T-Cell Immune Responses in Newborns and Long-Term Sequelae in Congenital Cytomegalovirus Infection (CYTRIC Study).

OBJECTIVE: The objective of this study was to assess the role of T-lymphocyte immune responses in newborns with congenital cytomegalovirus (CMV) infection (cCMV) and their potential association with the development of long-term sequelae. STUDY DESIGN: A multicenter, prospective study from 2017 to 2022 was conducted across 8 hospitals in Spain. Blood samples were collected within the first month of life from neonates diagnosed with cCMV. Intracellular cytokine staining was employed to evaluate the presence of CMV-specific interferon-gamma (IFN-γ)-producing CD8+ and CD4+ T lymphocytes (CMV-IFN-γ-CD8+/CD4+) using flow cytometry. The development of sequelae, including hearing loss and neurologic impairment, was assessed during follow-up. RESULTS: In total, 64 newborns were included; 42 infants (65.6%) had symptomatic cCMV. The median age at the last follow-up visit was 25.3 months (IQR 20.1-34.4). Eighteen infants had long-term sequelae (28.1%), predominantly hearing loss (20.3%) and neurologic disorders (15.6%). No relationship was observed between total count or percentage of CMV-specific IFN-γ-CD8+ or CD4+ lymphocytes and long-term sequelae. Multivariable analysis demonstrated an association between lower total lymphocyte count and long-term sequelae (aOR 0.549, 95% CI: 0.323-0.833), which requires further study. CONCLUSIONS: CMV-specific IFN-γ-CD4+ and CD8+ T-lymphocyte responses in neonates with cCMV were not predictive of long-term sequelae.

Changes of CD8+CD28- Tregs and Gamma-Delta-T-cells in a Neonate with Intrauterine Cytomegalovirus Infection: A Case Report.

BACKGROUND: Congenital cytomegalovirus (cCMV) infection can lead to a range of adverse outcomes. The majority of cCMV neonates with clinical symptoms are infected postnatally; however, established cases of intrauterine infection are uncommon, resulting in a paucity of reports on clinical findings and lymphocytes expression in CMV-infected neonates. CASE PRESENTATION: We followed a neonate with cCMV infection from the onset of hospitalization to several months of follow-up. This infant was intrauterine CMV-positive in the amniotic fluid of the mother at 21 weeks' gestation and received intravenous ganciclovir infusion and sequential oral valganciclovir after birth. The typical clinical signs manifested in the nervous system, liver, and peripheral blood and were documented during the hospitalizaion period and up to the follow-up visit. Flow cytometry was employed to examine the expression of T cells, their subsets, and the associated cytokines in peripheral blood samples at various time points. The flow data for the cCMV neonate were compared with those of the controls at each time point. Following treatment, clinical symptoms improved and the infant became CMV negative. However, developmental delays occurred later in life. The proportion of CD8+CD28- Tregs in the peripheral blood of the neonate with congenital CMV infection was higher than that in the controls at the three time points. The expression levels of perforin and granzyme B secreted by γδ T cells (Vδ1 and Vδ2 T cells), increased during the course of hospitalization until follow-up and were higher than those in the controls at the three time points. CONCLUSIONS: Despite the alleviation of clinical symptoms, developmental delay in later life remains inevitable in this intrauterine cCMV neonate. CD8+CD28- Tregs and Vδ1 and Vδ2 T cells secreting perforin and granzyme B may be involved in congenital CMV infection, although this hypothesis requires validation in a larger study. This report may contribute to our understanding of the effect of current treatment and the immune status of intrauterine cCMV-infected neonates.

Endothelial Cell Infection by Guinea Pig Cytomegalovirus Is a Lytic or Persistent Infection Depending on Tissue Origin but Requires Viral Pentamer Complex and pp65 Tegument Protein.

