Histological Analysis of Term Placentas from Hyperimmune Globulin-Treated and Untreated Mothers with Primary Cytomegalovirus Infection.

BACKGROUND: The Congenital Human Cytomegalovirus Infection Prevention (CHIP) study, a randomized, blinded, placebo-controlled trial, demonstrated that the efficacy of hyperimmune globulin (HIG) was not different from that of placebo regarding transmission of cytomegalovirus (CMV) from mothers to newborns. Our aim was to analyze histologically HIG effects on placentas collected for the CHIP study. MATERIALS AND METHODS: Virological and histological analyses were performed on 40 placentas from transmitter and nontransmitter HIG-treated and untreated mothers by assessing the number of CMV-positive cells, tissue viral load, tissue damage, and compensatory mechanisms. RESULTS: The HIG and placebo groups showed no significant differences in the number of CMV-positive cells (median number in 10 fields at 10 high-power fields: 2.5 vs. 2, p = 0.969) and viral load (median load: 5 copies/5 ng vs. 10.5 copies/5 ng, p = 0.874). Regarding histological examination, the scores of parameters related to tissue damage and hypoxic parenchymal compensation were higher in transmitters except for chorangiosis, with statistically significant differences observed for chronic villitis (p = 0.007), calcification (p = 0.011), and the total score of tissue damage (p < 0.001). The HIG and placebo groups showed no significant differences for all tissue damage and compensation parameters and overall scores. DISCUSSION: HIGs are not able to reduce placental viral load and histological damage, which was significantly associated only with infection.

The pentameric complex of human Cytomegalovirus: cell tropism, virus dissemination, immune response and vaccine development.

Between the 1980s and 1990s, three assays were developed for diagnosis of human cytomegalovirus (HCMV) infections: leuko (L)-antigenemia, l-viremia and l-DNAemia, detecting viral protein pp65, infectious virus and viral DNA, respectively, in circulating leukocytes Repeated initial attempts to reproduce the three assays in vitro using laboratory-adapted strains and infected cell cultures were consistently unsuccessful. Results were totally reversed when wild-type HCMV strains were used to infect either fibroblasts or endothelial cells. Careful analysis and sequencing of plaque-purified viruses from recent clinical isolates drew attention to the ULb' region of the HCMV genome. Using bacterial artificial chromosome technology, it was shown by both gain-of-function and loss-of-function experiments that UL131-128 genes are indispensable for virus growth in endothelial cells and virus transfer to leukocytes. In addition, a number of clinical isolates passaged in human fibroblasts had lost both properties (leuko-tropism and endothelial cell-tropism) when displaying a mutation in the UL131-128 locus (referred to as UL128L). In the following years, it was shown that pUL128L was complexed with gH and gL to form the pentameric complex (PC), which is required to infect endothelial, epithelial and myeloid cells. The immune response to PC was studied extensively, particularly its humoral component, showing that the great majority of the neutralizing antibody response is directed to PC. Although anti-HCMV antibodies may act with other mechanisms than mere neutralizing activity, these findings definitely favour their protective activity, thus paving the way to the development of a potentially protective HCMV vaccine.

A randomized trial of hyperimmune globulin to prevent congenital cytomegalovirus.

BACKGROUND: Congenital infection with human cytomegalovirus (CMV) is a major cause of morbidity and mortality. In an uncontrolled study published in 2005, administration of CMV-specific hyperimmune globulin to pregnant women with primary CMV infection significantly reduced the rate of intrauterine transmission, from 40% to 16%. METHODS: We evaluated the efficacy of hyperimmune globulin in a phase 2, randomized, placebo-controlled, double-blind study. A total of 124 pregnant women with primary CMV infection at 5 to 26 weeks of gestation were randomly assigned within 6 weeks after the presumed onset of infection to receive hyperimmune globulin or placebo every 4 weeks until 36 weeks of gestation or until detection of CMV in amniotic fluid. The primary end point was congenital infection diagnosed at birth or by means of amniocentesis. RESULTS: A total of 123 women could be evaluated in the efficacy analysis (1 woman in the placebo group withdrew). The rate of congenital infection was 30% (18 fetuses or infants of 61 women) in the hyperimmune globulin group and 44% (27 fetuses or infants of 62 women) in the placebo group (a difference of 14 percentage points; 95% confidence interval, -3 to 31; P=0.13). There was no significant difference between the two groups or, within each group, between the women who transmitted the virus and those who did not, with respect to levels of virus-specific antibodies, T-cell-mediated immune response, or viral DNA in the blood. The clinical outcome of congenital infection at birth was similar in the two groups. The number of obstetrical adverse events was higher in the hyperimmune globulin group than in the placebo group (13% vs. 2%). CONCLUSIONS: In this study involving 123 women who could be evaluated, treatment with hyperimmune globulin did not significantly modify the course of primary CMV infection during pregnancy. (Funded by Agenzia Italiana del Farmaco; CHIP ClinicalTrials.gov number, NCT00881517; EudraCT no. 2008-006560-11.).

