Rubella virus infections and immune status among pregnant women before the introduction of rubella vaccine in Amhara Regional State, Ethiopia

International Journal of Infectious Diseases 76 (2018) 14–22

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Rubella virus infections and immune status among pregnant women before the introduction of rubella vaccine in Amhara Regional State, Ethiopia Yitayih Wondimeneha,* , Moges Tiruneha , Getachew Feredea , Birhanu Aberab , Meseret Workinehc , Meseret Birhanied , Belay Tessemaa a Department of Medical Microbiology, School of Biomedical and Laboratory Sciences, College of Medicine and Health Sciences, University of Gondar, P.O. Box 196, Gondar, Ethiopia b Department of Gynecology and Obstetrics, School of Medicine, College of Medicine and Health Sciences, University of Gondar, P.O. Box 196, Gondar, Ethiopia c Department of Immunology, School of Biomedical and Laboratory Sciences, College of Medicine and Health Sciences, University of Gondar, P.O. Box 196, Gondar, Ethiopia d Department of Medical Parasitology, School of Biomedical and Laboratory Sciences, College of Medicine and Health Sciences, University of Gondar, P.O. Box 196, Gondar, Ethiopia

A R T I C L E I N F O

A B S T R A C T

Article history: Received 9 June 2018 Received in revised form 24 July 2018 Accepted 27 July 2018 Corresponding Editor: Eskild Petersen, Aarhus, Denmark

Background: Rubella and its associated congenital anomalies have been greatly reduced in most developed countries through use of the rubella vaccine. However, the magnitude of the problem is underestimated and there are no well-established rubella/congenital rubella syndrome prevention and control strategies in many developing countries, including Ethiopia. The aim of this study was to determine the prevalence of rubella virus infections among pregnant women and their immune status before the introduction of rubella vaccine in Amhara Regional State, Ethiopia. Methods: A prospective cross-sectional study was conducted among pregnant women in Dessie, FelegeHiwot, and University of Gondar referral hospitals, from December 2015 to February 2017. After obtaining written informed consent, socio-demographic data, reproductive history, clinical manifestations, and the possible risk factors for rubella virus infections were collected using a structured questionnaire. The laboratory analysis of rubella-specific antibodies was done using an enzyme-linked immunoassay method on venous blood samples. Data were entered and analyzed using IBM SPSS Statistics version 20. Binary logistic regression was used to determine the strength of association between the dependent variables and covariates. Results: A total of 600 pregnant women were included in the study. Their mean age was 26.4  5 years (range 16–40 years). The overall seroprevalence of rubella infection was 89%. Of the total study participants, 9.5% were positive for rubella-specific IgM antibody, which indicates acute/recent rubella virus infection. In contrast, 79.5% of them had protective levels of rubella-specific IgG antibody and were immune as a result of previous wild-type rubella infection. However, 11% of the pregnant women were negative for both rubella-specific antibodies; these women represent the susceptible group. Conclusions: A large number of pregnant women had acute/recent rubella virus infections at the time of data collection, indicating that the virus is endemic in the study area. More than a tenth of pregnant women were found to be susceptible to acquiring the infection in future pregnancies, with the possible risk of rubella-associated congenital anomalies. Hence screening of all women of child-bearing age before conception and during pregnancy might reduce the devastating effects of the virus on the developing fetus. © 2018 The Author(s). Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/).

Keywords: Rubella virus Pregnant women Immune status

* Corresponding author. E-mail addresses: [email protected] (Y. Wondimeneh), [email protected] (M. Tiruneh), [email protected] (G. Ferede), [email protected] (B. Abera), [email protected] (M. Workineh), [email protected] (M. Birhanie), [email protected] (B. Tessema). https://doi.org/10.1016/j.ijid.2018.07.024 1201-9712/© 2018 The Author(s). Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Introduction

