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

Open AccessPublished:April 12, 2016DOI:https://doi.org/10.1016/j.ajog.2016.04.003

      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.

      Key words

      Click Supplemental Materials under article title in Contents at ajog.org

      Introduction

      Congenital cytomegalovirus (CMV) infection affects 0.7% of live births and is the leading cause of congenital neurological disease of infectious origin.
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      This leaves a window of opportunity for treatment of symptomatic fetuses without brain involvement.
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      Maternal administration of valacyclovir in symptomatic intrauterine cytomegalovirus infection.
      Vaccination is not available
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      and no prenatal treatment of congenital CMV has yet been validated. The use of CMV-specific hyperimmune globulin to prevent transmission from mother to fetus has produced conflicting results.
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      but these drugs, highly genotoxic in vitro, are not approved in pregnancy. Although valacyclovir is less effective than ganciclovir against CMV in vitro,
      • Tyms A.S.
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      The in vitro activity of acyclovir and related compounds against cytomegalovirus infections.
      high-dosage valacyclovir has proven clinically efficient to prevent CMV disease in transplant recipients.
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      Valacyclovir for the prevention of cytomegalovirus disease after renal transplantation. International Valacyclovir Cytomegalovirus Prophylaxis Transplantation Study Group.
      The mechanism of acyclovir’s anti-CMV activity in clinical settings remains unexplained. Valacyclovir also has the best safety profile of the anti-CMV drugs. Neither cell transformation nor increased risk of neoplasia has been reported in vitro, and no increased risk of birth defects has been detected in the offspring of thousands of women exposed during pregnancy.
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      Finally, valacyclovir is well tolerated with rare side effects. In a pilot study, we found that oral administration of high-dosage valacyclovir to mothers significantly decreased viral load and produced therapeutic concentrations in fetal blood with a mean fetal blood plasma concentration of >17 μmol/L. These results suggested the value of a clinical trial to investigate this therapeutic option further.
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      • Yamamoto M.
      • Costa J.-M.
      • et al.
      Maternal administration of valacyclovir in symptomatic intrauterine cytomegalovirus infection.
      We failed to complete a randomized controlled trial comparing prenatal treatment with valacyclovir against placebo in moderately symptomatic infected fetuses due to failure to recruit (Cymeval NCT01037712). In this open-label phase II trial with 1 arm we show that high-dosage valacyclovir given in pregnancy is safe and appears effective for improving the outcome of moderately symptomatic fetuses.

      Materials and Methods

      Patients

      Eligible women were pregnant with an infected fetus identified by a positive CMV polymerase chain reaction assay in amniotic fluid, sampled by amniocentesis >21 weeks,
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      A thoughtful algorithm for the accurate diagnosis of primary CMV infection in pregnancy.
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      • Stamilio D.M.
      • Macones G.A.
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      • Guerra B.
      • Simonazzi G.
      • Banfi A.
      • et al.
      Impact of diagnostic and confirmatory tests and prenatal counseling on the rate of pregnancy termination among women with positive cytomegalovirus immunoglobulin M antibody titers.
      • Leruez-Ville M.
      • Sellier Y.
      • Salomon L.J.
      • Stirnemann J.J.
      • Jacquemard F.
      • Ville Y.
      Prediction of fetal infection in cases with cytomegalovirus immunoglobulin M in the first trimester of pregnancy: a retrospective cohort.
      together with the presence of ≥1 extracerebral ultrasound features compatible with CMV infection and/or 1 isolated cerebral abnormality and/or 1 of the following laboratory findings in fetal blood: fetal platelet count <100,000/mm3 or CMV DNA viral load >3000 copies/mL (Table 1). The presence of severe ultrasound brain abnormalities (Table 2) and the absence of any ultrasound feature of infection or laboratory abnormality in fetal blood were exclusion criteria. The detailed eligibility criteria are listed in the supplementary Appendix. Fetal blood sampling by cordocentesis under ultrasound guidance was offered to all participants to evaluate fetal platelets and viral DNA load to help refine the fetal prognosis.
      • Benoist G.
      • Salomon L.J.
      • Jacquemard F.
      • Daffos F.
      • Ville Y.
      The prognostic value of ultrasound abnormalities and biological parameters in blood of fetuses infected with cytomegalovirus.
      Cordocentesis was not, however, required for study eligibility, as this invasive procedure was not the standard of care in all participating centers.
      Table 1Main inclusion criteria
      At least 1 extracerebral abnormality compatible with fetal CMV infection
      Fetal growth restriction
      • Hadlock F.P.
      • Harrist R.B.
      • Sharman R.S.
      • Deter R.L.
      • Park S.K.
      Estimation of fetal weight with the use of head, body, and femur measurements–a prospective study.
      Abnormal amniotic fluid volume
      Ascites and/or pleural effusion
      Skin edema
      Hydrops
      Placentomegaly >40 mm
      • Grannum P.A.
      Ultrasound examination of the placenta.
      Hyperechogenic bowel
      Since diagnosis of hyperechogenic bowel can be subjective and associated with high interoberver and intraobserver variability, diagnosis of hyperechogenic bowel was only considered for grade-2 or grade-3 hyperechogenic bowel46; this semiquantitative analysis was chosen to limit subjectivity sometimes associated with ultrasound findings.
      Hepatomegaly >40 mm
      • Vintzileos A.M.
      • Neckles S.
      • Campbell W.A.
      • Andreoli J.W.
      • Kaplan B.M.
      • Nochimson D.J.
      Fetal liver ultrasound measurements during normal pregnancy.
      Splenomegaly >30 mm
      • Hata T.
      • Deter R.L.
      A review of fetal organ measurements obtained with ultrasound: normal growth.
      Liver calcifications
      And/or 1 isolated cerebral abnormality
      Moderate isolated ventriculomegaly (<15 mm)
      Isolated cerebral calcification
      Isolated intraventricular adhesion
      Vasculopathy of lenticulostriate vessels
      And/or laboratory findings of generalized CMV infection in fetal blood
      Fetal viremia >3000 copies/mL
      Fetal platelet count <100,000/mm3
      All ultrasound examinations leading to inclusion in study were reviewed by principal investigator at each center. Measurements of spleen, liver, and placenta were standardized according to literature.
      • Grannum P.A.
      Ultrasound examination of the placenta.
      • Vintzileos A.M.
      • Neckles S.
      • Campbell W.A.
      • Andreoli J.W.
      • Kaplan B.M.
      • Nochimson D.J.
      Fetal liver ultrasound measurements during normal pregnancy.
      • Hata T.
      • Deter R.L.
      A review of fetal organ measurements obtained with ultrasound: normal growth.
      CMV, cytomegalovirus.
      Leruez-Ville et al. In utero treatment of congenital cytomegalovirus infection. Am J Obstet Gynecol 2016.
      a Since diagnosis of hyperechogenic bowel can be subjective and associated with high interoberver and intraobserver variability, diagnosis of hyperechogenic bowel was only considered for grade-2 or grade-3 hyperechogenic bowel
      • Slotnick R.N.
      • Abuhamad A.Z.
      Prognostic implications of fetal echogenic bowel.
      ; this semiquantitative analysis was chosen to limit subjectivity sometimes associated with ultrasound findings.
      Table 2Main exclusion criteria
      Presence of at least 1 severe cerebral ultrasound abnormality among following:
      Ventriculomegaly ≥15 mm
      Periventricular hyperechogenicity
      Hydrocephaly
      Microcephaly <3 SD
      Mega-cisterna magna >10 mm
      Vermian hypoplasia
      Porencephaly
      Lissencephaly
      Periventricular cysts
      Abnormal corpus callosum
      Or absence of any ultrasound feature of infection or laboratory abnormality in fetal blood
      Leruez-Ville et al. In utero treatment of congenital cytomegalovirus infection. Am J Obstet Gynecol 2016.

