Quantitative Cytomegalovirus Polymerase Chain Reaction Research Articles

Colonic cytomegalovirus DNA detection by polymerase chain reaction does not influence outcomes in inflammatory bowel disease and immunosuppressed cohorts.

Cytomegalovirus (CMV) colitis is associated with negative outcomes in inflammatory bowel disease (IBD) and immunosuppressed cohorts and therefore requires timely recognition for appropriate management. We aimed to evaluate the diagnostic tools for CMV colitis and their associations with clinical outcomes. A retrospective cohort study of patients in a metropolitan health service with colonic samples analysed for CMV between 2012 and 2022, stratified into IBD and non-IBD groups, was performed. The main outcome measures were the prevalence of positive and negative results for each CMV test, as well as need for colectomy, use of antiviral and hospital length of stay. Five hundred eighty-two biopsies from 418 patients were included; the median age was 36 years (interquartile range, 24-52 years) and 223 (53.3%) were men. Four hundred sixty-one (79.2%) biopsies were from patients with IBD and 121 (20.8%) were from those without IBD. There were similar proportions of positive CMV histology (IBD 5.9% and non-IBD 7.4%) and tissue CMV polymerase chain reaction (PCR) in the two groups (IBD 5.6% and non-IBD 5.0%), but within each group, results were discordant. Positive CMV histology was significantly associated with need for colectomy in the IBD group, while positive tissue CMV PCR was not. Positive CMV histology, and tissue and serum CMV PCR were all significantly associated with antiviral use. Positive serum CMV PCR was significantly associated with colectomy. Histopathology remains the most predictive tool in assessing CMV colitis, while qualitative tissue CMV PCR was found to have limited utility. Quantitative serum CMV PCR may be useful but requires further evaluation.

A case of breast milk-acquired cytomegalovirus infection in an extremely low birth weight infant.

Although breast milk is considered the optimal nutrition for infants, it is also the primary cause of postnatal cytomegalovirus (CMV) infection. Preterm infants with postnatal CMV infections are susceptible to a variety of life-threatening conditions. Twin male infants were delivered via emergency caesarian section at 27 weeks' gestation secondary to maternal complete uterine rupture. The Apgar scores at 1 and 5 min were 1 and 1 for the older twin (Twin A) and 0 and 3 for the younger twin (Twin B). Their birth weights were 1203 g (+ 0.65SD) and 495 g (- 3.79SD) respectively. On day 41, laboratory blood test results for Twin B showed a moderate elevation in C-reactive protein (CRP), thrombocytopenia. CMV quantitative polymerase chain reaction (qPCR) tests in Twin B's urine and blood as well as in the mother's breast milk were positive, but stored, dried umbilical cord CMV qPCR tests were negative. Twin B was diagnosed with a postnatal CMV infection secondary to infected breast milk and ganciclovir was commenced on day 52. Treatment was switched to valganciclovir at 74 days of age, but a negative CMV-DNA level in the blood was not achieved. Postnatal CMV infection in this infant led to an exacerbation of pre-existing bronchopulmonary dysplasia (BPD) and he demised at 182 days of age. Postnatal cytomegalovirus infections may lead to exacerbations of BPD. Early use of raw breast milk in preterm infants should be done with careful consideration of this potential complication.

Prognostic Value of Amniotic Fluid Viral Load to Predict Adverse Outcome in Pregnancies Complicated by Congenital Cytomegalovirus Infection: A Multicenter Study

Introduction: The aim of the study was to report the prognostic value of cytomegalovirus (CMV) viral load in the amniotic fluid (AF) in predicting the outcome of infected pregnancies. Methods: Multicenter retrospective study involving 11 Italian referral centers from 2012 to 2021 was conducted. Inclusion criteria were fetuses with confirmed congenital CMV infection. The primary outcome was the prognostic value accuracy of CMV quantitative polymerase chain reaction (qPCR) in AF in predicting the risk of additional anomalies detected either at follow-up ultrasound or fetal magnetic resonance imaging (MRI). The secondary outcome was prediction of postnatal clinical symptoms related to CMV infection. Multivariate logistic regression and area under the curve (AUC) analyses were used to analyze the data. Results: 104 fetuses were included. Associated anomalies detected at follow-up ultrasound or fetal MRI were detected in 14.4% of cases (15/104). Mean AF CMV viral load was significantly higher in fetuses with additional anomalies compared to those without additional anomalies at follow-up ultrasound or fetal MRI (3,346,634.27 ± 402,582.95 vs. 761,934 ± 222513,2 p < 0.001). At multivariate logistic regression analysis, CMV AF viral load was independently associated with the presence of additional anomalies at follow-up ultrasound or MRI, with an OR of 1.07 (p = 0.010), while maternal age (p = 0.24), trimester at maternal infection (p = 0.97), and type of infection (primary vs. non-primary) (p = 0.12) were not. CMV AF viral load had AUC of 0.755 for the occurrence of anomalies due to CMV infection, with an optimal cut-off point of >1,310,520 copies/mL, a sensitivity of 66.7%, a specificity of 84.3%, and a positive likelihood ratio of 4.24. Once excluding fetuses with anomalies at ultrasound or MRI, the diagnostic performance of qPCR in identifying fetuses with symptomatic infection after birth was low, with an AUC of 0.586. Conclusion: CMV viral load at second trimester amniocentesis has a moderate accuracy for the occurrence of CMV-related anomalies in fetuses with congenital infection and normal ultrasound at the initial diagnosis. Conversely, prediction of symptomatic infection is low.

Effects of tissue cytomegalovirus quantitative polymerase chain reaction in the management of ulcerative colitis flare-ups: Should we wave aside?

