Feasibility of predicting the outcome of fetal infection with cytomegalovirus at the time of prenatal diagnosis.

BACKGROUND: Congenital cytomegalovirus infection occurs in 0.7% of live births with 15-20% of infected children developing long-term disability including hearing loss and cognitive deficit. Fetal cytomegalovirus infection is established by viral DNA amplification by polymerase chain reaction in amniotic fluid obtained by amniocentesis following maternal seroconversion or after the diagnosis of ultrasound features suggestive of fetal infection. Severe brain ultrasound anomalies are associated with a poor prognosis. The prognosis of an infected fetus showing either no ultrasound features or nonsevere ultrasound anomalies is difficult to establish up until late in the second or third trimester of pregnancy. OBJECTIVE: We sought to evaluate the prognostic value of fetal ultrasound, amniotic fluid, and fetal blood analysis at the time of prenatal diagnosis of fetal infection. STUDY DESIGN: We reviewed all cases of fetal cytomegalovirus infection with a sample of amniotic fluid positive for viral DNA and/or fetal blood analyzed in our laboratory from 2008 through 2013. Prenatal ultrasound features along with cytomegalovirus DNA loads in amniotic fluid and in fetal blood and fetal platelet counts were reviewed in relation to gestational age at maternal infection, neonatal examination, and postnatal follow-up or postmortem examination. RESULTS: In all, 82 fetuses were infected following maternal infection mainly in the first trimester. At the time of prenatal diagnosis at a median of 23 weeks, 19, 22, and 41 fetuses showed severe brain ultrasound abnormalities, nonsevere ultrasound features, and normal ultrasound examination, respectively. Nonsevere ultrasound features, higher DNA load in amniotic fluid, fetal platelet count ≤114,000/mm(3), and DNA load ≥4.93 log10 IU/mL in fetal blood were associated with a symptomatic status at birth in univariate analysis (P < .001, P = .001, and P = .018, respectively). Bivariate analysis combining ultrasound results and either adjusted viral load in amniotic fluid or fetal blood profile showed that these were independent prognostic factors of a symptomatic status at birth. Both fetal blood parameters were better predictors than amniotic fluid viral load. At the time of prenatal diagnosis, the ultrasound negative predictive value for symptoms at birth or at termination of pregnancy was 93%. The combined negative predictive values of ultrasound and viral load in amniotic fluid and that of ultrasound and fetal blood parameters were 95% and 100%, respectively. In fetuses presenting with nonsevere ultrasound features, the positive predictive values of ultrasound alone and in combination with amniotic fluid viral load or with fetal blood parameters were 60%, 78%, and 79%, respectively. CONCLUSION: Risk assessment of infected fetuses for being symptomatic at birth is possible as early as the time of diagnosis by using a combination of targeted ultrasound examination along with viral load in amniotic fluid and in fetal blood together with platelet count. The advantage of using amniotic fluid is that it is available at prenatal diagnosis. One may wonder if increasing the negative predictive value of the overall assessment of an infected fetus from 95-100% is worth the additional risk of cordocentesis for fetal blood sampling. This can only be an individual decision made by well-informed women and it seems therefore appropriate to use the figures presented here and their confidence intervals for counseling.

In utero treatment of congenital cytomegalovirus infection with valacyclovir in a multicenter, open-label, phase II study.