      Procedures

      Study design

      The trial was a multicenter, open-label, phase II study with 1 arm, based on 1 of Simon’s
      • Simon R.
      Optimal two-stage designs for phase II clinical trials.
      optimal 2-stage designs. All participants received oral valacyclovir (2 g, 4 times a day, therefore 8 g daily). The medication was continued until delivery or 24 treatment weeks, whichever was sooner. The study drug was purchased from GlaxoSmithKline (Marly Le Roi, France), which had no other role in the study. Visits for clinical and ultrasound examinations, questions about clinical side effects (headache, nausea, neurologic effects listed in the product monograph), and adherence assessment by pill count were scheduled every 2 weeks until delivery. Maternal plasma levels of aspartate aminotransferase, alanine aminotransferase, and creatinine were assessed once a month during treatment. All newborns were examined between days 4 and 7 of life by a trained pediatrician according to a standardized clinical evaluation. Auditory brainstem responses, cranial ultrasound, fundoscopy, and laboratory tests (see criteria for primary endpoint in supplementary data) were also performed.
      The ethics committee of Poissy-Saint Germain Hospital approved the study (2011-001610-34). Participants gave written informed consent before inclusion. Study oversight was provided by an independent data and safety monitoring board (Clinical Research Unit, Cochin-Necker, Paris).

      Study endpoints

      The primary study endpoint was the proportion of asymptomatic neonates born to women treated with valacyclovir. An asymptomatic neonate was a neonate without growth restriction (that is, with birthweight ≥10th percentile), normal clinical examination, normal laboratory findings, no severe features of infection on cerebral imaging, normal funduscopic examination, and normal audiology findings (see the supplementary Appendix for details).
      The secondary endpoints included adverse events related to the study medication and adherence to treatment. CMV DNA levels and platelet counts were compared in pretreatment fetal blood when available and cord blood at birth and in symptomatic and asymptomatic neonates. The effect of the duration of maternal treatment was also assessed.

      Historical comparator group

      Our systematic review on PubMed using 3 key words (“cytomegalovirus,” “congenital,” “ultrasound”) yielded 216 articles, but only 3 were written in English and included detailed tables that described both prenatal ultrasound findings and neonatal outcome and also included postmortem findings in termination of pregnancy cases.
      • Jacquemard F.
      • Yamamoto M.
      • Costa J.-M.
      • et al.
      Maternal administration of valacyclovir in symptomatic intrauterine cytomegalovirus infection.
      • Picone O.
      • Simon I.
      • Benachi A.
      • Brunelle F.
      • Sonigo P.
      Comparison between ultrasound and magnetic resonance imaging in assessment of fetal cytomegalovirus infection.
      • Guerra B.
      • Simonazzi G.
      • Puccetti C.
      • et al.
      Ultrasound prediction of symptomatic congenital cytomegalovirus infection.
      These 3 suitable studies included 724 pregnancies with a maternal CMV primary infection, 217 with an infected fetus. The review of the ultrasound symptoms of these 217 fetuses showed no ultrasound abnormalities in 142 of them and severe cerebral ultrasound abnormalities in 28. Ultrasound abnormalities matching our inclusion criteria were therefore seen in 47 cases, which formed the historical comparator group. Among them 20 (42.55%) neonates were born asymptomatic. Twenty fetuses were terminated and all underwent postmortem examination showing both macroscopic and microscopic evidence of brain damage in all cases. We therefore assumed that these fetuses would have been born symptomatic and classified then into the symptomatic group. See Table S1 for details in the supplementary Appendix.