Assessing Immunity to Rubella Virus: a Plea for Standardization of IgG (Immuno)assays.

Immunity to rubella virus (RV) is commonly determined by measuring specific immunoglobulin G (RV IgG). However, RV IgG results and their interpretation may vary, depending on the immunoassay, even though most commercial immunoassays (CIAs) have been calibrated against an international standard and results are reported in international units per milliliter. A panel of 322 sera collected from pregnant women that tested negative or equivocal for RV IgG in a prior test (routine screening) was selected. This panel was tested with two reference tests, immunoblotting (IB) and neutralization (Nt), and with 8 CIAs widely used in Europe. IB and Nt gave concordant results on 267/322 (82.9%) sera. Of these, 85 (26.4%) sera were negative and 182 (56.5%) sera were positive for both tests. All 85 IB/Nt-negative samples were classified as negative with all CIAs. Of the 182 IB/Nt-positive samples, 25.3 to 61.5% were classified as equivocal and 6 to 64.8% were classified as positive with the CIAs. Wide variations in titers in international units per milliliter were observed. In our series, more than half of the women considered susceptible to RV based on CIA results tested positive for RV antibodies by IB/Nt. Our data suggest that (i) sensitivity of CIAs could be increased by considering equivocal results as positive and (ii) the definition of immunity to RV as the 10-IU/ml usual cutoff as well as the use of quantitative results for clinical decisions may warrant reconsideration. A better standardization of CIAs for RV IgG determination is needed.

Slow increase in IgG avidity correlates with prevention of human cytomegalovirus transmission to the fetus.

Following primary human cytomegalovirus (HCMV) infection, virus-specific IgG antibody shift from low to high avidity with individual variations in the rate of avidity maturation. The kinetics of the avidity maturation of IgG specific for HCMV nuclear antigen in pregnant women with primary infection was investigated. Absorbance values used for avidity index calculation of 286 sequential sera collected from 69 pregnant women with primary HCMV infection were retrieved. Percent difference in absorbance values of IgG antibody bound to the solid phase after urea treatment (IgG avidity) between early (T1, 0-90, median 31 days) and late (T2, 91-180, median 136 days) serum samples was calculated for each woman. Three groups of women were identified: 24/69 (34.8%) women showed high (>100%) avidity increase between T1 and T2 (pattern H), 29/69 (42%) low (<50%) increase (pattern L), and 16/69 (23.2%) intermediate increase (pattern I). Avidity values in T1 samples were significantly higher in women with pattern L compared to women with pattern H (P=0.01). Altogether, 28/69 (40.6%) women transmitted HCMV infection to their fetuses. Fetal infection preferentially occurred (P<0.01) in women with pattern H (15/24, 62.5%) compared with women with pattern L (7/29, 24.1%). In conclusion, different patterns of IgG avidity maturation can be detected following primary HCMV infection. Pregnant women with pattern H (rapid IgG avidity increase) appear to be at higher risk for fetal infection, whereas, pregnant women developing early antibody with high avidity appear to be at a lower risk of vertical transmission.

Isolation of human monoclonal antibodies that potently neutralize human cytomegalovirus infection by targeting different epitopes on the gH/gL/UL128-131A complex.

Human cytomegalovirus (HCMV) is a widely circulating pathogen that causes severe disease in immunocompromised patients and infected fetuses. By immortalizing memory B cells from HCMV-immune donors, we isolated a panel of human monoclonal antibodies that neutralized at extremely low concentrations (90% inhibitory concentration [IC(90)] values ranging from 5 to 200 pM) HCMV infection of endothelial, epithelial, and myeloid cells. With the single exception of an antibody that bound to a conserved epitope in the UL128 gene product, all other antibodies bound to conformational epitopes that required expression of two or more proteins of the gH/gL/UL128-131A complex. Antibodies against gB, gH, or gM/gN were also isolated and, albeit less potent, were able to neutralize infection of both endothelial-epithelial cells and fibroblasts. This study describes unusually potent neutralizing antibodies against HCMV that might be used for passive immunotherapy and identifies, through the use of such antibodies, novel antigenic targets in HCMV for the design of immunogens capable of eliciting previously unknown neutralizing antibody responses.

