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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.

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.

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

Congenital cytomegalovirus (CMV) infection has potentially severe consequences in newborns. The testing of pregnant women for CMV-specific antibodies may be useful for the identification of women at risk of transmitting the infection to the fetus. The determination of CMV IgG avidity helps to establish the timing of infection as IgG avidity matures during the course of infection. This study examines the performance of the Elecsys® CMV IgG Avidity assay using preselected samples from patients at different phases of CMV infection. The Elecsys® CMV IgG Avidity assay was tested at three sites using sequential samples from patients with recent primary CMV infection, as well as single samples from patients with recent primary or past CMV infection. The Elecsys® assay discriminated well between early (low avidity) and late (high avidity) phases of infection in sequential serum samples. Overall, 98.8 % of low-avidity samples corresponded to infection onset 90 days before sampling. The assay’s sensitivity was 90–97 %, with specificity ranging from 89 to 100 %, depending on the consideration of gray-zone avidity values. Single samples from recent primary or past infection showed similar distributions of avidity results. The Elecsys® CMV IgG Avidity assay results are in agreement with preselected samples from patients with primary or past CMV infection, showing that the test is an adequate predictor of the phase of infection.

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 (

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.

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.

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.

Please cite this paper as: Fabbri E, Revello M, Furione M, Zavattoni M, Lilleri D, Tassis B, Quarenghi A, Rustico M, Nicolini U, Ferrazzi E, Gerna G. Prognostic markers of symptomatic congenital human cytomegalovirus infection in fetal blood. BJOG 2011;118:448–456. Objective To identify fetal cord blood prognostic markers of symptomatic congenital human cytomegalovirus infection (HCMV). Design Retrospective observational study. Setting Fetal medicine unit in Milan and Medical virology unit in Pavia, Italy. Population HCMV-infected and -uninfected fetuses of mothers with primary HCMV infection during the period 1995–2009. Methods Overall, 94 blood samples from as many fetuses of 93 pregnant women experiencing primary HCMV infection were examined for multiple immunological, haematological and biochemical markers as well as virological markers. Congenital HCMV infection was diagnosed by detection of virus in amniotic fluid, and symptomatic/asymptomatic infections were determined by ultrasound scans, nuclear magnetic resonance imaging, histopathology or clinical examination at birth. Blood sample markers were retrospectively compared in symptomatic and asymptomatic fetuses with congenital infection. Main outcome measures A statistical analysis was performed to determine the value of each parameter in predicting outcome. Results Univariate analysis showed that most nonviral and viral markers were significantly different in symptomatic (n = 16) compared with asymptomatic (n = 31) fetuses. Receiver operator characteristics analysis indicated that, with reference to an established cutoff for each marker, the best nonviral factors for differentiation of symptomatic from asymptomatic congenital infection were β2-microglobulin and platelet count, and the best virological markers were immunoglobulin M antibody and DNAaemia. β2-Microglobulin alone or the combination of these four markers reached the optimal diagnostic efficacy. Conclusions The determination of multiple markers in fetal blood, following virus detection in amniotic fluid samples, is predictive of perinatal outcome in fetuses with HCMV infection.

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.

Background. The development of human cytomegalovirus (HCMV)-specific T cell immunity after primary infection and its correlation with virus transmission to the fetus were investigated. Methods. The membrane phenotype (CCR7 and CD45RA expression) of and intracellular cytokine (interferon [IFN]-γ and interleukin-2) production by HCMV-specific T cells (stimulated with HCMV-infected dendritic cells) were investigated in 21 immunocompetent pregnant women (12 transmitters and 9 nontransmitters) and in 5 non-pregnant subjects during the first year after infection. Results. IFN-γ-producing CD4 + and CD8 + T cells were readily detected during the first month, and their levels did not significantly change with time. CCR7 expression was negligible during both the early and the late stage of infection. Among CCR7 - cells, those reexpressing CD45RA progressively increased until they reached median levels of 33% (range, 7%-51%) and 51% (range, 22%-76%) for HCMV-specific CD4 + and CD8 + T cells, respectively, similar to those observed in subjects with remote infection. CD45RA reexpression correlated with HCMV disappearance from blood. The level of HCMV-specific CD45RA + T cells during the first months after infection was significantly lower in mothers who were transmitters than in those who were nontransmitters. Conclusions. After primary infection, circulating HCMV-specific effector T cells revert to the CD45RA + phenotype, which appears to be associated with control of viremia and vertical transmission. Thus, these cells may represent long-lived true memory lymphocytes in the HCMV-specific pool.

