Cytomegalovirus DNA Polymerase Chain Reaction Research Articles

Human Cytomegalovirus Infection Among Patients Living With AIDS in a Tertiary Level Hospital in India

Detecting the prevalence of human cytomegalovirus (CMV) coinfection in HIV-infected individuals attending a tertiary care hospital in Delhi by using 3 laboratory tests namely pp65 antigenemia assay, CMV DNA polymerase chain reaction (PCR), and immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA) for anti-CMV antibody. HIV seropositive patients with CD4 count < or = 100 cells/mm(3) were included as cases and HIV-negative healthy individuals as controls. Blood samples were collected from all participants and 3 tests were performed. pp65 antigenemia assay and IgM ELISA were performed by commercially available kits; PCR was performed by an in-house nested PCR method. In all, 7.1%, 5.7%, and 2.9% were positive for pp65 antigenemia, CMV DNA, and IgM antibody against CMV, respectively. On the basis of pp65 and/or PCR positivity, it can be said that active CMV coinfection was present in 8.57% of the cases. In the post-antiretroviral therapy (ART) era, CMV coinfection in HIV-infected patients has significantly declined.

Immunizations and infectious diseases in pediatric liver transplantation

Liver transplantation (LTx) is an established and effective treatment for children with end-stage and metabolic liver disease. Improved surgical techniques and immunosuppressive regimens have led to enhanced short-term and long-term survival rates, which now approach or exceed 90%. Consequently, more children are being referred for LTx, and an increasing numbers of pediatric centers routinely perform this procedure. Infectious complications are an important cause of morbidity and mortality in children undergoing LTx. However, improvements in immune suppression management along with the availability of new antimicrobial agents and diagnostic tools have resulted in improved treatment and preventative regimens, thus reducing the impact of infectious complications on these children. This review provides an overview of the infectious complications and prevention strategies for children undergoing LTx.

Primary CMV Colitis in an Immunocompetent Infant, Successfully Treated by Gancyclovir

Primary CMV Colitis in an Immunocompetent Infant, Successfully Treated by Gancyclovir

Relationship between Toll-Like Receptor 2 Polymorphism and Cytomegalovirus Disease after Liver Transplantation

Experimental data suggest that cytomegalovirus (CMV) initiates innate immunity through the activation of Toll-like receptor 2 (TLR2). To assess the clinical relevance of this experimental observation, we assessed the association between the specific single-nucleotide polymorphism that results in the substitution of arginine for glutamine in position 753 of TLR2 (the TLR2 Arg753Gln polymorphism) and CMV replication and disease after liver transplantation. Ninety-two liver transplant recipients with chronic hepatitis C were screened for the presence of the TLR2 Arg753Gln polymorphism. CMV load was determined in serially collected blood samples using CMV DNA polymerase chain reaction. Kaplan-Meier estimation and univariable and multivariable stepwise Cox proportional hazard models were used to assess associations. The degree of CMV replication, as measured by CMV load, was significantly higher in patients who were homozygous (mean maximum viral load, 37,059 copies/mL) and heterozygous (mean maximum viral load, 29,718 copies/mL) for this polymorphism, compared with patients without the TLR2 Arg753Gln polymorphism (mean maximum viral load, 3252 copies/mL; P=.003). Kaplan-Meier survival analysis demonstrated an association between being homozygous for the TLR2 Arg753Gln polymorphism and CMV disease (P=.04). A multivariate Cox proportional hazard model demonstrated a trend towards a higher risk of CMV disease among patients who were homozygous for the TLR2 Arg753Gln polymorphism (hazard ratio, 1.91 [95% confidence interval, 0.91-3.40]; P=.08) after adjusting for patient age, CMV serostatus, and allograft rejection. TLR2 Arg753Gln polymorphism is possibly associated with CMV replication and disease after liver transplantation. This novel clinical observation supports the potential role of TLR2 in the immunologic control of CMV infection in humans.