Human cytomegalovirus-specific CD4+ and CD8+ T cell responses in primary infection of the immunocompetent and the immunocompromised host.

The kinetics of primary human cytomegalovirus (HCMV) infection and specific T-cell responses were investigated in 16 immunocompetent pregnant women and 8 solid-organ transplant recipients (SOTR). T-cell responses to whole HCMV and to pp65 and IE-1 peptides were determined by flow cytometry evaluation of IFNgamma production. HCMV-specific CD4(+) and CD8(+) T-cells appeared earlier and simultaneously in immunocompetent subjects, whereas specific CD8(+) T-cells preceded CD4(+) T-cells in half of the SOTR examined. The magnitude of the HCMV-specific T-cell pool was comparable. HCMV load reached peak levels 100-1000 times higher in SOTR than in immunocompetent women, while the virus persisted for months in blood of both groups. T-cells directed to pp65 and IE-1 were only detected in a portion of subjects developing a full T-cell response to the whole virus. Thus, the development of cell-mediated immune response in primary HCMV infection may be missed when looking at pp65 and IE-1 peptide-stimulated T-cells only.

Molecular epidemiology of primary human cytomegalovirus infection in pregnant women and their families.

The source of human cytomegalovirus (HCMV) infection was investigated in 29 pregnant women with primary HCMV infection by comparing DNA sequences of UL146, UL144 and a portion of UL55 gene of HCMV strains circulating within each family. Thirteen families were identified in which the pregnant woman, the husband and/or a child were shedding HCMV. In three of these families, both the woman and the husband suffered from a concomitant primary HCMV infection. Phylogenetic analysis of UL146, UL144, and UL55 genes indicated that strains circulating within each family were identical, whereas strains from different families appeared to be distinct. However, identical UL146, UL144, and UL55 DNA sequences were observed sporadically among unrelated strains. A child rather than the husband was the virus source for the great majority of pregnant women. No association was observed between UL144 polymorphisms and intrauterine transmission.

Rising antigenemia levels may be misleading in pre-emptive therapy of human cytomegalovirus infection in allogeneic hematopoietic stem cell transplant recipients.

BACKGROUND AND OBJECTIVES: Hematopoietic stem cell transplant (HSCT) recipients after show rising levels of antigenemia during pre-emptive ganciclovir treatment of human cytomegalovirus (HCMV) infection. This raises some doubts about the therapeutic decisions to be taken. DESIGN AND METHODS: Three groups of HSCT recipients with HCMV infection undergoing anti-viral treatment were identified: group A, showing increasing antigenemia and decreasing viremia and DNAemia; group B, with simultaneous increases in antigenemia, viremia, and DNAemia; and group C, with decreasing levels of all 3 viral markers. Viral load, determined as levels of antigenemia, viremia and DNAemia, was monitored for 3 months post-transplantation in all groups. RESULTS: Group A HSCT recipients showed antigenemia peaks 2-11 days after the onset of treatment, reaching negative levels only 25-30 days thereafter, whereas viremia and DNAemia started to drop earlier. Group B patients, mainly including HSCT recipients with grade II-IV acute GvHD treated with steroids prior to and during antiviral treatment, showed increasing levels of all three viral parameters until 5-10 days after the start of treatment; the levels dropped to negative values 25-30 days thereafter. Group C patients, who acted as controls, progressively cleared virus from blood as an early result of antiviral therapy. INTERPRETATION AND CONCLUSIONS: Antigenemia is not the best assay to guide pre-emptive therapy. Group A patients, who have an isolated increase of antigenemia, do not require a change of the ongoing antiviral therapy. Whether better control of infection could be obtained in group B patients by either reducing immunosuppressive therapy (when possible) or adopting combination therapy remains to be determined.

Prevention of Primary Cytomegalovirus Infection in Pregnancy.