The guinea pig is the only small animal model for congenital cytomegalovirus (CMV) but requires species-specific guinea pig cytomegalovirus (GPCMV). Infection of epithelial cells and trophoblasts by GPCMV requires the viral glycoprotein pentamer complex (PC) and endocytic entry because of the absence of platelet-derived growth factor receptor alpha (PDGFRA). Endothelial cells represent an important cell type for infection, dissemination in the host, and disease but have been poorly evaluated for GPCMV. Novel endothelial cell lines were established from animal vascular systems, including aorta (EndoC) and placental umbilical cord vein (GPUVEC). Cell lines were characterized for endothelial cell protein markers (PECAM1, vWF, and FLI1) and evaluated for GPCMV infection. Only PC-positive virus was capable of infecting endothelial cells. Individual knockout mutants for unique PC components (GP129, GP131, and GP133) were unable to infect endothelial cells without impacting fibroblast infection. Ectopic expression of PDGFRA in EndoC cells enabled GPCMV(PC-) infection via direct cell entry independent of the PC. Neutralizing antibodies to the essential viral gB glycoprotein were insufficient to prevent endothelial cell infection, which also required antibodies to gH/gL and the PC. Endothelial cell infection was also dependent upon viral tegument pp65 protein (GP83) to counteract the IFI16/cGAS-STING innate immune pathway, similar to epithelial cell infection. GPCMV endothelial cells were lytically (EndoC) or persistently (GPUVEC) infected dependent on tissue origin. The ability to establish a persistent infection in the umbilical cord could potentially enable sustained and more significant infection of the fetus in utero. Overall, results demonstrate the importance of this translationally relevant model for CMV research. IMPORTANCE Congenital CMV is a leading cause of cognitive impairment and deafness in newborns, and a vaccine is a high priority. The only small animal model for congenital CMV is the guinea pig and guinea pig cytomegalovirus (GPCMV) encoding functional HCMV homolog viral glycoprotein complexes necessary for cell entry that are neutralizing-antibody vaccine targets. Endothelial cells are important in HCMV for human disease and viral dissemination. GPCMV endothelial cell infection requires the viral pentamer complex (PC), which further increases the importance of this complex as a vaccine target, as antibodies to the immunodominant and essential viral glycoprotein gB fail to prevent endothelial cell infection. GPCMV endothelial cell infection established either a fully lytic or a persistent infection, depending on tissue origin. The potential for persistent infection in the umbilical cord potentially enables sustained infection of the fetus in utero, likely increasing the severity of congenital disease.

Correlation of cytomegalovirus viral load between whole blood and plasma of congenital cytomegalovirus infection under valganciclovir treatment.

BACKGROUND: Congenital cytomegalovirus (CMV) infection (cCMV) can cause sensorineural hearing loss and neurodevelopmental disabilities in children. Oral valganciclovir (VGCV) therapy has been reported to improve long-term audiological and neurodevelopmental outcomes in patients with cCMV. The levels of CMV DNA in whole blood have been monitored in previous studies. However, quantitative methods using whole blood have not been standardized. Recently, the plasma viral load has been standardized and widely used in CMV-associated diseases. METHODS: CMV viral loads in whole blood and plasma were serially measured in 24 patients with a confirmatory diagnosis of cCMV during oral VGCV therapy using an in-house real-time PCR assay. Plasma samples were assayed using the Cobas 6800 system (Roche Diagnostics) in addition to an in-house assay. RESULTS: Plasma CMV viral loads were remarkably decreased at the end of therapy compared to before therapy. A significant correlation of CMV levels between whole blood and plasma was observed (Spearman's ρ = 0.566). The levels of CMV DNA before therapy were significantly correlated with the period of decreasing the viral loads to below the detection limit, not only in whole blood (Spearman's ρ = 0.901) but also in plasma (Spearman, ρ = 0.804). Finally, CMV viral loads between the in-house assay and commercially available standardized assay in 75 plasma samples with positive PCR results for CMV were compared; a significant correlation was observed between the results of both assays. CONCLUSIONS: There was a significant correlation between the two assays (Spearman, ρ = 0.882), suggesting that CMV plasma viral loads measured by the standardized assay are widely used to monitor the levels of CMV DNA in patients with cCMV during oral VGCV therapy.

