7%) was suspected to have invasive disease. The median (IQR) duration of antiviral treatment was 7 (7?C12) days. Of these patients with CMV infection, 14 (24.1%) patients developed acute rejection peri-anti-CMV treatment and 36 (62.1%) developed other infectious complications.

Conclusion

Preemptive treatment is an effective way to halt the progression of asymptomatic CMV infection. A brief course of antiviral treatment is enough for seropositive patients with CMV infection after liver transplantation.     

CMV infection of liver transplant recipients: comparison of antigenemia and molecular biology assays

Background  

CMV is a major clinical problem in transplant recipients. Thus, it is important to use sensitive and specific diagnostic techniques to rapidly and accurately detect CMV infection and identify patients at risk of developing CMV disease. In the present study, CMV infection after liver transplantation was monitored retrospectively by two molecular biology assays - a quantitative PCR assay and a qualitative NASBA assay. The results were compared with those obtained by prospective pp65 antigenemia determinations.     

Response to peginterferon-alpha 2b and ribavirin in Japanese patients with chronic hepatitis C genotype 1

Purpose

Patient age and gender may be associated with response to peginterferon alpha plus ribavirin, the current standard of care (SOC) for chronic hepatitis C genotype 1. We queried whether there was an association between age, gender, and treatment response to SOC in Japanese patients infected with hepatitis C virus (HCV) genotype 1.

Methods

Between 2006 and 2009, HCV-infected Japanese patients treated with peginterferon alpha-2b plus ribavirin for 48 weeks were enrolled. Patients were allocated into four groups according to age and gender, and epidemiological data and treatment outcomes were retrospectively analyzed. HCV RNA was measured with COBAS AMPLICOR HCV Monitor Test v. 2.0.

Results

The overall sustained virological response (SVR) rate was 49.8%: patients aged ≤65 and >65 years, 50.9 and 44.0%, respectively; male and female, 56.5 and 39.0%. SVR rates of SOC against HCV genotype-1 females aged >65 years (19.0%) were inferior to those in males aged >65 years (57.8%) in Japan. Multivariate logistic regression analysis showed that SVR was attained independently of adherence 80/80/80 in all groups.

Conclusions

Adherence to medication is also a key factor for the eradication of HCV in patients aged >65 years. As the SVR rate of patients aged ≤65 years was similar to that of patients aged >65 years, SOC could be useful for treating some of the elderly patients.     

A stringent preemptive protocol reduces cytomegalovirus disease in the first 6 months after kidney transplantation

Background

The optimal strategy to prevent cytomegalovirus (CMV) disease after kidney transplantation continues to be open to debate. The preemptive approach requires regular determination of CMV viremia and prompt initiation of therapy.

Methods

We retrospectively compared the incidence of CMV disease during two periods at our center: A first phase (P1, n?=?84 kidney recipients), during which time the intensity of surveillance was determined by the responsible physician, was compared to a second phase (P2, n?=?74), when a stringent protocol of CMV surveillance was required for all patients. The preemptive approach was applied for all CMV risk groups; prophylaxis was optional in the case of treatment for rejection or delayed graft function in the intermediate- and high-risk group. Follow-up was truncated at 6?months after transplant surgery. CMV syndrome was differentiated from asymptomatic replication by the presence of at least one systemic symptom, while diagnosis of CMV end-organ disease required histological confirmation.

Results

Immunosuppression was similar in the two periods. CMV prophylaxis was used equally (26?%) in both periods. The probability for asymptomatic viremia episodes was not different for patients in P1 and P2 regardless of the prevention strategy. For patients following the preemptive strategy, the probability for CMV disease was increased during P1 (p?=?0.016), despite fewer PCR assays being performed in phase 2. Protocol violations were only observed during P1.

Conclusions

The probability of CMV disease episodes (CMV syndrome and CMV end-organ disease) was substantially reduced using a very stringent protocol. This study highlights the crucial importance of a stringent protocol with optimal adherence by all caregivers if the preemptive strategy is to be successful.     

