First-Trimester Universal One-Time Serology Screening for Cytomegalovirus: A Pilot Study at Two Tertiary Referral Centers in Barcelona (Catalunya, Spain)

Congenital cytomegalovirus (cCMV) is the most common congenital infection worldwide, with an estimated prevalence of 5–7 per 1,000 live births. cCMV represents a major cause of long-term sequelae including sensorineural hearing loss, neurological impairment, and developmental delay [1, 2].

A recent meta-analysis has shown that cCMV should be considered an embryopathy since the risk of fetal injury occurs almost exclusively after maternal infections acquired during the first-trimester or periconceptional period [3]. Early disruption in the development of the central nervous system in the embryonic phase produces impaired neuronal proliferation and cell death [4].

The risk of fetal transmission during this risk period is around 30% after a primary infection and much lower after a non-primary one, although the latter is a main cause of cCMV, especially in countries with high maternal seroprevalence [2]. CMV seroprevalence in childbearing-aged women varies widely, from 45% to 94%, and correlates with country of origin and socioeconomic status [5]. In high-income countries, such as those from Western Europe, seroprevalence is in the low range. Nevertheless, there is a lack of current epidemiological data in some countries, for instance Spain, with relevant recent immigrational movements. The latest data come from the study by de la Calle et al. [6], in Madrid, who reported a seroprevalence of 62.2%; nevertheless, this research was carried out as an opportunistic rather than a universal screening program.

Despite the burden of the infection, only a few consensus guidelines and scientific societies recommend routine serological screening during pregnancy [2, 7, 8]. The main reasons are that screening is inapplicable to non-primary infections and that in the absence of validated treatment options, there are concerns regarding its benefits. However, a recent randomized controlled trial by Shahar-Nissan et al. [9] showed relevant results regarding maternal treatment. The administration of valacyclovir (VCV) to pregnant women with primary CMV infection acquired in the first-trimester or periconceptional period has been associated with a significant reduction of fetal infection, from 30% to 11%. Following confirmation of these results by an additional observational study that demonstrated timely antiviral therapy was effective in reducing vertical transmission rates [10], we decided to launch a pilot program of universal first-trimester screening at our setting.

The aim of this study was to evaluate the results of the first 2 years of implementation of a universal first-trimester CMV screening program in a cohort of pregnant women attending two tertiary hospitals in Barcelona. The secondary outcome was to describe the maternal seroprevalence and the burden of primary infection during the period of fetal risk in this population.

We conducted an epidemiological surveillance study between February 2021 and August 2023 in two tertiary care hospitals in Barcelona: Hospital Clinic and Hospital Sant Joan de Déu which are linked for clinical and research purposes as a single center: BCNatal.

This study was started in parallel with the universal CMV screening program for diagnosis of maternal primary infections in the first-trimester or periconceptional period for prevention of cCMV that was launched in both hospitals. Serological determination of IgG and IgM antibodies was added to the established first-trimester laboratory testing.

We included all first-trimester pregnant women attended between 8+0 and 13+6 gestational weeks who underwent the combined test for aneuploidies at our institutions. Hospital Clinic of Barcelona and Hospital Sant Joan de Déu are two tertiary hospitals, with an average annual volume of 6,000 deliveries of which around 20% are attended from the first-trimester. Our research focused specifically on women who underwent our universal first-trimester CMV screening. Therefore, those referred from private or other public health institutions with an already first-trimester CMV serology performed, as well as those with it carried out outside the first-trimester, were excluded.

All women underwent both IgG and IgM serology for CMV specific antibodies determined by Elecsy CMV (Roche^®). In cases with positive IgG and negative IgM antibodies, a recent primary CMV infection was ruled out, as well as in pregnant women with negative IgG and IgM antibodies.