The socio-economic burden associated with congenital human cytomegalovirus (HCMV) infection is well recognized. Nevertheless, women are neither informed nor tested for HCMV antibody, and in the absence of screening, HCMV infections proceed mostly undiagnosed during pregnancy.Knowledge has accumulated concerning both the natural history of HCMV infection and the availability of diagnostic tests.Different assays for the diagnosis in the mother, fetus and newborn are well established. In addition, positive and negative predictive values of different assays for prenatal diagnosis have improved over time.Without an HCMV vaccine, diagnosis of primary infection in the mother and of congenital infection in the fetus/newborn are key factors for management of congenital HCMV infection.

Background. The T cell-mediated immune response to human cytomegalovirus (HCMV) after primary infection, as well as the determinants of intrauterine transmission, are poorly understood. Methods. Sequential peripheral blood leukocyte samples from 74 pregnant women and 29 nonpregnant individuals with primary infection were examined for HCMV-specific CD4 + T cells by cytokine flow cytometry (CFC) and lymphoproliferative response (LPR) analysis. Immunological results for 19 transmitter and 21 nontransmitter mothers were compared. Results. Comparison of CFC and LPR analysis results showed that (1) there was no difference between pregnant and nonpregnant individuals; (2) HCMV-specific CD4 + T cells were detected by CFC, in the absence of an LPR to HCMV, in the great majority or the totality (according to different intervals) of samples collected from both pregnant and nonpregnant individuals during follow-up; and (3) LPR to HCMV was significantly (P

Coding sequences of the UL131A, UL130, and UL128 genes of human cytomegalovirus (HCMV) were found to be highly conserved among 34 field isolates from pregnant women with primary HCMV infection and their fetuses or newborns, as well as from solid organ transplant recipients and patients with AIDS. No strain clustering was observed. In contrast, sequencing of UL55 (gB coding gene) allowed the 34 isolates to be clustered into 4 genotypes. The conservation of the UL131A-UL128 locus is consistent with the conclusion that the three encoded proteins are all essential for growth of HCMV in endothelial cells and virus transfer to leukocytes.

Immature dendritic cells (DC) infected with an endotheliotropic (Huv(+)) and leukotropic (Leuk(+)) human cytomegalovirus (HCMV) strain were used as a stimulus to determine functional HCMV-specific CD4(+) and CD8(+) T cells. Infected DC were co-cultured with autologous peripheral blood mononuclear cells and both arms of T cell activation were determined by intracellular flow cytometry analysis of IFN-gamma production. Efficient stimulation of HCMV-specific CD4(+) and CD8(+) T cell responses was achieved using DC productively infected with Huv(+) Leuk(+) VR1814 strain. On the contrary, a negligible CD8(+) T cell response was obtained when HCMV strains unable to infect DC, or DC pulsed with inactivated viral antigen, were used. HCMV specificity of the T cell response was confirmed in 46 HCMV-seropositive and 8 HCMV-seronegative healthy subjects. A cut-off was established to discriminate between immune and nonimmune subjects. The novel ex vivo assay enables the simultaneous evaluation of HCMV-specific CD4(+) and CD8(+) T cell responses and may be a useful tool for monitoring HCMV-specific T cell activity in immunocompromised transplanted patients.

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).

SUMMARYHuman cytomegalovirus (HCMV) is the leading cause of congenital viral infection and mental retardation. HCMV infection, while causing asymptomatic infections in most immunocompetent subjects, can be transmitted during pregnancy from the mother with primary (and also recurrent) infection to the fetus. Hence, careful diagnosis of primary infection is required in the pregnant woman based on the most sensitive serologic assays (immunoglobulin M [IgM] and IgG avidity assays) and conventional virologic and molecular procedures for virus detection in blood. Maternal prognostic markers of fetal infection are still under investigation. If primary infection is diagnosed in a timely manner, prenatal diagnosis can be offered, including the search for virus and virus components in fetal blood and amniotic fluid, with fetal prognostic markers of HCMV disease still to be defined. However, the final step for definite diagnosis of congenital HCMV infection is detection of virus in the blood or urine in the first 1 to 2 weeks of life. To date, treatment of congenital infection with antiviral drugs is only palliative both prior to and after birth, whereas the only efficacious preventive measure seems to be the development of a safe and immunogenic vaccine, including recombinant, subunit, DNA, and peptide-based vaccines now under investigation. The following controversial issues are discussed in the light of the most recent advances in the field: the actual perception of the problem; universal serologic screening before pregnancy; the impact of correct counseling on decision making by the couple involved; the role of prenatal diagnosis in ascertaining transmission of virus to the fetus; the impact of preconceptional and periconceptional infections on the prevalence of congenital infection; and the prevalence of congenitally infected babies born to mothers who were immune prior to pregnancy compared to the number born to mothers undergoing primary infection during pregnancy.

