Objective
To determine contribution of reinfection with new strains of cytomegalovirus in cytomegalovirus seromimmune women to incidence of congenital cytomegalovirus infection.
Study Design
In 7848 women studied prospectively for congenital cytomegalovirus infection from a population with near universal cytomegalovirus seroimmunity, sera from 40 mothers of congenitally infected infants and 109 mothers of uninfected newborns were analyzed for strain-specific anticytomegalovirus antibodies.
Results
All women were cytomegalovirus seroimmune at first prenatal visit. Reactivity for 2 cytomegalovirus strains was found in 14 of 40 study mothers and in 17 of 109 control mothers at first prenatal visit ( P = .009). Seven of 40 (17.5%) study women and 5 of 109 (4.6%) controls ( P = .002) acquired antibodies reactive with new cytomegalovirus strains during pregnancy. Evidence of infection with more than 1 strain of cytomegalovirus before or during current pregnancy occurred in 21 of 40 study mothers and 22 of 109 controls ( P < .0001).
Conclusion
Maternal reinfection by new strains of cytomegalovirus is a major source of congenital infection in this population.
Human cytomegalovirus (CMV) is the most common viral infection transmitted to the developing fetus with rates of infection ranging from 0.2-2.0% of live births. Importantly, congenital CMV infection is a major cause of sensorineural hearing loss in infants and children. Studies of prophylactic vaccines have suggested that prevention of transmission to offspring of previously nonimmune women could be effective. However, findings from studies in maternal population with high CMV seroprevalence have demonstrated that intrauterine infection and disease occurs not infrequently in the offspring of women with existing immunity, so called nonprimary infections. Thus, preconceptional immunity against CMV provides only partial protection against congenital infection and in maternal populations with high CMV seroprevalence, most congenital CMV infections follow nonprimary maternal infections. Studies from Brazil, the Ivory Coast, India, as well as urban African American populations in the United States, have demonstrated a direct relationship between maternal CMV seroprevalence and the incidence of congenital CMV infection. Proposed mechanisms for nonprimary maternal infections include reactivation of an existing persistent infection or reinfection with new strain of CMV. Only inferential evidence supports the first mechanism; however, this mechanism is consistent with lifelong persistence of CMV infection. Thus, reactivations from latency or a chronic infection could result in recurrent infections in previously infected women. Alternatively, reinfections with new strains of CMV have been documented in immunocompetent and immunocompromised patients. Mechanisms leading to intrauterine CMV transmission and congenital infection remain undefined in maternal populations in the developing world with seroprevalences approaching 100%. Because infection with more than 1 CMV strain in immunocompetent pregnant women can lead to fetal damage, reinfection could contribute significantly to the natural history of congenital CMV infections.
In the current study, we analyzed serum samples obtained at the initiation of prenatal care and at delivery from women prospectively enrolled in a study of congenital CMV infections in a highly seroimmune maternal population. Women delivering congenitally infected infants and control women delivering uninfected infants from the same population were studied for CMV strain-specific serological responses to determine the contribution of maternal reinfection during pregnancy to congenital CMV infection in this population with near universal preconceptional CMV seroimmunity.
Materials and Methods
Study population and design
Forty mothers of infants with congenital CMV infection and 109 mothers of uninfected infants were enrolled in the study. These subjects were selected from 7848 mothers of 8047 infants born at 2 maternity hospitals in the municipality of Ribeirão Preto, Brazil, whose infants were screened (85% all live births) for congenital CMV infection (1.1% rate of congenital CMV infection). Among 84 mothers of 87 infants (3 twins) who were identified with congenital CMV infection, 58 (69%) were residents and received prenatal care in Ribeirão Preto. Of these 58 women, 40 (74%) had prenatal serum specimens stored in a central repository and represented the study population. The control mothers were selected from women delivering uninfected infants at the same hospital, residents of Ribeirão Preto, matched for gestational age of their newborn infants, and had prenatal serum specimen stored in the central repository. The study and control population were derived from a maternal population with an overall CMV seroprevalence of 96%, thus it was not unexpected that all the women in this study were seropositive for CMV at entry into the study. The study protocol was approved by the Research Ethics Committee of the University Hospital (processes no. 4782/2002 and 9145/2003).
Diagnosis of congenital infection was based on the detection of CMV DNA in saliva and/or urine samples by polymerase chain reaction (PCR) and confirmed by virus isolation from 2 urine and/or saliva samples collected before 3 weeks of life. Infants with clinical findings, including petechiae, purpura, jaundice with direct bilirubin >2 mg/dL, hepatosplenomegaly, microcephaly, and chorioretinitis within the first 15 days of life were classified as having a symptomatic congenital CMV infection.
Determination of maternal CMV serostatus
Sequential serum specimens (first prenatal visit and at delivery) from mothers were assayed for anti-CMV IgG antibodies by a conventional ELISA and anti-CMV IgG avidity indicies were determined in all prenatal serum specimens (VIDAS CMV IgG Avidity, Biomérieux, France). An IgG avidity index of >80% is strongly suggestive of an infection that occurred at least 12 weeks earlier; however, the original data indicated that an avidity index of as low as 73% excluded 93% of CMV infections of <12 weeks’ duration.