Background and Study AimsThe role of cytomegalovirus (CMV) infection for disease reactivation in ulcerative colitis (UC) patients remains controversial and diagnostic tests are yet to be standardized. We aimed to define the clinical relevance of CMV detection by mucosal polymerase chain reaction (PCR) in UC patients by comparing the clinical course of UC in CMV-treated and CMV-untreated groups in tissue CMV-PCR positive cases. Patients and MethodsIn this retrospective study, 141 patients diagnosed with moderate-to-severe UC admitted to our clinic with disease flare, colonic tissue CMV PCR was assessed. ResultsThe median age of the study population was 39 years, and 99 (70.2%) patients were male. Eighty-eight (62.4%) patients were CMV-PCR (+) and 53 (37.6%) were CMV PCR (-). The CMV-PCR (+) and CMV PCR (-) groups showed no significant difference concerning age, sex, disease duration, site of involvement and disease activity and administered treatments. The median tissue CMV-PCR was 41,098 IU/mL (IQR:2,344.25–136,192). Thirty-four of 88 CMV-PCR (+) patients received antiviral therapy. The tissue CMV-PCR level of patients who received antiviral therapy was 124,381 IU/mL (IQR: 19,309–412,335), and it was 6,292 IU/mL (IQR: 997–71,154) in patients who did not receive antiviral therapy; (p < 0.001). Sixteen (47.1%) of 34 patients who received antiviral therapy achieved remission. Two of the non-responders underwent colectomy (one because of dysplasia and one who did not respond subsequent biologic agent either). Remaing 16 achieved remission by escalating the immunsuppresive/biologic agent therapy. ConclusionCMV infection is responsible for only a minority of cases of UC flares and all are steroid-resistant cases. Most of the patients non-responsive to antiviral treatment respond to increased anti-inflammatory treatment. Hesitancy in the decision of escalating immunsuppresive treatment rather than CMV disease may be responsible for worsening disease course and increased colectomy rate in a significant number of the patients who are tissue CMV-PCR (+).

High mucosal cytomegalovirus DNA helps predict adverse short-term outcome in acute severe ulcerative colitis.

Background/AimsPredictors of short-term outcome of intravenous (IV) steroid therapy in acute severe ulcerative colitis (ASUC) have been well described, but the impact of cytomegalovirus (CMV) infection as a predictor of outcome remains debatable. We investigated the role of quantitative CMV polymerase chain reaction (PCR) as a predictor of short-term outcome in patients with ASUC.MethodsConsecutive patients with ASUC satisfying Truelove and Witts criteria hospitalized at All India Institute of Medical Sciences (AIIMS) from May 2016 to July 2019 were included; all received IV steroid. The primary outcome measure was steroid-failure defined as the need for rescue therapy (with ciclosporin or infliximab) or colectomy during admission. AIIMS’ index (ulcerative colitis index of severity >6 at day 1+fecal calprotectin >1,000 μg/g at day 3), with quantitative CMV PCR on biopsy samples obtained at initial sigmoidoscopy were correlated with the primary outcome.ResultsThirty of 76 patients (39%) failed IV corticosteroids and 12 (16%) underwent surgery. Patients with steroid failure had a significantly higher mucosal CMV DNA than responders (3,454 copies/mg [0–2,700,000] vs. 116 copies/mg [0–27,220]; P<0.01). On multivariable analysis, mucosal CMV DNA load >2,000 copies/mg (odds ratio [OR], 10.2; 95% confidence interval [CI], 2.6–39.7; P<0.01) and AIIMS’ index (OR, 39.8; 95% CI, 4.4–364.4; P<0.01) were independent predictors of steroid-failure and need for colectomy. The combination correctly predicted outcomes in 84% of patients with ASUC.ConclusionsHigh mucosal CMV DNA (>2,000 copies/mg) independently predicts failure of IV corticosteroids and short-term risk of colectomy and it has an additional value to the established markers of disease severity in patients with ASUC.

Cost minimization analysis of an in-house molecular test for cytomegalovirus in relation to a commercial molecular system

IntroductionCytomegalovirus may cause severe disease in immunocompromised patients. Nowadays, quantitative polymerase chain reaction is the gold-standard for both diagnosis and monitoring of cytomegalovirus infection. Most of these assays use cytomegalovirus automated molecular kits which are expensive and therefore not an option for small laboratories, particularly in the developing world. ObjectiveThis study aimed to optimize and validate an in-house cytomegalovirus quantitative polymerase chain reaction test calibrated using the World Health Organization Standards, and to perform a cost-minimization analysis, in comparison to a commercial cytomegalovirus quantitative polymerase chain reaction test. Study designThe methodology consisted of determining: optimization, analytical sensitivity, analytical specificity, precision, curve variability analysis, and inter-laboratorial reproducibility. Patients (n=30) with known results for cytomegalovirus tested with m2000 RealTime System (Abbott Laboratories, BR) were tested with the in-house assay, as well as patients infected with other human herpes virus, in addition to BK virus. A cost-minimization analysis was performed, from a perspective of the laboratory, assuming diagnostic equivalence of the methodologies applied in the study. ResultsThe in-house assay had a limit of detection and quantification of 60.3IU/mL, with no cross-reactivity with the other viral agents tested. Moreover, the test was precise and had a R2 of 0.954 when compared with the m2000 equipment. The cost analysis showed that the assay was economically advantageous costing a median value of 37.8% and 82.2% in comparison to the molecular test in use at the hospital and the m2000 equipment, respectively. ConclusionsThese results demonstrated that in-house quantitative polymerase chain reaction testing is an attractive alternative in comparison to automated molecular platforms, being considerably less expensive and as efficacious as the commercial methods.

