Cytomegalovirus: congenital infection and clinical presentation in infants with respiratory distress syndrome

México

Communication with: Angélica María Martínez-Contreras

Email: angy1040n@prodigy.net.mx

Background: Respiratory distress syndrome (RDS) is a multifactorial and common disease that varies from 15 to 50 % in the newborn, causing 50 % of mortality. The RDS may be associated with bacterial and viral infections, and one of the most common viral agents is the cytomegalovirus (CMV). In the neonatal period the virus incidence goes from 0.4 to 2.5 % with a seroprevalence of 50 to 75 %; the incidence of infection in newborn with RDS is unknown. The objective was to determine the frequency of CMV infection in neonates with RDS and identify the risk factors associated with infection.

Methods: The CMV-DNA was identified in plasma by quantitative PCR; maternal and neonatal variables that defined the clinical findings were analyzed by logistic regression.

Results: The frequency of CMV infection in 197 infants with RDS was 8.6 % (95 % CI, 4.7-12.5). The significant variables in newborn were: neutropenia (p = 0.012), thrombocytopenia (p = 0.021), mottled skin (p = 0.03), and the maternal significant variable was cervicovaginitis (p = 0.05).

Conclusions: We reported for the first time the highest frecuency of CMV infection in newborns with RDS and the association of various risk factors with CMV infection.

Keywords: Newborn respiratory distress syndrome; Newborn infant; Cytomegalovirus; Polymerase chain reaction

Respiratory distress syndrome (RDS) is one of the most common diseases in newborns (NB). It occurs in developed and developing countries, with a frequency ranging from 15 to 50%.¹ The morbidity and mortality is variable and is related to the heterogeneity of treatment and level of hospital care.¹ RDS can be associated with diseases such as hyaline membrane disease, transient tachypnea of ​​the newborn, cardiopulmonary malformations, and bacterial and viral infections.² These latter, mainly acquired  congenitally, may manifest as pneumonia and pulmonary complications.³ The viral agents that may be most commonly associated with pneumonia and RDS are those of the herpes group, including cytomegalovirus (CMV). This virus is the most common cause of congenital infection in the United States and Europe. In the neonatal period it has an incidence of 0.4 to 2.5% and seroprevalence of 50 to 75%.⁴ Between 10 and 20% of patients suffering from congenital infection have neurological and sensory sequelae or hearing loss, eye disorders, and impaired motor and cognitive functions.⁵ The clinical presentation of the infection in newborns varies: it may be asymptomatic or may present serious symptoms of sepsis, pneumonia, pneumonitis^6,7 and bleeding that can be deadly in 20 to 30%,⁸ so in the case of newborns with RDS, the infection should be identified.

The detection of CMV infection in newborns has limitations due to the availability and cost of tests for screening and diagnosis in hospitals. In developing countries, the quantitative determination of IgG and IgM antibodies against cytomegalovirus (IgG-CMV and IgM-CMV) is used most for the diagnosis of CMV infection in newborns. The positivity of IgG-CMV with high levels or positivity of IgM-CMV, coupled with the clinical status of the newborn and maternal history of positivity of IgG-CMV and/or IgM-CMV, are indicators of CMV infection;⁹ however, the sensitivity of IgG-CMV is less than 70% and IgG decrease can be presented at one month old,¹⁰ so it is recommended to combine with other more sensitive tests, such as detection of CMV-DNA using real-time PCR (qPCR/CMV-DNA).¹¹ One of the genes used in this technique to detect CMV viral material is the UL55 gene. This gene encodes glycoprotein B (gB), which is the predominant envelope glycoprotein. The test has a sensitivity and a specificity of up to 98%.^9,12

In Mexico there have been two studies in newborns with congenital CMV infection, which was identified in saliva culture and filter paper. The incidences obtained have been low: 0.68 and 0.89%, respectively.^13,14 These results are similar to those reported in other countries like the United States and Spain.^4,5,9 However, there are no studies that analyze RDS frequency in relation to CMV infection, to give us a current overview of this disease, which is considered a global public health problem because of the economic impact on health systems.^5,9

Therefore, an investigation was done of CMV infection in newborns with RDS treated at the Hospital de Gineco-Pediatría con Medicina Familiar 31 in Mexicali, Baja California.

The objective was to determine the frequency of congenital CMV infection in newborns with RDS, to analyze clinical and laboratory characteristics, and to identify risk factors associated with infection.