BACKGROUND: Cytomegalovirus (CMV) is the leading infectious agent causing congenital sensorineural hearing loss and psychomotor retardation. CMV vaccine is currently unavailable and treatment options in pregnancy are limited. Susceptible pregnant women caring for children are at high risk for primary infection. CMV educational and hygienic measures have the potential to prevent primary maternal infection. METHODS: A mixed interventional and observational controlled study was conducted to investigate the effectiveness of hygiene information among pregnant women at risk for primary CMV infection for personal/occupational reasons. In the intervention arm, CMV-seronegative women, identified at the time of maternal serum screening for fetal aneuploidy at 11-12 weeks of gestation, were given hygiene information and prospectively tested for CMV until delivery. The comparison arm consisted of women enrolled at delivery who were neither tested for nor informed about CMV during pregnancy, and who had a serum sample stored at the screening for fetal aneuploidy. By design, groups were homogeneous for age, parity, education, and exposure to at least one risk factor. The primary outcome was CMV seroconversion. Acceptance of hygiene recommendations was a secondary objective and was measured by a self-report. FINDINGS: Four out of 331 (1.2%) women seroconverted in the intervention group compared to 24/315 (7.6%) in the comparison group (delta = 6.4%; 95% CI 3.2-9.6; P < 0.001). There were 3 newborns with congenital infection in the intervention group and 8 in the comparison group (1 with cerebral ultrasound abnormalities at birth). Ninety-three percent of women felt hygiene recommendations were worth suggesting to all pregnant women at risk for infection. INTERPRETATION: This controlled study provides evidence that an intervention based on the identification and hygiene counseling of CMV-seronegative pregnant women significantly prevents maternal infection. While waiting for CMV vaccine to become available, the intervention described may represent a responsible and acceptable primary prevention strategy to reduce congenital CMV.

Human cytomegalovirus pp67 mRNAemia versus pp65 antigenemia for guiding preemptive therapy in heart and lung transplant recipients: a prospective, randomized, controlled, open-label trial.

BACKGROUND: Preemptive therapy of human cytomegalovirus (HCMV) infections has gained popularity in transplantation centers. However, standardized protocols are not available. In particular, whether a qualitative molecular assay for detection of a late (pp67) HCMV mRNA represents a valuable alternative to quantitative antigenemia remains to be defined. METHODS: Overall, 82 heart (HTR) and lung (LTR) transplant recipients were randomized into two arms, where therapy was guided by qualitative pp67 mRNA NASBA (40 patients) or quantitative antigenemia (42 patients). In the NASBA arm, both primary and recurrent infections were treated upon first confirmed positive NASBA result. In the antigenemia arm, primary infections were treated upon first confirmed positive result, while recurrent infections were treated upon cutoff of 100 pp65-positive leukocytes. In both arms, therapy was stopped upon virus disappearance. Primary endpoint was duration of therapy. RESULTS: The number of treated/infected patients was significantly higher in the NASBA arm (25/30 vs. 15/39; P=0.015), as was the number of treated/relapsing patients (5/8 vs. 1/11; P=0.040), whereas the number of HCMV-infected/total number of patients was significantly higher in the antigenemia arm (39/42 vs. 30/40; P=0.026). Thus, in the NASBA arm, although the median duration of therapy was shorter compared to antigenemia (17 vs. 21 days, P>0.05), the overall number of days of therapy was significantly higher. No patient developed HCMV disease. CONCLUSION: pp67 mRNA NASBA can safely replace antigenemia, with some apparent advantages (semiautomation and objectivity of test results) and disadvantages (overtreatment of patients and greater duration of overall treatment).

Pathogenesis and prenatal diagnosis of human cytomegalovirus infection.

Congenital human cytomegalovirus (HCMV) infection is the leading infectious cause of mental retardation and sensorineural deafness. Intrauterine transmission and adverse outcome are mainly related to primary maternal infection. Mechanisms of intrauterine transmission are slowly being unraveled and compelling evidence of the importance of using HCMV clinical strains rather than laboratory-adapted strains for in vitro studies is growing. In the absence of a vaccine or a specific antiviral therapy which could be safely administered to pregnant women with primary HCMV infection, the option of prenatal diagnosis has a crucial role in the management of pregnancy complicated by primary HCMV infection. Reliability of prenatal results, however, is still a major concern presenting the risk of either false-negative or false-positive results. However, as more light is shed on the natural history of HCMV infection during pregnancy and fetal life, the predictive value of negative prenatal diagnosis results is becoming more defined, thus improving the quality of counseling. In addition, the availability of different assays for detection of HCMV in both fetal blood and amniotic fluid samples will eventually reduce the risk of false-positive results. Finally, the identification of reliable prognostic markers of fetal disease remains the ultimate goal and a major challenge.

