Maria Paola Landini

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.

Cytomegalovirus-induced immunopathology and its clinical consequences

Human cytomegalovirus (CMV) is a ubiquitous DNA virus that causes severe disease in patients with immature or impaired immune systems. During active infection, CMV modulates host immunity, and CMV-infected patients often develop signs of immune dysfunction, such as immunosuppression and autoimmune phenomena. Furthermore, active viral infection has been observed in several autoimmune diseases, and case reports have linked primary CMV infection and the onset of autoimmune disorders. In addition, CMV infection promotes allograft rejection and graft-versus-host disease in solid organ and bone marrow transplant recipients, respectively, further implicating CMV in the genesis and maintenance of immunopathological phenomena. The mechanisms by which CMV could induce inhibition of host defense, inflammation, and autoimmunity are discussed, as is the treatment of virus-induced immunopathology with antivirals.

West Nile virus in Europe: emergence, epidemiology, diagnosis, treatment, and prevention

West Nile virus (WNV), a mosquito-borne flavivirus in the Japanese encephalitis antigenic group, has caused sporadic outbreaks in humans, horses and birds throughout many of the warmer regions of Europe for at least 20 years. Occasional cases of West Nile encephalitis have also been associated with infected blood transfusions and organ donations. Currently, WNV appears to be expanding its geographical range in Europe and causing increasing numbers of epidemics/outbreaks associated with human morbidity and mortality. This brief review reports on the current epidemic situation regarding WNV in Europe, highlighting the clinical, diagnostic and preventive measures available for controlling this apparently emerging human pathogen.

Human Cytomegalovirus Differentially Controls B Cell and T Cell Responses through Effects on Plasmacytoid Dendritic Cells

Plasmacytoid dendritic cells (PDCs), the main producers of type I IFN in response to viral infection, are essential in antiviral immunity. In this study, we assessed the effect of human CMV (HCMV) infection on PDC function and on downstream B and T cell responses in vitro. HCMV infection of human PDCs was nonpermissive, as immediate-early but not late viral Ags were detected. HCMV led to partial maturation of PDCs and up-regulated MHC class II and CD83 molecules but not the costimulatory molecules CD80 and CD86. Regardless of viral replication, PDCs secreted cytokines after contact with HCMV, including IFN-α secretion that was blocked by inhibitory CpG, suggesting an engagement of the TLR7 and/or TLR9 pathways. In the presence of B cell receptor stimulation, soluble factors produced by HCMV-matured PDCs triggered B cell activation and proliferation. Through PDC stimulation, HCMV prompted B cell activation, but only induced Ab production in the presence of T cells or T cell secreted IL-2. Conversely, HCMV hampered the allostimulatory ability of PDCs, leading to decreased proliferation of CD4+ and CD8+ T cells. These findings reveal a novel mechanism by which HCMV differentially controls humoral and cell-mediate immune responses through effects on PDCs.

Update on the prevention, diagnosis and management of cytomegalovirus infection during pregnancy.

Human cytomegalovirus (CMV) is the leading cause of congenital infection, with morbidity and mortality at birth and sequelae. Each year approximately 1-7% (Rev Med Virol 2010; 20: 311) of pregnant women acquire a primary CMV infection. Of these, about 30-40% transmit infection to their fetuses. The risk of serious fetal injury is greatest when maternal infection develops in the first trimester or early in the second trimester. Between 10 and 15% of congenitally infected infants are acutely symptomatic at birth and most of the survivors have serious long-term complications. Until a few years ago, laboratory testing was not possible to precisely define the maternal immune status, the recent development of advanced serological tests (IgG avidity test, IgM immunoblot and neutralizing antibody testing) allow us to identify, among pregnant women with suspected CMV, those with primary infection who are therefore at high risk of transmitting CMV to the fetus. This is done with the use of a screening test. As most maternal infections are asymptomatic, the only way to disclose primary infection is to implement specific serological testing as early in pregnancy as possible (before week 12-16 of gestation). Given the high risk of mother-fetus transmission and fetal damage, prenatal diagnosis is recommended to women with primary CMV infection contracted in the first half of pregnancy and in case of fetal abnormalities suggestive of infection. The correct interpretation of serological and virological tests followed by appropriate counselling by an expert physician is an effective tool to reduce the number of unnecessary pregnancy terminations by over 70%.