Study participants

Rubella virus is an important human pathogen that causes an acute and contagious disease known as rubella, little red, 3-day measles, or German measles (Fokunang et al., 2010). Humans are the only reservoir for this virus (Mounerou et al., 2015). The virus has an incubation period of 2–3 weeks. The route of transmission is air-borne in postnatal cases and transplacental during pregnancy (Kolawole et al., 2014). The disease caused by this virus commonly occurs in childhood and is characterized by a maculopapular rash associated with a low-grade fever, lymphadenopathy, and malaise (Al-Rubai et al., 2010). It can also cause joint pains, headache, and conjunctivitis in adults (Lezan, 2015). Transient arthralgia or arthritis may also occur (Heggie and Robbins, 1969). It is also a rare cause of thrombocytopenic purpura and encephalitis (Sherman et al., 1965). However, up to 50% of rubella cases are subclinical (Horstmann et al., 1965). Rubella infection is considered relatively benign, and in the absence of pregnancy, the infection is usually mild and self-limiting (CDC, 2001). However, during pregnancy, it has a devastating effect on the developing fetus (Adam et al., 2013; Cradock-Watson et al., 1981) and this represents a major health concern worldwide (Mirambo et al., 2015). Currently, there is no specific treatment for the virus (Olajide et al., 2015). However, its burden can be minimized through use of the live attenuated rubella vaccine (Alleman et al., 2016; Demicheli et al., 2012; WHO, 2014). The control of rubella and congenital rubella syndrome (CRS) relies on a high population level of immunity (Gilbert et al., 2017). The World Health Organization (WHO) proposed the introduction of rubella vaccine in each country in the year 2000 (Robertson et al., 2003) and different efforts are undergoing in different WHO regions (Martínez-Quintana et al., 2015; CDC, 2010; WHO, 2008). As a result, the burden has declined, although mostly in industrialized countries (Adewumi et al., 2014). However, rubella vaccine is still not available in many developing countries (Njeru et al., 2015) and it is not included in their immunization programs (WHO, 2012). Rubella is an under-recognized public health problem (Cutts and Vynnycky, 1999; Mamvura et al., 2015). In Africa, few countries have included rubella vaccine in their national immunization programs and data on the seroprevalence of the virus are very limited (Martínez-Quintana et al., 2015). Although Ethiopia has planned to introduce the rubella vaccine (WHO, 2015), it is currently not included in the national immunization program (Getahun et al., 2016). There are only a few reports on rubella in the country (Cutts et al., 2000a; Gebreselassie and Almaz, 1985), with most containing very old information (Gebreselassie and Almaz, 1985; Sandow et al., 1982) or reporting on suspected cases of measles among children (Mitiku et al., 2011; Getahun et al., 2016; Shiferaw et al., 2016). There is only one recently published report on rubella among pregnant women (Tamirat et al., 2017) and one case report on CRS (Mekonnen, 2017) in the country. All of these indicate that there is scarcity of data and that the magnitude of rubella and its consequences is largely unknown. Hence, the aim of this study was to determine rubella virus infections and immune status among pregnant women before the introduction of rubella vaccine in Amhara Regional State, Ethiopia.

The study participants were pregnant women who visited the respective antenatal care clinics of the referral hospitals during the study period and gave informed consent and the required amount of blood sample for laboratory analysis.

Materials and methods Study design, area, and period A prospective cross-sectional study was conducted in three referral hospitals in Amhara Regional State, namely Dessie, FelegeHiwot, and University of Gondar referral hospitals, from December 2015 to February 2017.

Sample size and sampling technique The study participants were selected using a simple random sampling technique and the sample size was calculated using a single population proportion formula by considering a 95% confidence interval, 4% margin of error, and 50% proportion. The sample size was proportionally allocated to the selected referral hospitals based on the previous flow of pregnant women visiting the antenatal care clinics of the respective referral hospitals. Pregnant women who gave informed consent and the required amount of blood sample were included in the study. Pregnant women who were seriously sick at the time of data collection and those who visited the respective referral hospitals for the second time during the study period were excluded from the study. Data collection After obtaining written informed consent from each study participant, socio-demographic data, clinical information, and information on reproductive history and possible risk factors of the pregnant women were collected using a structured and pre-tested questionnaire. Blood collection and handling Using a plain tube, 5 ml of venous blood was collected aseptically from each pregnant woman for the determination of rubella antibodies. Blood was allowed to clot for an hour at room temperature, centrifuged at 3500 rpm for 5 min, and then serum was separated and collected in sterile storage vials to be stored at 70  C until laboratory analysis. Laboratory analysis and interpretation of results Rubella IgM and IgG antibodies were determined using an enzyme-linked immunoassay (ELIA) method as per the manufacturer’s instructions (Linear Chemicals SL, Spain). The results were read in a micro-well reader at 450 nm and compared in a parallel manner with calibrators and controls. For rubella-specific IgM, the qualitative result was interpreted as positive if the rubella IgM index was >1.1, negative when the index was <0.9, and equivocal when the index was 0.9 and 1.1. The quantitative rubella IgG result was expressed in international units per milliliter (IU/ml). In accordance with the manufacturer’s instructions, the IgG result was interpreted as positive when the IgG index value was >10 IU/ ml, as equivocal at 5–10 IU/ml, and as negative at <5 IU/ml. Quality assurance mechanisms The rubella test kits (IgM and IgG EIA kits) have their own quality control materials that can be run in parallel with patient samples, and all test procedures were done strictly following the manufacturer’s instructions. In addition, standard operational procedures were strictly followed and the questionnaire was pretested in non-selected health institutions. Training was given for data collectors and they were also regularly supervised by the research team. In addition, the inclusion and exclusion criteria were given to the data collectors.