Cytomegalovirus Humoral Response Against Epithelial Cell Entry-Mediated Infection in the Primary Infection Setting After Hematopoietic Cell Transplantation.

The influence of humoral immunity on the prevention of primary cytomegalovirus (CMV) infection after hematopoietic cell transplantation (HCT) is poorly understood. To determine whether neutralizing antibodies (nAbs) against CMV pentameric complex (PC)-mediated epithelial cell entry decrease CMV infection after HCT, samples were analyzed from a randomized controlled trial of CMV intravenous immunoglobulin (IVIG) prophylaxis. Weekly serum from 61 CMV donor-positive/recipient-negative (D+/R-) HCT patients (33 control, 28 CMV IVIG) was tested using a PC-entry nAb assay and quantitative CMV polymerase chain reaction (PCR). There was a trend toward higher weekly PC-entry nAb titers (P = .07) and decreased CMV infection by PCR at viral load cutoffs of ≥1000 and ≥10 000 IU/mL in the CMV IVIG arm. High nAb titers were not significantly protective against CMV infection later after HCT in both study arms. Among CMV-infected patients, each log2 increase in nAb titer was associated with an average 0.2 log10 decrease in concurrent CMV viral load after infection (P = .001; adjusted for study arm). This study provides initial support that CMV IVIG prophylaxis moderately enhances PC-entry nAB activity in D+/R- HCT recipients.

Determination of the Biological Form of Human Cytomegalovirus DNA in the Plasma of Solid-Organ Transplant Recipients.

Whether cytomegalovirus (CMV) DNA exists in plasma as virion-associated or free DNA is uncertain. An assay combining DNase I digestion and CMV quantitative polymerase chain reaction (DNase-CMV-qPCR) was developed to differentiate free naked DNA from virion DNA. One hundred three frozen and 10 fresh CMV DNA-positive plasma samples from solid-organ transplant recipients (SOTRs) were tested. Three sets of paired qPCR (P-qPCR) assays with amplicons of variable length were used to study CMV DNA fragmentation in 20 SOTR plasma samples, viral stocks (Towne, Merlin, AD169) and the first World Health Organization (WHO) international standard (IS) for CMV DNA. In all plasma samples, 98.8%-100% of CMV DNA was free DNA; this was the only form in 93 of 103 (90.3%) frozen and all 10 fresh samples tested using DNase-CMV-qPCR. Low levels of virion CMV DNA were found in 10 of 103 (9.7%) samples with higher total DNA load. Cytomegalovirus DNA results were highly reproducible for 3 CMV virus stocks and WHO IS (P > .80), tested by three sets of paired q-PCR. However, for the 20 SOTR plasma samples, the smaller amplicon assay result was 2.6-fold, 3.4-fold, and 6.5-fold higher than the longer amplicion result (P < .001). Cytomegalovirus DNA in SOTR plasma is almost exclusively free DNA, highly fragmented, and not virion associated.

How to develop an in-house real-time quantitative cytomegalovirus polymerase chain reaction: Insights from a cancer centre in Eastern India

Development of a reliable, cost-effective cytomegalovirus quantitative polymerase chain reaction (QPCR) is a priority for developing countries. Manufactured kits are expensive, and availability can be inconsistent. Development of an in-house QPCR kit that is reliable and quality assured requires significant effort and initial investment. However, the rewards of such an enterprise are manifold and include an in-depth understanding of molecular reactions, and expertise in the development of further low-cost molecular kits. The experience of an oncology centre in Eastern India has been shared. Hopefully, this would provide a brief roadmap for such an initiative. Staff with adequate understanding of molecular processes are essential along with vital infrastructure for molecular research and development.

CMV Viremia Is Associated With a Decreased Incidence of BKV Reactivation after Kidney and Kidney-Pancreas Transplantation

Cytomegalovirus (CMV) and BK virus (BKV) infections can cause significant morbidity after kidney and kidney-pancreas transplant. There are limited data on the epidemiology and interactions between these two viral pathogens. We prospectively screened 609 kidney or kidney-pancreas transplant recipients from January 2007 to June 2011 for BKV and/or CMV viremia. This included 7453 quantitative BKV polymerase chain reaction and 15,496 quantitative CMV polymerase chain reaction tests. We evaluated risk factors and timing of these infections and the impact of treatment of one infection on the other. Among 609 recipients, 108 (17.7%) developed CMV viremia, of which 95 (88%) were asymptomatic, 5 (5%) had CMV syndrome, and 8 (7%) developed CMV tissue invasive disease at a median of 5.6 months after transplantation. Risk factors for CMV infection using multivariable analysis were D+R- serogroup (P≤0.0001), donor age >50 years (P=0.013), and higher mean tacrolimus (P=0.0009) and mycophenolate mofetil (P=0.01) blood levels. The incidence of BKV infection in the total population was 163 of 609 (26.7%), of which 150 (92%) occurred in patents without antecedent CMV viremia. Such patients demonstrated a higher rate of subsequent BKV viremia than patients with antecedent CMV viremia (P=0.003; hazard ratio, 2.05; 95% confidence interval, 1.2-3.4). Moreover, we found that only symptomatic CMV viremia had a significant negative impact on graft survival when compared with asymptomatic CMV viremia and those without CMV viremia (relative risk, 3.5; 95% confidence interval, 1.06-8.9; P=0.04). CMV viremia may indirectly protect against subsequent BK viremia possibly due to a reduction of intensity of immunosuppression after diagnosis of CMV viremia.

