Seroprevalence of rubella hemagglutination inhibition and rubella-specific IgM antibodies and the significance of rubella-specific IgM antibody measurement in pregnant Japanese women: A retrospective study

DOI: https://doi.org/10.21203/rs.3.rs-2339538/v1

Abstract

Background

Cases of congenital rubella syndrome are reported annually in Japan. Rubella-specific IgM antibody titers can show false positivity or remain positive for a long period. Furthermore, the seroprevalence of rubella hemagglutination inhibition (HI) and IgM antibodies is unknown, and the relevance of measuring rubella-specific IgM antibodies in asymptomatic individuals is unclear. Hence, this study aimed to assess the validity of rubella-specific IgM antibody titers as a diagnostic marker, by retrospectively assessing the prevalence of rubella HI and IgM antibodies among pregnant Japanese women. The study also aimed to estimate the incidence of rubella among rubella IgM antibody-positive pregnant women.

Methods

This single-center, retrospective study measured rubella HI and IgM antibody titers of pregnant Japanese women during prenatal visits from January 2007 to December 2020 at Yokohama City University Medical Center Perinatal Center for Maternity and Neonates. Rubella-specific IgM antibody titers were measured by enzyme immunoassay. Values > 1.2 and ≤ 1.2 were defined as positive and negative, respectively.

Results

A total of 14965 pregnant women were included in the study, of whom 186 (1.2%) were IgM-positive. One patient (0.5%) was clinically diagnosed with rubella. Of the IgM-positive patients, 136 (73.1%) had rubella HI antibody titers of < 1:256. The patient with clinically diagnosed rubella had a fever and skin rash and showed a high titer value for both rubella HI (1:2048) and rubella-specific IgM. The patient decided to terminate the pregnancy without a repeat blood examination. The correlation coefficient between rubella-specific IgM and rubella HI antibody titers was 0.2527 (p < 0.0001), indicating a weak positive correlation.

Conclusion

This study showed that almost all asymptomatic rubella-specific IgM-positive patients measured in the context of high rubella HI antibody titers were not acutely infected regardless of whether the rubella-specific IgM antibody titer was high or low.

Background

Rubella is an acute infectious disease caused by the rubella virus. Rubella infection during pregnancy can cause congenital rubella syndrome. Several countries have active programs to control rubella, and it was officially eradicated in the Americas in 2015 [1]. Despite established guidelines on rubella prevention by the Japanese Government to eradicate rubella by 2020, rubella outbreaks have occurred during the past few years (Table 1) [2] because of differences in rubella vaccination strategies between Japan and the U.S. In the U.S., the rubella vaccination program was established in 1969 and universal vaccination of children was initiated, resulting in a steady decrease in the number of rubella infections [3, 4]. However, in Japan, a routine vaccination program was started in 1977 for girls in junior high schools, with a focus on preventing infection among pregnant women. Since then, the target population has expanded to include men, women, and infants. In 2006, the measles-rubella vaccination schedule was changed to two doses (1st dose for children aged 1 year and 2nd one for young children) [57]. Although a vaccine program was provided for unvaccinated individuals because of the gap in the vaccination schedule, the coverage was not high [7]. As a result, rubella outbreaks were seen mainly in adults who have low antibody prevalence due to exclusion from the vaccination program, and cases of congenital rubella syndrome continue to be reported [2, 8]. Therefore, screening and diagnosis of congenital rubella syndrome during pregnancy remains important in Japan.

The 2020 Japanese guideline for obstetric practice recommends that pregnant women with hemagglutination inhibition (HI) antibody titers ≥ 1:256 in their first trimester should have paired serum HI antibody titers and rubella-specific IgM antibody titers measured [9]. However, owing to the presence of persistent IgM antibodies for a long period [9, 10], not all IgM antibody-positive results indicate recent infection. Additionally, given the lack of large-scale studies, the prevalence of persistent IgM antibody carriers is unknown. Furthermore, the validity of the rubella IgM antibody assay as a diagnostic marker of rubella infection in asymptomatic individuals is unclear. Hence, the current study aimed to assess the validity of rubella-specific IgM antibody titers as a diagnostic marker of rubella infection by retrospectively examining the prevalence of rubella HI and IgM antibodies among pregnant women in Japan. The study also aimed to estimate the incidence of rubella among rubella IgM antibody-positive pregnant women.

