Liliana Gabrielli

Neonatal Cytomegalovirus Blood Load and Risk of Sequelae in Symptomatic and Asymptomatic Congenitally Infected Newborns

OBJECTIVE. Human cytomegalovirus (CMV) is a ubiquitous human-specific DNA virus and is the main cause of congenital virus infection in developed countries leading to psychomotor impairment and deafness. Diagnostic techniques for CMV detection have greatly improved during recent years with the advent of sophisticated serological and virological methods. The aim of the present study was to assess the diagnostic and prognostic value of detection and quantification of virus in neonatal blood samples of symptomatic and asymptomatic newborns with CMV congenital infection. METHODS. Between January 1997 and December 2003, we studied 99 newborns who were born to women with primary, recurrent, and undefined CMV infection during pregnancy. CMV congenital infection was identified by isolation of the virus in urine within the second week of life. Fifty-eight of 99 infants were infected and were assessed clinically for disease in the newborn period and classified as having symptomatic or asymptomatic infection on the basis of physical, instrumental, and laboratory findings. The infants were followed up from birth according to a protocol of the tertiary NICU at the University of Bologna in a prospective study of long-term sequelae of congenital infection. Forty-seven blood samples were obtained from 47 infants in the neonatal period: 34 were examined for pp65 antigenemia test and 44 for qualitative and quantitative polymerase chain reaction (PCR and qPCR). Sequelae at 12 months were evaluated in a group of 50 infants. RESULTS. Antigenemia was positive in only 10 of 34 samples of infected newborns (29.4% sensitivity). PCR was performed in 44 samples of infected newborns and was positive in all (100% sensitivity). qPCR showed a finding of ≥100 copies per 105 of polymorphonuclear leukocytes (PMNLs) in 39 of 44 samples; in the other 5 cases, the number of copies per 105 PMNLs was <100. Between symptomatic and asymptomatic newborns, the mean values of viral blood load determined by qPCR turned out to be significantly higher in symptomatic newborns. Mean values of neonatal blood viral load were statistically higher in newborns who developed sequelae than in those who did not. Of 20 children with a neonatal viral blood load of <1000 copies per 105 PMNLs, 19 did not develop sequelae (negative predictive value: 95%), whereas 2 of 3 with a viral blood load of >10000 copies did develop sequelae. CONCLUSIONS. Different viremia value ranges are correlated to a different risk of sequelae: ∼70% sequelae were found in newborns with a qPCR higher than 10000 copies per 105 PMNLs. Low neonatal viral blood load detected by pp65 antigenemia test and qPCR was highly predictive of absence of sequelae: DNAemia <1000 copies per 105 PMNLs has a negative predictive value of 95%. As an independent predictive factor of outcome, neonatal viremia is another useful element for neonatal counseling and therapeutic choices in symptomatic and asymptomatic newborns.

Hydroxymethyl-Glutaryl Coenzyme A Reductase Inhibition Limits Cytomegalovirus Infection in Human Endothelial Cells

Background—Statins exert anti-inflammatory effects independently of cholesterol-lowering properties. Cytomegalovirus (CMV) infection appears to be implicated in the pathophysiology of atherosclerosis by inducing inflammatory modifications in endothelial cells, especially in immunosuppressed patients. We investigated whether the activity of statins can inhibit replication of CMV in human endothelial cells. Methods and Results—Human umbilical vein endothelial cells (HUVECs) were infected with CMV and coincubated with fluvastatin at 0.1 and 0.2 &mgr;mol/L. Fluvastatin inhibited (P <0.001) CMV antigen expression, and this effect was dose related (P <0.001). Quantitative polymerase chain reaction showed that CMV DNA concentration was consistently lower in supernatants from fluvastatin-treated cells than in infected controls, and viral particle concentration was up to 30 times lower in 0.2 &mgr;mol/L fluvastatin-treated cells than in infected controls (10.5±0.9 versus 0.34±0.03 per 103 pfu/mL, P <0.001). Addition of mevalonate to treated cultures almost completely abolished fluvastatin inhibition of viral growth. Electrophoretic mobility shift assay showed that fluvastatin reduced nuclear factor-&kgr;B binding activity in CMV-infected cells. Conclusions—HMG-CoA inhibition by fluvastatin restrains CMV replication in HUVECs by inhibiting viral antigen expression, DNA synthesis, and viral particle production, conceivably by involving a reduction of nuclear factor-&kgr;B binding activity.