Human cytomegalovirus inhibits the migration of immature dendritic cells by down‐regulating cell‐surface CCR1 and CCR5

Dendritic cells (DC) play a key role in the host immune response to infections. Human cytomegalovirus (HCMV) infection can inhibit the maturation of DC and impair their ability to stimulate T cell proliferation and cytotoxicity. In this study, we assessed the effects of HCMV infection on the migratory behavior of human DC. The HCMV strain TB40/E inhibited the migration of immature monocyte‐derived DC in response to inflammatory chemokines by 95% 1 day after infection. This inhibition was mediated by early viral replicative events, which significantly reduced the cell‐surface expression of CC chemokine receptor 1 (CCR1) and CCR5 by receptor internalization. HCMV infection also induced secretion of the inflammatory chemokines CC chemokine ligand 3 (CCL3)/macrophage inflammatory protein‐1α (MIP‐1α), CCL4/MIP‐1β, and CCL5/regulated on activation, normal T expressed and secreted (RANTES). Neutralizing antibodies for these chemokines reduced the effects of HCMV on chemokine receptor expression and on DC migration by ∼60%. Interestingly, the surface expression of the lymphoid chemokine receptor CCR7 was not up‐regulated after HCMV infection on immature DC, and immature‐infected DC did not migrate in response to CCL19/MIP‐3β. These findings suggest that blocking the migratory ability of DC may be a potent mechanism used by HCMV to paralyze the early immune response of the host.

Diagnosis of bloodstream infections in immunocompromised patients by real-time PCR

OBJECTIVES The diagnosis of bloodstream infections (BSIs) in immunocompromised patients, such as patients with cancer, is challenging. Although blood culture (BC) is considered the standard diagnostic tool for BSIs, it takes several days to yield results and has low sensitivity in these patients. Here, we tested a novel method for diagnosing BSIs in a large cohort of immunodepressed patients. METHODS Real-time PCR (LightCycler SeptiFast Test M(GRADE), Roche Diagnostics) was compared with BC for its ability to detect bacteria and fungi in blood samples from 100 immunocompromised patients (98 with cancer) in whom sepsis was suspected. RESULTS In concordant samples (79.2% of total cases), real-time PCR identified the presence or absence of microbes significantly faster than BC (p=3.7x10(-49), t-test). Furthermore, in 6 cases, SeptiFast distinguished contamination of BCs by coagulase-negative staphylococci. SeptiFast, however, failed to detect 5 cases of clinically relevant BSI that tested positive by BC. CONCLUSIONS SeptiFast rapidly diagnosed BSIs in our cohort of immunosuppressed patients. The results of this study suggest that SeptiFast can be used in conjunction with, but cannot replace, BC to better identify the etiology of fever in immunocompromised patients.

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.

Human cytomegalovirus structural proteins.

Introduction. Members of the Herpesviridae family are viruses with a core containing the viral DNA in the form of a torus, a capsid (100 nm in diameter and composed of 162 capsomers), a tegument, which is a structure of variable thickness between the capsid and the envelope, and an envelope. The envelope is the trilaminar outer covering which contains numerous protrusions of spikes consisting of virally encoded glycoproteins. The resulting diameter of the virion varies from 120 to 300 nm. Herpesviruses have been divided into three subfamilies on the basis of biological properties. The Alphaherpesvirinae are herpesviruses with a variable host range, short replication cycle, rapid spread in cultured cells, a lytic effect on infected cells and a tropism for establishing a latent infection in sensory ganglia. The Betaherpesvirinae have a restricted host range, a longer replication cycle, an infection which progresses more slowly in cultured cells and a latent state that can be established in numerous cells and tissues.

HHV-6A in Syncytial Giant-Cell Hepatitis

Syncytial giant-cell hepatitis is a rare but severe form of hepatitis that is associated with autoimmune diseases, drug reactions, and viral infections. We used serologic, molecular, and immunohistochemical methods to search for an infectious cause in a case of syncytial giant-cell hepatitis that developed in a liver-transplant recipient who had latent infection with variant B of human herpesvirus 6 (HHV-6B) and who had received the organ from a donor with variant A latent infection (HHV-6A). At the onset of the disease, the detection of HHV-6A (but not HHV-6B) DNA in plasma, in affected liver tissue, and in single micromanipulated syncytial giant cells with the use of two different polymerase-chain-reaction (PCR) assays indicated the presence of active HHV-6A infection in the patient. Expression of the HHV-6A-specific early protein, p41/38, but not of the HHV-6B-specific late protein, p101, was demonstrated only in liver syncytial giant cells in the absence of other infectious pathogens. The same markers of HHV-6A active infection were documented in serial follow-up samples from the patient and disappeared only at the resolution of syncytial giant-cell hepatitis. Neither HHV-6B DNA nor late protein was identified in the same follow-up samples from the patient. Thus, HHV-6A may be a cause of syncytial giant-cell hepatitis.