In women with positive IgG and IgM antibodies, the IgG avidity test was determined simultaneously by the microbiology laboratory. An automatic alert system was established by which any case with IgG avidity determination was notified immediately to the professionals involved in the circuit, with an elapsed time between maternal blood testing and result notification of fewer than 5 days. The time of maternal primary infection was categorized in each of the cases according to their IgG avidity cut-off value established by the manufacturer Elecsy CMV (Roche^®). Detection of a low avidity index (<40%) is a strong indication of a primary infection of less than 3 months, whereas a high-avidity index (>65%) is a strong indication of primary infection of more than 3 months. Intermediate avidity (40–65%) suggests that the time of infection may have occurred between those two periods, and has been shown to correspond to a primary infection in the previous 3 months in 10–15% of cases, and between 3 and 6 months in 30–70% [11].

All CMV seronegative women, and those with positive IgG and negative IgM, or positive IgG and IgM with high avidity titers, were followed-up according to standard prenatal care. In those with a positive IgG and IgM with a low or intermediate avidity, high-dose oral VCV 2 g/6 h was offered from the moment of serologic diagnosis until the time of amniocentesis. Amniocentesis was recommended to diagnose/discard fetal infection and was scheduled from 17 weeks of pregnancy, and at least 8 weeks after presumed maternal infection. Maternal renal function was monitored before the onset of treatment, and every fortnight, due to the minor risk of reversible acute kidney failure previously described with high-dosage VCV [12].

Fetal infection was assessed by a positive CMV-PCR in amniotic fluid and confirmed by a CMV-PCR in neonatal urine within the first 2 weeks of life. In those women with negative CMV-PCR in amniotic fluid, VCV was ceased, and in cases with a positive result it was continued until the end of pregnancy according to our clinical protocol (https://portal.medicinafetalbarcelona.org/protocolos/es/patologia-materna-obstetrica/infecciones-torch.html).

In pregnant women declining amniocentesis, our protocol establishes maintaining VCV until the time of amniocentesis schedule, with assessment of cCMV status in neonatal urine. Follow-up of infected fetuses and those with a rejected amniocentesis consisted of serial ultrasound scans and magnetic resonance imaging at 32 weeks (Fig. 1).

The Statistical Package for Social Sciences for MacOS, version 29.0 (IBM Corp., Armonk, NY, USA) was used to enter and analyze the data. Demographic data were categorized to calculate frequencies and percentages. Quantitative variables were assessed using the Shapiro-Wilk test for normality. The independent sample t test was employed to evaluate the difference between seropositive and seronegative women. All tests were two-tailed, and a statistically significant association was declared at a p value 0.05. Listwise deletion was used as a method to handle missing data.

From February 2021 to August 2023, 2,777 women participated in our CMV screening program. Serology was undertaken at a mean gestational age of 10.6 ± 1 weeks. The mean maternal age at screening was 34.2 ± 5.1 years. Out of 2,777 women, 1,961 showed positive IgG antibodies, representing a seroprevalence of 70.6%. When comparing the baseline characteristics according to CMV serostatus, we observed that seronegative women were older (p ≤ 0.001) and showed a nonsignificant trend toward a higher educational level (Table 1).

Among seropositive women, 22 (0.8%) presented positive IgM antibodies simultaneously. Out of these, 18 had a high avidity index ruling out a recent primary infection, while 4 (0.14% of the whole population) presented low (n = 2) or intermediate avidity (n = 2), corresponding to a primary infection during first-trimester or periconceptional period, shown in Figure 2. Secondary prophylaxis with VCV was offered and accepted by all of them with complete adherence and none reporting side effects. Periodical blood monitoring was within normal ranges, and amniocentesis was accepted and performed between 17 and 21 weeks.

Out of these four amniocenteses, three (75%) were negative; therefore, treatment with VCV was suspended. Three asymptomatic neonates were born in whom urine samples were collected with negative PCR for CMV, ruling out vertical transmission. In the remaining case with a primary infection in the first-trimester and a CMV-PCR positive in amniotic fluid at 21 weeks, targeted ultrasound and neurosonography at the time of diagnosis of fetal infection were performed with normal results. Nevertheless, the parents requested termination of pregnancy (TOP). In this case, that took place during the first weeks of the study, there was a failure in the alert system with the avidity test, resulting in a 21 days delay from maternal serology to initiation of VCV treatment (Table 2).