Fulminant gastrointestinal graft-versus-host disease concomitant with cytomegalovirus infection: Case report and literature review

Here, we report a case of fulminant gastrointestinal graft-versus-host disease (GI-GVHD) with cytomegalovirus (CMV) infection in 44-year-old woman. Despite the difficulties associated with the treatment of GI-GVHD and GI-CMV disease, the mucosal findings and the clinical course showed marked improvements during long-term clinical observation. The endoscopic findings were remarkable, with diffuse sloughing mucosa in the stomach and highly active inflammation and deep discrete ulcers throughout the colon. Changes in the CMV quantitative polymerase chain reaction results were correlated with the endoscopic mucosal findings and were useful for assessing the efficacy of the treatment. Although a definite diagnosis of GI-GVHD is generally made by endoscopy with biopsy, the gross appearance of this disease can vary depending on the endoscopy. In this paper, we also conduct a literature review of patients with GI-GVHD.

Artesunate is Ineffective in Controlling Valganciclovir-Resistant Cytomegalovirus Infection

To the Editor—Cytomegalovirus (CMV) disease is a common pathogen and represents a difficult clinical problem in transplant recipients. CMV-naive patients receiving a CMV-positive allograft may experience complications, such as retinitis, gastritis, colitis, and pneumonitis, which may force clinicians to reduce immunosuppressive agents, thereby risking rejection. However oral agents, such as valganciclovir, are effective in controlling such disease. Unfortunately, CMV resistance to oral valganciclovir is an emerging problem and poses particular management difficulties in renal transplant recipients, because alternative agents, such as foscarnet and cidofovir, are renal toxic [1]. Recently, the antimalarial agent arteseunate was reported as an effective agent against both wild-type and ganciclovir-resistant CMV [2]. Unlike ganciclovir, cidofovir, and foscarnet, artesunate is not nephrotoxic and is believed to inhibit viral replication via an alternate mechanism of action to DNA polymerase. We recently trialled artesunate in a renal transplant recipient with documented valganciclovir resistance mutations in CMV (suscepitible to cidofovir and foscarnet). Our CMV-naive, 47year-old female patient received a CMVpositive renal allograft 12 years previously and presented with a relapse of CMV colitis, with a high CMV load. She experienced 1 previous episode of CMV colitis documented with positive histopathology 2 years prior. Her medications at admission included oral valganciclovir (450 mg twice daily), diltiazem CD (240 mg twice daily), mycophenolate (1 g twice daily), prednisolone (10 mg daily), fenofibrate (48 mg daily), trandolapril (8 mg twice daily), pravastatin (80 mg daily), sertraline (50 mg twice daily), and esomeprazole (40 mg daily). Initial CMV quantitative polymerase chain reaction at admission was .1.1 3 10 copies/mL, exceeding the laboratory CMV disease threshold of 5000 copies/ mL. Her estimated glomerular filtration rate (modification of diet in renal disease formula) was 27 mL/min, thus limiting use of standard CMV antiviral agents. Initial therapy with CMV immunoglobulin and intravenous ganciclovir therapy failed to improve viral load. Immunosuppression was modulated by substituting mycophenolate with everolimus (.75 mg twice daily), and prednisolone therapy (10 mg daily) was continued. Intravenous artesunate (180 mg daily) was commenced, and the dosage was increased to 240 mg daily on day 7. A total of 20 days of intravenous artesunate therapy failed to significantly reduce CMV load on serial quantitative polymerase chain reaction assay. Subsequent therapy with foscarnet rapidly suppressed CMV load to undetectable levels. We found that artesunate was ineffective against this valganciclovir-resistant CMV strain. Alternate nonrenal toxic agents are needed for the treatment of these cases.

Comparison of QIAGEN Automated Nucleic Acid Extraction Methods for CMV Quantitative PCR Testing

We examined the effect of nucleic acid extraction methods on the analytic characteristics of a quantitative polymerase chain reaction (PCR) assay for cytomegalovirus (CMV). Human serum samples were extracted with 2 automated instruments (BioRobot EZ1 and QIAsymphony SP, QIAGEN, Valencia, CA) and CMV PCR results compared with those of pp65 antigenemia testing. Both extraction methods yielded results that were comparably linear and precise, whereas the QIAsymphony SP had a slightly lower limit of detection (1.92 log(10) copies/mL vs 2.26 log(10) copies/mL). In both cases, PCR was more sensitive than CMV antigen detection, detecting CMV viremia in 12% (EZ1) and 21% (QIAsymphony) of antigen-negative specimens. This study demonstrates the feasibility of using 2 different extraction techniques to yield results within 0.5 log(10) copies/mL of the mean value, a level that would allow for clinical comparison between different laboratory assays.

Prevalence, Risk Factors, and Impact on Outcome of Cytomegalovirus Replication in Serum of Cambodian HIV-Infected Patients (2004-2007)

In developing countries, the study of cytomegalovirus (CMV) coinfection in HIV-infected patients remains neglected. Quantitative CMV polymerase chain reaction (PCR) is the gold standard diagnostic tool for analyzing serum CMV replication and for predicting CMV disease. We estimated the prevalence of replicating CMV in sera of newly diagnosed HIV-infected Cambodian patients and examined its impact on mortality. This cohort study was based on 2 highly active antiretroviral therapy treatment programs in Cambodia between 2004 and 2007. Quantitative CMV PCR was performed on baseline serum samples of 377 HIV-infected patients. The prevalence of serum CMV DNA was 55.2% (150 of 272) in patients with CD4 count <100/mm. In multivariate analysis, hemoglobin <9 g/dL, CD4 count <100/mm, and Karnofsky index <50 were independently associated with positive serum CMV DNA at baseline. During a 3-year follow-up period, CMV viral load >or=3.1 log10 copies per milliliter was significantly associated with death independently of CD4 count, other opportunistic infections, and highly active antiretroviral therapy. As in industrialized countries, serum CMV replication is highly prevalent among HIV-infected Cambodian patients and is associated with increased mortality. This underscores the importance of diagnostic CMV infection by PCR in sera of HIV-infected patients with CD4 count <100/mm and treating this opportunistic infection to reduce its associated mortality.