The development of a human cytomegalovirus (HCMV) vaccine for the prevention of perinatal disease is urgently needed. However, markers of HCMV attenuation are not defined at present. An in vitro model for the study of interactions of HCMV-infected human fibroblasts and peripheral blood polymorphonuclear leukocytes showed that (1) known laboratory-adapted HCMV strains as well as cell culture-adapted HCMV isolates were not transmitted to polymorphonuclear leukocytes; (2) each of the 80 HCMV recent isolates was consistently transmitted to polymorphonuclear leukocytes as both infectious virus and viral components (nucleic acid and proteins); and (3) all 15 polymorphonuclear leukocyte-tropic strains tested thus far were also endothelial cell-tropic. The in vitro transmissibility to polymorphonuclear leukocytes (and endothelial cells) is proposed as a surrogate marker of pathogenicity of HCMV strains. It seems reasonable to assume that a HCMV strain candidate for a vaccine be verified as deprived of the transfer property to polymorphonuclear leukocytes (and endothelial cells) before involvement in clinical trials assessing safety and immunogenicity.

Evaluation of human cytomegalovirus (HCMV)-specific T-helper immunity could contribute in optimizing anti-HCMV therapy in human immunodeficiency virus (HIV)-infected patients. Testin the lymphoproliferative response (LPR) is the standard technique used to evaluate T-helper response, but its use in the routine diagnostic laboratory setting can be problematic. The most promising new alternative technique is the determination of HCMV-specific CD4 + T-cell frequency by flow cytometry detection of intracellular cytokine production after short-term antigen-specific activation of peripheral blood mononuclear cells. HCMV-specific LPR and CD4 + T-cell frequency were compared in a group of 78 blood samples from 65 HIV-infected patients. The results showed concordance in 80.7% of samples. In addition, comparative analysis of sequential blood samples from 13 HIV-infected patients showed that while in about half of patients the T-helper HCMV-specific immune response remained stable during highly active antiretroviral therapy (HAART), in the other half declining levels of the HCMV-specific CD4 + -mediated immune response were determined by either one or both assays. In conclusion, our data suggest that the determination of HCMV-specific CD4 + T-cell frequency can be considered a valuable alternative to the LPR test for the detection of HCMV-specific T-helper response in HIV-infected patients. It could facilitate wider screening of anti-HCMV T-helper activity in HIV-infected patients, with potential benefits for clinicians in deciding strategies of discontinuation or maintenance of anti-HCMV therapy.

Human cytomegalovirus (HCMV) immediate-early messenger RNA (IEmRNA) in sequential blood samples from 32 pregnant women with primary infection and from 14 congenitally infected newborns was qualitatively investigated by nucleic acid sequence-based amplification. IEmRNA was detected in 100%, 75%, 36.3%, 22.2%, and 0% of samples collected 1, 2, 3, 4-6, and >6 months after onset of primary HCMV infection, respectively, showing 83.7% sensitivity and 92.2% specificity, compared with results of quantitative DNAemia (detection of viral DNA in blood). In infected newborns, IEmRNA was positive in 100% of samples collected 1-7 days (median, 1.5 days) and in 46.4% of samples collected 27-260 days (median, 88 days) after birth, showing 75.7% sensitivity and 100% specificity, compared with DNAemia results. IEmRNA was not detected in HCMV-immune individuals with remote or recurrent HCMV infection or in uninfected newborns. IEmRNA determination appears to be a valuable tool for early diagnosis of both primary and congenital HCMV infection.

