Janaina Fortes Lino ·Lilian Martins Oliveira Diniz ·Débora Marques de Miranda , ·Daniela Valad?o Freitas Rosa ·Nathália Gualberto Souza e Silva ·Eduardo de Souza Nicolau ·Larissa Gon?alves Rezende ·Lais Silva Carvalho ·Marianna Fischer de Paula Lopes ·Luísa Petri Correa ·Gabriela Mafra de Oliveira ·Flávia Miranda da Silva Alves ·Lorena Batista Pascoal ·érika Lima Dolabella Teixeira da Costa ·Lêni Márcia Anchieta ·Roberta Maia de Castro Romanelli

Cytomegalovirus is the most frequent agent of congenital viral infections,affecting approximately 0.2%—2.4% of live births in different countries [1— 7].Universal screening for congenital cytomegalovirus infection in newborns is not a standard practice considering cost—benefit issues and viability of diagnostic tests.Besides,most newborns are asymptomatic and there is no effective treatment for this population[2—5].

On the other hand,early neonatal diagnosis enables early access to rehabilitation,with a better prognosis in adulthood.Infants with congenital cytomegalovirus infection may present premature birth,low birth weight,or may be small for gestational age [7— 9].However,the prevalence of this congenital infection in these high-risk infants is uncertain,and data are controversial and scarce [2— 5,8— 10].

In this context,screening of newborns who are premature,low birth weight,or small for gestational age would enable diagnosis of patients with a high risk of infection.In addition,cytomegalovirus-infected newborns exhibit prolonged viral replication,which may reflect their inability to produce an adequate immune response and consequently lead to a worse prognosis [11—13].Understanding the role of the inflammatory cascade and cytokines can help to clarify patient prognosis and indicate the need to search for new treatments.

The aim of the present study was to determine the prevalence of congenital cytomegalovirus infections in preterm,low birth weight,or small for gestational age newborns without a defined cause,compared with healthy term-born babies and to describe their cytokine profiles.

This cross-sectional study was conducted in Belo Horizonte,State of Minas Gerais,Brazil,from 2016 to 2020.Newborn samples were collected from four maternity hospitals:Hospital das Clínicas/UFMG,Maternidade Odete Valadares/FHEMIG,Hospital Sofia Feldman,and Hospital Risoleta Tolentino Neves.

Inclusion criteria included newborns born at a gestational age ≥ 28 weeks and birth weight of ≥ 1.000 g,premature infants and/or low birth weight,and/or small for gestational age infants,without a defined cause.The control group consisted of full-term newborns,with adequate weight for their gestational age,without comorbidities,and whose mothers did not present with any underlying disease or were on any relevant medications in the antenatal period or during delivery.Exclusion criteria included newborns with asphyxia,neonatal sepsis,or other comorbidities and patients whose mothers had obstetrical or peripartum complications.Patients whose mothers used medication during pregnancy also were excluded because these conditions may cause newborns to have low birth weight,small for gestational age or premature birth.Eligible patients were identified from the daily delivery list.

For classification,definitions of small for gestational age[14],low birth weight [15],and premature newborns [16]were based on the World Health Organization.For sample calculation,a previous study by Oliveira et al.[17] was considered,which identified 7.14% viral infections in 70 placentas.Possible losses associated with sample processing and storage were considered,with an estimated 100 patients in each group.

Blood (2 mL) was collected from each patient and centrifuged.Plasma was stored at -80 °C until processing,and non-viable or thawed samples were rejected.Cytomegalovirus-polymerase chain reaction was performed using nested polymerase chain reaction [18].The primers,as described by McIver et al.[19],were produced by IDT—integrated DNA Technologies.Basic local alignment search tool (BLAST)was used to assess the sensitivity and specificity of the primers.For DNA extraction,the Purelink Viral RNA/DNA Mini kit (Thermo Fisher Scientific-USA) was used.Samples for positive (ATG-73) and negative (ATG-126) control of Cytomegalovirus (Cytomegalovirus/Manufacturer Controllab) also were extracted.For the qualitative polymerase chain reaction results,1% agarose gel electrophoresis was performed.