CMV quantitative PCR in the diagnosis of CMV disease in patients with HIV-infection – a retrospective autopsy based study

BackgroundPatients with advanced HIV infection at the time of diagnosis and patients not responding to antiretroviral therapy are at risk of cytomegalovirus (CMV) disease. Earlier studies of patients with HIV infection have demonstrated that the diagnosis is often first made post-mortem. In recent years new molecular biological tests have become available for diagnosis of CMV disease. Although clinical evaluation of tests for diagnosis of CMV disease in HIV-infected individuals is suboptimal without autopsy, no results from such studies have been published. The aim of this study was to explore the diagnostic utility of CMV quantitative polymerase chain reaction (PCR) in plasma from HIV and CMV seropositive patients who died during the period 1991–2002 and in whom autopsy was performed.MethodsAutopsy was performed in all cases, as part of routine evaluation of HIV-infected cases followed at Ullevaal University Hospital. Of 125 patients included, 53 had CMV disease, 37 of whom were first diagnosed at autopsy. CMV disease was diagnosed either by ophthalmoscopic findings typical of CMV retinitis, biopsy or autopsy. One or two plasma samples taken prior to the first diagnosis of CMV disease (alive or at autopsy) or death without CMV disease were analysed by CMV quantitative PCR. Sensitivity, specificity, positive and negative predictive values were calculated for different CMV viral load cut-offs and according to detection of viraemia in one versus two samples.ResultsTwenty-seven of 53 patients with CMV disease (51%) and 10 of 72 patients without CMV disease (14%) had detectable viraemia in at least one sample. Sensitivity and negative predictive value (NPV) of the test, maximised with a cut-off at the test's limit of detection of CMV viraemia (400 copies/mL), were 47% and 70%, respectively. With cut-off at 10 000 copies/mL, specificity and positive predictive value (PPV) were 100%. With a requirement for CMV viraemia in two samples, specificity and PPV were 100% in patients with CMV viraemia above the limit of detection.ConclusionOur results indicate that quantitative CMV PCR is best used to rule in, rather than to rule out CMV disease in HIV-infected individuals at high risk.

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.

Periodic Myoclonus Due to Cytomegalovirus Encephalitis in a Patient With Good Syndrome

Good syndrome (GS) refers to a constellation of thymoma and adult-onset immunodeficiency characterized by low or absent B cells, hypogammaglobulinemia, and variable defects in cell-mediated immunity with an inverted CD4/CD8(+) T-cell ratio. Patients may develop severe or chronic infections as a result of this immunodeficiency. To describe a patient with GS who developed cytomegalovirus (CMV) encephalitis and showed a periodic electroencephalographic pattern and myoclonus. Case report. Outpatient neurology clinic at a university medical center. Patient A 64-year-old man who developed periodic myoclonus involving the right half of his body and the left arm. Five years previously, the patient had undergone resection of a pathologically confirmed epithelial thymoma. Quantitative CMV polymerase chain reaction of the serum and cerebrospinal fluid showed strongly positive results (1:10 000), allowing the diagnosis of CMV encephalitis. To the best of our knowledge, myoclonus and periodic electroencephalographic pattern have not previously been reported in CMV encephalitis. Opportunistic CMV infection should be considered early in the evaluation of patients with GS or a history of thymoma who develop unusual neurological symptoms.

Laboratory-based risk factors for cytomegalovirus retinitis

Background: Very few studies have investigated risk factors for cytomegalovirus (CMV) retinitis. Identifying these risk factors will have many benefits, including helping establish screening regimens, examination frequency regimens, and targeted prophylaxis with oral therapy with valganciclovir or other anti-CMV agents. The purpose of this study was to determine the laboratory-based risk factors for CMV retinitis in patients with AIDS.Methods: We conducted a case-control study involving 120 patients in whom CMV retinitis had been diagnosed in 1990-99 and 159 patients without CMV retinitis from the same period.The sampling was from a primary study base in eastern Ontario and western Quebec of patients with AIDS and CD4 counts less than 50 cells/μL at the time of diagnosis of retinitis in the case subjects or an analogous date for the control subjects.There were two components to the study. In the first component (n = 279) we examined standard-of-care laboratory tests (hematologic and nutrition variables) done during the study period. In the second component (n = 57), which was a subset of the first, we examined laboratory tests (HLA type, qualitative and quantitative CMV polymerase chain reaction [PCR] and HIV load) on stored blood samples from the eastern Ontario site. Multivariate logistic regression was used to model the data and control for confounding. We developed a systematic model-building strategy, from assumption testing to model building to model checking.Results: A low hemoglobin concentration was a statistically significant predictor of CMV retinitis (odds ratio [OR] 0.96, 95% confidence interval [CI] 0.94–0.98). Both qualitative CMV PCR (OR 21.71, 95% Cl 1.80–261.67) and quantitative CMV PCR (OR 33.03,95% CI 2.32–469.39) were strong predictors of CMV retinitis. Among the 80 HLA types tested, HLA-Bw4 (OR 11.68, 95% CI 1.29–105.82) and HLA-DRBI 15 (OR 9.34, 95% CI 1.14–76.41) were significant predictors of CMV retinitis, whereas HLA-Cw7 was protective against CMV retinitis (OR 0.09, 95% CI 0.01–0.67).Interpretation: We have identified laboratory variables that elevate (or decrease) the risk of CMV retinitis.These findings may be useful to clinicians and health policy experts in developing rational guidelines for screening, examination frequency and targeted prophylaxis for patients with AIDS.