Table 1

Number of rubella cases by year in Japan and Yokohama City

Year

Rubella cases

 

Japan [27] (per million population)

Yokohama [28] (per million population)

2008

294 (2.31)

12 (3.29)

2009

147 (1.16)

4 (1.09)

2010

90 (0.70)

3 (0.82)

2011

378 (2.98)

15 (4.07)

2012

2386 (18.8)

113 (30.6)

2013

14344 (113)

621 (168)

2014

319 (2.51)

37 (9.97)

2015

163 (1.28)

6 (1.61)

2016

126 (1.00)

3 (0.80)

2017

91 (0.72)

9 (2.41)

2018

2946 (23.4)

171 (45.7)

2019

2306 (18.3)

135 (36.1)

2020

100 (0.79)

3 (0.80)

Methods

Study population and design

This study included all the pregnant women in their first trimester who visited the Perinatal Center for Maternity and Neonates, a Japanese tertiary perinatal center at the Yokohama City University Medical Center, between January 2007 and December 2020. The data were retrospectively collected from the patient’s medical records and analyzed. The hospital is a comprehensive perinatal center and provides medical care for pregnant women with or without pregnancy-related complications living in and around Yokohama. The hospital is located in the center of the city of Yokohama, which is located approximately 30 km from the metropolitan city of Tokyo and has a population of approximately 3.7 million. The study protocol was approved by the Ethics Committee of the Yokohama City University Medical Center Hospital (EC approval number: F211100044). We used the opt-out method instead of obtaining informed consent from individuals.

Outcome ascertainment

The rubella HI and rubella-specific IgM antibody titers were measured in all pregnant women who visited the hospital in the first trimester of their pregnancy. Rubella HI and rubella-specific IgM antibody titers were measured using enzyme immunoassays (EIAs). The rubella HI and IgM antibody titer values > 1.2 and ≤ 1.2 were defined as positive and negative, respectively. The study retrospectively assessed the participants for the following: IgM positivity, HI antibody titer distribution, IgM positivity by HI antibody titer, and correlation between rubella HI and rubella-specific IgM antibody titers. Additionally, we assessed IgM-positive patients for the incidence of rubella, congenital rubella syndrome, miscarriage, and distribution of rubella-specific IgM and rubella HI antibody titers. As shown in Table 1, 2013 is considered an epidemic year because the number of reported rubella cases was approximately 10 times higher than that in other years; therefore, we also assessed the effect of the rubella epidemic on the study participants.

Statistical analyses

Categorical data were reported as frequency (percentage) and continuous data were reported as medians and interquartile ranges. The correlation between rubella-specific IgM and rubella-specific HI antibody titers was analyzed using Spearman’s rank correlation coefficient. P values < 0.05 were considered statistically significant. The statistical analyses were performed using JMP Pro 15 (version 15.0.0, SAS Institute Inc., Cary, North Carolina, USA).

Results

The study included a total of 14965 pregnant women, of whom 186 (1.2%) were positive for rubella-specific IgM antibody.

Characteristics of rubella-specific IgM-positive patients

Among the rubella-specific IgM-positive patients, 1 patient (0.5%) was clinically diagnosed with rubella infection, 8 (4.3%) had miscarriages at less than 22 weeks of gestation, and 177 (95.2%) gave birth at 22 weeks of gestation or later. There were no cases of miscarriage due to rubella infection or congenital rubella syndrome diagnosed after birth. Figure 1 shows the rubella-specific IgM antibody titers distribution among the IgM-positive patients. The median (interquartile range) rubella-specific IgM antibody titer was 1.72 (1.45–2.40). The distribution of rubella HI antibody titers among rubella-specific IgM-positive patients is shown in Fig. 2. Of the rubella-specific IgM-positive patients, 136 (73.1%) had rubella HI antibody titers of < 1:256. The patient with clinically diagnosed rubella had a fever and skin rash and high titer values for both rubella HI (1:2048) and rubella-specific IgM (10). The patient decided to terminate the pregnancy without a repeat blood test.

Rubella HI and rubella-specific IgM antibody titers

Figure 3 shows the distribution of HI antibody titers among all pregnant women tested, and Table 2 shows the IgM positivity by HI antibody titer. Of the patients who tested IgM positive, the HI antibody titer was ≥ 1:256 in 2.55%, ≥ 1:128 in 1.95%, ≥ 1:64 in 1.72%, ≥ 1:32 in 1.50%, ≥ 1:16 in 1.37%, and ≥ 1:8 in 1.30%. The correlation coefficient between rubella-specific IgM and rubella HI antibody titers was 0.2527 (p < 0.0001), indicating a weak positive correlation (Fig. 4).