      Laboratory assays

      CMV serology and viral load quantification were centralized and analyzed at the Necker Hospital virology laboratory. Maternal CMV primary infection was diagnosed by seroconversion or the concomitant presence of CMV-specific IgM antibodies and low IgG avidity.
      • Leruez-Ville M.
      • Sellier Y.
      • Salomon L.J.
      • Stirnemann J.J.
      • Jacquemard F.
      • Ville Y.
      Prediction of fetal infection in cases with cytomegalovirus immunoglobulin M in the first trimester of pregnancy: a retrospective cohort.
      After extracting DNA from fetal whole blood, cord whole blood, and neonatal urine with the MagnaPure LC platform (Roche Diagnostic, Meylan, France), we performed quantitative polymerase chain reaction for CMV with the CMV-R gene kit (Argene, BioMerieux, Marcy-L’Etoile, France). The limit of detection was 446 copies (or 178 IU)/mL.

      Statistical analysis

      To estimate the sample size according to Simon
      • Simon R.
      Optimal two-stage designs for phase II clinical trials.
      optimal 2-stage design, we assumed that a proportion of asymptomatic neonates of <60% was not clinically relevant in relation to valacyclovir efficacy, while a proportion of >80% was deemed acceptable. With a type I error fixed at 0.05 and a power of 80%, we needed 11 infected fetuses for the first stage: if at least 8 cases in stage 1 had a good outcome (were asymptomatic), then 32 additional infected fetuses would be included in stage 2 for a total of 43. If at least 31 of the 43 cases were asymptomatic at birth, valacyclovir would be judged to have a positive effect.
      Analyses of efficacy data were performed for all included fetuses (intention to treat). The proportion of asymptomatic neonates and its 95% confidence interval were calculated with an unbiased estimator.
      • Jung S.-H.
      • Kim K.M.
      On the estimation of the binomial probability in multistage clinical trials.
      In the historical comparator group, we used a random effects model to calculate the overall proportion of asymptomatic neonates and a 95% confidence interval for all 47 cases from the 3 studies.
      We used nonparametric tests for all secondary analyses.
      Platelet counts at birth and in fetal blood were compared to the gestational age-specific reference range published by Meher-Homji et al.
      • Meher-Homji N.J.
      • Montemagno R.
      • Thilaganathan B.
      • Nicolaides K.H.
      Platelet size and glycoprotein Ib and IIIa expression in normal fetal and maternal blood.
      The average individual deviations of values at birth and in fetal blood from the published mean in noninfected fetuses were compared to 0. We also compared individual differences in platelet counts between fetal blood and birth to the slope of the reference mean in noninfected fetuses. Two-sided t tests were used for these analyses.
      R software (Version 2.15; R Foundation for Statistical Computing, Vienna, Austria) was used for all statistical analyses and its “meta” package for the metaanalysis.

      Results

      Enrollment and baseline characteristics

      From January 2012 through December 2014, 41 pregnant women and their 43 fetuses (39 singletons and 4 twins from 2 pairs of dichorionic twins) were enrolled at 6 centers, as described in Figure 1. Table 3 summarizes their baseline characteristics. The median gestational age at inclusion was 26 (range 22-35) weeks. All but 1 woman had a primary infection at a median gestational age of 10 weeks. In all, 54% of all fetuses presented with at least 2 eligible ultrasound symptoms, 39% with only 1, and 3 showed only viremia >3000 copies/mL (4500; 90,000; and 101,000 copies/mL, respectively).
      Figure thumbnail gr1
      Figure 1Flowchart of enrollment and outcomes
      Enrollment and outcomes.
      Leruez-Ville et al. In utero treatment of congenital cytomegalovirus infection. Am J Obstet Gynecol 2016.
      Table 3Characteristics of population at baseline
      CharacteristicsMedian [interquartile range] or n (%)
      Women (N = 41)
       Age at inclusion, y31.2 [28.6–33.9]
       Body mass index before pregnancy21.6 [19.8–23]
       Parity
      011 (26.8)
      ≥130 (73.2)
       No. of pregnancies2 [2–3]
       Primary infection
      Forty women had primary infection and 1 woman was diagnosed with secondary infection.
      40 (97.6)
       Gestational age at primary infection, wk10 [7.8–16.2]
       Gestational age at inclusion, wk25.9 [24.1–31.7]
       Interval between primary infection and inclusion, wk16 [12.3–18.6]
      Fetuses (N = 43)
      Only 1 symptom at ultrasound17 (39.5)
      >1 Symptom at ultrasound23 (53.5)
      Fetal blood CMV DNA load >3000 copies/mL3 (7)
       Fetal growth restriction3 (7)
       Abnormal amount of amniotic fluid3 (7)
       Ascites and/or pleural effusion1 (2.3)
       Placentomegaly13 (30.2)
       Hyperechogenic bowel25 (58.1)
       Hepatomegaly6 (14)
       Splenomegaly9 (20.9)
       Liver calcification1 (2.3)
       Moderate cerebral abnormality5 (11.6)
      CMV, cytomegalovirus.
      Leruez-Ville et al. In utero treatment of congenital cytomegalovirus infection. Am J Obstet Gynecol 2016.
      a Forty women had primary infection and 1 woman was diagnosed with secondary infection.