Using a recently developed model for in vitro generation of pp65-positive polymorphonuclear leukocytes (PMNLs), we demonstrated that PMNLs from immunocompetent subjects may harbor both infectious human cytomegalovirus (HCMV) and viral products (pp65, p72, DNA, and immediate-early [IE] and pp67 late mRNAs) as early as 60 min after coculture with human umbilical vein endothelial cells (HUVEC) or human embryonic lung fibroblasts (HELF) infected with a clinical HCMV isolate (VR6110) or other wild-type strains. The number of PMNLs positive for each viral parameter increased with coculture time. Using HELF infected with laboratory-adapted HCMV strains, only very small amounts of viral DNA and IE and late mRNAs were detected in PMNLs. A cellular mRNA, the vascular cell adhesion molecule-1 mRNA, which is abundantly present in both infected and uninfected HUVEC, was detected in much larger amounts in PMNLs cocultured with VR6110-infected cells than in controls. Coculture of PMNLs with VR6110-infected permissive cells in the presence or absence of RNA, protein, and viral DNA synthesis inhibitors showed that only IE genes were transcribed in PMNLs during coculture. Synthesis of IE transcripts in PMNLs was also supported by the finding that only the copy number of IE mRNA (and not the DNA or the pp67 mRNA) per infected PMNL increased markedly with time, and the pp67 to IE mRNA copy number ratio changed from greater than 10 in infected HUVEC to less than 1 in cocultured PMNLs. Fluorescent probe transfer experiments and electron microscopy studies indicated that transfer of infectious virus and viral products from infected cells to PMNLs is likely to be mediated by microfusion events induced by wild-type strains only. In addition, HCMV pp65 and p72 were both shown to localize in the nucleus of the same PMNLs by double immunostaining. Two different mechanisms may explain the virus presence in PMNLs: (i) one major mechanism consists of transitory microfusion events (induced by wild-type strains only) of HUVEC or HELF and PMNLs with transfer of viable virus and biologically active viral material to PMNLs; and (ii) one minor mechanism, i.e., endocytosis, occurs with both wild-type and laboratory strains and leads to the acquisition of very small amounts of viral nucleic acids. In conclusion, HCMV replicates abortively in PMNLs, and wild-type strains and their products (as well as cellular metabolites and fluorescent dyes) are transferred to PMNLs, thus providing evidence for a potential mechanism of HCMV dissemination in vivo.

Human cytomegalovirus (HCMV) infection was monitored retrospectively by qualitative determination of immediate-early (IE) mRNA by nucleic acid sequence-based amplification (NASBA) in a series of 51 bone marrow transplant (BMT) recipients. The qualitative results for IE mRNA obtained by NASBA were compared with those obtained by prospective quantitation of HCMV viremia and antigenemia and retrospective quantitation of DNA in blood (DNAemia) by PCR as well as by qualitative determination of late pp67 mRNA by NASBA. On the whole, of the 39 HCMV-positive patients (all asymptomatic), HCMV was detected in 14 (35.9%) by quantitation of viremia, 15 (38.5%) by detection of pp67 mRNA by NASBA, 32 (82.1%) by quantitation of DNAemia, and 33 (84.6%) by quantitation of antigenemia, while HCMV was detected in 38 (97.4%) patients by detection of IE mRNA by NASBA. In the immunocompetent host, IE mRNA was not detected by NASBA in 100 blood donors or during reactivated infections in 30 breast-feeding mothers. Likewise, NASBA did not detect IE mRNA in 56 solid-organ transplant recipients in the first 21 days after transplantation. By using NASBA for detection of IE mRNA as the reference standard for detection of HCMV infection in blood samples, the diagnostic sensitivities were 67.7% for quantitation of DNAemia, 59.0% for quantitation of antigenemia, 18.3% for detection of pp67 mRNA by NASBA, and 16.0% for quantitation of viremia. Specificities and negative and positive predictive values were >90.0, >70.0, and >80.0%, respectively, for all four assays. The mean times to first HCMV detection after bone marrow transplantation were 37.7 ± 15.4 days for detection of IE mRNA by NASBA, 39.6 ± 15.6 days for quantitation of antigenemia, 40.9 ± 15.2 days for quantitation of DNAemia, and 43.7 ± 16.3 or 43.7 ± 17.5 days for quantitation of viremia and detection of pp67 mRNA by NASBA, respectively. On the whole, 31 BMT recipients received preemptive therapy by using confirmed antigenemia positivity as a cutoff, while 35 patients could have been treated by using NASBA positivity as a cutoff and 31 could have been treated by using quantitation of DNAemia as a cutoff. In single patients, IE mRNA was detected in every episode of active HCMV infection, mostly preceding and sometimes accompanying antigenemia and DNAemia, whereas pp67 mRNA was detected only concomitantly with the highest peaks of infection. HCMV IE mRNA detection may represent a useful parameter for initiation of preemptive therapy in BMT recipients.