Who is at risk for developing cytomegalovirus (CMV) infection following liver transplantation?

Background Experimental data suggest that cytomegalovirus (CMV) initiates innate immunity through the activation of Toll-like receptor 2 (TLR2). To assess the clinical relevance of this experimental observation, we assessed the association between the specific single-nucleotide polymorphism that results in the substitution of arginine for glutamine in position 753 of TLR2 (the TLR2 Arg753Gln polymorphism) and CMV replication and disease after liver transplantation. Methods Ninety-two liver transplant recipients with chronic hepatitis C were screened for the presence of the TLR2 Arg753Gln polymorphism. CMV load was determined in serially collected blood samples using CMV DNA polymerase chain reaction. Kaplan-Meier estimation and univariable and multivariable stepwise Cox proportional hazard models were used to assess associations. Results The degree of CMV replication, as measured by CMV load, was significantly higher in patients who were homozygous (mean maximum viral load, 37,059 copies/mL) and heterozygous (mean maximum viral load, 29,718 copies/mL) for this polymorphism, compared with patients without the TLR2 Arg753Gln polymorphism (mean maximum viral load, 3252 copies/mL; P = .003). Kaplan-Meier survival analysis demonstrated an association between being homozygous for the TLR2 Arg753Gln polymorphism and CMV disease (P = .04). A multivariate Cox proportional hazard model demonstrated a trend towards a higher risk of CMV disease among patients who were homozygous for the TLR2 Arg753Gln polymorphism (hazard ratio, 1.91 [95% confidence interval, 0.91–3.40]; P = .08) after adjusting for patient age, CMV serostatus, and allograft rejection. Conclusions TLR2 Arg753Gln polymorphism is possibly associated with CMV replication and disease after liver transplantation. This novel clinical observation supports the potential role of TLR2 in the immunologic control of CMV infection in humans.

Cytomegalovirus Viremia, Mortality, and End-Organ Disease Among Patients With AIDS Receiving Potent Antiretroviral Therapies

To determine the association of cytomegalovirus (CMV) viremia with CMV disease and death in patients with AIDS. Prospective, observational cohort study conducted at a university hospital. A cohort of 190 subjects with AIDS who were CMV seropositive and had no history or evidence of CMV disease were longitudinally evaluated for signs and symptoms of CMV disease and CMV viremia with plasma CMV DNA polymerase chain reaction (PCR) and whole blood CMV hybrid capture. A total of 187 subjects had at least 1 study visit following entry. At baseline, the median CD4 cell count and plasma HIV RNA level were 110/microL (range = 3-620/microL) and 47,973 copies/mL (<30- >750,000 copies/mL), respectively. Highly active antiretroviral therapy (HAART) use increased from 87.5% during the 1st study year to 98.5% by the end of the study. During a median follow-up of 334 days, 16% (30) of the subjects died and 2 (6%) developed CMV disease. No deaths were attributable to CMV disease; 4 subjects who died developed CMV prior to death. Baseline HIV viral load and final CD4 cell count were significantly and independently associated with mortality. Detectable plasma CMV DNA PCR was an independent predictor of death even after adjusting for HIV RNA level and CD4 cell count prior to death (P = 0.038). In contrast, whole blood CMV hybrid capture did not predict mortality. The CMV assays neither collectively nor individually were found to be associated with the few cases of CMV disease. In patients with AIDS and seropositive for CMV, detection of CMV viremia with plasma CMV DNA PCR was predictive of death and provided additional prognostic information on the risk of all cause-mortality beyond that obtained with CD4 cell count and HIV viral load testing alone. Detection of CMV viremia by plasma with CMV DNA PCR in patients with AIDS, particularly those with low CD4 cell counts, provides additional rationale for optimization of antiretroviral therapy and consideration for preemptive anti-CMV therapy.

