Maternal IgG avidity, IgM and ultrasound abnormalities: combined method to detect congenital cytomegalovirus infection with sequelae

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Abstract

Objective:

We used maternal immunoglobulin M (IgM), immunoglobulin G (IgG) avidity index (AI) and fetal ultrasonography (US) to effectively detect a congenital cytomegalovirus-infected fetus that would suffer neurological sequelae after birth.

Study Design:

The detecting method was prospectively adapted to 1163 unselected pregnant women. IgM, IgG and IgG-AI were measured at the first prenatal examination (10.8±2.2 weeks of gestation). Advanced US was performed for the IgM-positive women at our center. The urine of 1163 neonates was examined via PCR. All infected neonates were followed for neurological development.

Result:

Most women (83.3%) were seropositive. Among them, 40 (4.1%) were IgM positive. Nine of forty (22.5%) had low AI, of which one showed abnormal US and suffered severe sequelae. The remaining eight had a normal US; however, one infant had hearing impairment. There were another three infected infants with normal development. Their mothers’ serological results were: IgM positive with high AI (n=1); IgG positive; IgM negative with high AI (n=1); and both IgG and IgM negative (n=1).

Conclusion:

This method enabled us to detect infected fetuses having severe sequelae. However, the problem remains of detecting infected fetuses that only have a hearing impairment.

Introduction

Human cytomegalovirus (CMV) is known to be one of the most common fetomaternal viral infections; it occurs in 0.3–2.3% of all live births.1, 2 Congenital CMV infections can cause permanent physical sequelae or impairments that result in disabilities such as hearing loss, visual impairment and mental retardation.3, 4 Nevertheless, no nation performs systematic screening for CMV infection among pregnant women5 because no vaccine or treatment has been proven effective.

Although the Japan Society of Obstetrics and Gynecology does not recommend universal screening for CMV primary infection among pregnant women, random screening has been conducted in some hospitals. However, misinterpreted test results have a strong influence on a woman’s decision to terminate her pregnancy; thus, this may cause a serious social problem. Furthermore, screening could help prevent congenital infections because seronegative pregnant women could be given basic information on how to avoid them.6, 7

Ultrasound is widely used to screen for various congenital anomalies and to evaluate fetal well-being. Ultrasound may be a useful diagnostic tool for pregnant women with CMV infection. However, Guerra et al. conducted a retrospective cohort study and concluded that ultrasound abnormalities predict symptomatic congenital infection in only a one-third of cases.8 New ultrasound parameters to identify infected fetuses at risk of symptoms after birth have not yet been devised. Furthermore, prospective studies to identify infected fetuses with neurological impairments using a combination of maternal serological tests and fetal ultrasound have rarely been reported.

The amniotic fluid is subjected to a direct search for CMV in culture and the viral genome can be identified by PCR. Positive results in the amniotic fluid identify CMV-infected fetuses but do not discriminate those infants who will have symptoms after birth. As amniocentesis is an invasive procedure, it might best be reserved for high-risk fetuses likely to have abnormal neurological development.

Mother-to-child transmission is mainly the result of primary maternal CMV infection, which carries a risk of transmission varying from 14.2 to 52.4%.9 The immunoglobin G (IgG) avidity assay can help distinguish primary from non-primary human CMV infection. This assay is based on the observation that virus-specific IgG of low avidity is produced during the first months after onset of infection, whereas subsequently a maturation process occurs by which IgG antibody of increasingly higher avidity is generated.10, 11

In view of the foregoing, we performed a pilot study whether antenatal screening using maternal CMV immunoglobin M (IgM), IgG avidity and fetal ultrasonography (US) can detect an infected fetus likely to have abnormal neurological development. Furthermore, we measured CMV IgM antibody and IgG avidity in sera from 1163 pregnant Japanese women to determine the prevalence and demographic trends of these values.

Materials and methods

The prospective cohort study was approved by the Institutional Review Board of the Faculty of Medicine at the University of Miyazaki, and written informed consent was obtained from all study participants.