Risk factors associated with elevated blood cytomegalovirus pp65 antigen levels in patients with autoimmune diseases

Objectives

To further assess the relationship between elevated levels of cytomegalovirus (CMV) pp65 antigen in blood, as indicative of viral load, during treatment-free follow-up and CMV diseases in patients with autoimmune diseases and to identify any risk factors associated with elevated viral loads.

Methods

This was a retrospective review of the electronic medical charts of 148 patients with autoimmune diseases who tested positive for CMV pp65 antigen in the blood.

Results

A total of 106 patients were analyzed. During follow-up, elevated viral loads were detected in 35 patients who were not on antiviral therapy, of whom five developed CMV diseases. Elevated viral load was significantly associated with CMV diseases [5/35 vs. 0/71 (no elevated viral load); P = 0.001). Multivariate analysis revealed that lymphopenia [lymphocyte numbers <700/mm³, odds ratio (OR) 34.44, 95 % confidence interval (CI), 7.82–151.66; P < 0.001], systemic lupus erythematosus (SLE) (OR 6.71, 95 % CI, 1.23–36.49; P = 0.028), and polymyositis/dermatomyositis (PM/DM) (OR 10.62, 95 % CI 1.41–79.77; P = 0.022) were significantly associated with elevated viral load.

Conclusions

Elevated viral load was significantly associated with CMV diseases. Patients with SLE or PM/DM and lymphopenia would therefore benefit from a detailed viral load follow-up and careful physical examination.     

Real-time PCR assays based on distinct genomic regions for cytomegalovirus reactivation following hematopoietic stem cell transplantation

Real-time PCR has many advantages compared with antigenemia and qualitative PCR assays for detecting cytomegalovirus (CMV) infection in patients following SCT. However, the procedure used in each report was not standardized. This study compares the CMV load detected by real-time PCR assays amplifying distinct genomic regions. Real-time PCR assays based on US17, UL65, immediate early protein (IE) and glycoprotein B(gB) were selected and comparisons were made between each genomic region, and with antigenemia and nested PCR (IE region) in 18 SCT patients. The CMV load detected by real-time PCR using all combinations of primers targeting distinct genomic regions and by antigenemia assays correlated well. However, US17 and UL65-PCR could detect CMV earlier than gB-PCR, antigenemia and nested PCR assays. In longitudinal analysis, gB-PCR demonstrated a trend for showing a lower viral load in some patients than US17-, UL65- and IE-PCR. Moreover, the results suggest that a cutoff level of 500 copies/ml might be used to decide when to initiate treatment. We propose that monitoring should be carried out using real-time PCR assays targeting the US17 region and that a CMV load of 500 copies/ml could be used as a cutoff value for initiating treatment in patients following SCT, receiving immunoglobulin prophylaxis.     

Response to Peginterferon-alfa 2b and Ribavirin in Japanese Patients with Chronic Hepatitis C Genotype 2

Background

The current standard treatment for patients infected with hepatitis C virus (HCV) of genotype 2 is the combination of peginterferon (PEG-IFN) plus ribavirin (RBV) for 24 weeks.

Aims

We assessed the sustained virological response (SVR) rates in HCV genotype 2-infected Japanese patients in relation to the duration of treatment.

Methods

Between 2006 and 2009, among 147 patients with HCV genotype 2-infection in Chiba Prefecture, 138 consecutive patients were finally enrolled. Twenty-one, 97 and 20 patients were treated with PEG-IFN-alfa 2b plus RBV for 16, 24 and 48 weeks, respectively. Epidemiological data and treatment outcomes were retrospectively evaluated. HCV RNA was measured with COBAS AMPLICOR HCV Monitor Test v. 2.0.

Results

The overall SVR rate was 82.6% (114 of 138): treatment-naïve patients, 86.4% (89 of 103); patients with history of previous treatment, 71.4% (25 of 35). Patients treated for 16, 24 and 48 weeks obtained SVR rates of 66.6% (14 of 21), 86.5% (84 of 97) and 80.0 (16 of 20), respectively.

Conclusions

The SVR rates of PEG-IFN-alfa 2b plus RBV in Japanese patients were similar to those in previous studies. Combination treatment for 24 weeks for some patients infected with HCV genotype 2 may be superior to that for 16 weeks. More precise patient selection will be needed to shorten the combination treatment.     