Effectiveness and safety of prenatal valacyclovir for congenital cytomegalovirus infection: systematic review and meta-analysis.

OBJECTIVE: Universal screening for cytomegalovirus (CMV) infection in pregnancy is not recommended in most countries. One of the major deterrents is the lack of effective prenatal therapy. The role of valacyclovir therapy in reducing the risk of vertical transmission, symptomatic congenital CMV infection and adverse outcome is controversial. The main aim of this systematic review and meta-analysis was to investigate the safety and effectiveness of prenatal valacyclovir therapy in pregnancies with maternal CMV infection. METHODS: MEDLINE, EMBASE and Cochrane databases and ClinicalTrials.gov were searched. The inclusion criteria were pregnancy with confirmed maternal CMV infection, treated or untreated with valacyclovir. The primary outcome was the incidence of congenital CMV infection confirmed by a positive CMV polymerase chain reaction result of the amniotic fluid. The secondary outcomes were symptomatic and asymptomatic infection, perinatal death, termination of pregnancy, anomalies detected on follow-up ultrasound, on fetal magnetic resonance imaging or at birth, severe and mild-to-moderate symptoms due to congenital CMV infection, neurological, visual and hearing symptoms, and adverse events related to valacyclovir. Risk of bias was assessed using the revised Cochrane risk-of-bias tool for randomized trials (RoB 2) or Risk Of Bias In Non-randomized Studies of Interventions (ROBINS-I) tool, as appropriate. Head-to-head meta-analyses were used to compare the risk of each of the explored outcomes according to whether pregnancies with maternal CMV infection were treated with prenatal valacyclovir therapy. RESULTS: Eight studies (620 women) were included. Pregnancies treated with valacyclovir had a significantly lower risk of congenital CMV infection compared with those not receiving valacyclovir (three studies; 325 fetuses; pooled odds ratio (OR), 0.37 (95% CI, 0.21-0.64); I2 = 0%; P < 0.001). When stratifying the analysis according to gestational age at maternal infection, the risk of vertical transmission was significantly lower in pregnancies receiving valacyclovir following first-trimester maternal infection (three studies; 184 fetuses; pooled OR, 0.34 (95% CI, 0.15-0.74); I2 = 20.9%; P = 0.001), while there was no significant difference between the two groups in those acquiring CMV infection in the periconceptional period or in the third trimester of pregnancy. Only one study reported on the risk of vertical transmission in women infected in the second trimester, demonstrating a lower risk of congenital infection in women taking valacyclovir, although this was based on a small number of cases. Pregnancies treated with valacyclovir therapy had an increased likelihood of asymptomatic congenital CMV infection compared with those not receiving valacyclovir (two studies; 132 fetuses; pooled OR, 2.98 (95% CI, 1.18-7.55); I2 = 0%; P = 0.021), while there was no significant difference between the two groups in the risk of perinatal death (P = 0.923), termination of pregnancy (P = 0.089), anomalies detected at follow-up imaging assessment during pregnancy or at birth (P = 0.934) and symptoms due to CMV infection in the newborn (P = 0.092). The occurrence of all adverse events in pregnant individuals taking valacyclovir was 3.17% (95% CI, 1.24-5.93%) (six studies; 210 women), with 1.71% (95% CI, 0.41-3.39%) experiencing acute renal failure, which resolved after discontinuation of the drug. On GRADE assessment, the quality of evidence showing that valacyclovir reduced the risk of congenital CMV infection and adverse perinatal outcome was very low. CONCLUSIONS: Prenatal valacyclovir administration in pregnancies with maternal CMV infection reduces the risk of congenital CMV infection. Further evidence is needed to elucidate whether valacyclovir can affect the course of infection in the fetus and the risk of symptomatic fetal or neonatal infection. © 2022 International Society of Ultrasound in Obstetrics and Gynecology.