Phantom cytomegalovirus infection in vasculitis patients: what it means and what to do

We report our experience and hypothesis on the diagnosis and treatment of patients with vasculitis who are simultaneously diagnosed with serum-positive cytomegalovirus (CMV) immunoglobulin (Ig)M antibodies and negative CMV DNA polymerase chain reaction (PCR). It remains unknown how to treat this kind of "phantom" CMV infection. In a patient diagnosed with Henoch-Schönlein vasculitis, CMV IgM titers were increased while angiitis and renal function deteriorated. Empiric treatment of phantom CMV infection with ganciclovir in this CMV IgM-positive and PCR-negative patient resulted in complete vasculitis remission, serum CMV antibody seroconversion, and renal function improvement. These results imply something more than coincidence.

Cytomegalovirus (CMV) DNA load in plasma for the diagnosis of CMV disease before engraftment in hematopoietic stem-cell transplant recipients.

Among hematopoietic stem-cell transplant (HSCT) recipients, cytomegalovirus (CMV) disease before engraftment is rare but often fatal, and cell-based diagnostic tests have low sensitivity in this clinical setting. We used the quantitative real-time polymerase chain reaction (PCR) assay to test for CMV DNA in plasma samples from 15 HSCT recipients who developed CMV disease before engraftment and from 33 matched control patients. CMV DNA was detected in plasma in 14 (93.3%) of the 15 patients who had CMV disease before engraftment, compared with 5 (15.2%) of 33 control patients (P<.001). CMV DNA was detected a median of 13 days before the onset of CMV disease (range, 0-35 days). The maximum CMV virus load in plasma was >1 log(10) higher among case patients than among control patients (median, 1700 [range, 50 to 5.5x107] vs. <50 [range, <50-350] CMV DNA copies/mL plasma, respectively; P<.001). Quantitative PCR for CMV DNA in plasma appears to be useful for the identification of HSCT recipients at risk for CMV disease before engraftment.

Cytomegalovirus Infection in a Seroendemic Renal Transplant Population: A Longitudinal Study of Virological Markers

Background/Aims: The detection of viremia by polymerase chain reaction (PCR) in cytomegalovirus (CMV) infection in renal allograft recipients has been shown to have a predictive value for disease. However, its diagnostic utility in a population with high background seropositivity has not been defined. This prospective study was undertaken to assess the relationship of CMV DNAemia, and/or IgM seropositivity to CMV disease in a seroendemic transplant population. Methods: Consecutive patients undergoing renal transplantation between August 1997 and February 1998 were enrolled. Blood was sampled before transplantation from the donors and recipients for CMV serology and nested PCR for CMV DNA, and after transplantation from the recipients only at monthly intervals until 6 months. Patients were observed for the development of any CMV-like illness during follow-up. CMV DNA was quantitated using limiting dilution PCR on samples obtained from symptomatic patients at the time of illness and from asymptomatic patients at the end of their 6-month follow-up. Results: A total of 57 recipient-donor pairs were recruited. Immunosuppression was cyclosporine-based in 55 of 57 (95.6%). The CMV serologic status was D+R+ in 55 of 57 and D+R– in 2 of 57 pairs. PCR positivity indicating viremia increased from 5% before transplantation to 95% at 6 months after transplantation. Similarly IgM positivity reached 80% at 3 months and thereafter; positivity for any marker was 100% by 6 months. Viremia was sustained in over half the patients. The incidence of CMV-attributable disease peaked at 3 months, and was predominantly mild and self-limiting. Tissue-invasive disease appeared later in 4 patients (7%). Asymptomatic viremia was seen in 60–70% of patients at each sampling point. The positive predictive value (PPV) of PCR positivity for disease was 35–40%, and the negative predictive value (NPV), 90–100%. However, the high NPV was of use only in the early post-transplant period, negativity for markers declining rapidly with time. Quantitative assay showed significantly higher levels of CMV DNA in symptomatic patients (p = 0.01). A cutoff of 0.001 µg had a specificity of 95% and a PPV of 92.3% for symptomatic CMV disease. Conclusion: Qualitative tests to detect CMV DNAemia and IgM, although useful markers of viremia and active infection, have limited utility for the diagnosis of disease in a seroendemic transplant population. Quantitation of CMV DNAemia may play an important role in diagnosis in such a setting.