      Primary endpoint

      At the planned interim analysis, 8 of the first 11 women included had given birth to an asymptomatic neonate. This number exceeded the number of asymptomatic neonates (≥7) required to continue to step 2, which included 32 additional fetuses, for a total of 43. The final analysis showed 34 asymptomatic neonates, more than the 31 required to suggest efficacy according to the Simon
      • Simon R.
      Optimal two-stage designs for phase II clinical trials.
      optimal 2-stage design in the population studied (Table 4).
      Table 4Primary endpoint and efficacy
      First stepSecond step
      Outcome no.
      Asymptomatic neonates834
      Symptomatic neonates or termination of pregnancy39
      Total1143
      Leruez-Ville et al. In utero treatment of congenital cytomegalovirus infection. Am J Obstet Gynecol 2016.
      Comparison of results of our trial with those of the historical cohort indicates that valacyclovir significantly increased the proportion of asymptomatic neonates, from 43% (range, 29–57) without treatment to 82% (range, 67–88) with treatment (and without any overlapping of confidence intervals) (Figure 2).
      Figure thumbnail gr2
      Figure 2Valacyclovir effect compared to historical control group
      Proportion of asymptomatic neonates and 95% confidence interval (CI) were calculated with unbiased estimator.
      • Jung S.-H.
      • Kim K.M.
      On the estimation of the binomial probability in multistage clinical trials.
      In historical control group, we used random effects model to calculate overall proportion of asymptomatic neonates and 95% CI for 43 cases reported in 3 suitable published studies. *Unbiased estimation of binomial probability in multistage design.
      Leruez-Ville et al. In utero treatment of congenital cytomegalovirus infection. Am J Obstet Gynecol 2016.
      The 9 symptomatic cases include 2 terminations of pregnancy performed because a severe brain abnormality subsequently appeared on ultrasound (Table 5). There was only 1 significant difference in maternal and fetal characteristics at baseline (Table 6a, 6b) between the cases with good (asymptomatic neonates) and poor (symptomatic neonates or termination of pregnancy) outcomes: fetal platelet count at inclusion, which was significantly lower in the poor outcome group (P < .007) (Table 6b). The duration of maternal treatment did not differ significantly between the 2 groups (P = .236) (Table 6a).
      Table 5Characteristics of 9 cases with poor outcome
      CaseGestational age at primary infection, wkGestational age at inclusion, wkInclusion criteriaDays of treatmentSymptoms at birth or termination of pregnancy
      11023Oligohydramnios, hyperechogenic bowel92Bilateral hearing loss
      223Hyperechogenic bowel, intraventricular adhesion18Termination of pregnancy
      Two terminated fetuses underwent postmortem examination and in both cases severe brain lesions were identified.
      31335Intraventricular adhesion5Termination of pregnancy
      Two terminated fetuses underwent postmortem examination and in both cases severe brain lesions were identified.
      4932Fetal growth restriction placentomegaly, intraventricular adhesion6Bilateral hearing loss
      5925Hyperechogenic bowel, fetal thrombocytopenia (55,000/mm3)97Thrombocytopenia (59,000/mm3)
      6623Hyperechogenic bowel99Unilateral hearing loss
      7724Hyperechogenic bowel, fetal thrombocytopenia (66,000/mm3)103Unilateral hearing loss
      8822Hyperechogenic bowel15Unilateral hearing loss
      9Secondary maternal infection26Fetal growth restriction68Growth restriction
      Leruez-Ville et al. In utero treatment of congenital cytomegalovirus infection. Am J Obstet Gynecol 2016.
      a Two terminated fetuses underwent postmortem examination and in both cases severe brain lesions were identified.
      Table 6Comparison of maternal and fetal characteristics at baseline between cases with good (asymptomatic neonate) and poor (symptomatic neonate or termination of pregnancy) outcomes
      Table 6a. Women’s characteristics
      TotalGood outcomePoor outcomeP
      Median [interquartile range]Median [interquartile range]Median [interquartile range]
      (N = 41)(N = 33)(N = 8)
      Maternal age at inclusion, y31.2 [28.6–33.9]31.5 [28.8–33.9]30.2 [28.1–33.5].717
      Body mass index before pregnancy21.6 [19.8–23]20.4 [19.4–22.7]22.9 [21.8–23.9].058
      Parity, n (%)
       011 (26.8)8 (24.2)3 (37.5).744
       120 (48.8)17 (51.5)3 (37.5)
       29 (22)7 (21.2)2 (25)
       31 (2.4)1 (3)0 (0)
      No. of pregnancies2 [2–3]2 [2–3]2 [1.8–2.5].73
      Primary infection, n (%)40 (97.6)33 (100)7 (87.5).195
      Gestational age at primary infection, wk10 [7.8–16.2]11 [8–17]9 [7.5–9.5].199
      Gestational age at inclusion, wk25.9 [24.1–31.7]27 [24.6–31.7]24.6 [23.8–27.6].411
      Interval between primary infection and inclusion, wk16 [12.3–18.6]15.9 [12.1–18.6]17.1 [15.9–20].182
      Treatment interruption, n (%)2/39 (5.1)1/33 (3)1/6 (16.7).287
      Duration of treatment, d89 [41–102]89 [43–102]80 [15–97.5].236
      Table 6b. Fetal characteristics
      TotalGood outcomePoor outcomeP
      Median [interquartile range]Median [interquartile range]Median [interquartile range]
      (N = 43)(N = 34)(N = 9)
      Inclusion criteria, n (%)
       Only 1 symptom at ultrasound17 (39.5)15 (44.1)2 (22.2).332
       Fetal blood DNA load >3000 copies/mL3 (7)3 (8.8)0 (0)
       >1 Symptom at ultrasound23 (53.5)16 (47.1)7 (77.8)
      Fetal growth restriction, n (%)3 (7)1 (2.9)2 (22.2).106
      Abnormal amount of amniotic fluid, n (%)3 (7)2 (5.9)1 (11.1).515
      Ascites and/or pleural effusion, n (%)1 (2.3)1 (2.9)0 (0)1
      Placentomegaly, n (%)13 (30.2)11 (32.4)2 (22.2).699
      Hyperechogenic bowel, n (%)25 (58.1)19 (55.9)6 (66.7).712
      Hepatomegaly, n (%)6 (14)5 (14.7)1 (11.1)1
      Splenomegaly, n (%)9 (20.9)7 (20.6)2 (22.2)1
      Liver calcifications, n (%)1 (2.3)0 (0)1 (11.1).209
      Moderate cerebral anomalies,
      Moderate isolated ventriculomegaly (<15 mm) or isolated cerebral calcification or isolated intraventricular adhesion or vasculopathy of lenticulostriate vessels.
      n (%)
      5 (11.6)2 (5.9)3 (33.3).054
      Fetal viremia at inclusion in log10 IU/mL4.4 [4–5]4.3 [3.8–4.8]5.1 [4.4–5.9].1
      Fetal platelet count at inclusion/mm3174,000 [145,000–208,000]177,500 [155,250–208,000]67,000 [61,000–88,000].006
      Leruez-Ville et al. In utero treatment of congenital cytomegalovirus infection. Am J Obstet Gynecol 2016.
      a Moderate isolated ventriculomegaly (<15 mm) or isolated cerebral calcification or isolated intraventricular adhesion or vasculopathy of lenticulostriate vessels.
      Table S2 reports all details on pregnancy outcome (gestational age at delivery, birthweight, clinical examination, imaging, and laboratory data).