Human cytomegalovirus (HCMV) load and virus-specific IgM were quantified in blood of 36 fetuses from mothers with primary HCMV infection. Nineteen fetuses were congenitally infected and 17 were uninfected as diagnosed by virus isolation from and DNA detection in amniotic fluid. Sensitivity of antigenemia was 57.9%; of viremia, 55. 5%; of leukoDNAemia, 82.3%; and of IgM, 57.9%; specificity was 100% for all assays. When amniocentesis was performed, 4 HCMV-infected fetuses (group A) showed abnormal ultrasound and biochemical/hematologic findings, 8 (group B) had elevated gamma-glutamyl transferase values, and 7 (group C) had normal ultrasound and biochemical findings. Virus loads were higher in groups A and B than in group C. In group A, no pregnancy went to term, in group B, 3 of 6 newborns were symptomatic at birth, and in group C, the 6 newborns were subclinically infected. Taken together, virologic, laboratory, and ultrasound findings may contribute to a better prognostic definition of fetal HCMV infection.

Background: Diagnosis of congenital human cytomegalovirus (HCMV) infection relies on virus isolation from urine collected in the first 3 weeks of life. However, very little is known about the presence, levels and duration of HCMV pp65 antigenemia, viremia and DNAemia in congenitally infected newborns. Objectives: To investigate the diagnostic and prognostic value of HCMV load determination in blood of newborns/infants with congenital HCMV infection. Study design: HCMV pp65 antigenemia, viremia and DNAemia were investigated in 116 sequential peripheral blood leukocytes (PBL) samples from 41 newborns/infants with congenital HCMV infection and in 34 PBL samples from 34 uninfected newborn. Virus-specific IgM were determined in parallel on 145 sequential serum samples. Results: Compared to virus isolation from urine, sensitivities of DNAemia, antigenemia, viremia, and IgM determination were 100, 42.5, 28.2, and 70.7%, respectively. Specificity was 100% for all assays. Antigenemia, viremia and DNAemia levels were significantly higher and persisted longer in newborns with symptomatic infection compared to subclinically infected babies, whereas no difference was observed for virus-specific IgM antibody between the two groups. Conclusions: (i) determination of viral DNA in blood at birth appears to be a sensitive and specific marker for diagnosis of congenital HCMV infection; (ii) significantly higher levels of HCMV load were detected in infants with symptomatic HCMV infection; and (iii) virus clearance from blood occurs spontaneously both in symptomatic and subclinically infected infants. However, the process takes longer in infants presenting with symptoms at birth.

Two major variables may cause false-negative results in prenatal diagnosis of congenital human cytomegalovirus (HCMV) infection: sensitivity of the technique(s) used; and time elapsed between maternal infection and antenatal testing. Previous results indicated that rapid HCMV isolation from amniotic fluid samples and viral DNA detection in amniotic fluid by nested polymerase chain reaction (nPCR) had comparable levels of sensitivity (69.2% and 76.9%, respectively). The nPCR protocol was reviewed following two additional false-negative antenatal diagnosis in a twin pregnancy during which two procedures were performed at 18 and 23 weeks of gestation, respectively. In the new assay, multiple (instead of single) and 100 (instead of 20) μl amniotic fluid aliquots were individually amplified and tested by nPCR. By using this approach, low DNA levels (1–10 genome equivalents) were detected in 1–5/8 replicates of amniotic fluid samples taken from both twins during both procedures. In addition, viral DNA was detected in 5/6 replicates from two amniotic fluid samples still available from two previous false-negative cases. However, nPCR on multiple amniotic fluid replicates did not anticipate positive prenatal results in a retrospective case, which required two procedures for correct diagnosis and, when prospectively employed, did not avoid one additional false-negative prenatal diagnosis 8 weeks after maternal infection. Thus, delayed intrauterine transmission of the infection may be a potential cause of false-negative results. However, the combination of a very sensitive technique with appropriate timing of prenatal testing can substantially increase the reliability of prenatal diagnosis results. J. Med. Virol. 56:99–103, 1998. © 1998 Wiley-Liss, Inc.