Monitoring of human cytomegalovirus (HCMV)-specific CD4+ T cell frequency by cytokine flow cytometry as a possible indicator for discontinuation of HCMV secondary prophylaxis in HAART-treated AIDS patients.

OBJECTIVE: Absolute CD4+ T cell count and human cytomegalovirus (HCMV)-specific CD4+ T cell frequency (as determined by cytokine flow cytometry, CFC) were compared for their ability to predict HCMV disease and safe discontinuation of HCMV secondary prophylaxis. STUDY DESIGN: Three groups of AIDS patients with previous nadir CD4+ T cell count <100/microl were studied. Group A included 48 HAART-treated patients with no HCMV disease. Group B included 11 HAART-treated patients with previous HCMV disease who discontinued HCMV prophylaxis. Group C included 23 HAART-treated (n = 16) or -naive (n = 7) patients with previous HCMV disease either continuing or starting HCMV prophylaxis. Patients underwent follow-up for detection of HCMV viremia or disease (groups A and B) and for discontinuation of HCMV secondary prophylaxis on the basis of either HCMV-specific or absolute CD4+ T cell count (group C). RESULTS: During follow-up, while some patients showed a stable HCMV-specific CD4+ T cell response, others had a fluctuating response (unstable responders) or showed no response at all. In detail, 13/48 group A patients were either HCMV non-responders or unstable responders and 2 of them developed HCMV viremia; 3/11 group B patients were unstable responders, none developing either HCMV viremia or disease; finally, 9 group C patients discontinued HCMV prophylaxis based on absolute CD4+ T cell count > 150 cells/microl, but in 2 of them lacking HCMV-specific response HCMV retinitis relapsed. None of the seven group C patients discontinuing HCMV prophylaxis on the basis of CFC showed HCMV disease relapse. CONCLUSIONS: CFC may support absolute CD4+ T cell count for both guiding HCMV prophylaxis discontinuation and better monitoring HCMV infection in AIDS patients with no previous HCMV disease or having discontinued HCMV prophylaxis.

Cytomegalovirus DNAemia in pregnant women.

BACKGROUND: Cytomegalovirus (CMV) transmission from mother to fetus occurs at a much greater rate following primary rather than reactivated infections and CMV dissemination in the mother is considered a key step in the pathogenesis of fetal infection. However, knowledge of CMV DNAemia in CMV-seropositive pregnant women is very limited. OBJECTIVE: Major objective of this study was to assess the prevalence and diagnostic value of CMV DNAemia in a large population of seropositive pregnant women. STUDY DESIGN: Serologic and DNAemia results obtained from 2211 blood samples of 1371 consecutive pregnant women referred to our Institution for suspected CMV infection in the period 2001-2010 were reviewed. RESULTS: DNAemia was detected in 452/597 (75.7%) women with serologic evidence of primary CMV infection and in 4/774 (0.5%) women without evidence of primary infection. CONCLUSION: In pregnant women, CMV DNAemia is detected primarily during primary infection. CMV DNAemia determination may be helpful in the diagnosis of primary infection.

Role of human cytomegalovirus (HCMV)-specific antibody in HCMV-infected pregnant women.

Maternal preconception immunity confers substantial protection against HCMV infection and disease to the unborn child. However, the protective role played by single components of virus-specific humoral and cellular immunity is poorly defined. Recently, it was discovered that UL128-131 gene products are essential for the virus to exert endothelial/epithelial cell tropism during natural infection. This, together with the finding that the gH-gL-UL128-131 complex can elicit early, highly potent, and long-lasting neutralizing antibody response as well as other antibodies involved in cell-to-cell spreading and virus transfer from endothelial cells to leukocytes, indicate that antibodies may indeed potentially control virus dissemination in vivo and play a role in mother-to-fetus transmission as well. Additionally, passive immunization of pregnant women with primary HCMV infection has been reported to be highly beneficial for both prevention and therapy of congenital infection in nonrandomized studies. Recently, a phase IIB, randomized, double blind, hyperimmunoglobulin vs placebo trial (CHIP study) showed a lower, although not significant, rate of transmission in the hyperimmunoglobulin arm. Ongoing phase III controlled trials as well as laboratory investigations will hopefully help in better defining the protective role of maternal antibodies.