      Secondary endpoints

      Adverse events

      Valacyclovir was well tolerated. Only 2 women reported headaches, and treatment was suspended for 10 days in only 1. Although maternal alanine aminotransferase and aspartate aminotransferase levels increased after 3 months of treatment, this increase was not clinically relevant; all values were <40 IU/L, and creatinine levels did not change throughout treatment (Figure S1). Adherence in the subgroup of 27 women evaluated for it was >90% (Table S3).

      Viral loads and platelet counts

      Although the duration of maternal treatment was not correlated with CMV DNA levels in either cord blood (P = .65) or neonatal urine (P = .24), longer duration was associated with a higher platelet count at birth (P = .018) (Figure S2 and Table S4). Neither viral loads nor platelet counts at birth were correlated with the time of maternal infection in pregnancy and therefore with the duration of fetal infection (Table S4). Symptomatic and asymptomatic neonates did not differ significantly for viral DNA load levels in either neonatal cord blood (P = .391) or neonatal urine (P = .081), but the neonatal platelet count was significantly lower in symptomatic neonates (P < .001) (Table S5). Blood viral load decreased and platelet count increased, both significantly, between the fetal blood obtained in utero before maternal treatment began and the cord blood sampled at birth (P = .01 and P < .001, respectively) (Table 7). The mean increase in platelet counts from the beginning to the end of valacyclovir treatment was significantly higher than the expected corresponding increase in noninfected fetuses (P = .008) (Figure S3).
      Table 7Correlation of fetal and neonatal laboratory indicators (viral DNA load and platelet count, from cord blood, compared with viral DNA load and fetal platelet count obtained in utero before inclusion)
      Fetal blood before beginning maternal treatmentNeonatal cord bloodDifferences
      Differences between values obtained for same case in utero in fetal blood before maternal treatment and in neonatal cord blood after treatment. Participants with viral DNA load less than threshold value of 178 IU were considered to have viral DNA load of 89 IU (1.94 IU).
      P
      Viral DNA in blood, log10 IU/mL
       Median (interquartile range)4.0 (3.55–4.6)3.05 (2.57–3.92)–0.5 (–2.075 to –0.075).01
       N283224
      Platelet count/mm3
       Median (interquartile range)173,000 (141,500–201,500)245,000 (193,000–274,000)101,000 (47,500–122,000)<.001
       N274127
      Leruez-Ville et al. In utero treatment of congenital cytomegalovirus infection. Am J Obstet Gynecol 2016.
      a Differences between values obtained for same case in utero in fetal blood before maternal treatment and in neonatal cord blood after treatment. Participants with viral DNA load less than threshold value of 178 IU were considered to have viral DNA load of 89 IU (1.94 IU).
      The 5-year follow-up of the children is obviously not completed. They have currently been followed up for a median of 12 (range 4-36) months. Among the 33 children who were asymptomatic at birth, all were still asymptomatic at 12 months of age. All 7 neonates with symptoms were treated with valganciclovir for 6 weeks and none showed subsequent worsening of neurosensory hearing loss.