BACKGROUND: The emergence of a ganciclovir (GCV)-resistant human cytomegalovirus (HCMV) strain in a heart transplant recipient (HTR) coinfected by multiple HCMV strains was investigated. METHODS: A HTR with primary HCMV infection was treated with three induction courses of intravenous GCV followed by a 2-month maintenance treatment with oral GCV. HCMV antigenemia, viremia, and leukoDNAemia levels were monitored. GCV susceptibility was analyzed by an immediate-early antigen plaque reduction assay and by a rapid screening assay performed using peripheral blood leukocytes (PBL) as viral inoculum. The viral population in blood was investigated by restriction analysis of multiple genome regions. UL97 and UL54 genes were sequenced in parallel in both HCMV isolates and the relevant PBL samples. A rapid molecular assay for detection and quantitation of the GCV-resistant mutant was developed. RESULTS: The emergence of a GCV-resistant UL97 mutant (Cys-607 --> Tyr) was responsible for treatment failure during oral GCV therapy. The genetic analysis of the HCMV population showed the sequential appearance in blood of two unrelated strains (referred to as A and B). Strain A most likely derived from the transplanted organ and strain B from a subsequent blood transfusion. The resistant variant (Br) emerged from strain B and became predominant "in vivo" under the GCV pressure. However, after foscarnet administration, the resistant mutant disappeared in viral isolates, whereas it was still present as a minor proportion in PBL samples. CONCLUSION: (a) Oral GCV may select resistant strains in transplanted patients; (b) results of the rapid screening assay were clinically useful for shifting to an alternative treatment, thus avoiding the appearance of HCMV disease; (c) virus isolation may not be the most reliable approach to detection of HCMV drug-resistant strains; (d) a novel molecular assay for rapid detection of UL97 Cys-607 --> Tyr mutation directly in clinical specimens was developed, allowing earlier "in vivo" detection of the resistant mutant.

Chemokine receptors (CR), which can mediate migration of immune cells to the site of inflammation, also function as coreceptors for human immunodeficiency virus (HIV) entry into CD4+ T lymphocytes and antigen-presenting cells. We demonstrate here that interferon-γ (IFN-γ) increases the expression of chemokine receptors CCR1, CCR3, and CCR5 in monocytoid U937 cells as detected by cell surface molecule labeling and mRNA expression, as well as by intracellular calcium mobilization and cell migration in response to specific ligands. The increased expression of these chemokine receptors also results in an enhanced HIV-1 entry into cells. Our data provide evidence for a relationship of cellular pathways that are induced by IFN-γ with those that regulate chemokine receptor expression.

Chemokine receptors (CR), which can mediate migration of immune cells to the site of inflammation, also function as coreceptors for human immunodeficiency virus (HIV) entry into CD4+ T lymphocytes and antigen-presenting cells. We demonstrate here that interferon-γ (IFN-γ) increases the expression of chemokine receptors CCR1, CCR3, and CCR5 in monocytoid U937 cells as detected by cell surface molecule labeling and mRNA expression, as well as by intracellular calcium mobilization and cell migration in response to specific ligands. The increased expression of these chemokine receptors also results in an enhanced HIV-1 entry into cells. Our data provide evidence for a relationship of cellular pathways that are induced by IFN-γ with those that regulate chemokine receptor expression.

In a group of 29 AIDS patients with biopsy-proven human cytomegalovirus (HCMV) gastrointestinal disease (GID), HCMV GID was shown to correlate mostly with systemic HCMV infection. The antiviral induction treatment (IT) with either ganciclovir (GCV) or foscarnet (PFA) caused a significant reduction in the level of HCMV antigenemia, viremia and leukoDNAemia, and a complete virus clearance or a sharp drop of viral load in the blood of 13/13 patients and in the gastrointestinal (GI) mucosa of 12/13 (92%) patients in the GCV arm, and in the blood of 13/14 (93%) patients and in the GI mucosa of 10/12 (83%) patients in the PFA arm of the study, respectively. Similarly, the clinical response was good in 13/15 (87%) patients in the GCV arm and in 13/14 (93%) patients in the PFA arm. In addition, the finding that 2/6 patients positive for HCMV isolated from both GI mucosa and blood prior to IT were still positive in the GI tract after IT, suggested that IT could be prolonged to clear the virus from GI tract. In conclusion, both GCV and PFA showed a remarkable systemic and local antiviral effect in the treatment of HCMV GID in AIDS patients.

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 (P0.01) 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.

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.