BACKGROUND: Congenital infection with human cytomegalovirus is a major cause of morbidity and mortality. A randomized controlled trial showed that high-dosage valacyclovir prevents cytomegalovirus disease in transplant recipients. Fetuses showing ultrasound features of infection are at high risk of being symptomatic at or before birth. In a pilot study, oral administration of high-dosage valacyclovir to mothers significantly decreased viral load and produced therapeutic concentrations in the blood of infected fetuses. A randomized controlled trial comparing prenatal treatment with valacyclovir against placebo in infected fetuses failed to recruit because women declined randomization. Randomized controlled trials in fetal medicine have often proven unacceptable by women who decline termination of pregnancy and are not prepared to resign themselves to the odds of the natural history of the disease. OBJECTIVE: We evaluated the efficacy of oral valacyclovir, 8 g daily, for pregnant women carrying a symptomatic cytomegalovirus-infected fetus, targeting a high-risk group for developing both neurosensory and neurological impairment. STUDY DESIGN: We designed a multicenter, open-label, phase II study with 1 arm, using one of Simon's optimal 2-stage designs. Symptomatic fetuses were defined by the presence of measurable extracerebral or mild cerebral ultrasound symptoms. They were treated in utero from prenatal diagnosis at a median of 25.9 weeks' gestation until delivery or termination of pregnancy. Fetuses with severe brain anomalies on ultrasound were not included as were cases completely asymptomatic at presentation, because treatment was unlikely to modify either outcome. The primary endpoint was the proportion of asymptomatic neonates born to treated mothers. RESULTS: At the interim analysis, 8 of 11 women delivered an asymptomatic neonate (required: ≥7). In step 2, 32 additional cases were included for a total of 43; the final number of asymptomatic neonates was 34, more than the 31 required to indicate efficacy according to the Simon 2-stage design. They remained asymptomatic at 12 months. High-dosage valacyclovir given for a median of 89 days to pregnant women carrying a moderately infected fetus was efficient at giving birth to asymptomatic neonates. Fetal blood viral loads decreased and platelet counts increased, both significantly (P = .01 and P < .001, respectively), between treatment initiation and birth after treatment completion, regardless of duration of fetal infection. Compared with a historical cohort obtained by a metaanalysis of the literature, the use of valacyclovir (8 g daily) significantly increased the proportion of asymptomatic neonates from 43% without treatment to 82% with treatment. Although the pill burden was high (16 pills a day) adherence to treatment was >90%. Finally, valacyclovir at this high dosage was extremely well tolerated. CONCLUSION: Our results indicate that high-dosage valacyclovir given in pregnancy is effective for improving the outcome of moderately symptomatic infected fetuses. Although this study is not a randomized controlled trial, this is the first study reporting the efficacy of an antiviral drug to treat cytomegalovirus-infected fetuses. Moreover, this first study will allow new trials to be conducted, using valacyclovir as a baseline safe and effective treatment in pregnancy, to be compared to the new emerging and more potent anticytomegalovirus drugs that have not currently been tested in pregnancy.

Prediction of fetal infection in cases with cytomegalovirus immunoglobulin M in the first trimester of pregnancy: a retrospective cohort.

BACKGROUND: Interpretation of positive cytomegalovirus (CMV) immunoglobulin M (IgM) in the first trimester of pregnancy is ill-defined. We aimed to quantify the risk of fetal transmission in women with positive CMV IgM in the first trimester. METHODS: A retrospective cohort of women (2009-2011) was tested for CMV immunoglobulin G (IgG) and IgM before 14 weeks of gestation. IgG avidity was tested with 2 assays (LIAISON and VIDAS). CMV polymerase chain reaction (PCR) was done in maternal serum, amniotic fluid, or neonatal urine at birth. RESULTS: A total of 4931 consecutive women were screened; 201 presented with positive or equivocal IgM and with high, intermediate, or low IgG avidity in 58.7%, 18.9%, and 22.3%, respectively. In 72 women with low or intermediate avidity, fetal transmission was 23.6%. In multivariate analysis, positive CMV PCR in maternal serum, decreasing avidity index with both LIAISON and VIDAS, and low IgG titers were all associated with fetal transmission (odds ratio [OR], 12.38 [95% confidence interval {CI}, 1.77-86.33], P = .011; OR, 0.16 [95% CI, .03-.95], P = .044; OR, 0.54 [95% CI, .11-.88], P = .028; and OR, 0.27 [95% CI, .29-.84], P = .010, respectively). CONCLUSION: This study demonstrates a significant association between the risk of vertical transmission and the avidity index combined with CMV PCR in maternal serum or IgG titers. This allows calculation of incremental risk of fetal transmission upon which informed choice can be based and could lead to a better pickup rate of fetal infection while decreasing unnecessary invasive procedures.

Management of pregnancies with confirmed cytomegalovirus fetal infection.

Systematic screening for cytomegalovirus (CMV) maternal infection is not recommended in most countries. Nevertheless, primary CMV infection will occur in around 1% of women. The vertical transmission rate is estimated to be around 30-50%. Newborns with congenital CMV infection remain asymptomatic in the majority of cases and around 10% will present with a wide range of abnormalities. Fetal infection can be diagnosed by amniocentesis with amplification of the viral genome in the amniotic fluid by polymerase chain reaction. This prenatal diagnosis is mainly performed when ultrasound abnormalities are observed. The purpose of this mini-review is to describe the management options when a fetus is known to be infected.

[Prenatal risk calculation: comparison between Fast Screen pre I plus software and ViewPoint software. Evaluation of the risk calculation algorithms]. / Calcul du risque de trisomie 21 fœtale : comparaison du logiciel Fast Screen pre I plus avec le logiciel ViewPoint. Évaluation des algorithmes de calcul.