      Historical cohort

      Analysis of the historical group showed that any proportion of asymptomatic neonates <60% would be unacceptable for Simon
      • Simon R.
      Optimal two-stage designs for phase II clinical trials.
      optimal 2-stage design. Moreover, comparison of our trial results with those of the historical cohort indicates that valacyclovir significantly increased the proportion of asymptomatic neonates, from 43% without treatment to 82% with treatment (and without any overlapping of confidence intervals) (Figure 2).

      Comment

      This is the first trial of antiviral treatment of symptomatic fetuses with CMV disease. This approach followed a pilot study conducted from 2003 through 2005 that suggested treatment with high-dosage valacyclovir given orally to pregnant women carrying a symptomatic fetus infected with CMV could decrease viral DNA loads and achieve therapeutic concentrations in fetal blood.
      • Jacquemard F.
      • Yamamoto M.
      • Costa J.-M.
      • et al.
      Maternal administration of valacyclovir in symptomatic intrauterine cytomegalovirus infection.
      A phase II randomized placebo-controlled trial was planned to test the efficacy of valacyclovir given to women with a symptomatic infected fetus (Cymeval NCT01037712) in 2008. It failed, however, to recruit enough participants, including only 6 women over 2 years. To avoid what appeared to be an unacceptable placebo arm, we shifted to a trial based on a Simon
      • Simon R.
      Optimal two-stage designs for phase II clinical trials.
      optimal 2-stage design with only 1 arm–treated patients (Cymeval II NCT01651585). Severe fetal cerebral lesions are known to have a dismal prognosis and are unlikely to be reversible under treatment.
      • Gaytant M.A.
      • Steegers E.A.P.
      • Semmekrot B.A.
      • Merkus H.M.M.W.
      • Galama J.M.D.
      Congenital cytomegalovirus infection: review of the epidemiology and outcome.
      Their odds ratio for a poor outcome was 40.6
      • Benoist G.
      • Salomon L.J.
      • Jacquemard F.
      • Daffos F.
      • Ville Y.
      The prognostic value of ultrasound abnormalities and biological parameters in blood of fetuses infected with cytomegalovirus.
      ; we therefore excluded them. Women carrying an infected fetus that appeared completely asymptomatic on ultrasound and had normal laboratory results were similarly excluded, because their outcome is known to be generally good.
      • Benoist G.
      • Salomon L.J.
      • Jacquemard F.
      • Daffos F.
      • Ville Y.
      The prognostic value of ultrasound abnormalities and biological parameters in blood of fetuses infected with cytomegalovirus.
      • Benoist G.
      • Salomon L.J.
      • Mohlo M.
      • Suarez B.
      • Jacquemard F.
      • Ville Y.
      Cytomegalovirus-related fetal brain lesions: comparison between targeted ultrasound examination and magnetic resonance imaging.
      • Picone O.
      • Vauloup-Fellous C.
      • Cordier A.G.
      • et al.
      A series of 238 cytomegalovirus primary infections during pregnancy: description and outcome.
      • Farkas N.
      • Hoffmann C.
      • Ben-Sira L.
      • et al.
      Does normal fetal brain ultrasound predict normal neurodevelopmental outcome in congenital cytomegalovirus infection?.
      In a previous study, we showed that odds ratio for a poor outcome was 4.4 when there was any noncerebral ultrasound abnormality and 1.13 for each 10,000/mm3 decrease in fetal platelet count <100,000/mm3.
      • Benoist G.
      • Salomon L.J.
      • Jacquemard F.
      • Daffos F.
      • Ville Y.
      The prognostic value of ultrasound abnormalities and biological parameters in blood of fetuses infected with cytomegalovirus.
      Another observational study showed that a fetal platelet count <50,000/mm3 had 80% predictive value for poor outcome.
      • Fabbri E.
      • Revello M.G.
      • Furione M.
      • et al.
      Prognostic markers of symptomatic congenital human cytomegalovirus infection in fetal blood.
      We therefore enrolled women with either extracerebral or nonsevere cerebral ultrasound features, a low platelet count, or a high viral load in blood sampled in utero.
      • Benoist G.
      • Salomon L.J.
      • Jacquemard F.
      • Daffos F.
      • Ville Y.
      The prognostic value of ultrasound abnormalities and biological parameters in blood of fetuses infected with cytomegalovirus.
      • Fabbri E.
      • Revello M.G.
      • Furione M.
      • et al.
      Prognostic markers of symptomatic congenital human cytomegalovirus infection in fetal blood.
      • Boppana S.B.
      • Fowler K.B.
      • Pass R.F.
      • et al.
      Congenital cytomegalovirus infection: association between virus burden in infancy and hearing loss.
      These criteria were selected specifically to target a group at high risk for progressive damage, on the hypothesis that prenatal treatment could lower this risk that could lead to neonatal neurodevelopmental impairment.
      According to Simon
      • Simon R.
      Optimal two-stage designs for phase II clinical trials.
      optimal 2-stage design, high-dosage valacyclovir given orally to pregnant women was effective in improving the outcome of pregnancies with a fetus infected with symptomatic CMV, shown by either extracerebral or nonsevere cerebral ultrasound features or abnormal fetal laboratory results. Moreover, when compared with a historical cohort of similar cases collected from the literature,
      • Jacquemard F.
      • Yamamoto M.
      • Costa J.-M.
      • et al.
      Maternal administration of valacyclovir in symptomatic intrauterine cytomegalovirus infection.
      • Picone O.
      • Simon I.
      • Benachi A.
      • Brunelle F.
      • Sonigo P.
      Comparison between ultrasound and magnetic resonance imaging in assessment of fetal cytomegalovirus infection.
      • Guerra B.
      • Simonazzi G.
      • Puccetti C.
      • et al.
      Ultrasound prediction of symptomatic congenital cytomegalovirus infection.
      • Liesnard C.
      • Donner C.
      • Brancart F.
      • Gosselin F.
      • Delforge M.L.
      • Rodesch F.
      Prenatal diagnosis of congenital cytomegalovirus infection: prospective study of 237 pregnancies at risk.
      • Lipitz S.
      • Hoffmann C.
      • Feldman B.
      • Tepperberg-Dikawa M.
      • Schiff E.
      • Weisz B.
      Value of prenatal ultrasound and magnetic resonance imaging in assessment of congenital primary cytomegalovirus infection.
      an unbiased estimation confirmed the efficacy of valacyclovir compared to no treatment. Among the 43 symptomatic fetuses treated in utero, there were only 4 cases with an indisputably poor outcome (2 terminations of pregnancy for severe brain abnormalities and 2 cases of bilateral hearing loss), while the other 5 infants symptomatic at birth showed only mild disease (3 with unilateral hearing loss, 1 with isolated thrombocytopenia, and another with growth restriction). The clinical impact of valacyclovir may be explained by its direct inhibition of CMV replication. In renal transplantation and in HIV infection, high-dose regimens of valacyclovir are effective in preventing CMV disease and suppressing CMV viremia.
      • Lowance D.
      • Neumayer H.H.
      • Legendre C.M.
      • et al.
      Valacyclovir for the prevention of cytomegalovirus disease after renal transplantation. International Valacyclovir Cytomegalovirus Prophylaxis Transplantation Study Group.
      • Griffiths P.D.
      • Feinberg J.E.
      • Fry J.
      • et al.
      The effect of valacyclovir on cytomegalovirus viremia and viruria detected by polymerase chain reaction in patients with advanced human immunodeficiency virus disease. AIDS Clinical Trials Group Protocol 204/Glaxo Wellcome 123-014 International CMV Prophylaxis Study Group.