Cytomegalovirus (CMV) is a leading cause of physical and neurological abnormalities in newborns. Hence, the diagnosis of CMV infection in pregnant women is necessary in order to allow appropriate management of their pregnancy. New assays have been developed for the Roche Elecsys® immunoassay platform that detect CMV-specific immunoglobulin (Ig)M and IgG, with the IgM assay designed to target IgM produced at the start of infection rather than IgM persisting later in infection. This study aimed to evaluate the performance of the new assays compared with other commercial kits widely distributed in laboratories. The performance of the Elecsys and comparator CMV IgM and IgG assays was assessed using 967 preselected patient samples characterised by CMV infection status, as well as being compared using 1,668 unselected clinical samples. The Elecsys CMV IgM and IgG assays performed consistently with comparator assays using the preselected samples. The Elecsys CMV IgM assay showed improved sensitivity compared with the Enzygnost® assay in primary infection (91.2 % vs. 79.4 %) and improved specificity over the Architect® assay in potentially cross-reacting samples (94.1 % vs. 82.4 %). The Elecsys IgM assay reported fewer positive results in the later stages of CMV infection compared with ETI-CYTOK-M ELISA, while the Elecsys IgG assay reported slightly fewer negative results in the early stages of infection compared with ETI-CYTOK-G ELISA. There was good agreement between Elecsys and comparator assays using unselected clinical samples (range 90.4–99.4 %). The Elecsys CMV IgM and IgG assays compare well with routinely used assays and are suitable for clinical use.

Fifteen fetuses at risk of congenital human cytomegalovirus (HCMV) infection underwent prenatal diagnosis at 16-30 weeks' gestation by a combination of amniocentesis and fetal blood sampling. HCMV was isolated from the amniotic fluid in six patients, but HCMV-specific IgM was detected in only three of them. Two of the nine neonates, who were delivered following a negative prenatal diagnosis, had congenital HCMV infection diagnosed by virus isolation in the urine. The interval from infection to prenatal testing was 3 and 4 weeks in the two false-negative cases andor = 7 weeks in the true-positive cases. Although timely testing for HCMV infection allows the option of termination of pregnancy, it may be flawed by false-negative results.

Giant cells fully permissive for human cytomegalovirus (HCMV) were found to circulate, at a variable proportion, in peripheral blood of 21 out of 25 immunocompromised patients with disseminated HCMV infection. Circulating endothelial giant cells (EGC) were identified by a specific monoclonal antibody of endothelial origin and shown to express immediate-early, early, and late viral proteins. Immunostaining patterns of different viral proteins were comparable to those detected in vitro in cultured human umbilical vein endothelial cells. EGC counts > 10 were associated with high levels (> 100) of HCMV viremia and antigenemia, as well as with an overt clinical syndrome in transplanted patients, and to an untreated long lasting organ localization in AIDS patients. On the other hand, EGC counts were < 10 during disseminated HCMV infections of both transplant recipients with no apparent organ syndrome and AIDS patients with recent organ involvement. In tissue sections from AIDS patients, infected endothelial cells were found to progressively enlarge till detaching from the small vessel wall and entering blood stream. HCMV-infected EGC represent a new systemic parameter suitable for the diagnosis of HCMV organ involvement and for the study of the pathogenesis of disseminated infections.

Background The interpretation of a positive IgM antibody result to human cytomegalovirus (HCMV) in a pregnant woman is of major importance for the correct management of the pregnancy. Determination of HCMV-specific IgG avidity is considered an useful approach for distinguishing IgM antibody due to primary HCMV infection from IgM antibody elicited during non-primary infection. Objective Comparative evaluation of eight commercial HCMV IgG avidity assays currently available in Europe. Study design A panel of 198 sequential samples collected from 65 pregnant women at 0–90, 91–180, and >180 days after the onset of primary HCMV infection was retrospectively tested by Abbott, BioMerieux, Bio-Rad, DiaSorin, Diesse, Euroimmun, Radim, and Technogenetics HCMV IgG avidity assays according to the manufacturer's instructions. Results None of the 198 samples tested yielded identical scores by the kits under evaluation. The Euroimmun and Radim assays showed the best correlation with expected results in terms of low (0–90 days), intermediate (90–180 days) and high (>180 days) avidity results, respectively. The best accuracy in diagnosing a recent ( 180 days after the onset) primary HCMV infection was shown by Radim followed by Euroimmun and Diesse. The best correlation with a well established in-house developed HCMV IgG avidity assay was shown by Radim. Conclusions HCMV IgG avidity kits need to be improved and standardized. In the meantime, highly specific IgM assays are preferable for screening purposes in pregnant women.

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.

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.