Quantification of the levels of 45 cytokines in plasma was performed using the Milliplex-MAP human high sensitivity t-Cell assay (HSTCMAG-28 K/EMD-Millipore).The following analytes were evaluated:MIP-1alpha,SDF-1alpha,IL-27,LIF,IL-1beta,IL-2,IL-4,IL-5,IP-10,IL-6,IL-7,IL-8,IL-10,PIGF-1,eotaxin,IL-12p70,IL-13,IL-17A,IL-31,IL-1RA,SCF,RANTES,IFN-gamma,GM-CSF,TNF-α,HGF,MIP-1beta,IFN-alpha,MCP-1,IL-9,VEGF-D,TNFbeta,bNGF,EGF,BDNF,GRO-alpha,IL-1alpha,IL-23,IL-15,IL-18,IL-21,FGF-2,IL-22,PDGF-BB,and VEGFA in 96-well plates.Each sample was measured in duplicate.All procedures and analyses of peripheral blood were performed at the molecular laboratory of the INCT—MM.

Data were analyzed using the Statistical Package for Social Sciences (SPSS) 19.0 (IBM,Armonk,NY,USA).Descriptive analysis was performed with frequency and percentage of categorical variables and with mean and standard deviation of quantitative variables.A comparative analysis of the variables between case and control groups and between newborns infected and not infected with cytomegalovirus included Chi-square or Fisher’s exact test for categorical variables and

t

test or Mann—Whitney test for quantitative variables.Statistical significance was considered with

P

≤0.05.The study was approved by the Research Ethics Committee of all the institutions,and informed consent was obtained from parents or legal guardians.

Data were collected from the following 213 newborns who met the inclusion criteria:118 patients and 95 controls.Appropriate samples of adequate volume were selected and matched for case—control comparison.Thus,cytomegalovirus-polymerase chain reaction and cytokine profiling were performed for 75 samples that included 39 from the case group and 36 from the control group.

When comparing 75 newborns with selected samples submitted to cytomegalovirus-polymerase chain reaction and cytokine dosage to the other 138 newborns,the only variable with a statistical difference was prenatal care,with a lower number of consultations in the group of 75 patients(OR=0.016,95% CI 0.03—0.86).

There was no statistical difference when comparing maternal age,prenatal care,number of consultations,previous pregnancies,abortions,type of delivery,or sex of the newborn.The mean number of ultrasounds performed during prenatal care was higher in the case group (

P

＜0.001).Investigation of HIV,syphilis,hepatitis C,rubella,or cytomegalovirus during prenatal care did not show a statistical difference.A greater number of tests was performed in the case group for toxoplasmosis (

P

=0.04) and hepatitis B(

P

＜0.001).Gestational age,weight,length,and newborn head circumference were lower in the case group.Cytomegalovirus-polymerase chain reaction was positive in the peripheral blood of 7 patients with an overall prevalence of 9%:3 (8%) in the control group and 4 (10%)in the case group,without statistical significance (

P

=1.00).Fisher’s exact test presented

P

=1.00 and an odds ratio of 1.25 (95% CI 0.20—9.21).While comparing cytomegalovirus-infected patients(7 patients) and cytomegalovirus-uninfected patients (68 patients),it was found that more women in the latter group had cytomegalovirus test (

P

=0.02).In the cytomegalovirusinfected group (

n

=7),1 pregnant woman did not undergo an antenatal ultrasound,and of the six women who did,no abnormalities were detected.Finally,the number of females was greater in the infected group (

P

=0.03).Table 1 describes clinical characteristics of each infected newborn.However,a Bonferroni adjustment for 22 variables defined significance if