Maternal CMV strain-specific serologic responses
Sequentially obtained maternal samples were tested for CMV strain-specific serologic responses based on the polymorphism within an antibody binding site on glycoprotein H (gH) between 2 prototypic laboratory strains of CMV, AD169 (gH-AP86) and Towne (gH-TO86), and a second polymorphic site for antibody reactivity on glycoprotein B (gB) that has been defined on AD169 (gB-AD54) and Towne (gB-TO54) virus strains. Both antibody binding sites are defined by a linear sequence of amino acids. As there is no known linkage between serologic reactivity against linear epitopes on gH and gB, 7 different patterns of antibody reactivities are possible for each study participant, including lack of recognition of the gH or gB-specific serologic determinants ( Figure ). Reactivity for both polymorphic antigenic sites on gH or gB indicated exposure to >1 strain of virus. The detection of new antibody reactivity to either epitope on gH or gB in delivery serum samples of seropositive women was considered as seroconversion and infection with a new virus strain (reinfection) during pregnancy.
CMV strain-specific ELISA
This assay is described in a recent report and uses recombinant peptides encoding the AD169 gH or the Towne gH and by the AD169 gB or the Towne gB antigens. The N-terminal region of gH was expressed as beta-galactosidase fusion protein in Escherichia coli . A 106 amino acid fragment from the amino-terminal region of gB was his-tagged by cloning into pET21a(+) (EMD, Gibbstown, NJ) vector, expressed in E. coli , and purified using TALON Superflow columns (Clonetech, Mountain View, CA). A positive control used the highly conserved and dominant antigenic domain (AD-1) from gB cloned into both vectors. Reactivity against empty vectors expressing fusion protein alone or unrelated proteins of mouse origin were used as negative controls. A positive result was defined as 3 standard deviations (SD) above the OD value obtained from serum from a CMV seronegative individual.
Sequence analysis of viruses recovered from infected infants
CMV DNA was extracted from peripheral blood, saliva, and urine from infected infants as described. Viral DNA was amplified (Fusion; New England Biolabs, Beverly, MA) using primers to amplify a 460 base pair (bp) product from the 5′ end of the UL75 orf (gH) (nucleotides 110,603-111,063) or a 300-bp product from the UL55 orf (gB) (nucleotides 84,117-84,423, AD169). Gel-purified amplimers were sequenced directly or in some cases cloned into the pCRBlunt vector (Invitrogen, Carlsbad, CA) and propagated in E. coli . Approximately 10-12 clones were selected and plasmid DNA sequenced. Nucleotide sequences were analyzed using Vector NTi software (Invitrogen).
Statistical analysis
Statistical analysis was performed using the EPI INFO software program, v. 6.4 (Center for Disease Prevention and Control). The proportion of strain-specific serologic responses to different epitopes in study and control groups were compared using χ 2 test or Fisher’s exact test.
Results
Mothers of infected and uninfected infants did not differ in age (median, 20 vs 22 years), years of formal education (median, 8 years vs 9 years), exposure to children <2 years of age (14/40 vs 23/109), age of sexual debut (median, 15 vs 16 years), or number of sexual partners (median, 2). When exposure to young children was extended to include children ≤3 years, significantly more mothers of infected infants cared for young children (23/40 vs 37/109; P = .01).
The median gestational age at which the prenatal sample was obtained for study and control women was 13 weeks (range, 4–27 weeks). The median interval between prenatal and delivery serum specimens was 24 weeks (range, 8–32 weeks) in both groups. Serum from the first prenatal visit from all 40 mothers of infected offspring and 109 control mothers contained CMV IgG antibodies, a finding consistent with the CMV seroprevalence of this population. Anti-CMV IgG antibodies of high avidity index could be demonstrated in serum specimens from women in the study group (median, 96%; range, 74–100%) and the control group (median, 94%; range, 76–100%).
CMV strain-specific antibody responses to gH and gB epitopes in the serum samples obtained during pregnancy
The strain-specific response to each CMV epitope on gH, gB, and combinations of reactivity at first prenatal visit and at delivery of mothers of infected infants and control mothers are shown in Table 1 . Reactivity to at least 1 CMV polymorphic site on gH or gB was observed in the serum specimens obtained during pregnancy in all but 1 of the 40 women who delivered congenitally infected infants (97.5%) but in only 84 of 109 (77%) mothers of non-infected infants ( P = .003).
| Acquisition of new serotypic reactivity during pregnancy b | |||||||
|---|---|---|---|---|---|---|---|
| gH reactivity c | gB reactivity c | gH reactivity c | gB reactivity | ||||
| AP86↔TO86 | Neg→pos | AD54↔TO54 | Neg→pos | AP86↔TO86 | Neg→pos | AD54↔TO54 | Neg→pos |
| 2 (5%) | 3 (7.5%) | 2 (5.0%) | 2 (5.0%) | 1 (1.4%) | 2 (5.3%) | 0 (0%) | 2 (3.1%) |
| Total 4 (10.0%) | Total 3 (7.5%) 3 | Total 3 (2.7%) | Total 2 (1.8%) | ||||
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