In the absence of a cytomegalovirus (CMV) vaccine, other strategies for prevention of primary infection in pregnancy should be considered. Behavioural interventions have been reported to significantly decrease seroconversion rate among seronegative pregnant women. We report here on a recently completed controlled study in which seronegative women at high risk of infection because of close contacts with children

Cytomegalovirus (CMV) infection remains a significant cause of morbidity and mortality in young children. We have previously shown that CD8+ T cell responses to CMV pp65 or IE1 protein were readily detectable in children with congenital or postnatal CMV infection. Here, we have further characterized the evolution of the peptide specificity of these responses in 7 infants

Objective. We sought to study the development of human cytomegalovirus (HCMV)–specific T cell–mediated immune responses during primary HCMV infection in pregnancy. Methods. The HCMV-specific lymphoproliferative response (LPR) and intracellular cytokine (interferon [IFN]– g and interleukin [IL]–2) production were investigated during the first year after primary infection in 49 pregnant women and 9 nonpregnant control subjects. An HCMV-specific CD4 + and CD8 + T cell LPR was detected by the 5,6-carboxyfluorescein diacetate succinimidyl ester dilution method, and a cell-division index was calculated. Results. The CD4 + T cell LPR developed slightly earlier than the CD8 + T cell LPR. However, CDI values for both T cell subpopulations were lower than those of seropositive control subjects in both pregnant and nonpregnant individuals. During the first month after infection, IFN-g–producing CD4 + and CD8 + T cells were consistently observed, whereas IL-2–producing T cells were very rarely detected in blood. A correlation between the development of HCMV-specific LPR and virus clearance from blood was observed. A significantly delayed development of the CD4 + T cell LPR was observed in infected mothers who transmitted virus to the fetus, compared with those who did not. Conclusions. The development of adaptive T cell immunity after primary HCMV infection appears to be a complex and slow process until a memory T cell response develops. The T cell immune response appears to influence vertical HCMV transmission.

The risk of vertical transmission of human cytomegalovirus (HCMV) was investigated in 14 women who had primary HCMV infection 2-18 weeks before their last menstrual period during 2001-2004. One (8.3%) of 12 newborns examined at birth was found to be subclinically infected. Preconceptional primary HCMV infection has a transmission rate that is significantly higher than that observed in an unselected newborn population but significantly lower than that associated with maternal infection during pregnancy. Although a safe time interval between primary infection and initiation of pregnancy still needs to be defined, documented information can now be provided during counseling.

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.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.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.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.

Summary. From 2001 through 2004, 808 pediatric patients admitted to hospital because of acute respiratory infections were examined for presence of respiratory viruses by either direct fluorescent staining using monoclonal antibodies or RT-PCR during three consecutive winter-spring seasons. On the whole, 336 (42%) patients were detected as positive for one or more respiratory viruses. The most widely circulating virus was human respiratory syncytial virus (hRSV) infecting 50% of positive patients, followed by human metapneumovirus (hMPV) found in 13% of patients, and then by influenza virus type A, human parainfluenzaviruses and coinfections. Significant variations in the circulation rate of hRSV, hMPV and influenzavirus type A were observed during the individual seasons. In addition, the circulation rates of the different types of hMPV changed yearly. In 2001–2002 and 2002–2003 hMPV circulated at a significant lower proportion than hRSV, while in 2003–2004 the circulation rates of the two viruses were closer. In conclusion, the 4 hMPV subtypes circulated yearly in Northern Italy flanking hRSV as major respiratory pathogens in the infantile patient population.

Presented here are the details of a rubella outbreak that occurred in 2002 in the Lombardy region of northern Italy followed by a discussion of rubella vaccination policy in this country. From 13 maternal cases of rubella infection, congenital rubella infection was diagnosed in three fetuses and three newborns. Of the three infected fetuses, one was aborted and two died in utero, while of the three infected newborns, two were born with severe disease and one was subclinically infected. Follow-up revealed that one of the two symptomatic newborns had died at 4 months of age with disseminated rubella infection, while the other suffered from bilateral blindness and deafness and was severely retarded at 15 months of age. The remaining infant remained asymptomatic at 14 months. Congenital rubella remains a serious health problem in Italy and a successful vaccination strategy is required.

Clinical evaluation of a novel fully automated chemiluminescence immunoassay for determination of immunoglobulin G avidity to human cytomegalovirus (HCMV) showed 92.8% sensitivity and 84.7% specificity in detecting a recent (≤90 days) primary HCMV infection. The assay appears useful for accurately diagnosing recent primary HCMV infections.