Our study shows the preliminary results of a universal first-trimester CMV screening program during routine first-trimester maternal blood test. It demonstrates that the program is feasible and provides the opportunity to detect and treat primary maternal infections for prevention of vertical transmission at the time of fetal greatest risk. Nevertheless, due to the high seroprevalence observed, the number of primary infections detected was too low to draw conclusions regarding screening efficacy.

The growing evidence supporting the effectiveness of VCV in preventing vertical transmission of CMV in primary infections in the periconceptional period and during the first-trimester has been confirmed in a recent meta-analysis by Chatzakis et al. [12], who concluded that vertical transmission was reduced by 70%. They also described the value of VCV as a tertiary prevention treatment in women in whom vertical transmission had not been avoided. The authors observed a significant reduction of TOP due to severe fetal central nervous system findings among cases treated with VCV [12]. Such a window of opportunity to reduce the burden of cCMV will probably lead to the reconsideration of the recommendation against screening by most health authorities [11, 13]. Moreover, the earlier the treatment is started, the lower the vertical transmission rate will be [9, 10, 12]. For this reason, we have decided to establish a laboratory alert system to promote early identification and start VCV treatment as soon as possible. Although the alert system showed some failures at the beginning of the program, we consider that ensuring a consistent and coordinated pathway for screen-positive cases is a prerequisite prior to implementing a screening program.

VCV is safe during pregnancy, although at a high dose of 8 g/24 h, acute renal failure that resolves after discontinuation has been described in up to 2% of pregnant women [12]. This potential risk highlights the importance of prescribing VCV for secondary prevention exclusively after maternal infections in the first-trimester or periconceptional period and advises against performing the screening thereafter due to the low risk of fetal involvement [3].

Before engaging in a universal CMV screening program, in addition to treatment for the condition, there are other necessary prerequisites to lessen harm. There should be facilities for diagnosis of the infection such as reliable IgG avidity tests, PCR for diagnosis of fetal infection, and opportunities for a proficient fetal sonographic follow-up and MRI [14]. Educational efforts for serology interpretation and expert counseling of positive cases are of paramount importance for the reduction of TOP requested due to maternal anxiety [15]. Although we met all these requirements in our program, one woman requested TOP in a fetus without apparent anomalies at 21 weeks. This is perhaps inevitable with current clinical practice, nevertheless, it will probably become less frequent when it is confirmed that early treatment with VCV also aids as prevention of neonatal sequelae [12].

The 70% CMV seroprevalence that we found is higher than expected for a European country, and somewhat greater than the one reported recently in the study in Madrid (62.2%), although their data were not obtained from a universal screening program [6]. To date, our screening program has led us to detect only 4 cases of CMV primary infections up to a mean of 10.6 weeks of pregnancy (0.14%). This would represent a prevalence of 0.4–0.5% throughout the pregnancy, much lower than the 1–2% expected for a high-income country [16], and lower than the previously mentioned study with 1.35% of primary infections in the first-trimester [6]. Specific characteristics of our population, such as 22% of immigrants from non-European countries, could have influenced our results. Nevertheless, our data do not demonstrate a greater proportion of non-European patients in the CMV seropositive group. It is difficult to determine whether these data represent the current prevalence of primary infections in other areas of Barcelona or Spain. The seroprevalence, and consequently the prevalence of primary infections, might present large variations within the same country or even the same city because they are dependent on socioeconomic status and country of origin [14]. There are no previous data on CMV seroprevalence in Barcelona, and there are very few data on recent seroprevalence in Spain [17]. Hence, our study might depict the current prevalence of first-trimester primary infections in our area, further research, however, is needed to determine the influence of other factors such as recent educational interventions for primary prevention carried out in our setting. A broader universal first-trimester screening program (CITEMB study) has recently been launched at the primary health level in the Barcelona area [18]. Results from this study will probably provide more robust data on CMV maternal seroprevalence and burden of the infection in our region.