Methods

A transverse, prolective, analytical, and comparative study of two groups was done in a population of 237 newborns, starting at 25 weeks gestational age and with RDS, during the period from August 2010 to February 2012. Plasma and serum samples were obtained from neonates admitted. Newborns born in other institutions, those involved with transfers, and those without authorization for sampling were excluded from this study. Of 237 patients recruited, 40 were excluded, one for the mother's request to withdraw them from the study, and 39 patients who did not have full information, so the sample was of 197 RN.

The study was approved by the Local Health Research Committee of the IMSS Hospital General de Zona 30 and informed consent was requested of patients’ parents (record: R-2009-201-3).

Serum samples detected anti-CMV IgG and IgM using the equipment VITROS® ECiQ Immunodiagnostic from Ortho-Clinical Diagnostics, with the corresponding reagents. The concentration of IgG-CMV was reported in IU/mL with cutoff of IgG ≥ 8 IU/mL; the concentration of IgM-CMV was expressed in AU/mL, with cutoff of ≥ 1.0 AU/mL.¹⁵

DNA from plasma samples were extracted using the Favor PreTM Viral Nucleic Acid Extraction Kit I (FAVORGEN®) following manufacturer specifications. QPCR was standardized for identification of CMV-DNA with SYBR Green (SYBER Green I Master Roche) with real-time PCR equipment Light Cycler LC480 (Roche). The standard curve was prepared with a detection range of 106-103 copies/mL, using a CMV-DNA control (Vircell Microbiologists). The sequence of primers used was: Gene gB1-MF, direction 5'-GAGGACAACGAAATCCTGTTGGGCA-3', position 82663-82639 and gB2-MR opposite direction 5'-TCGACGGTGGAGATACTGCTGAGG-3', position 82515-82538, which amplify a 125bp fragment.¹⁶ The conditions for qPCR were as follows: 5 mL of Master Mix LC®480 SYBER Green I Master 2X (Roche), 0.75 mM of gB1-MF and gB2-MR, and 3.5mL of DNA for each reaction, with the amplification conditions of 95 °C for 5 minutes and 45 cycles of 10 seconds at 95 °C, 10 seconds at 60 °C and 10 seconds at 72 °C.

All qPCR samples were analyzed in triplicate. Data analysis was performed using LC® 480 software, version 1.2 (Roche).  

For evaluation the following variables were collected on admission of each newborn: gestational age; sex; weight; Apgar assessment; new Ballard; neurological, pulmonary, cardiac, gastrointestinal, renal, hematologic, and infectious clinical signs; additional diseases; blood count; liver function tests; and maternal biological, laboratory, anthropometric, and sociodemographic variables.

Descriptive statistics were used for neonatal and maternal clinical variables. Measures of association were analyzed using odds ratios (OR) with a confidence interval of 95% (CI 95%), Mantel-Haenszel chi-squared and Fisher’s exact test, with significance level of < 0.05. Unconditional binary logistic regression was done. SPSS statistical software program, version 17 was used.

Results

Identification of congenital CMV infection

Out of a total of 197 samples of newborns with RDS analyzed by qPCR/CMV-DNA, 17 positive samples (8.6%; 95% CI 4.7-12.5) were identified and considered as congenital infection in the present study. In serology, 93.9% (185 of 197; 95% CI, 90.6-97.2) were positive for IgG-CMV and IgM-CMV, 93.5% (173 of 185; 95% CI, 90.1-96.9) positive for IgG-CMV and 6.4% (12 of 185; 95% CI, 3-9.8) were positive for IgM-CMV. Of the 12 newborns who were serologically positive for IgM-CMV, CMV-DNA was not detected in any.

Serological, hematological, and liver function test results were analyzed in the group of newborns with RDS infected and uninfected by CMV; in both groups, the most frequent alterations were neutropenia and lymphocytosis (Table I).

Clinical characteristics of the group of newborns with RDS and infection

Median gestational age, weight, and length were reported for the 17 neonates in whom CMV-DNA was identified by qPCR. The range of weeks of gestational age was 25 to 41, median 34.4 weeks, weight 600-4720 g, median 2000 g, and length of 32-56 cm, median 44 cm. 76.4% were premature; females predominated with 64.7%, 70.5% of the newborns had adequate weight for gestational age, 29.4% had low weight for gestational age (SGA) 23.5% had low height; the most common route of birth was cesarean with 76.47%.