Healthcare costs of congenital cytomegalovirus (cCMV) disease in infants during the first two years of life: a retrospective German claims database analysis.

BACKGROUND: Congenital cytomegalovirus (cCMV) infection can cause severe neurological damage, growth retardation, hearing loss, and microcephaly in infants. We aimed at assessing healthcare costs of infants with recorded cCMV diagnosis in an administrative claims database in the first 2 years of life. METHODS: We conducted a retrospective, controlled cohort study using German claims data from the Institute for Applied Health Research Berlin (InGef) database. Incremental healthcare costs during the first and second year of life were assessed by matching (1:60) infants with cCMV diagnoses ≤ 90 days after birth (cCMV90 cohort) to infants without cCMV diagnosis ("representative" controls) and infants with cCMV diagnoses ≤ 21 days after birth plus specific symptoms (cCMV21-S) to infants without cCMV and any ICD-10-GM records (besides Z00-Z99) until 4th preventive health check-up ("healthy" controls). Due to missing data, mean imputation was applied for aids and remedies costs. RESULTS: We identified 54 and 24 infants born 2014-2018 for the cCMV90 and cCMV21-S cohorts, respectively. During the first year, mean (median) healthcare costs were significantly higher in cCMV90 cases vs. "representative" controls (€22,737 (€9759) vs. €3091 (€863), p < 0.001), with 87.2% inpatient costs. Healthcare costs for cCMV21-S cases compared to "healthy" controls were €34,498 (€20,924) vs. €680 (€569), p < 0.001. Differences decreased for both comparisons in the second year but remained statistically significant. CONCLUSIONS: cCMV comprises a considerable economic burden for the German healthcare system (€19,646 to €33,818 higher mean costs for infants with recorded cCMV diagnosis in the first year of life). Attempts should be made to reduce this burden.

Two-sample Mendelian randomization study does not reveal a significant relationship between cytomegalovirus (CMV) infection and autism spectrum disorder.

BACKGROUND: Autism spectrum disorder (ASD) is a neurodevelopmental disorder affecting ~ 2% of children worldwide and is characterized by repetitive, stereotypical behaviours and impaired expressive communication. Cytomegalovirus (CMV) is considered a risk factor for ASD; however, published studies are usually limited by covering too few events and have different conclusions, indicating that the relationship between CMV infection and ASD remains elusive. METHODS: To investigate the association between CMV infection and ASD, we conducted this 2-sample Mendelian randomization (MR) study using genome-wide association studies (GWAS) summary data from FinnGen and the IEU Open GWAS project. RESULTS: Our results showed no significant relationship between all 3 CMV infections (unspecified cytomegaloviral diseases, anti-CMV IgG levels, and maternal CMV) and ASD. CONCLUSIONS: Our results indicate that CMV infection does not significantly increase ASD risk. These results show that the relationship between CMV infection and ASD remains elusive and needs to be further clarified.

Is CMV PCR of inner ear fluid during cochlear implantation a way to diagnose CMV-related hearing loss?