Evaluation of fully automated assays for the detection of Rubella IgM and IgG antibodies by the Elecsys(®) immunoassay system.

Screening for acute rubella infection in pregnancy is an important element of antenatal care. This study compared the sensitivity, specificity and reproducibility of two new, fully automated Elecsys(®) Rubella IgM and IgG immunoassays designed for the Elecsys 2010, Modular Analytics E170, COBAS e-411 and COBAS e-601 and e602 analytical platforms, with current assays using serum from patients with primary rubella infections, vaccinated patients, patients with potentially cross-reacting infections and on routine samples in clinical laboratories in France, Germany and Italy. Both assays showed good within-run and within-laboratory precision. A sensitivity of 79.8-96.0% was demonstrated for Elecsys IgM in primary, early acute infection, consistent with existing assays. In samples obtained from routine antenatal screening, the Elecsys Rubella IgM assay revealed high specificity (98.7-99.0%). A significantly (p<0.0001) lower reactivity was demonstrated in samples from previously infected patients where acute rubella infection was excluded, and the incidence of false positives in patients with potentially cross-reacting infections was lower with Elecsys Rubella IgM compared with other. The Elecsys Rubella IgG assay exhibited a relative sensitivity of 99.9-100.0% and specificity of 97.4-100.0% in samples from routine antenatal screening. The Elecsys Rubella IgM and IgG assays allow convenient, rapid and reliable determination of anti-rubella antibodies. Sensitivity, specificity and reproducibility were comparable with existing assay systems. Assay results were available in approximately half the time required for currently employed methods and the assays are compatible with widely used analytical platforms.

Fetal human cytomegalovirus transmission correlates with delayed maternal antibodies to gH/gL/pUL128-130-131 complex during primary infection.

Primary human cytomegalovirus (HCMV) infections during pregnancy are associated with a high risk of virus transmission to the fetus. To identify correlates of intrauterine HCMV transmission, serial serum samples from HCMV transmitter and non-transmitter pregnant women with primary HCMV infection were analyzed for the presence of neutralizing antibodies against different glycoproteins and glycoprotein complexes, which are known to mediate entry into distinct types of host cells. Neutralizing activity was detected in the sera early after primary infection; absorption with a soluble pentameric complex formed by gH/gL/pUL128-131, but not with gH/gL dimer or with gB, abolished the capacity of sera to neutralize infection of epithelial cells. Importantly, an early, high antibody response to pentamer antigenic sites was associated with a significantly reduced risk of HCMV transmission to the fetus. This association is consistent with the high in vitro inhibition of HCMV infection of epithelial/endothelial cells as well as cell-to-cell spreading and virus transfer to leukocytes by anti-pentamer antibodies. Taken together, these findings indicate that the HCMV pentamer complex is a major target of the antibody-mediated maternal immunity.

Role of prenatal diagnosis and counseling in the management of 735 pregnancies complicated by primary human cytomegalovirus infection: a 20-year experience.

BACKGROUND: The burden of congenital human cytomegalovirus (HCMV) infection is well recognized. However, screening for maternal infection remains controversial in view of diagnostic challenges, counseling difficulties, and absence of medical treatment. OBJECTIVE: To assess the role of prenatal diagnosis and counseling in the management of pregnancy complicated by primary HCMV infection. STUDY DESIGN: Retrospective study aimed at investigating diagnostic features, options, and pregnancy outcome in 735 women with primary HCMV infection over a period of 20 years (1990-2009). RESULTS: Overall, 25.6% women were found to be seronegative before the actual pregnancy. However, none were informed about HCMV infection and potential prevention strategies. Diagnosis of primary HCMV infection was achieved by seroconversion in 44.4% cases and by different combinations of virus-specific IgM, low IgG avidity, and DNAemia in 43.9% cases. Non-specific symptoms and/or haematological/biochemical alterations were recalled by 73.5% women. The onset of infection could be established, and counseling adjusted accordingly in >90% cases. The overall rate of vertical transmission was 37.1%, ranging from 5.6% for preconceptional infections to 64.1% for third trimester infections. Amniocentesis was chosen by 43.1% women, whereas pregnancy termination was requested by 15.6%. CONCLUSIONS: Reference virology centers and ad hoc trained and experienced physicians are required for accurate diagnosis of primary infection in pregnancy and ensuing counseling. Prenatal diagnosis has a central role in the management of pregnancies complicated by primary HCMV infection. HCMV-seronegative women should receive adequate information.