This study was conducted from 1 January to 31 August 2008. The flow chart to detect congenital CMV infection in this study is presented in Figure 1. We collected maternal serum at the first prenatal visit, when blood tests were obtained; we measured CMV IgM and IgG by ELISA (SRL, Tokyo, Japan). Cutoff values of CMV IgG and CMV IgM were 2.0 and 0.8, respectively. Maternal sera were stored for the measurement of CMV-IgG avidity index (AI). Women who were CMV seronegative (for both IgG and IgM) had blood samples drawn at the second and third trimester to examine seroconversion. Detailed hygiene information to protect CMV infection was not systematically given to the seronegative women. Routine ultrasound examinations were performed by obstetricians at each clinic during the second and third trimesters for all pregnant women.

Figure 1
figure1

Flow chart to detect an intrauterine cytomegalovirus-infected infant with sequelae. AI, avidity index; ABR, auditory brain stem response; IgG, immunoglobulin G; IgM, immunoglobulin M; US, ultrasonography. *When AIs were measured, maternal sera collected at first trimester were used.

Women who were CMV IgM positive were referred to the university hospital. Women positive for CMV IgM were counseled about fetal risk, the advisability of ultrasound monitoring, and the possibility to diagnose fetal infection by amniocentesis at 20–21 weeks’ gestation. They were then screened for ultrasound abnormalities suggestive of congenital infection such as intrauterine growth restriction, hydrops or ascites, hyperechogenic bowel, pleural or pericardial effusion, hepatosplenomegaly, oligohydramnios or polyhydramnios, placental enlargement or central nervous system abnormalities.

We performed a PCR using urine from all newborns within 5 days following birth to confirm the congenital infection. All infants were systematically examined including the eyes, ears and heart. For all congenitally infected infants, auditory brainstem response audiometry, cranial US and ophthalmoscope examinations were performed. All congenitally infected infants were carefully followed for neurological development.

CMV IgG avidity testing

CMV-IgG-positive sera were tested for CMV-IgG AI. IgG avidity tests were carried out as previously described,12 with slight modification: a CMV ELISA kit (Enzygnost anti-CMV, Dade Behring, Germany) was used. The antibody AI (%) was calculated as the mean absorbance at 450 nm (OD 450) of the urea-washed wells divided by the mean OD 450 of the control wells without urea washing; the index35% was defined as low IgG avidity.10, 12

In women with CMV-IgM positive, the result of CMV-IgG AI was referred on the occasion of counseling. CMV-IgG AI was also measured for investigation of correlation with the presence of CMV IgM.

Statistical analysis

Multiple group comparisons were performed by analysis of variance, followed by post-hoc paired t-test with Bonferroni correction if a significant F ratio was obtained. Correlations between low AI and congenital CMV infection were examined with the χ2 test and the Fisher test. Simple linear regression was used to determine the correlation between IgM values and AI (StatView software; SAS Institute, Cary, NC, USA). A probability value <0.05 was considered statistically significant. Data are presented as mean±s.d.

Results

CMV IgG and IgM prevalence

Pregnant women enrolled in this study had their first blood sampling drawn at 10.8±2.2 weeks of gestation. Overall, 83.3% of the women (969/1163) were CMV IgG seropositive (Figure 2). Among them, 40 (4.1%) were CMV IgM positive at the first trimester. There were 194 (16.7%) seronegative women. One (0.5%) of the 194 IgG-negative women showed seroconversion during pregnancy (Figure 2).

Figure 2
figure2

Serological prevalence and patient flow diagram. Ab, abnormal; AI, avidity index; G, cytomegalovirus IgG; M, cytomegalovirus IgM; Nor, normal; US, ultrasonography.

Table 1 shows CMV IgG and IgM prevalence according to age. The CMV seropositivity rate steadily increased with age. In women younger than 20 years of age, approximately half (45.5%) were CMV IgG seronegative. A significant difference was observed in the prevalence of seronegative women, when comparing women younger than 20 years of age with all other age groups.