Given the low prevalence of CMV primary maternal infections, the implementation of universal screening could be called into question since most cases of cCMV at our setting would come from non-primary infections, non-amenable to diagnosis with the current testing methods [19]. Nonetheless, we are committed to continuing our screening program considering it allows the detection of more cases than other usual screenings such as toxoplasma that have been carried out for many years and whose secondary prevention treatment provides lower results [20].

This study has several strengths. To the best of our knowledge, it is the first to show the results of a prospective universal first-trimester screening applied to clinical practice including administration of a treatment for prevention of fetal infection. Moreover, it has allowed the high maternal seroprevalence to be demonstrated at our setting and represents a first step toward the decision to implement it nationwide. Finally, the diagnostic and clinical pathways that we have displayed are an example that could be replicated in other institutions.

The study has also some limitations since the population could be biased as first-trimester pregnant women who attend our tertiary health institutions mostly represent high-risk groups (e.g., history of preeclampsia, intrauterine growth restriction, perinatal death, twin pregnancies, and IVF) which could lead to an overestimation of maternal seroprevalence. However, such an analysis is beyond the objective of the current study. In any case, if it were indeed a group with higher seroprevalence, in a lower seroprevalence population more cases could have been identified, therefore increasing the effectiveness of the screening. Moreover, in our pilot study we implemented the easiest strategy with a single blood test at the time of the combined test for aneuploidy. One could argue that this approach may have missed the opportunity for timely medical prophylaxis in cases of very early pregnancy infection with the greatest fetal risk [3], together with first trimester infections after the test. However, regarding very early infections, a reasonable effect of this strategy can be expected, since in the study by Shahar-Nissan et al. [9] the sequence event from maternal to fetal infection was considered to take about 7 weeks allowing a time margin before treatment. A more comprehensive strategy, as suggested by the emergent body of evidence, involves retesting seronegative women every 4 weeks until 14 weeks of gestation [11]. Finally, we did not evaluate the cost-effectiveness of the program since as a pilot program this was beyond our scope.

Universal first-trimester CMV screening at the time of fetal risk is feasible, even though it requires facilities for diagnosis, adequate coordination to initiate timely treatment, and expert advice for positive cases. A higher than expected maternal seroprevalence, and lower than expected prevalence of primary infections, were observed in our population. Larger studies will confirm whether it is cost-effective in our setting.

We are indebted to the midwives, obstetricians, and microbiology laboratory technicians, especially Alex Navarro Rivera, for their outstanding role in the application of the CMV screening program in Hospital Clinic and Sant Joan de Déu.

This study protocol was reviewed and approved by the Hospital Clinic Ethics Committee (Reg. HCB/2024/0511) and was conducted in accordance with the Declaration of Helsinki. Verbal informed consent was obtained from participants prior to the study. The requirement for written informed consent to participate was waived by our Ethics Committee and was not mandated in accordance with local guidelines. This consent procedure was reviewed and approved by the Ethics and Drug Research Committee of the Hospital Clinic in Barcelona (Approval: Reg. HCB/2024/0511), with the decision made on May 28, 2024. Given the increasing evidence supporting screening for congenital CMV infection during first-trimester, this pilot program was accepted by consensus as a clinical standard of care within our institutions. Prior to implementing the program, the clinical protocol was transcribed to our guidelines for the management of CMV infections during pregnancy.

Prof. Francesc Figueras is an associate editor of Fetal Diagnosis and Therapy. The rest of the authors have no conflicts of interest to declare.

This study was not supported by any sponsor or funder.

A.G. and F.F. contributed to the design of the research and implementation of the screening program; M.D.G.-R., E.C.F., M.A.M., and I.M. contributed to the implementation of the screening program; K.P.C., L.G., A.C., Q.L., and C.M. performed the data collection; A.G., K.P.C., F.F., and M.L. contributed to the analysis of the results and the writing of the manuscript.

The data that support the findings of this study are not publicly available due to their containing information that could compromise the privacy of research participants but are available from the corresponding author upon reasonable request.