Cytomegalovirus (CMV)–Encoded UL144 (Truncated Tumor Necrosis Factor Receptor) and Outcome of Congenital CMV Infection

BACKGROUND Cytomegalovirus (CMV) is the most common congenital infection in humans. The effect of viral strains on the outcome of congenital CMV is debated. We evaluated whether UL144 polymorphisms in amniotic fluid from CMV-infected Italian women were associated with terminations of pregnancy, subsequent disease in their offspring, or viral load. METHODS The study was nested within a prenatal CMV program. We sequenced the UL144 gene from 66 amniotic-fluid samples, without knowledge of pregnancy outcome. We performed data analyses on 56 samples for which all information was available. RESULTS Genotype C was associated with termination of pregnancy (P=.03). Genotype B was associated with fewer terminations of pregnancy (P=.003). A possible association was found between genotype C and symptomatic disease in newborns (odds ratio, 8.81 [95% confidence interval, 0.48-164.02]; P=.05). There was no association between specific genotype and the viral load in amniotic fluid. Symptomatic newborns who had the most common UL144 genotype (B) were more likely to have higher viral loads than were asymptomatic infants (P=.003). CONCLUSIONS UL144 polymorphisms may be associated with the outcome of congenital CMV infection. Larger studies should be conducted to confirm this association, before genotype analysis can be used, along with other factors, in considering terminations of pregnancy.

Congenital cytomegalovirus infection: patterns of fetal brain damage

Cytomegalovirus (CMV) is the most prevalent infectious agent causing neurological dysfunction in the developing brain. This study analysed the different patterns of tissue damage, particularly in the brain, of fetuses with documented CMV infection. We studied 45 fetuses at 20-21 weeks of gestation with congenital CMV infection documented by invasive positive prenatal diagnosis. At the time of amniocentesis, abnormal ultrasound findings had been recorded for 13 of the 45 fetuses (29%). Histological and immunohistochemical characterization was performed on the placenta, brain, heart, lung, liver, kidney, and pancreas. The different degrees of brain damage were correlated with tissue viral load, inflammatory response, placental functionality, and extramedullary haematopoiesis. Even though a high CMV load was detected in all amniotic fluids, brain infection occurred in only 62% of the fetuses and with different degrees of severity. Tissues with a low viral load showed a globally weak inflammatory response, and fetuses had only mild brain damage, whereas tissues with a high CMV load showed prominent infiltration of the activated cytotoxic CD8(+) T-lymphocytes responsible for immune-mediated damage. Furthermore, severe placental infection was associated with diffuse villitis and necrosis, consistent with functional impairment and possible consequent hypoxic cerebral damage. Brain injury induced by CMV congenital infection may be the result of uncontrolled viral replication, immune-mediated damage by cytotoxic CD8(+) T-lymphocytes, and, in the presence of placental insufficiency, fetal hypoxia.

New advances in the diagnosis of congenital cytomegalovirus infection

With the advances in anticytomegalovirus (anti-CMV) serology, the new recombinant IgM tests seem likely to become the screening tests for pregnant women whose prepregnancy serological status for CMV is unknown. When a woman is found to be IgM-positive, further diagnostic evaluation focused on determining whether this is due to a primary infection should be carried out. Maternal primary infections that were difficult to determine until a few years ago unless documented by seroconversion can now be readily diagnosed from the presence of low-avidity anti-CMV antibody which persists for approximately 20 weeks after primary infection. In primarily infected mothers prenatal diagnosis can be performed between 21 and 23 weeks of gestation, and the amniotic fluid (AF) represents the pathological material of choice to determine intrauterine virus transmission. In AF, the virus can be detected by culture and/or PCR. Both procedures differentiate uninfected from infected fetuses, but cannot predict fetal outcome. The determination of the viral load in AF carried out by quantitative PCR is more promising and could represent an important starting point for preemptive fetal therapy.

Histological findings in foetuses congenitally infected by cytomegalovirus.

BACKGROUND Congenital cytomegalovirus (CMV) infection is a major cause of central nervous system damage leading to sensorineural hearing loss, mental retardation and cerebral palsy. OBJECTIVES Identify the type of organ involvement and understand the histopathogenesis of damage in foetuses of women with a CMV-highly positive amniotic fluid. STUDY DESIGN 34 foetuses with congenital CMV infection documented by prenatal diagnosis were studied. Three foetuses died in utero. The remaining pregnancies were electively terminated at 20-21 weeks gestation. RESULTS Foetal organs positive for CMV antigens were: placenta (100%), pancreas (100%), lung (87%), kidney (87%), liver (71%), brain (55%) and heart (44%). Inflammatory infiltrate was almost always present in CMV-infected foetal organs and the severity of the inflammatory response was correlated with the organ damage. Brain damage with necrosis was observed in 33% (9/27) and a mild telencephalic leukoencephalopathy in 22% (6/27) of foetuses studied. CONCLUSIONS Focal necrosis was observed very frequently in organs such as pancreases, livers, hearts and kidneys. However the damage in these organs is likely to be resolved by parenchymal regeneration. Brain damage, which seems to be the results of a combined effect of viral infection, inflammatory infiltration and hypoxia due to severe placentitis, is less likely to be resolved because of the low regeneration ability of this organ.

