Seroprevalence of rubella virus infection among antenatal care clients of Halaba Town public health facilities, southern Ethiopia

Rubella virus infection during pregnancy has several effects on the developing fetus. However, little is known about the epidemiology of the infection in Ethiopia. A cross-sectional study was conducted to assess the seroprevalence of rubella virus infection on consecutive 299 pregnant women attending antenatal care clinics in public health facilities in Halaba Town, Southern Ethiopia. Structured questionnaires were used to collect information on socio-demographic and reproductive characteristics. Venous blood samples were collected, and sera were tested for anti-rubella IgM and IgG using the enzyme-linked immunosorbent assay. Anti-rubella IgG and IgM were detected in 265 (88.6%) and 15 (5.0%) of 299 participants, respectively. Pregnant women in their first trimester [crude odds ratio (cOR) = 4.26; 95% CI (1.47, 12.4)] were at increased risk of having anti-rubella IgM compared to those in their second and third trimesters. Urban residents [cOR = 4.06; 95% CI (1.94, 8.47)] were with a higher percentage of IgG positivity compared to rural residents. Anti-rubella IgG positivity was higher in housewives [cOR = 2.94; 95% CI (1.07, 8.04)] compared to self-employed women. Our findings showed a high prevalence of rubella virus exposure, and considerable percentages of recent infection and susceptible women to contracting the infection, emphasizing the importance of congenital rubella syndrome in the research area.


Study population. Pregnant women who visited the antenatal clinics at the General Hospital and Health
Centre in the town during the study period constituted the study population. Pregnant women older than 18 years were included. However, those who were critically ill at the time of enrolment and refused to participate in the study were excluded.
Sample size and sampling technique. The sample size was determined using a single population proportion formula (n = z 2 p (1 − p)/d 2 ), assuming an anti-rubella IgG seroprevalence of 86.3% among pregnant women based on a recent report from Hawassa 9 , aiming for a 95% confidence level with 4% precision. Assuming a non-response rate of 5%, the final sample size was estimated to be 299. Consecutive pregnant women were enrolled from the health facilities until the target sample size is obtained.
Data collection techniques. Socio-demographic and reproductive characteristics. Nurses with a 4-years degree gathered data on socio-demographic characteristics (age, residence, marital status, women's educational and occupational status, and estimated monthly income) as well as reproductive characteristics (gestational age, gravidity, parity, and histories of stillbirth and spontaneous abortion) using pretested and structured questionnaire.
Serological analysis. Venous blood samples (about 5 ml) were collected from every study participant, and sera were stored at − 20 °C for a month. Samples were transported to the Public Health Laboratory Institute of SNNPR using a cold box, and stored at − 70 °C until tested. All samples were tested for anti-rubella IgM and IgG using enzyme-linked immunosorbent assay (ELISA) test kits. The Anti-Rubella Virus Glycoprotein (IgM) (EUROIMMUN Medical Laboratory Diagnostics AG, Lubeck, Germany) and the Rubella IgG test kits (DIALAB Diagnostics, Wiener Neudorf, Austria) were used and performed according to the respective manufacturer's instructions.
Definitions. Past exposure to rubella virus infection: pregnant women whose blood is tested positive for anti-rubella IgG; thus, protective immunity against the infection.
Recent rubella virus infection: pregnant women who tested positive for IgM antibody.
Data analysis. Data were double-entered into EpiData version 3.1 12 and analyzed using SPSS version 20 (IBM Corp., Armonk, NY). Descriptive statistics results were presented using percentages. Binary logistic regression analysis was used to assess the association between socio-demographic and reproductive characteristics and anti-rubella IgM and IgG serostatus. Variables found to have a p value < 0.05 were considered significant differences.
Ethics approval and consent to participate. Ethical

Discussion
In these serological analyses of rubella infection in pregnant women, we found anti-rubella IgM positivity of 5%, indicating a recent rubella virus infection (or re-infection), and anti-rubella IgG positivity of 88.6%, indicating past exposure to rubella virus infection with development of protective immunity. While a recent rubella infection was more frequently detected among pregnant women in their first trimester, past rubella virus infection was more likely among urban residents, housewives, and multigravida women. The observed proportion of anti-rubella IgM positivity in this study was comparable with pooled seroprevalence of rubella among pregnant women in sub-Saharan Africa (89.0%) including a single report from southern Ethiopia (86.3%) 9 . Compared to our finding, slightly lower proportions were reported in studies in China (83.3%) 13 and India (83.4 and 82.3%) 14,15 , as also in northern Ethiopia (79.5%) 10 . Despite the observed variability in the prevalence of rubella infections, studies generally showed high proportions of exposure to rubella infections among pregnant women in various geographical regions where a rubella vaccination has not been introduced or has recently been introduced. Most children aged under 15 years in Africa develop immunity as a result of natural infection 1 . The observed seronegativity of anti-rubella IgG (11.4%) in our study indicates an intermediate level of susceptibility (10-20%) and a medium risk of CRS in the study setting 16 . Our study showed a higher proportion of seronegativity compared to the WHO threshold of < 5% in a childbearing-age woman 17 , which was below the target to be achieved in Africa by 2030 18 .
Our finding of seroprevalence of IgM was higher compared to a result from southern Ethiopia (2.1%) 9 . However, the pooled prevalence of recent rubella infection in sub-Saharan Africa (5.1%) was consistent with our current observation, as also reported recently from Cameroon (5.5% and 5%) 19,20 . On the other hand, the proportion of IgM positivity was lower than that reported in northern Ethiopia (9.5%) 10 and Nigeria 7.8-38.8% [21][22][23] . The observed difference in proportions of IgM positivity among the studies might be due to variable endemicity of rubella, testing methods, and population density.
A higher proportion of IgM positivity among pregnant women who were in their first trimester was consistent with studies from Ethiopia 24 and Tanzania 25 , and it is a major concern in light of an increased risk of developing CRS with decreasing gestational age 3 . Our finding of the higher proportion of anti-rubella IgG positivity among urban dwellers compared to rural ones was consistently reported by several studies in African countries 21,[26][27][28] . The possible reason might be crowded living conditions in urban populations increase the risk of rubella transmission.  www.nature.com/scientificreports/ Our study has the strength of being one of few such investigations of rubella virus infection among pregnant women in Ethiopia. Findings from this study should inform decision-makers on the prevalence of recent rubella virus infection and the status of susceptibility to rubella virus infection to plan intervention efforts aimed at reducing the incidence of CRS. However, the study faced some limitations. Since participants were recruited from health facilities, findings may not be compared to all pregnant women in the study area. Further, our study might not have been powered adequately to identify socio-demographic and reproductive characteristics associated with rubella infection. The study participants were interviewed in health facilities by health workers, and data collection relied on participants' self-reports, which might be subject to social desirability, recall, and information bias.

Conclusion
The high proportion of pregnant women with exposure to the rubella virus, particularly in urban areas, indicates the endemicity of the infection in the Ethiopian context. The observed proportion of recent infection, particularly in pregnant women in their first trimester, may reflect the significance of CRS in the study area. Our findings call for interventions to reduce the risk of CRS, including vaccinating susceptible women of childbearing against the rubella virus and introducing vaccines to the routine childhood immunization programme.

Data availability
The datasets generated and/or analysed during the current study are available from the corresponding author, upon reasonable request and with the Institutional Review Board of the Hawassa University College of Medicine and Health Sciences. Restrictions apply to the availability of these clinical data, which caregivers had consented for the collected information to be used for our research study only, and so are not publicly available.