P

＜ 0.002 for each test.Thus,none of the perinatal or clinical characteristics differed significantly between CMV positive and CMV negative infants.The mean cytokine levels of cytomegalovirus-infected patients (

n

=7) and uninfected patients (

n

=68) are described in Table 2.Higher levels of IL-10 (

P

=0.04),IFN-α (

P

=0.02),and TNF-β (

P

=0.01) were observed in the infected group comparing with the control group.However,a Bonferroni adjustment for 45 variables defined significance if

P

＜0.001 for each test.Thus,none of theserum cytokine levels differed significantly between CMV positive and CMV negative infants.

Table 1 Perinatal and clinical characteristics of newborns with and without congenital cytomegalovirus infection,reference maternity hospitals in Belo Horizonte-MG,Brazil,2016—2020

standard deviation, cytomegalovirus.* Mann—Whitney test; ? Student’s Test; ? Chi-square Pearson’s test

Table 2 Mean dosage of cytokines in the blood of newborns,groups with and without congenital cytomegalovirus infection,reference maternity hospitals in Belo Horizonte-MG,Brazil,2016—2020

* Mann—Whitney test; ? Student’s test;Bold letter means ＜0.05

In this study,the overall prevalence of congenital cytomegalovirus was 9.0% (7/75),which is higher than that reported in literature.The cytomegalovirus prevalence ranges from 0.2 to 2.4% in different countries and is more frequent in regions with lower socioeconomic conditions[5,7].Santos et al.[20] found a prevalence of 6.8% for congenital cytomegalovirus infection in newborn infants at the same institution.

The gold standard diagnostic technique for congenital cytomegalovirus infection is viral isolation in urine [20];however,the urine polymerase chain reaction is the most used method considering the ease of running the exam,the high sensitivity and specificity [21].The use of peripheral blood to perform polymerase chain reaction for diagnosis[2,8,22] and follow-up [13,21] of newborns with congenital infections has already been described;however,it has not been validated as a diagnostic method.

In the present study,urine collection was not performed in time owing to difficulties in obtaining urine samples before newborn discharge.Difficulties in the collection and transport of blood samples were highlighted,which led to the exclusion of some patient samples.Considering the importance of congenital cytomegalovirus infection,especially the possibility of neurological sequelae and limitations in establishing the diagnosis due to difficulties in performing the standard test in a timely manner,newer diagnostic methods are needed.Ross et al.studied the diagnosis of congenital cytomegalovirus through urine polymerase chain reaction on cotton balls [23] and Koyano et al.[24] studied the diagnosis through urine polymerase chain reaction on filter paper;however,neither technique was validated.

Although a higher prevalence of congenital cytomegalovirus was seen in this study than that described in the literature,one must also consider the possibility of underdiagnosis because testing was done on peripheral blood and infected newborns will not necessarily have viremia [9,22]at the time of testing.

The higher prevalence of congenital cytomegalovirus infection found in this study can be attributed to the low socioeconomic level of the population.In Brazil,Mussi-Pinhata et al.found a seroprevalence of 97% among lowincome and urban pregnant women [25].Cytomegalovirus can be transmitted to the fetus when a pregnant woman has a primary infection,reactivation,or reinfection [26,27].

When comparing the case (

n

=39) and control (

n

=36)groups,it was found that a higher number of cases were screened for hepatitis B and toxoplasmosis in the case group.This can be attributed to different antenatal protocols [28];however,the difference was not observed in the general group (

n

=213).

Several authors have studied the importance of congenital cytomegalovirus infection in prematurity,low birth weight,and small for gestational age infants with variable prevalences,differing in country,population,and method of diagnosis [2,3,7— 10].This is the first study investigating congenital cytomegalovirus infection through blood polymerase chain reaction in premature,low birth weight or small for gestational age newborns.In previous studies the highest prevalence was described by Panhani and Heinonen [10],who identified four infections (4.8%) in 83 Finnish preterm newborns before 34 weeks of gestational age.Urine virus culture,which is considered the gold standard,was used for confirmation of diagnosis of congenital infections allowing for additional diagnoses.However,the small sample size may have influenced the results.Turner et al.[7] also used urine viral culture to investigate congenital cytomegalovirus infections and found a prevalence of 0.39% (18/4594) in American preterm newborns with very low birth weight.