Herpes virus in Alzheimer's disease: relation to progression of the disease

Studies regarding different viruses of the herpes family, such as cytomegalovirus (CMV), Epstein-Barr virus (EBV), or human herpes virus 6 (HHV-6) in Alzheimers disease (AD) are scarce. DNA from peripheral blood leukocytes (PBL) and brain samples were analyzed for the presence of CMV, EBV, or HHV-6. All samples were negative for CMV. EBV positivity was 6% in AD brains, whereas 45% of PBL samples from AD patients and 31% from controls were positive for EBV (p = 0.05). HHV-6 showed a 23% positivity in PBL samples from AD and 4% from controls (p = 0.002). 17% of AD brains were HHV-6 positive. Within a group of elderly individuals, followed up for 5 years, EBV-positive or HHV-6-positive PBL increased in those who developed clinical AD. Virus serological positivity was also investigated, and IgG levels for CMV and EBV antigens were also increased in those subjects who developed AD during the follow-up. Our findings suggest that EBV and HHV-6 may be environmental risk factors for cognitive deterioration and progression to AD in elderly persons.

Role of cerebral ultrasound and magnetic resonance imaging in newborns with congenital cytomegalovirus infection

PURPOSE To assess the diagnostic and prognostic value of cerebral magnetic resonance imaging (cMRI) in comparison with that of cerebral ultrasound (cUS) in predicting neurodevelopmental outcome in newborns with congenital cytomegalovirus (CMV) infection. METHODS Forty CMV-congenitally infected newborns underwent cUS and cMRI within the first month of life. Clinical course, laboratory findings, visual/hearing function and neurodevelopmental outcome were documented. RESULTS Thirty newborns showed normal cMRI, cUS and hearing/visual function in the first month of life; none showed CMV-related abnormalities at follow-up. Six newborns showed pathological cMRI and cUS findings (pseudocystis, ventriculomegaly, calcifications, cerebellar hypoplasia) but cMRI provided additional information (white matter abnormalities in three cases, lissencephaly/polymicrogyria in one and a cyst of the temporal lobe in another one); cerebral calcifications were detected in 3/6 infants by cUS but only in 2/6 by cMRI. Four of these 6 infants showed severe neurodevelopmental impairment and five showed deafness during follow-up. Three newborns had a normal cUS, but cMRI documented white matter abnormalities and in one case also cerebellar hypoplasia; all showed neurodevelopmental impairment and two were deaf at follow-up. One more newborn showed normal cUS and cMRI, but brainstem auditory evoked responses were abnormal; psychomotor development was normal at follow-up. CONCLUSIONS Compared with cUS, cMRI disclosed additional pathological findings in CMV-congenitally infected newborns. cUS is a readily available screening tool useful in the identification of infected newborns with major cerebral involvement. Further studies with a larger sample size are needed to determine the prognostic role of MRI, particularly regarding isolated white matter lesions.

Delayed Acquisition of High-Avidity Anti-Cytomegalovirus Antibody Is Correlated with Prolonged Antigenemia in Solid Organ Transplant Recipients

Previous studies have demonstrated that maturation of cytomegalovirus (CMV)-specific antibodies in solid organ transplant recipients is delayed after primary CMV infection. To investigate the clinical significance of this finding, the avidity indices of anti-CMV antibody were determined in parallel with other serologic and virologic parameters in serial serum samples from 24 solid organ transplant recipients who had primary CMV infection that began during the first 3 months after transplantation. The data obtained show that a delay in antibody maturation is significantly correlated with a long persistence of positive antigenemia.

Multicenter comparative study of Epstein–Barr virus DNA quantification for virological monitoring in transplanted patients

BACKGROUND EBV-related post-transplant lymphoproliferative diseases are usually accompanied by increased EBV DNA in peripheral blood. Monitoring EBV DNAemia is the basis for weighing decisions regarding initiation of pre-emptive or anti-EBV-related tumor therapy. However, the definition of clinically relevant cut-off values is hampered by the lack of standardization in EBV DNA testing. OBJECTIVES To estimate inter-laboratory variability and to evaluate the impact of different matrices in EBV DNA load determination in Italian laboratories involved in monitoring of virus infections in transplanted patients. STUDY DESIGN Two different proficiency panels were distributed among seven centers: the first contained cell-associated and cell-free EBVs; the second was prepared by spiking both cell-associated and cell-free EBVs in EBV DNA-negative whole blood from EBV seropositive healthy donors. Samples were extracted and amplified with different methods. Intra-laboratory and inter-laboratory variabilities was evaluated. RESULTS 337 EBV DNA determinations were performed. Sensitivity was 100% for both panels, specificity was 100% for the first and 74% for the second panel, where whole blood was utilized as the matrix. Discrepant results in the second panel were restricted to samples containing low copy numbers. Quantification fell within ±0.5 log in 73% of the determinations. Values for cell-associated samples tended to be more heterogeneous than those obtained from cell-free samples. Good overall linearity was observed for each sample type; inter-laboratory variability ranged from 4.71% to 12.86%. CONCLUSIONS The results of this multicenter study indicate that EBV DNAemia may be reliably quantified by different laboratories using a variety of commercial and in-house molecular assays.