To evaluate the diagnostic performance of cytomegalovirus (CMV) polymerase chain reaction (PCR) on inner ear fluid collected during cochlear implantation and to assess its interest in current practice. This monocentric prospective study included consecutive children presenting with severe to profound sensorineural hearing loss (SNHL) who were candidates for unilateral and/or bilateral cochlear implantation. The etiology of the SNHL was determined before cochlear implantation when possible. During the surgery, drop-like samples of inner ear fluid and blood were collected. CMV PCR was then performed on both samples. Between January 2017 and September 2021, 113 children with severe to profound SNHL underwent cochlear implantation with inner ear fluid collection. Among these children, 77 of them presented with a known cause of SNHL (68%) and 36 of them had an unknown cause of SNHL at the time of surgery (32%). Sensitivity and specificity of the CMV PCR on inner ear fluid were 60% (95% CI: [49-71]) and 98% (95% CI: [96-100]), respectively. Positive and negative predictive values were 90% (95% CI: [83-97]) and 92% (95% CI: [86-98]), respectively. A sensitivity analysis according to age at cochlear implantation showed a decrease with age. CONCLUSION: Sampling of inner ear fluid during cochlear implant surgery is an interesting, simple and safe way to diagnose CMV-related hearing loss, especially when the diagnosis of congenital infection can no longer be confirmed. However, the sensitivity decreases with age. TRIAL REGISTRATION: NCT04724265 What is Known: • Congenital cytomegalovirus infection is the leading infectious cause of neurological disabilities and sensorineural hearing loss in children. In the absence of systematic screening at birth, many cCMV infections go undetected and are often undiagnosed despite the development of sensorineural sequelae.  • Nearly 40% of indications for cochlear implantation are of unknown etiology. WHAT IS NEW: • Performing CMV PCR on inner ear fluid at the time of cochlear implantation is a safe way with high diagnostic performance (PPV = 90%, NPV = 92%) to detect a CMV-related hearing loss. • This sample may be interesting in cases of unknown cause of hearing loss in order to identify undiagnosed cCMV infections.

Is there room for prompt routine ophthalmic examination in neonates with congenital cytomegalovirus (CMV) infection during the neonatal period?

PURPOSE: To establish the clinical necessity of routine targeted ophthalmic examination of newborns with congenital cytomegalovirus (CMV) infection during the neonatal period. METHODS: This retrospective study included consecutive neonates that were referred for ophthalmological screening within the context of a proven congenital CMV infection. The presence of CMV-related ocular and systemic findings was determined. RESULTS: Among the 91 patients included in this study, 72 (79.12%) were symptomatic with one or more of the following manifestations: abnormal brain ultrasound (42; 46.15%), small for gestational age (29; 31.87%), microcephaly (23; 25.27%), thrombocytopenia (14; 15.38%), sensory neural hearing loss (13; 14.29%), neutropenia (12; 13.19%), anemia (4; 4.4%), skin lesions (4; 4.4%), hepatomegaly (3; 3.3%), splenomegaly (3; 3.3%), direct hyperbilirubinemia (2; 2.2%). Not one single neonate in this cohort had any of the ocular findings surveyed. CONCLUSION: The presence of ophthalmological findings among neonates with congenital CMV infection during the neonatal period is infrequent, suggesting that routine ophthalmological screening may be safely deferred for the post-neonatal period.

In Utero Activation of Natural Killer Cells in Congenital Cytomegalovirus Infection.

BACKGROUND: Congenital cytomegalovirus (CMV) infection is the most common infectious cause of birth defects and neurological damage in newborns. Despite a well-established role for natural killer (NK) cells in control of CMV infection in older children and adults, it remains unknown whether fetal NK cells can sense and respond to CMV infection acquired in utero. METHODS: Here, we investigate the impact of congenital CMV infection on the neonatal NK-cell repertoire by assessing the frequency, phenotype, and functional profile of NK cells in cord blood samples from newborns with congenital CMV and from uninfected controls enrolled in a birth cohort of Ugandan mothers and infants. RESULTS: We find that neonatal NK cells from congenitally CMV infected newborns show increased expression of cytotoxic mediators, signs of maturation and activation, and an expansion of mature CD56- NK cells, an NK-cell subset associated with chronic viral infections in adults. Activation was particularly prominent in NK cell subsets expressing the Fcγ receptor CD16, indicating a role for antibody-mediated immunity against CMV in utero. CONCLUSIONS: These findings demonstrate that NK cells can be activated in utero and suggest that NK cells may be an important component of the fetal and infant immune response against CMV. CLINICAL TRIALS REGISTRATION: NCT02793622.