Clinical presentation of CMV infection in newborns

The clinical manifestations of the 17 newborns were classified as consistent with sepsis. The most frequent were lethargy (70.5%; 95% CI 64-77), jaundice (64.7%; 95% CI 58-71), hyperthermia (52.9%; 95% CI 46-60), mottled skin (35.8 95% CI 29-42%), hypoglycemia (35.3%; 95% CI 28-42), hypothermia, and pale skin (29.4%; 95% CI 23-35). When gastrointestinal involvement was detected, demonstrations were intestinal loop disorder and gastric residuals; children with impairment of the hepatobiliary system more frequently showed unconjugated hyperbilirubinemia; neurologically, lethargy and apnea were predominant; for cardiopulmonary conditions, hypoventilation and crackling rales; hematologically, infection manifested in petechiae and bleeding at different levels, such as upper gastrointestinal (8 of 17), intraventricular hemorrhage (3 of 17) and pulmonary hemorrhage (1 of 17).

Table II summarizes percentages and 95% CI of the clinical manifestations. The most frequent early clinical manifestations were neurologic, hepatobiliary, hemorrhage, and cardiopulmonary.

In patient R-009 Staphylococcus epidermidis was detected in umbilical catheter. In patient R-071 Candida albicans was detected in gastric secretion and Staphylococcus epidermidis in umbilical catheter.

Table III describes clinical diagnosis of each patient admitted to the different neonatal units; the most common diagnoses were: prematurity 76.4%; early sepsis and multifactorial jaundice 52.94%; pneumonia, hyaline membrane disease and upper gastrointestinal bleeding 47.05%.

Congenital birth defects in infected newborns  

A patient was detected with Down’s syndrome and bilateral cryptorchidism (R-009), another with hydrocephalus (R-071), and another with gastroschisis (R-229). In patient R-009 ductus arteriosus, atrial septal defect, and ventricular septal defect were detected by echocardiography, along with tricuspid insufficiency and severe pulmonary hypertension. Patient R-028 presented patent ductus arteriosus, patent foramen ovale, tricuspid insufficiency, and moderate pulmonary arterial hypertension. Patient R-048 presented ductus arteriosus and permeable foramen ovale.
Other findings in radiological imaging studies showed that patients R-009 and R-164 had Grade I intraventricular hemorrhage (ependymal), patient R-071 had grade IV intraventricular hemorrhage (parenchymal) and hydrocephalus detected by transfontanelar ultrasonography. Cranial CT of patient R-028 showed signs of brain edema by asphyxiation.

As for the results of chest radiography of the newborns, the main findings were: diffuse interstitial infiltrate 88.2% (suggesting pneumonitis), and localized alveolar infiltrate 70.5% (suggesting pneumonia), increase in size of the cardiac silhouette 64.70%, dense focal consolidation 23.5% and atelectasis 17.6%.

Analysis of maternal risk factors for congenital CMV infection

Table IV describes risk variables inherent to the mother that are more frequent for congenital CMV infection in newborns with RDS. Among the most common factors were infections, low socioeconomic status, work outside the home, preterm birth, and that the mother was a passive smoker. The mothers of patient R-048 and patient R-229 were positive for IgG-CMV and IgM-CMV.

Table V describes the important perinatal history that may influence CMV infection. The mothers of patients R-048, R-086, R-101 and R-229 were exposed to children under 4 years (increased risk of excreting CMV in urine) due to their type of work, for example, working in day care, pediatric hospitals, and restaurants.¹⁷

In most of the mothers, pregnancy was concluded by caesarean section. The main indications were for preeclampsia and premature rupture of the amniotic membranes (29.41%), acute fetal distress (23.52%) and preterm labor, severe oligohydramnios and chorioamnionitis, including fetid amniotic fluid and septic delivery (17.64%). Patient R-073’s mother had twin births and the second twin was stillborn.

In univariate analysis of the newborns’ variables, neutropenia (OR 3.47, 95% CI 1.09-11.07, p = 0.05), lymphocytosis (OR 3.28, 95% CI 1.11-9.70, p = 0.04), thrombocytopenia (RM 3.05, 95% CI 1.04-8.94, p = 0.07), hyperthermia (OR 2.84, 95% CI 1.04-7.78, p = 0.06) and mottled skin (OR 3.23, 95% CI 1.14-9.14, p = 0.04) were associated with congenital CMV infection and were identified with OR > 2.

In the unconditional binary logistic regression, significant variables for CMV infection were neutropenia p = 0.012, thrombocytopenia p = 0.021, and mottled skin p = 0.032 in newborns, and cervicovaginitis p = 0.050 as a maternal factor.