Table 1 CMV IgG and IgM prevalence according to age among 1163 women

Relationship between IgM antibody levels and IgG avidity

CMV IgM-negative sera (n=929) and IgM-positive sera (n=40) of various IgG antibody levels were examined for IgG avidity. The prevalence of low avidity among CMV IgM-negative and IgM-positive sera were 69/929 (7.4%) and 9/40 (22.5%), respectively (Figure 2).

The relationship between IgM antibody levels and IgG avidity among women who were CMV IgM positive (n=41) during pregnancy was analyzed. No correlation was seen between IgM levels and IgG avidity (R2=0.157). The relationship between IgM antibody levels and IgG avidity was also analyzed among CMV IgG-positive and CMV IgM-negative women (n=929); no significant correlation was found (R2=0.005). The incidence of presence of IgM antibody, congenital CMV infection and sequelae are presented in Table 2. IgM antibody was positive for various IgG AIs. Although congenital CMV infection occurred even in cases with high IgG AI, infants with sequelae were observed only in cases with low IgG AI. The incidence of congenital CMV infection in women with <35% of AI was significantly higher, when compared with that in women with any other AI values (P<0.01).

Table 2 The incidence of presence of IgM, infection and sequelae according to IgG Al

The method to detect the intrauterine infection with sequelae

Our method to detect congenital CMV infection with sequelae was adapted to the 1163 pregnant women (Figure 2). Overall, five women delivered congenital CMV-infected infants.

In nine women with IgM positive with a low IgG AI, there were two women who delivered infants with neurological sequelae (Figure 2). One (11%) of nine women showed abnormal ultrasonographic findings (ventricular dilatation, hyperechogenic bowel, oligohydramnios) and the infant had cerebral palsy, mental retardation and bilateral hearing loss. The remaining eight had normal ultrasonographic findings (Figure 2); however, one infant had bilateral hearing loss.

Among women with IgM-positive and high IgG AI at first serological test (n=31), one had abnormal ultrasonographic finding (mega cistern magna); however, she was not infected (Figure 2). The remaining 30 women had normal ultrasonographic finding; one infant had an asymptomatic infection.

Amniocentesis was discussed with all pregnant women who were IgM positive; seven (17%) of them underwent amniocentesis.

Among 929 women who were IgG positive and IgM negative, 860 had a high IgG AI and normal ultrasonographic findings (Figure 2); one infant had an asymptomatic infection. The remaining 69 infants (7.5%) had low IgG AIs and normal ultrasonographic findings; none were infected (Figure 2).

During the study period, 1 out of 194 (0.5%) woman with IgG negative and IgM negative had seroconversion at 17 weeks’ gestation. PCR using amniotic fluid at 20 weeks’ gestation was negative; however, PCR using the newborn’s urine was positive. Ultrasound monitoring during pregnancy did not reveal abnormalities. One infant had an asymptomatic infection.

Discussion

We performed a pilot study to find out whether antenatal screening using maternal CMV IgM, IgG avidity and fetal US can detect an infected fetus likely to have abnormal neurological development, because the prevalence of congenital CMV infection is low. In this study, we prospectively applied a method to detect congenitally infected fetuses with sequelae among 1163 unselected pregnant women. Candidates for congenitally infected infants with sequelae comprised nine women (0.8%) with IgM positive and low IgG AI, namely primary maternal infection. The measurement of IgM and IgG AI enabled us to select nine women from all the pregnant women in this study. The women with low IgG AI had a risk of congenital CMV infection in this study. Furthermore, IgG AI may be useful in detecting congenital CMV infection, when it is used with IgM. Lazzarotto et al. followed a cohort of 1520 pregnant women considered at risk of transmitting the virus. They showed that of 316 pregnant women who had both CMV IgM and low IgG AI, 286 fetuses/infants were examined, and 87 (30.4%) of them were infected.13 Munro prospectively performed screening for 600 pregnant women. They showed that 7 of 33 women with CMV IgM positive had low IgG AI and 2 (28.6%) had infected infants.14 Picone et al. conducted a prospective study with 3665 pregnant women who agreed to screening. Nine of ninety-eight women with CMV IgM positive had low IgG AI and one (11.1%) had an infected infant.15 Leruez-Ville performed a retrospective cohort of 4931 women. Forty-five of two hundred and one women with positive or equivocal IgM had low IgG AI. Forty-one fetuses/infants were examined and fifteen (36.5%) were infected.16 We think that it is important to measure both CMV IgM and IgG AI in order to detect women at high risk of congenital CMV infection.