The CARI guidelines. CMV disease and kidney transplant: treatment of cytomegalovirus disease in renal transplant recipients.

NephrologyVolume 9, Issue S3 p. S37-S40 Free Access 4. Treatment of Cytomegalovirus disease in renal transplant recipients First published: 06 October 2004 https://doi.org/10.1111/j.1440-1797.2004.00309.xCitations: 1AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat GUIDELINES No recommendations possible BACKGROUND Cytomegalovirus (CMV) disease is characterized by clinical symptoms and signs of disease coupled with microbiological evidence of CMV infection. Disease occurs in 8% of renal transplant recipients compared with liver or pancreas (29%) and heart-lung (39%) recipients. Patients at greatest risk of developing disease include D+/R– recipients and those who have been given methyl-prednisolone or antilymphocyte antibody. The diagnosis may be complicated by the fact that CMV infection is frequently associated with superinfection with other bacteria, fungi, protozoa and viruses. Ganciclovir is the treatment of choice for CMV disease and should be administered intravenously with the dose modified according to creatinine clearance. Treatment should be continued for at least 2 weeks or until CMV-DNA is not detectable. While immunosuppressive therapy is frequently reduced in patients with CMV disease, this measure has not been subjected to a clinical trial. Oral ganciclovir has been used successfully to treat mild to moderate CMV disease. The recent introduction of valganciclovir, with 10 times the bioavailability of oral ganciclovir and kinetics comparable to IV ganciclovir, may provide an alternative to IV therapy. Valganciclovir has the potential to reduce hospital stay and the complications associated with prolonged IV access. Ganciclovir resistance has been reported in up to 7% of solid organ transplants and may be treated with second-line agents such as foscarnet and cidofovir. Both of these agents are associated with significant nephrotoxicity and side-effects. There is no evidence to support the use of acyclovir, valaciclovir and CMV-Ig in renal transplant recipients. Failure to respond to therapy may be due to inadequate treatment, ganciclovir resistance or superinfection. SEARCH STRATEGY Databases searched: MeSH terms and text words for CMV were combined with MeSH terms and text words for solid organ transplantation, including kidney transplantation, then combined with MeSH terms and text words for interventions for CMV, and then combined with the Cochrane highly sensitive search strategy for randomised controlled trials. The search was carried out in Medline (1966 – June Week 1 2002). The Cochrane Renal Group Trials Register was also searched for trials not indexed in Medline. Date of search/es: 28 June 2002. WHAT IS THE EVIDENCE? There are no randomised controlled trials (RCTs) on this topic. SUGGESTIONS FOR CLINICAL CARE (Suggestions are based on Level III and IV evidence) • Ganciclovir is the treatment of choice for CMV disease. Features include: – IV administration – Dose modification for renal impairment – Duration of treatment is 2–3 weeks or until CMV-DNA is not detectable in leucocytes. (Level III evidence) • Treatment failure may be due to inadequate treatment, superinfection or ganciclovir resistance. (Level III evidence) • Foscarnet and cidofovir remain second-line agents. (Level III evidence) Which patients are at greatest risk of CMV disease? While many patients will have evidence of CMV infection following transplantation, Sagedal et al. identified donor positive- and recipient-negative CMV status and acute rejection episodes as significant risk factors associated with the development of CMV disease.1 A total of 477 consecutive renal transplant recipients (397 first transplants and 80 retransplants) operated on between October 1994 and July 1997 were included in the study. The patients were followed prospectively for 3 months with serial measurements of CMV pp65 antigen being made to detect CMV infection. The incidence of CMV infections in first transplants was 68% in D+ R– and D+/R+ serostatus groups, whereas the incidence of CMV disease was higher in D+ R– (56%) than in D+/R+ patients (20%, p < 0.001). No difference in severity of CMV disease in D+ R– and D+/R+ was seen except for an increased incidence of hepatitis in primary infections. Cox regression analysis showed that rejection (RR 2.5, p < 0.01) and serostatus group D+ R– (RR 3.9, p < 0.001) were significant risk factors for the development of CMV disease. Intravenous ganciclovir There are over 30 uncontrolled, non-randomised studies using ganciclovir to treat CMV disease. The study by Rondeau et al. was one of the first reported and recommended ganciclovir dose reductions in patients with renal impairment. In this study, 32 renal transplant recipients with proven CMV disease were treated with ganciclovir for 14 days. The mean time to CMV disease was 49 days following transplantation. CMV disease was diagnosed based on the combination of clinical signs and histological/virological studies.2 Twenty-nine patients, 10 of whom had moderate to severe disease were improved by treatment. Three deaths occurred, with two dying from combined severe pulmonary and hepatic CMV disease. Adverse effects of ganciclovir therapy included leukopenia (n = 7), thrombocytopenia (n = 2) and abdominal pain (n = 1). CMV was no longer found in virological samples from 80% of patients. The clearance of ganciclovir was linearly correlated with creatinine clearance. Oral ganciclovir The inconvenience associated with the administration of IV ganciclovir, requiring hospitalization and frequent intravenous cannulation, has led to a search for effective oral therapy. Jordan et al. describe a small study using oral ganciclovir for the treatment of moderate CMV disease using quantitative PCR to monitor treatment.3 Fifteen patients with donor/recipient CMV status of D+/R– (53%), D+/R+ (40%) and D–/R+ (7%) were studied. All patients were commenced on oral ganciclovir 1 g tid with dose reduction in patients with impaired renal function. Treatment continued until the CMV-PCR copy number was negative and symptoms resolved. The mean number of CMV copies at the time of diagnosis was 580 (normal < 5) and fell to 65 copies after 5–7 days of treatment. A total of 14/15 patients responded to treatment and 1 non-responder was attributed to non-compliance. While the time to disease onset following transplantation is not indicated in the report, it can be inferred from the prophylactic protocol for D+/R– patients (comprising 1 week IV ganciclovir followed by 4 months of oral ganciclovir therapy) that CMV disease occurred more than 4 months after transplantation. Oral valganciclovir The development of valganciclovir resulted in a 10-fold increase in oral bioavailability. Pescovitz et al. compared valganciclovir at a dose of 900 mg, once daily, with IV ganciclovir in 28 healthy liver transplant recipients, using a 4-way randomised cross-over design. The AUC for IV ganciclovir was 47.61 mg/h/mL compared with 42.69 for valganciclovir. Thus, valganciclovir achieved 90% (90% CI: 83–97) of IV ganciclovir AUC in transplant recipients but it remains to be proven that valganciclovir absorption is maintained in sick patients with CMV disease.4 Disease recurrence One measure of treatment efficacy is the prevalence of recurrent CMV disease following standard IV ganciclovir therapy. Sawyer et al. address this issue in both kidney and combined kidney/pancreas recipients.5 A total of 619 transplant recipients (535 kidney [253 living related and 282 cadaveric donors] and 84 combined