Cytomegalovirus infection during immunosuppressive therapy for diffuse parenchymal lung disease

Background and objective: Cytomegalovirus (CMV) infection is a life‐threatening condition in patients with diffuse parenchymal lung diseases (DPLDs), who are receiving immunosuppressive therapy. The aim of this study was to describe the clinical features of CMV infection and to propose a strategy for managing CMV infection in patients with DPLD who are receiving immunosuppressive therapy. Methods: A retrospective longitudinal observational study was performed on 69 patients with DPLDs (39 with acute/subacute onset, 30 with chronic onset) who were receiving immunosuppressive therapy and were positive for CMV pp65 antigen (CMV‐pp65Ag) in peripheral blood leukocytes (PBLs). Results: Clinical CMV disease and subclinical CMV antigenaemia developed in 23 and 46 patients, respectively. The cut‐off level of CMV‐pp65Ag indicating clinical CMV disease, as determined by receiver operator characteristic curve analysis, was 7.5 cells per 5 × 10⁴ PBLs. Multivariate analysis revealed that early CMV infection was associated with acute/subacute onset of underlying DPLD and with respiratory dysfunction at the commencement of immunosuppressive therapy. Multivariate analysis also suggested that the acute/subacute onset of underlying DPLD, a CMV‐pp65Ag titre of >7.5 cells per 5 × 10⁴ PBLs, and C‐reactive protein levels ≥10 mg/L indicated a poor prognosis. Conclusions: We recommend that CMV‐pp65Ag antigenaemia of >7.5 cells per 5 × 10⁴ PBLs in patients with DPLD should be treated with ganciclovir. Patients with lower levels of CMV‐pp65Ag antigenaemia should be closely monitored or treated with ganciclovir if the clinical findings suggest a poor prognosis.     

New molecular assays to predict occurrence of cytomegalovirus disease in renal transplant recipients

Thirty renal transplant recipients, after transplantation, were tested weekly with the following assays: cytomegalovirus (CMV) antigenemia (pp65 Ag), plasma qualitative Amplicor CMV (P-AMP), plasma and peripheral blood leukocyte quantitative Amplicor CMV monitor (P- and PBL-CMM), peripheral blood leukocyte (PBL) quantitative Quantiplex bDNA CMV, version 2.0 (bDNA), and whole-blood Nuclisens pp67 CMV (pp67). Eleven patients developed symptomatic CMV disease, and 7 developed asymptomatic CMV infection. For prediction of CMV disease, the sensitivity, specificity, and positive and negative predictive values, respectively, were as follows: 100%, 63%, 61%, and 100% for pp65 Ag; 100%, 42%, 50%, and 100% for bDNA; 91%, 47%, 50%, and 90% for PBL-CMM; 55%, 74%, 55%, and 74% for P-AMP; 55%, 74%, 55%, and 74% for P-CMM; and 64%, 79%, 64%, and 79% for pp67. First positive results in PBL were obtained 9-10 days before symptoms of CMV disease, compared with 5-6 days in plasma and 0 days in whole blood. PBL assays appear to be more appropriate than plasma assays when pre-emptive therapy is required to prevent the rapid progression from the first detection of the virus to CMV disease.     

A Low Incidence of Cytomegalo Virus Infection Following Allogeneic Hematopoietic Stem Cell Transplantation Despite a High Seroprevalence