These women were targeted for further investigation with the aid of US. Although two of these nine women had infected infants, ultrasound abnormalities were only observed in one fetus that had severe neurologic sequelae. The other had hearing impairment and normal findings on ultrasound. Ultrasound is a noninvasive procedure that can disclose structural or growth abnormalities caused by a CMV infection. Guerra et al. conducted a retrospective study and reported that ultrasound had low sensitivity; it correctly identified only a third of the cases in a selected population.8 Conversely, Benoist et al. retrospectively analyzed data collected prospectively in antenatally diagnosed cases and found that the presence of ultrasound abnormalities (even in the absence of ultrasonographic features of brain involvement) was a significant and independent predictor of poor outcome, including sensorineural hearing loss.17 In our series, we reported a limitation of an ultrasound examination in a case with solitary hearing impairment at birth. Similarly, Endres et al. reported that there were cases without fetal ultrasound abnormalities, which manifested hearing loss at several months or years after birth.18 Moreover, prenatal assessment should account for the fact that fetal ultrasound anomalies can sometimes be revealed only late in the pregnancy. Furthermore, they may vary or even disappear over time.19, 20, 21 However, we did not observe any such cases in our series. Taking cost-effectiveness into consideration, at this point in time we think that ultrasound examination for detecting congenital CMV infection should be performed for women at risk based on serological results.

Maternal CMV seropositive prevalence was 83% in our study population. The reported seropositive prevalence varies from 44 to 94%.22 It depends on the reported year or the targeted population (eg, socio-economic status). Our study population was unselected and socio-economic status was average (primarily middle socio-economic status), according to Japanese standards. Nevertheless, the maternal seroprevalence was higher than other reports.22 A substantial drop in maternal seroprevalence from 85% in 1988 to 68% in 2000 was observed in Sapporo, Japan.23 A serological survey has been conducted in Nagasaki, Japan, which reported a seropositive prevalence (87%) as high as that in our study.24 It is unlikely that a significant difference in socioeconomic status was present among different regions in Japan. Our study also found that the seronegative prevalence in women younger than 20 of age was about 50%; however, it dramatically increased in women older than 20 years. We theorized that a change in lifestyle or social activities may be responsible for the high seropositive prevalence. Vauloup-Fellous et al. reported that hygiene counseling given during pregnancy reduces the CMV seroconversion rate.25 It appears to be important that basic hygiene information before conception as well as during pregnancy should be given; however, it remains to be determined how to best disseminate this information.

One limitation of our study is the small study population. Conversely, the strength of this study is that we studied an unselected population, prospectively observed the pregnancy course, including ultrasound findings for all women, and studied the neurological developments of all infected infants. Following the results of our pilot study, further studies are needed to confirm the usefulness of IgG AI and antenatal ultrasound in detecting intrauterine CMV infection.

In summary, with our protocol and algorithm, we were able to detect infected fetuses with severe sequelae. However, the problem remains regarding the detection of an infected fetus that has solitary hearing impairment.

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Acknowledgements

We thank Drs Shigeki Tanaka, Syunichi Noda, Hiroyuki Watanabe and Takashi Matsumura for collecting sera and providing the patient data. This work was partly funded by a grant (No.19591899) from the Japan Ministry of Education, Culture, Sports, Science, and Technology.

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Correspondence to M Kaneko.

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The authors declare no conflict of interest.

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Kaneko, M., Sameshima, H., Minematsu, T. et al. Maternal IgG avidity, IgM and ultrasound abnormalities: combined method to detect congenital cytomegalovirus infection with sequelae. J Perinatol 33, 831–835 (2013) doi:10.1038/jp.2013.87

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Keywords

  • congenital infection
  • cytomegalovirus
  • epidemiology
  • prognosis
  • ultrasound

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