Discussion

In Mexico there are few reports of CMV infection in newborns. The results of this study made it possible to determine the frequency of CMV infection and the characteristics of clinical manifestations in a population of newborns with RDS in a hospital in Mexicali. We detected the presence of CMV-DNA in the blood of 8.66% of the newborns from the 25^th week of gestation to term newborns of 41 weeks gestation with RDS. Seropositivity for IgG-CMV or IgM-CMV in newborns with RDS was 93.9% and none of the newborns infected with CMV had positive IgM, which shows the low diagnostic sensitivity of serology for the detection of congenital infection. The frequency of infection reported in this paper is considered high when compared with current figures for other hospitals in other parts of the world that handle infected and asymptomatic neonates (0.3 to 2%).^4,5,9 We confirmed that CMV infection was congenital¹⁸ and not postnatal by analyzing some of the risk factors of postnatal infection, according to which the termination of pregnancy was mostly by cesarean section (76.47%), only 17.64% were transfused with blood products, and only 2 newborns received maternal milk, without these data being significant in the analysis.¹⁹

In our study, the percentage of preterm newborns with RDS who were positive for CMV-DNA was 76.4%, higher than that reported in the literature (38%),18 while the rate of low weight for gestational age coincided with that reported by Kylat.^18,20 Prematurity and low weight for gestational age are the two conditions that increase the risk of congenital CMV infection due to immaturity of the immune system and the low concentration of maternal immunoglobulins.^19,20

The clinical manifestations due to the condition of the various organ systems determine a risk to the life of the newborn. The most common clinical signs in our cases were the same as those reported in the literature: prematurity, multifactorial jaundice, hepatomegaly, lethargy, hyperthermia, petechiae, and mottled skin.^8,18,20 The predominant pathologies in these patients were: hyaline membrane disease (HMD), early sepsis, liver cholestasis, upper gastrointestinal bleeding, and intrauterine pneumonia/pneumonitis; the latter are referred to in the literature as isolated cases.^6,7,8,20

In nine newborns with mild RDS we detected CMV infection with minimal clinical manifestations, a worrying situation if the infection goes unidentified because of 10 to 15% of asymptomatic children present neuromotor and sensory sequelae.^4,5,21

Of the most common laboratory findings in newborns with CMV and those mentioned in the literature, we found thrombocytopenia, high indirect and direct bilirubin, and high glutamic oxaloacetic transaminase, in addition to neutropenia, basophilia, lymphocytosis and leukocytosis. The presence of these hematological disorders indicates a high probability of symptomatic infection.^{5,8,9,18,20,21}

Throughout this study 5.88% mortality was seen, indicating low mortality compared with those reported by Kaneko et al.,8 which was 20 to 30% for symptomatic newborns.⁸

In the maternal serological diagnosis we do not know if seroconversion occurred during pregnancy, reinfection, or superinfection because of a lack of several serological determinations of IgG-CMV and IgM-CMV in mothers during pregnancy. 94.11% of mothers of newborns with RDS infected with CMV had positive IgG-CMV, compared with those reported in other Latin American countries, according to which 60 to 80% of women between 20 and 40 years and have been exposed to the virus.^11,22

In the literature sociocultural factors have been identified including low socioeconomic status, mostly working mothers, and low level of schooling, leading to a lack of health knowledge and inadequate prenatal monitoring.^{9,17,19,20,22,23} In our study we only identified cervicovaginitis as a significant variable.

In pediatric clinical practice CMV infection should be intentionally searched for in the group of newborns with perinatal risk factors and respiratory diseases at birth. It is also necessary to monitor newborns with congenital infection, especially in children of HIV-positive mothers, neonates born at less than 32 weeks and weighing less than 1500 g, and with low weight for gestational age.^9,24 This will enable the identification of a large group of infected newborns because the consequences are much more common in newborns who have symptoms at birth than in asymptomatic children, and one of the important aspects is to predict which children might have long-term sequelae and require admission to a multidisciplinary group for rehabilitation and to reduce the economic impact to the health system.^{5,9,19,20,21,25}

Conclusion

This is the first study reporting the highest frequency of CMV infection in newborns with RDS and identifying neonatal variables that define the clinical chart and maternal risk variables that can contribute to infection. Our results suggest the importance of identifying CMV infection in asymptomatic and symptomatic newborns, especially if they have risk factors.

Acknowledgements

We thank Doctors Ricardo de Leon Figueroa, Eduardo Romero Martinez, and Laura Monge Siordia; the laboratory personnel of the Hospital General de Zona 30; Ivet Borja and Distribuidor Científico Pallach Tijuana (DICIPA). This project was conducted with financial support from the Fondo de Investigación en Salud (FIS/IMSS/PROT/MD13/1255).

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