cadaveric kidney-pancreas transplants) were studied over a 3.5-year period. Of these, 114 (18.4%) developed tissue-invasive CMV disease and were treated with IV ganciclovir for 14–21 days. Twenty-eight (24.6%) developed recurrent disease after more than 30 days from first infection. Proportionally, more kidney-pancreas transplants than kidney transplant alone relapsed. All patients with recurrent disease were retreated with IV ganciclovir, achieving a cure rate at 30 days of 100%. With the introduction of molecular markers of disease activity, Sia et al. followed CMV-DNA load prospectively in patients with CMV infection and disease and demonstrated that DNA load is predictive of CMV relapse following transplantation. Twenty-four solid-organ transplant recipients (heart, liver, kidney) with CMV infection (42%) or disease (58%) were tested for CMV-DNA load before and after 14 days of IV ganciclovir therapy.6 Recurrent infection or disease occurred in 8/24 (33%) patients, with no significant difference between CMV infected and CMV disease groups. The median pretreatment viral loads were higher in the relapsing group than in the cured group. Most importantly, the relapsing group had detectable CMV-DNA after treatment, whereas the CMV-DNA was undetectable in the non-relapsing patients. Superinfection Patients with CMV disease are at risk of superinfection with a variety of microorganisms. Pneumocystis carinii pneumonia (PCP) in the early post-transplant period contributes significantly to early transplant morbidity and mortality. The link between CMV disease, allograft rejection episodes and PCP is not clearly defined. Arend et al. attempt to link these in a small study of 15 renal transplant recipients with proven PCP compared with 95 control patients.7 The number of rejection episodes correlated with increased risk of developing PCP. After 1, 2 or 3 rejection episodes, the relative risk of developing PCP increased to 1.7 (95% CI: 0.2–12.5), 4.8 (95% CI: 0.9–25.5), and 9.5 (95% CI: 1.6–56.4), respectively. Furthermore, CMV infection increased the relative risk of PCP five-fold (95% CI: 1.6–15.8) with the highest relative risk of 5.7 in the D + R– combination (95% CI: 1.4–22.3). Ganciclovir resistance Failure to respond to standard therapy, especially after prolonged exposure to ganciclovir, should raise the possibility of ganciclovir resistance, which can occur in up to 7% of patients.8 Second-line agents Only one study was found that had evaluated foscarnet in kidney transplant recipients. Ringden et al. described the pharmacokinetics, safety and their preliminary clinical experiences using foscarnet in the treatment of cytomegalovirus infections in bone marrow and renal transplant recipients.9 Fifty-seven episodes of severe CMV infection were treated with IV foscarnet in 13 bone marrow and 33 renal graft recipients. Multiple adverse effects were recorded including decreased haemoglobin, decreased renal function and increased serum calcium. Abnormal liver function tests, hallucinations and tremor were seen in one uraemic patient with foscarnet levels >400 mg/L. Improvements, such as eradication of CMV (8/14 assessable patients), resolution of fever (11/22), and improved laboratory values (13/23) were noted in 17/24 (70%) patients. WHAT DO THE OTHER GUIDELINES SAY? International Herpes Management Forum, 2000: In any transplant recipient, CMV disease should be treated for 2–4 weeks with intravenous ganciclovir. Acyclovir is ineffective in treating established CMV disease in transplant recipients.10 British Transplantation Society Guidelines, 2002: Patients with CMV disease should receive intravenous ganciclovir for at least 14 days. Consideration should be given to reduction in immunosuppression.11 Clinical Practice Guidelines of the American Society of Transplantation, 2000: No treatment recommendations. Aim to prevent disease with prophylaxis.12 European Best Practice Guidelines for Renal Transplantation (Part 1), 2000: All recipients with documented CMV disease (symptomatic CMV infection) must receive a curative treatment. Currently, the only validated treatment is IV ganciclovir at a daily dose of 5 mg/kg twice daily, adjusted for renal function, for at least 14 days. Alternatively, they may receive IV ganciclovir for at least 5 days followed by oral ganciclovir at a daily dose of 1000 mg three times daily for a longer period (2–12 weeks).13 IMPLEMENTATION AND AUDIT No recommendations. SUGGESTIONS FOR FUTURE RESEARCH 1 Perform a study to assess the efficacy of IV ganciclovir/introduction of new agents. All new anti-CMV therapy should be tested in RCTs with one arm receiving IV ganciclovir. 2 Run studies of molecular markers of disease activity to determine the optimal duration of therapy. 3 Investigate post-treatment prevention to reduce recurrence. Give oral therapy to high-risk patients? 4 Establish the relationship between ganciclovir exposure and prophylaxis and the emergence of ganciclovir resistance. REFERENCES 1 Sagedal S, Nordal KP, Hartmann A et al. A prospective study of the natural course of cytomegalovirus infection and disease in renal allograft recipients. Transplantation 2000; 70: 1166– 74. 2 Rondeau E, Farquet C, Fries D et al. Treatment of cytomegalovirus infections with ganciclovir in kidney transplant recipients. Clinical and pharmacokinetic study. Press. Med. 1991; 20: 2030– 2. 3 Jordan SC, Vo A, Bunnapradist S et al. Treatment of active cytomegalovirus disease with oral ganciclovir in renal allograft recipients: monitoring efficacy with quantitative cytomegalovirus polymerase chain reaction. Am. J. Transplant. 2002; 2: 671– 3. 4 Pescovitz MD, Rabkin J, Merion R et al. Valganciclovir provides ganciclovir plasma exposure similar to IV ganciclovir in transplant recipients. Transplantation 1999; 67 (Suppl.): S126 (Abstract). 5 Sawyer MD, Mayoral JL, Gillingham KJ et al. Treatment of recurrent cytomegalovirus disease in patients receiving solid organ transplants. Arch. Surg. 1993; 128: 165– 9. 6 Sia IG, Wilson JA, Groettum CM et al. Cytomegalovirus (CMV) DNA load predicts relapsing CMV infection after solid organ transplantation. J. Infect. Dis. 2000; 181: 717– 20. 7 Arend SM, Westendorp RG, Kroon FP et al. Rejection treatment and cytomegalovirus infection as risk factors for Pneumocystis carinii pneumonia in renal transplant recipients. Clin. Infect. Dis. 1996; 22: 920– 5. 8 Limaye AP, Corey L, Koelle DM et al. Emergence of ganciclovir-resistant cytomegalovirus disease among recipients of solid-organ transplants. Lancet 2000; 356: 645– 9. 9 Ringden O, Lonnqvist B, Paulin T et al. Pharmacokinetics, safety and preliminary clinical experiences using foscarnet in the treatment of cytomegalovirus infections in bone marrow and renal transplant recipients. J. Antimicrob. Chemother. 1986; 17: 373– 87. 10 International Herpes Management Forum. Management guidelines: The challenge of CMV infection and disease in transplantation. Cambridge: Cambridge Medical Publications, 2000. 11 Newstead CG, O'Grady J, Parameshwar J et al. British Transplantation Society Guidelines for the prevention and management of Cytomegalovirus disease after solid organ transplantation. London: British Transplantation Society, 2002. 12 Kasiske BL, Vazquez MA, Harmon WE et al. Clinical practice guidelines of the American Society of Transplantation. J. Am. Soc. Nephrol. 2000; 11 (Suppl. 1): S45– S46. 13 Berthoux F, Abramowicz D, Bradley B et al. European best practice guidelines for renal transplantation (Part 1). Nephrol. Dial. Transplant. 2000; 15 (Suppl. 7): S71– S74. Citing Literature Volume9, IssueS3October 2004Pages S37-S40 ReferencesRelatedInformation