In Brazil,Yamamoto et al.investigated cytomegalovirus infection in preterm and full-term newborns in a population under similar socioeconomic status [5].The authors performed polymerase chain reaction and urine culture which have high sensitivity and specificity [29].A positive rate of 2.1% in preterm newborns and 1.8% in full-term newborns was identified,without statistical difference.In this study,the prevalence of cytomegalovirus infection in the case group was 10% and 8% in the control group,which was higher than that reported in previous studies.

In this study,the cytokine profile did not present a difference between the cytomegalovirus-positive group and the non-cytomegalovirus group;however,the small sample size may have influenced the results.Chen et al.observed a significant increase in IL-33 levels in newborns with congenital cytomegalovirus infection compared to patients without infection [30].Numazaki et al.observed high levels of IFN-γ and TNF-α in two newborns with symptomatic congenital cytomegalovirus compared with two asymptomatic newborns [31].They also found higher cytomegalovirus-specific CD4+T lymphocytes in symptomatic patients than those in healthy,immune to cytomegalovirus and without urinary viral excretion.Although the association between cytokine levels and congenital infections seems important,studies on this topic are rare.

The presence of proinflammatory cytokines in children with positive cytomegalovirus-polymerase chain reaction must be studied further and followed-up to define consequences of exposure to the infection in fetal life.Infants exposed to microorganisms or other stimuli can present with fetal inflammatory response syndrome and have a high chance of presenting with early onset neonatal sepsis,intraventricular hemorrhage,periventricular leukomalacia,and death.Some complications may emerge as sequelae,including bronchopulmonary dysplasia,neurodevelopmental disorders,retinopathy of prematurity,and hearing loss,worsening the development and adaptability of these children [32].Potential pharmacological interventions could minimize the impact of the virus and a damaging proinflammatory response [33].

Cytomegalovirus-infected newborns may present prolonged viral replication [11— 13].Although the cytokine profile did not present differences between the two groups,new studies with a larger sample could help to define the prognosis and the potential treatments with immunomodulatory therapies.This is a preliminary study on small for gestational age,low birth weight and preterm newborns without any clinically apparent cause.

Cytomegalovirus was highly prevalent in this sample of tested newborns.Routine antenatal screening for cytomegalovirus needs to be considered particularly in the susceptible populations.Congenital infection by cytomegalovirus may modify inflammatory response;however,it was not observed in this study.This needs to be studied further to devise management protocols in infected newborns.

Acknowledgements

To Professor Dr Erna Kroon,Laboratory of Virology—Federal University of Minas Gerais,who provided samples for CMV-PCR controls.

Author contributions

All authors participated in substantial contributions to the conception or design of the work;or the acquisition,analysis,or interpretation of data for the work,drafting the work or revising it critically for important intellectual content,final approval of the version to be published,and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Funding

The study had financial resources obtained by the FAPEMIG Universal Notice and through the Institutional Scientific Initiation Scholarship UFMG/FAPEMIG and UFMG/CNPq.

Data availability

Patient data may not be available once the original project approved by the Ethics Committees did not predict the availability of the database.

Declarations

Conflict of interest

No benefits have been received or will be received from any author related directly or indirectly to the subject of this article.

Ethical approval

The study was approved by the Research Ethics Committee of all the institutions (CAAE 07,849,612.6.0000.5149;07,849,612.6.3001.5119;07,849,612.6.3002.5132).Informed consent to participate in the study have been obtained from parents or legal guardian of patients.