Table 2

Percentage of rubella-specific IgM positivity by hemagglutination inhibition (HI) antibody titer

HI antibody titer

Rubella-specific IgM positivity

 

2007–2020 overall, % (n)

2013 (the epidemic year), % (n)

4096

0 (0)

-

2048

25 (1)

-

1024

2.1 (1)

0 (0)

512

2.8 (15)

5.9 (3)

256

2.4 (33)

2.2 (3)

128

1.5 (44)

1.2 (3)

64

1.4 (51)

0.7 (2)

32

0.9 (30)

1.8 (4)

16

0.5 (8)

0 (0)

8

0.1 (1)

0 (0)

< 8

0.3 (2)

0 (0)

HI: hemagglutination inhibition

Characteristics of rubella-specific IgM-positive pregnant women during the epidemic year (2013)

During the epidemic year (2013), there were 1157 patients tested positive for rubella antibodies, including 15 (1.3%) patients who were IgM-positive, but there were no cases of congenital rubella syndrome. There were no cases of congenital rubella syndrome and no miscarriages at less than 22 weeks of gestation in the 15 patients who tested IgM-positive. Figure 5 shows the rubella-specific IgM distribution of antibody titers among the IgM-positive patients. The median (interquartile range) rubella-specific IgM antibody titer was 1.6 (1.4–3.65). Figure 6 shows the distribution of HI antibody titers among IgM-positive patients. Nine (60%) IgM-positive patients had HI antibody titers of < 1:256.

Rubella HI and rubella-specific IgM antibody titers during the epidemic year (2013)

Figure 7 shows the distribution of HI antibody titers among all pregnant women tested during the epidemic year, and Table 2 shows the rubella-specific IgM positivity by rubella HI antibody titer. No patients had a rubella HI antibody titer ≥ 1:2048. Of the patients who tested IgM positive in 2013, the rubella HI antibody titer ≥ 1:256 in 3.2%, ≥ 1:128 in 2.0%, ≥ 1:64 in 1.5%, ≥ 1:32 in 1.6%, ≥ 1:16 in 1.4%, and ≥ 1:8 in 1.4%. The correlation coefficient between rubella-specific IgM and rubella-specific HI antibody titers was 0.1579 (p < 0.0001) (Fig. 8).

Discussion

This study showed that the rubella-specific IgM antibody prevalence in Japanese pregnant women was approximately 1.0%, and almost all of them were not acutely infected. The study also showed that a few patients retained high rubella-specific IgM antibody titers. Furthermore, there was a weak positive correlation between rubella HI and rubella-specific IgM antibody titers; however, more than 70% of patients who tested positive for rubella-specific IgM had rubella HI antibody titers of less than 1:256.

Rubella-specific IgM antibodies are detectable 4 days after the onset of illness, persist for 1 month, and are used with IgG antibodies measured in paired sera to diagnose acute-phase infection [11]. False-positive IgM antibody test results can be detected in patients with other viral infections or rheumatoid factor positivity, and IgM antibodies can be persistently positive after immune acquisition [12], which is termed “persistent IgM” in the Japanese obstetric guidelines [9]. Given that the positive predictive value is relatively low because of the frequency of false-positive and persistent IgM antibody carriers and the low prevalence of rubella infection, it is important to assess the frequency of false-positive and persistent IgM antibody carriers for the diagnosis of rubella infection by using IgM antibodies. In our survey [10] of 1,850 deliveries between 2003 and 2006, the prevalence of rubella-specific IgM antibody positivity was reported at 1.1%. Although data on the number of rubella cases in Japan before 2007 are unavailable, the exact number of all cases of congenital rubella syndrome is known. There were 13 cases (an average of 3.25 cases/year) in the 4 years at that time and 53 cases (an average of 3.79 cases/year) in the 14 years of the present study period with no significant difference [13], suggesting that the rubella-specific IgM antibody positivity rate also remained unchanged. The rubella-specific IgM antibody positivity rate was 1.3% in the year 2013, which was slightly higher than that in the non-epidemic years. Therefore, it can be reasonably estimated that the rubella-specific IgM antibody positivity rate in pregnant women in Japan is approximately 1%, although it is expected to vary from time to time and among different regions, depending on the degree of an outbreak. In addition, the epidemic year 2013 did not significantly affect the rubella-specific IgM antibody positivity rate.

This study also showed that the rubella-specific IgM antibody titer of persistent IgM can be high.

In Japan, HI antibody titers have traditionally been used to diagnose rubella infection because of their simplicity and cost. Japanese obstetric guidelines recommend HI antibody measurement in the first trimester of pregnancy to alert pregnant women with low antibody titers for possible rubella infection or to diagnose rubella infection with high antibody titers [9]. In pregnant women with high HI antibody titers, rubella-specific IgM antibody measurement is recommended for the diagnosis of rubella infection for recent infection. However, owing to the false-positive/persistent IgM titers, a rubella-specific IgM positive result does not always indicate a recent infection. Although there were many reports of false-positive/persistent IgM [1419], the levels of IgM antibody titers of false-positive/persistent IgM are not known.