Cytomegalovirus (CMV) infection remains an important cause of morbidity and mortality following allogeneic stem cell transplantation (SCT). We wanted to study if the high sero-prevalence seen in our population translated into a high incidence of CMV infection following SCT. This is a retrospective analysis of patients who underwent allogeneic SCT between January 2008 and December 2012 at our centre. 475 patients underwent allogeneic SCT for malignant (46.5%) and non-malignant (53.5%) haematological disorders. 463 (97.4%) SCT recipients and 403 (84.8%) SCT donors were IgG seropositive for CMV. CMV reactivation within 100 days post SCT was seen in 174 (36.6%) at a median of 41 days (range 10–100) post SCT. Ganciclovir was used in 166 patients (95.4%) for a mean duration of 16 days (range 5–32). 157 patients (90%) responded to therapy. Sixty-six patients (42.3%) had secondary reactivation of the virus. Use of a male donor (p?=?0.000), donor and recipient age?>?15 (p?=?0.005 and 0.000), unrelated donor (p?=?0.000), degree of HLA mismatch (p?=?0.000), occurrence of acute GVHD (p?=?0.000) and steroid refractory acute GVHD (p?=?0.026) were identified as risk factors for CMV reactivation while early neutrophil recovery (<?15 days) was found to be protective (p?=?0.004). On multivariate analysis, male donor (p?=?0.042), degree of HLA mismatch (p?=?0.006), the occurrence of acute GVHD (p?=?0.000) and steroid refractory acute GVHD (p?=?0.031) continued to remain significant. 5-year overall survival was significantly better in patients without CMV reactivation compared to those who developed reactivation of CMV (68.9?±?3.7 vs 58.2?±?4.9% p?=?0.004). The incidence of CMV infection does not seem to be higher despite a high sero-prevalence of CMV. However, patients who developed CMV infection post SCT had inferior outcomes.     

Stable hepatitis C virus RNA detection by RT-PCR during four days storage

Background

Suboptimal specimen processing and storage conditions of samples which contain hepatitis C virus (HCV) RNA may result in a decline of HCV RNA concentration or false-negative results in the detection of HCV RNA in serum. We evaluated the stability of HCV RNA in serum and clotted blood samples stored at room temperature or at 4°C for 4 days with the aim of optimizing the standard procedures of processing and storage of samples.

Methods

Blood from five HCV RNA positive patients was collected in tubes with and without separator gel, centrifuged 1 or 6 hours after collection. Samples were then left 6, 24, 48, 72 or 96 h at room temperature (21.5 – 25.4°C) or at 4°C before determining their HCV RNA level using the COBAS AMPLICOR HCV MONITOR Test, vs 2.0 (Roche Diagnostic Systems).

Results

The logarithm of the HCV RNA level measurements remained within a 0.3 value of the means for 4 days at both temperatures (room temperature or 4°C).

Conclusions

We conclude that blood samples may be collected and aliquoted within 6 h of collection and can be stored at 4°C for 72 hours as proposed by the manufacturer without significant differences in measured HCV RNA level. Our results indicate that lapses in this scheme may still yield reliable results.

     

Analysis of cytomegalovirus (CMV) viremia using the pp65 antigenemia assay, the amplicor CMV test, and a semi-quantitative polymerase chain reaction test after allogeneic marrow transplantation

A pp65 antigenemia assay for polymorphonuclear leukocytes (PMNLs) (CINAkit Rapid Antigenemia), and a qualitative polymerase chain reaction (PCR) test for plasma 'PCR-P qual' (Amplicor cytomegalovirus [CMV] test) were performed for 126 samples (blood and plasma) obtained from 18 bone marrow transplant patients, over a 9-month surveillance period. Among those samples, 92 were assayed with a semi-quantitative PCR test for PMNLs 'PCR-L quant.' The number of samples with a positive CMV test for antigenemia and PCR-P qual assays was 20.63% and 12.7%, respectively, whereas the PCR-L quant assay was positive in 48 of the 92 samples assayed (52.17%). The rates of concordance of the results of PCR-P qual and antigenemia, PCR-P qual and PCR-L quant, antigenemia and PCR-L quant were 92%, 65.2% and 66.8%, respectively. The analysis of the results for the 92 specimens tested by all 3 methods showed a rate of concordance of 63% among all methods. Good agreement (kappa=0.72) was found only between pp65 Ag and PCR-P qual assays. Clinical disease correlates with an antigenemia high viral load. Three patients had CMV disease despite preemptive therapy, and all of them had graft-versus-host-disease (GVHD). PMNLs-based assays are more efficient in monitoring CMV reactivation, but for high-risk patients with GVHD, more sensitive assays (real-time PCR) must be done.     