The Fetal medicine foundation (FMF) has developed a new algorithm called Prenatal Risk Calculation (PRC) to evaluate Down syndrome screening based on free hCGß, PAPP-A and nuchal translucency. The peculiarity of this algorithm is to use the degree of extremeness (DoE) instead of the multiple of the median (MoM). The biologists measuring maternal seric markers on Kryptor™ machines (Thermo Fisher Scientific) use Fast Screen pre I plus software for the prenatal risk calculation. This software integrates the PRC algorithm. Our study evaluates the data of 2.092 patient files of which 19 show a fœtal abnormality. These files have been first evaluated with the ViewPoint software based on MoM. The link between DoE and MoM has been analyzed and the different calculated risks compared. The study shows that Fast Screen pre I plus software gives the same risk results as ViewPoint software, but yields significantly fewer false positive results.

Prognostic markers of symptomatic congenital cytomegalovirus infection.

The objective of this research was to identify maternal and fetal characteristics as prognostic markers of congenital cytomegalovirus (CMV) infection. This is a descriptive study of 13 cases of congenital CMV infection referred to Institute de Puericulture et Perinatologie de Paris (IPP) from January 2005 to October 2006. Amniotic fluid puncture was performed to research CMV polimerase chain reaction (PCR). Cordocentesis and cord blood samples at delivery were also analyzed to determinate fetal platelets count, GGT, ASAT, ALAT, CMV-DNA and IgM antibody. Variables of symptomatic and asymptomatic infants were then compared. Data were analyzed by SPSS--15.0. Mean gestational age of amniocentesis was 24.6 weeks and there was no difference of mean viral load in amniotic fluid considering infant features. Mean gestational age of cordocentesis was 26.1 weeks. There were no statistical differences of fetal viral load, IgM, platelets, GGT, ASAT and ALAT analyzed at cordocentesis samples, but at delivery, mean values of IgM and ASAT of fetal blood were increased in symptomatic ones (p= 0.03 for both parameters). When considering groups with normal and abnormal parameters, ASAT of cordon samples was also increased in symptomatic infants (p= 0.02). Sensibility, specificity, positive and negative predictive value of fetal ultrasound anomalies to detect symptomatic infants were, respectively, 80%, 62.5%, 57.1% and 83.3%. Thus, identification of markers of CMV symptomatic infants should be aimed. Prenatal diagnosis, identification and follow up of congenital CMV infected infants are important to consider treatment for symptomatic infants, trying to avoid or reducing some possible sequels.

Comparison of CD8+ T Cell responses to cytomegalovirus between human fetuses and their transmitter mothers.

BACKGROUND: The mechanisms responsible for the increased susceptibility of fetuses to cytomegalovirus (CMV) were studied by comparing CD8(+) T cell responses to the virus in susceptible fetuses to those in their comparatively more resistant mothers. METHODS: Included in the study were 16 transmitter mothers who underwent seroconversion during the first trimester of pregnancy as well as their fetuses, who were positive for CMV in amniotic fluid by polymerase chain reaction at 17-19 weeks of gestation. Fetal and maternal blood samples were collected between the 22nd and 39th week of gestation. Cytotoxic T lymphocytes (CTLs) that had activated (HLA-DR(+)), effector/memory (CD28(-)), and memory (CD18(high)) phenotypes; that stained with the HLA-A2/pp65 or the HLA-B7/pp65 multimer; and that secreted interferon (IFN)- gamma were enumerated by flow cytometry. Viral loads were determined simultaneously. RESULTS: The results showed (1) similar levels of activated, effector/memory, and memory CTLs in fetuses and mothers but a smaller pp65-specific CTL pool in fetuses (median, 0.015% vs. 0.99%; P=.003); (2) similar percentages of CTLs secreting IFN- gamma after stimulation with ionomycin/phorbol myristate acetate in fetuses and mothers but lower percentages of CTLs secreting IFN- gamma after stimulation with a CD3 monoclonal antibody in fetuses (median, 1% vs. 14%; P=.01); and (3) higher viral loads (mean, 17,290 vs. <250 genome equivalents/mL) in fetuses. CONCLUSION: Impaired viral clearance might be related to a defective expansion of the pp65-specific CTL pool and/or to the immaturity of IFN- gamma -secreting cells in fetuses.

Prenatal diagnosis of zygosity by fetal DNA analysis, a contribution to the management of multiple pregnancies. A series of 31 cases.