      • Emery V.C.
      • Sabin C.
      • Feinberg J.E.
      • Grywacz M.
      • Knight S.
      • Griffiths P.D.
      Quantitative effects of valacyclovir on the replication of cytomegalovirus (CMV) in persons with advanced human immunodeficiency virus disease: baseline CMV load dictates time to disease and survival. The AIDS Clinical Trials Group 204/Glaxo Wellcome 123-014 International CMV Prophylaxis Study Group.
      A sort of placebo effect may have also participated in the effect of valacyclovir. That is, although the participants were informed that the efficacy of valacyclovir in utero was unknown and that they could request a termination of pregnancy in accordance with French law, only 2 terminations were requested, both after fetuses developed severe cerebral symptoms visible on ultrasound. The availability of treatment may have alleviated parental anxiety and dissuaded women from requesting termination of pregnancy by offering them something more than helpless anxiety and expectant management.
      Although the valacyclovir dosage used in this study was much higher than that used for treatment of herpes simplex infection in pregnancy (8 g per day vs 1 g per day), the maternal clinical and laboratory tolerances were excellent and no adverse effect was observed in the neonates.
      Moreover, despite the burden of taking 16 tablets throughout the day, cumulative adherence to treatment was >90%.
      Overall, 40% of the women met the inclusion criterion of 1 ultrasound abnormality suggestive of CMV, 53% had >1, and 7% had only a high fetal blood viral load (>3000 copies/mL). The latter criterion was selected on the basis of previous work by Boppana et al,
      • Boppana S.B.
      • Fowler K.B.
      • Pass R.F.
      • et al.
      Congenital cytomegalovirus infection: association between virus burden in infancy and hearing loss.
      who reported that no neonates with a viral load <3000 copies developed hearing loss. However, a more recent study reports that fetal viral load >30,000 copies/mL is a predictive marker for poor outcome.
      • Fabbri E.
      • Revello M.G.
      • Furione M.
      • et al.
      Prognostic markers of symptomatic congenital human cytomegalovirus infection in fetal blood.
      Therefore, the cut-off of 3000 copies/mL might have been too low. Nonetheless, only 3 cases were included based on this criterion alone, and 2 of them had much higher viral loads (110,000 and 90,000 copies/mL). None of these 3 patients had a poor outcome.
      At birth, viral loads in cord blood were significantly lower and platelet counts significantly higher than in fetal blood obtained in utero before treatment began. The antiviral effects of valacyclovir can easily explain these changes. Nonetheless, both differences might also be due either to chance or the natural course of these markers since little is known about the spontaneous kinetics of blood viral loads or platelet counts over time after acute fetal infection. The similarity of blood viral DNA loads reported in neonates born to untreated mothers, regardless of the trimester of pregnancy of her primary infection,
      • Zavattoni M.
      • Lombardi G.
      • Rognoni V.
      • et al.
      Maternal, fetal, and neonatal parameters for prognosis and counseling of HCMV congenital infection.
      suggests that no spontaneous change in viral load is likely to be significant over the few weeks of intrauterine life. Postnatal decreases in viral load have been evaluated over a much longer period of time.
      • Forner G.
      • Abate D.
      • Mengoli C.
      • Palù G.
      • Gussetti N.
      High cytomegalovirus (CMV) DNAemia predicts CMV sequelae in asymptomatic congenitally infected newborns born to women with primary infection during pregnancy.
      Another reason for the differences in the prenatal and postnatal courses evolutions of viremia is the closed intrauterine circuit across the fetal-placental circulation. Moreover, the 1.0 log reduction we observed in blood viral load between fetal blood sampled before treatment and cord blood at birth after antiviral treatment is similar to the 1.3 log reduction reported in a controlled trial of HIV-infected patients also treated with high-dose valacyclovir.
      • Griffiths P.D.
      • Feinberg J.E.
      • Fry J.
      • et al.
      The effect of valacyclovir on cytomegalovirus viremia and viruria detected by polymerase chain reaction in patients with advanced human immunodeficiency virus disease. AIDS Clinical Trials Group Protocol 204/Glaxo Wellcome 123-014 International CMV Prophylaxis Study Group.
      We observed no correlation between the duration of fetal infection and neonatal platelet counts or viral loads. Although platelet counts are reported to increase during pregnancy, their increase in our study was correlated with the duration of valacyclovir treatment and was significantly higher than expected in a population of noninfected fetuses (17). A spontaneous decrease in fetal viral loads and an increase in platelet counts over time, independently of treatment, is very unlikely.
      Blood viral load levels have been reported to be significantly higher in symptomatic neonates.
      • Boppana S.B.
      • Fowler K.B.
      • Pass R.F.
      • et al.
      Congenital cytomegalovirus infection: association between virus burden in infancy and hearing loss.
      • Vauloup-Fellous C.
      • Ducroux A.
      • Couloigner V.
      • et al.
      Evaluation of cytomegalovirus (CMV) DNA quantification in dried blood spots: retrospective study of CMV congenital infection.
      • Walter S.
      • Atkinson C.
      • Sharland M.
      • et al.
      Congenital cytomegalovirus: association between dried blood spot viral load and hearing loss.
      In our study viral loads in neonatal blood and in neonatal urine showed only a trend to being higher in the symptomatic group. This could be due to the small number of symptomatic cases in the study (N = 7), which may not allow reaching statistical significance. One could also speculate that this gap in viral load levels between symptomatic and asymptomatic neonates during the natural history of the infection might have been reduced in a population of treated fetuses.
      The main limitation of our study is that it is not randomized. It therefore remains difficult to assess definitively the respective roles of true antiviral effect and a placebo effect to explain the positive effect of treatment demonstrated in this setting. However, conducting a randomized placebo-controlled trial in pregnant women with a symptomatic CMV-infected fetus proved utterly impracticable. This difficulty has previously been encountered for another rare fetal condition carrying a risk of death or severe handicap.
      • Morris R.K.
      • Malin G.L.
      • Quinlan-Jones E.
      • et al.
      Percutaneous vesicoamniotic shunting versus conservative management for fetal lower urinary tract obstruction (PLUTO): a randomized trial.
      This is especially relevant for women who choose not to terminate the pregnancy, at least as long as a potential good outcome may exist. Because a classic randomized controlled trial proved to be too difficult to achieve, a possibility to strengthen the results of the present study could be to follow the design described by Relton et al
      • Relton C.
      • Torgerson D.
      • O’Cathain A.
      • Nicholl J.
      Rethinking pragmatic randomized controlled trials: introducing the “cohort multiple randomized controlled trial” design.
      of a cohort multiple randomized controlled trial. In such trial, half of the eligible patients would be randomly selected to be treated while the other half would receive usual care; this would allow keeping comparable arms while avoiding the unacceptability of classic randomization.