Cytomegalovirus,inflammatory bowel disease,and anti-TNFα

Background and purpose

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.

Aims

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.

Methods

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).

Results

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: 19 months (3–36). During follow-up, 14 patients were under combination therapy with azathioprine and 5 did at least 1 cycle 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.

Conclusions

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.

     

TB PCR in the early diagnosis of tuberculous meningitis: evaluation of the Roche semi-automated COBAS Amplicor MTB test with reference to the manual Amplicor MTB PCR test. .

SETTING: Cecilia Makiwane Hospital, Mdantsane, Eastern Cape, Republic of South Africa. OBJECTIVE: To assess the role of the semi-automated Roche COBAS AMPLICOR(TM)Mycobacterium tuberculosis PCR test in the diagnosis of tuberculous meningitis (TBM). DESIGN: Eighty-three specimens of cerebrospinal fluid (CSF) were collected prospectively from 69 patients with suspected TBM. The COBAS AMPLICOR TB PCR test was compared with the manual AMPLICOR(TM)TB PCR test, clinical and cerebrospinal fluid (CSF) findings, direct ZN smear and radiometric TB culture. RESULTS: CSF from 7/40 (17.5%) patients treated for TBM were positive by TB COBAS AMPLICOR(TM). The sensitivity of the test was not significantly different (p=0.375) from the manual TB AMPLICOR(TM)PCR test. The comparative sensitivities of the TB COBAS AMPLICOR(TM)PCR and the manual AMPLICOR PCR for detecting cases of definite and probable TBM from CSF collected within 9 days of commencing antituberculosis treatment were 40% and 60% respectively. All 29 patients not treated for TBM were negative by COBAS AMPLICOR(TM), giving a specificity of 100%. CONCLUSION: The COBAS AMPLICOR(TM)TB PCR test is a rapid and highly specific diagnostic test for TBM. However, there was a non-significant trend favouring slightly greater sensitivity using the manual AMPLICOR(TM)TB PCR test.     

Use of multiple methods for genotyping Fusarium during an outbreak of contact lens associated fungal keratitis in Singapore

Background

Since African-Americans have twice the prevalence of cytomegalovirus (CMV) infections as age-matched Caucasians we sought to determine the ages and possible sources of infection of African-American children.

Methods

Subjects were 157 African-American healthy children and adolescents and their 113 household adults in Richmond VA. Families completed a questionnaire, provided saliva for antibody testing, and adolescents were interviewed regarding sexual activity.

Results

Regardless of age CMV seropositivity was not associated with gender, breast feeding, health insurance, sexual activity, or household income, education, or size. In the final regression model, prior CMV infection in adults was over two-fold higher than in children (chi-square = 18.8, p < 0.0001). At one year of age the CMV seropositivity rate was 11% (95%CI = 4% – 24%) and increased 1.8% each year until age 13 years. Between ages 13 and 20 years the CMV seropositivity rate remained between 22% and 33%. For adults, the CMV seropositivity rate was 84% in 21 year olds (95%CI = 69%–.92%). There was no association between CMV infections of the children and their mothers but CMV infections among siblings were associated.

Conclusion

We observed that African-American children had CMV seroprevalence rates by age 20 years at less than one-half of that of their adult mothers and caregivers. Sibling-to-sibling transmission was a likely source of CMV infections for the children. The next generation of African-American women may be highly susceptible to a primary CMV infection during pregnancy and may benefit from a CMV vaccine.     

CMV reactivation caused by methylprednisolone therapy for ARDS after esophagectomy

Background

Cytomegalovirus (CMV) infection is endemic worldwide. Although CMV reactivation often becomes a serious problem in immunocompromised patients, the prevalence of CMV reactivation caused by methylprednisolone therapy for ARDS after esophagectomy has yet to be determined.

Method

Among 175 consecutive patients with thoracic squamous cell esophageal cancer who underwent esophagectomy with extensive lymph node dissection at Akita University Hospital between 2007 and 2010, 11 patients (6.3?%) diagnosed with ARDS during the acute phase of esophagectomy were enrolled and treated with steroid pulse therapy, high-dose (15?C20?mg/kg/day) administration and tapering in this retrospective study.