Detection of CMV DNA in peripheral blood leukocytes as a long-range harbinger of CMV disease in HIV-infected patients

The incidence of cytomegalovirus (CMV) end-organ disease can be reduced in AIDS patients by oral ganciclovir. However, the cost effectiveness of this prophylaxis is low. Targeting prophylaxis to patients with the highest risk of developing CMV disease might be useful. Several studies have shown the potential of various polymerase chain reaction (PCR) assays and of the antigenemia assay to identify the subset of patients with a definitely higher risk of developing CMV disease. We studied the CMV viral load using quantitative PCR in the leukocytes and plasma (or serum) of 28 patients in the four years before they experienced a CMV event. We observed rising CMV DNA copy numbers in the patients' leukocytes a year before the event. In contrast, plasma or serum copy numbers rose later and in fewer patients. In a control population of 21 profoundly immunodeficient patients (median CD4+ T cell count: 31/mm 3 ) without history of CMV disease, only five had detectable CMV DNA in the leukocytes, three of whom had barely above-threshold levels. We suggest that, at the present time, leukocyte CMV DNA PCR might represent a sensitive test providing an early warning signal of increased risk of CMV disease. Performing it twice a year might identify patients at risk so that closer monitoring and targeted prophylaxis can be conducted.

Cost-effectiveness of cytomegalovirus (CMV) disease prevention in patients with AIDS: oral ganciclovir and CMV polymerase chain reaction testing.

To perform a cost-effectiveness analysis of strategies to prevent cytomegalovirus (CMV) disease. Markov model and published data. Hypothetical HIV-infected patients with CD4 cell counts < or = 50 x 10(6)/l and positive CMV serologies. Oral ganciclovir daily versus plasma CMV DNA polymerase chain reaction (PCR) testing every 3 months with oral ganciclovir for patients with positive tests. The number of CMV disease cases prevented by the interventions, life expectancy, disease-free life expectancy, and the cost to extend life by 1 year. Oral ganciclovir preventive therapy reduces the lifetime number of CMV disease cases by 50 per 1000 cohort, extends life expectancy by 5 days and disease-free life expectancy by 18 days, and costs US$ 1,762,517 per year of life extended. Periodic PCR testing reduces the lifetime number of CMV disease cases by eight per 1000 cohort, extends life expectancy by 1 day and disease-free life expectancy by 3 days, and costs US$ 495,158 per year of life extended. The prevention strategies could be acceptably cost effective only under a combination of optimistic assumptions and reduced costs. Oral ganciclovir preventive therapy and periodic plasma testing for CMV PCR with oral ganciclovir for those with positive tests result in small benefits at great cost. They are not cost-effective prevention strategies for persons with advanced HIV infection and positive CMV serologies.

A polymerase chain reaction to detect a spliced late transcript of human cytomegalovirus in the blood of bone marrow transplant recipients.

A reverse transcription (RT) nested polymerase chain reaction (PCR) procedure is described for detecting RNA to a spliced late gene (SLG) of human cytomegalovirus (CMV), the product of which (175 bp) is easily differentiated in agarose gels from the product when the target is unspliced viral RNA or DNA (258 bp). The SLG-RT-PCR has been compared against a semi-quantitative PCR for CMV DNA in buffy-coat specimens collected weekly after bone marrow transplantation from 3 patients and against the results of culturing these specimens for CMV both by conventional virus isolation, based on the detection of cytopathic effect, and by the early detection of infected cells by staining with virus-specific monoclonal antibodies. The detection of CMV RNA by SLG-RT-PCR correlated well with the detection of infective virus but only when the results of both culture methods were combined, in that neither culture method alone was as sensitive as the SLG-RT-PCR. The presence of SLG RNA in the circulation is of value as a marker of active CMV infection.