OBJECTIVE: To evaluate the contribution of prenatal diagnosis of zygosity by fetal DNA analysis to the management of multiple pregnancies. METHODS: Between March 1999 and March 2000, 31 same-sex multiple pregnancies, were referred to our Institute during their second trimester to have amniocentesis done. Fetal DNA variants were studied in addition to karyotype. The main indications for amniocentesis were fetal growth discordance, fetal malformations or selective pregnancy termination. Zygosity results were compared to the first-trimester ultrasound diagnosis. RESULTS: 21 out of the 31 multiple pregnancies were dizygous, and 10 out of the 31 pregnancies were monozygous. First-trimester ultrasound provided information on chorionicity only in 24 out of the 31 cases (77%). Of these 24 reports, 21 proved to be correct (87.5%). Moreover, in 2 cases among 14 pregnancies with growth discordance, DNA analysis ruled out a twin-to-twin transfusion syndrome, while ultrasound could not yield a conclusion. In 2 twin pregnancies with fetal malformations affecting one of the fetuses, DNA analysis made it possible to assess the risk for the other twin. In the 5 cases leading to selective termination, prenatal diagnosis of zygosity had a decisive value for the surviving fetus. CONCLUSION: Fetal DNA analysis is a simple and highly useful test for prenatal diagnosis of zygosity in the management of complicated multiple pregnancies.

Cellular immune response of fetuses to cytomegalovirus.

Primary infection with cytomegalovirus (CMV) in immunocompetent hosts is accompanied with activation and differentiation of naive CD8(+) T cells to effector/memory cells secreting interferon-gamma (IFN-gamma). Alteration of these responses during the perinatal period is suggested by a higher rate of CMV diseases in congenital infection. For addressing this issue, immunologic investigations were performed in 15 fetuses (22-36 wk of gestation) with documented congenital CMV infection. Results show that cellular immune responses can be detected as soon as the 22nd week of gestation (the youngest fetus analyzed). Compared with age-matched control subjects, infected fetuses evidence a dramatic increase in the percentages of activated and terminally differentiated CD8 T cells. Indeed, median percentages (interquartile range) of HLA-DR(+) and of CD28(-)CD8(+) T cells were 24% (19-34) and 38% (24-52), respectively in infected fetuses versus 3% (0-4) for each subset in control subjects. In addition, the percentages of T cells secreting IFN-gamma after in vitro stimulation with phorbol myristate acetate and ionomycin was significantly higher in infected fetuses [10% (5-25)] than in healthy fetuses [0.8% (0.6-1.2)] with IFN-gamma being mostly secreted by CD8(+) T cells and to a lesser extend by CD4(+) T cells. These cellular immune responses have clear similarities with responses previously reported in adults. Cellular immunity to CMV, however, might not be fully functional in fetuses. Indeed, the number of T cells capable of secreting IFN-gamma is strikingly lower after in vitro stimulation with the CMV-specific antigen than after in vitro stimulation with phorbol myristate acetate/ionomycin that bypasses signaling through the T-cell receptor.

Prognostic markers of symptomatic congenital cytomegalovirus infection

The objective of this research was to identify maternal and fetal characteristics as prognostic markers of congenital cytomegalovirus (CMV) infection. This is a descriptive study of 13 cases of congenital CMV infection referred to Institute de Puericulture et Perinatologie de Paris (IPP) from January 2005 to October 2006. Amniotic fluid puncture was performed to research CMV polimerase chain reaction (PCR). Cordocentesis and cord blood samples at delivery were also analyzed to determinate fetal platelets count, GGT, ASAT, ALAT, CMV-DNA and IgM antibody. Variables of symptomatic and asymptomatic infants were then compared. Data were analyzed by SPSS - 15.0. Mean gestational age of amniocentesis was 24.6 weeks and there was no difference of mean viral load in amniotic fluid considering infant features. Mean gestational age of cordocentesis was 26.1 weeks. There were no statistical differences of fetal viral load, IgM, platelets, GGT, ASAT and ALAT analyzed at cordocentesis samples, but at delivery, mean values of IgM and ASAT of fetal blood were increased in symptomatic ones (p= 0.03 for both parameters). When considering groups with normal and abnormal parameters, ASAT of cordon samples was also increased in symptomatic infants (p= 0.02). Sensibility, specificity, positive and negative predictive value of fetal ultrasound anomalies to detect symptomatic infants were, respectively, 80 percent, 62.5 percent, 57.1 percent and 83.3 percent. Thus, identification of markers of CMV symptomatic infants should be aimed. Prenatal diagnosis, identification and follow up of congenital CMV infected infants are important to consider treatment for symptomatic infants, trying to avoid or reducing some possible sequels.