      Acknowledgment

      We thank all the women who participated in the trial and Sylvain Goupil, the clinical research assistant. We are grateful to Mrs Jo Ann Cahn for her help in editing the manuscript.

      Supplementary Data

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      Linked Article

      • Efficacy of oral valacyclovir in cytomegalovirus-infected fetuses
        American Journal of Obstetrics & GynecologyVol. 216Issue 2
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          We read carefully the article of Leruez-Ville et al1 about the efficacy evaluation of oral valacyclovir for pregnant women carrying a symptomatic cytomegalovirus-infected fetus, who are at high risk for developing both neurosensory and neurological impairments. Symptomatic fetuses, defined by the presence of measurable extracerebral or mild cerebral ultrasound signs, were treated in utero from the prenatal diagnosis, performed at a median age of 25.9 weeks’ gestation, to the delivery or the termination of pregnancy.
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        American Journal of Obstetrics & GynecologyVol. 216Issue 2
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          We thank Drs Straface et al for their interest in our study. The placenta does play a pivotal role as an immunological barrier against congenital cytomegalovirus (cCMV) but also as a reservoir in which cytomegalovirus replicates. Invasive access to the placenta would only be focal and therefore histological samples unlikely to be consistently helpful in the absence of an available biomarkers of prognosis.
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