Treatment of Active Cytomegalovirus Disease with Oral Ganciclovir in Renal Allograft Recipients: Monitoring Efficacy with Quantitative Cytomegalovirus Polymerase Chain Reaction

Treatment regimens of patients with active cytomegalovirus (CMV) disease require 2-3 weeks of intravenous ganciclovir (GCV) with/without CMV hyperimmune globulin. Oral GCV is effective as a prophylactic agent in prevention of CMV disease. Here we explored the utility of oral GCV as a treatment of active CMV disease. Fifteen renal allograft recipients (CMV donor+/recipient- [53%], CMV donor+/recipient+ [40%] or CMV donor-/recipient+ [7%]) developed active CMV disease. Cytomegalovirus polymerase chain reaction (CMV-PCR) tests were performed at the time of presentation and patients were treated with oral ganciclovir 1 g tid (adjusted for renal function). Patients were monitored for efficacy of treatment by assessment of clinical symptoms and CMV-PCR. Treatment was continued until the CMV-PCR copy number was negative and symptoms resolved. The mean CMV-PCR copy number at the time of diagnosis was 580 copies/microg DNA (nl: < 5 copies/microg DNA). After 5-7 days of treatment, the mean copy number was 65 copies/microg DNA. Fourteen of 15 patients responded well to oral ganciclovir, with complete resolution of clinical symptoms and eradication of CMV-PCR positivity. One patient did not respond to oral ganciclovir therapy due to probable noncompliance. Our data suggest that oral ganciclovir treatment, coupled with careful CMV-PCR monitoring, may be a reasonable alternative to long-term intravenous ganciclovir.