Banatvala et al. [14] reported low levels of rubella-specific IgM positivity over a long period but did not provide specific antibody titers. In the present study, several pregnant women had rubella-specific IgM antibody titers as high as those at the start of rubella infection and rubella vaccination [20]. However, these patients did not show a recent clinical infection. This finding suggests that even patients with rubella-specific IgM antibody titers markedly higher than the cutoff, in the absence of risk factors for rubella infection, should be advised that persistent IgM or false-positive results are very likely. The measurement of rubella-specific IgM antibodies should be performed in women with a history of contact or when symptoms are observed during pregnancy [21], which is supported by the present study’s findings.

The study found a weak positive correlation between rubella HI antibody titer and rubella-specific IgM antibody titer; however, more than 70% of rubella-specific IgM-positive patients had rubella HI antibody titers less than 1:256. The HI assay measures total IgG, IgM, and IgA antibody titers, and immunity against rubella infection is associated with the levels of these antibodies [22]. The rubella HI assay is known to correlate with rubella-specific IgG antibody titers in the EIA assay [23]; however, in several patients rubella HI antibody titers did not correlate with rubella-specific IgG antibody titers in the EIA assay [24]. This may be due to high rubella-specific IgM antibody titers, as shown in this study, and measurement error in the HI assay [25]. The results of this study indicate that it is difficult to estimate the time point of rubella infection using only the combination of rubella HI antibody measurement and rubella-specific IgM antibody measurement. Hence, it is important to consider the medical history of the patient and the measurement of rubella-specific IgG antibody titers using paired sera for the diagnosis of rubella. The measurement of rubella-specific IgM antibody titers should be considered an auxiliary diagnostic method. In the absence of risk factors for rubella infection, the measurement of rubella-specific IgM antibodies based on the rubella HI antibody titers is not essential because almost all rubella-specific IgM antibody-positive patients and rubella-specific IgM antibody-high patients were not acutely infected and more than 70% of rubella-specific IgM positive patients have HI antibody titers of less than 1:256.

This is the largest study reporting rubella-specific IgM prevalence in pregnant women and is the first to report a correlation between rubella HI antibody titer and rubella-specific IgM antibody titer using rubella-specific IgM antibody levels, regardless of whether the rubella HI antibody titer was high or low.

There are a few limitations of this study. First, this study did not rule out rubella infection among patients who were rubella-specific IgM-negative, so it is not possible to calculate the sensitivity and specificity of rubella-specific IgM measurements for the diagnosis of rubella infection. During the study period, there were no cases of congenital rubella syndrome in our hospital, and none of the rubella-specific IgM-negative patients had an induced abortion because of rubella infection. However, the study could not confirm the presence or absence of rubella infection in patients with interrupted hospital visits who tested negative for rubella-specific IgM. Second, the study did not examine the subclinical infection. In Japan, 70 cases of congenital rubella syndrome were reported between 2000 and 2021, including 10 cases in women with no history of rubella during pregnancy, and 14 cases in women with an unknown history of rubella during pregnancy [13]. Congenital rubella syndrome caused by subclinical infection is still unclear [26], and it is unknown how serological changes are detected in subclinical infection. Therefore, further future studies are warranted in that direction to achieve the complete eradication of rubella in Japan.

Conclusion

This study showed that all asymptomatic rubella-specific IgM-positive cases, measured in the context of high rubella HI antibody titers, were not acutely infected, regardless of whether the rubella-specific IgM antibody titer was high or low. Creating awareness among pregnant women by informing them that almost all rubella-specific IgM-positive individuals without symptoms are not acutely infected, as supported by this study's findings, could decrease their anxiety and prevent unnecessary termination of pregnancies. Depending on the future trends in rubella outbreaks, it is essential to develop cost-effective, accurate, and efficient screening methods.

Abbreviations

EIA Enzyme immunoassay

HI Hemagglutination inhibition

Declarations

Ethics approval and consent to participate

The study protocol was approved by the Ethics Committee of the Yokohama City University Medical Center Hospital (EC approval number: F211100044). Informed consent for this retrospective analysis was obtained from all subjects. This study was conducted in accordance with the principles of the Declaration of Helsinki.

Consent for publication

Not applicable.

Availability of data and materials

The datasets generated and/or analyzed during the current study are not publicly available because of institutional regulations on restrictions on the disclosure of health information but are available from the corresponding author upon reasonable request.

Competing interests

The authors declare that they have no competing interests

Funding

No funding sources were used in the conception, composition, editing, or submission of this manuscript.

Authors' contributions

KH wrote the initial draft of the manuscript as the first author; RS and SA assisted in writing the manuscript; KH, RS, and SS contributed to the analysis and interpretation of data; MO and EM contributed to the review and final approval of the manuscript. All authors contributed to the data interpretation and critical revision of the manuscript and approved the final version of the manuscript.

Acknowledgments

Not applicable

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