Results

Seven of the 11 patients (63.6?%) were diagnosed with CMV reactivation based on CMV antigenemia assayed 19.1?days after the start of methylprednisolone administration and were treated with ganciclovir for 39.6?days. Six of the 7 patients (85.7?%) diagnosed with CMV reactivation were administered a total methylprednisolone dose of more than 4,000?mg. Though there was no significant difference between patients with and without CMV reactivation, there was a tendency that patients with CMV reactivation showed a lower minimum number of lymphocytes during the acute phase of esophagectomy (p = 0.051, Student??s t test, average 223.3 and 298.0/??l, respectively).

Conclusion

Though the number of study patients is small, the prevalence of CMV reactivation caused by high-dose methylprednisolone therapy for ARDS after esophagectomy is remarkably high. This result strikes a note of warning concerning the management of these patients and suggests the importance of screenings for CMV reactivation so as to make an accurate diagnosis and initiate treatment in a timely manner.     

CD4+CD28− T cell expansion in granulomatosis with polyangiitis (Wegener's) is driven by latent cytomegalovirus infection and is associated with an increased risk of infection and mortality

Objective

Expanded populations of CD4+CD28− T cells with a cytotoxic phenotype have been repeatedly reported in patients with granulomatosis with polyangiitis (Wegener's) (GPA). In healthy individuals expansion of this T cell population follows cytomegalovirus (CMV) infection. We undertook this study to investigate whether CMV infection may be responsible for driving the expansion of CD4+CD28− T cells in GPA patients and how this might relate to clinical features.

Methods

Forty‐eight GPA patients and 38 age‐matched healthy donors were included in the study. CMV‐specific IgG in serum was detected by enzyme‐linked immunosorbent assay. Flow cytometric analysis was used to study T cell populations and phenotype. The presence of CMV in renal biopsy tissue from GPA patients was investigated by immunohistochemistry and polymerase chain reaction (PCR). Clinical information was obtained from patient records.

Results

Populations of CD4+CD28− T cells were only expanded in CMV‐seropositive GPA patients and controls. In CMV‐seropositive GPA patients we observed negative correlations between the percentages of CD4+CD28− T cells and both the percentage of naive T cells and the glomerular filtration rate at presentation. There was a significant association between the percentage of CD4+CD28− T cells and risk of infection and mortality. CMV could not be detected in renal tissue by PCR or immunohistochemistry. CMV seropositivity itself was not a risk factor for infection in a cohort of 182 patients with antineutrophil cytoplasmic antibody–associated vasculitis who had been recruited into clinical trials performed by the European Vasculitis Study Group.

Conclusion

The expansion of CD4+CD28− T cells in GPA patients is associated with CMV infection and leads to a reduction in the number of naive T cells in peripheral blood. Patients with expanded CD4+CD28− T cells have significantly increased mortality and risk of infection.

     

Evaluation of the Murex CMV DNA Hybrid Capture assay (version 2.0) for early diagnosis of cytomegalovirus infection in recipients of an allogeneic stem cell transplant

Early diagnosis of CMV infection based on sensitive diagnostic assays has helped to reduce CMV-related mortality after allogeneic stem cell transplantation (SCT). In this study, the commercialized Murex CMV DNA Hybrid Capture assay (version 2.0) (HCS) was prospectively compared to an in-house CMV-DNA PCR assay from whole blood in patients after allogeneic stem cell transplantation. Overall, a high concordance between HCS and PCR was documented (kappa = 0.686; n = 385). The HCS assay was found to be as sensitive as the PCR indicating active CMV infection at a median of 35 and 34 days after transplantation, respectively. None of the HCS-negative patients developed CMV-related symptoms (negative predictive value 100%). Declining CMV DNA load in the blood was found to be an indicator for effective antiviral therapy, whereas persistence of a high viral load was associated with fatal CMV disease. In conclusion, the Hybrid Capture CMV DNA assay (v 2.0) allows early diagnosis of CMV infection after allogeneic SCT and assessment of the efficacy of antiviral therapy.