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Data analysis procedure Data were checked for completeness, cleaned manually, and entered into and analyzed using IBM SPSS Statistics version 20.0 (IBM Corp., Armonk, NY, USA). Data were summarized using frequency tables and graphs. For continuous variables, the range and mean  standard deviation (SD) were used. In the case of two categorical variables, univariate and multivariate analysis with a 95% confidence interval (CI) was performed to measure the association, and p-value of less than 0.05 was considered statistically significant. Results Socio-demographic characteristics of the pregnant women A total of 600 pregnant women were included in the study. The mean age of the participants was 26.4  5 years (range 16–40 years). Approximately a third of the study participants were in the age group of 25–29 years (n = 224, 37.3%), the majority were married (n = 587, 97.8%), and approximately two-thirds lived in an urban area (n = 386, 64.3%). One hundred and seventy (28.3%) of the study participants had a certificate and above level of education and 317 (53.0%) had an occupation of housewife (Table 1).

(79.5%, 95% CI 76.3–82.5%) were positive only for rubella IgG antibodies. This indicates that the overall number of rubellaspecific IgM-positive pregnant women at the time of data collection was 57 (9.5%, 95% CI 7.3–11.7%). According to the present study, 66 (11.0%, 95% CI 8.7–13.7%) of the pregnant women were negative for both rubella IgM and IgG antibodies; these women represent the susceptible group (Figure 1). Rubella IgM and IgG antibodies in relation to socio-demographic characteristics In the present study, eight (12.9%) of the IgM-positive study participants were in the age group of 35 years (p = 0.688), one (20.0%) was divorced (p = 0.433), 18 (10.8%) had a high school level of educational attainment (p = 0.417), and 34 (10.7%) had an occupation of housewife (p = 0.591). Similarly, 51 (82.3%) of the IgG-positive pregnant women were in the age group of 35 years (p = 0.100), 468 (79.7%) were married (p = 0.294), 313 (81.1%) lived in an urban area (p = 0.196), 17 (89.5%) had an occupation of student (p = 0.209), and 120 (82.2%) had no formal education (p = 0.922). There was no significant association for any of these sociodemographic factors. However, the pregnant women living in urban settings had IgM positivity two times (95% CI 1.05–3.78) that of the women living in rural settings (p = 0.036) (Table 1).

Overall prevalence of rubella IgM and IgG antibodies

Rubella IgM and IgG antibodies in relation to reproductive characteristics

The overall seroprevalence of rubella was 89.0% (n = 534) (95% CI 86.3–91.3%). Of the total study participants, 49 (8.2%, 95% CI 6.2– 10.2%) were positive for both IgM and IgG antibodies at the same time. However, eight (1.3%, 95% CI 0.5–2.2%) of the pregnant women were positive only for rubella IgM antibodies and 477

At the time of data collection, 211 (35.2%) of the women were in the first trimester of pregnancy, 191 (31.8%) in the second trimester, and 198 (33.0%) in the third trimester. With regard to the relationship of rubella antibodies with the study participants’ reproductive characteristics, 26 (13.1%) of the IgM-positive

Table 1 Rubella-specific IgM and IgG antibodies in relation to socio-demographic characteristics and trimesters of pregnancy of women in Amhara Regional State referral hospitals, Ethiopia, December 2015 to February 2017. Socio-demographic characteristics

Number tested

IgM-positive

Only IgG-positive

Positive, n (%)

COR (95% CI)

p-Value

Positive, n (%)

COR (95% CI)

p-Value

0.103 0.117 0.115 0.100

Age group <20 years 20–24 years 25–29 years 30–34 years 35 years

30 186 224 98 62

3 (10.0) 13 (7.0) 22 (9.8) 11 (11.2) 8 (12.9)

1 0.7 (0.18–2.53) 1.0 (0.28–3.50) 1.1 (0.30–4.4) 1.3 (0.33–5.43)

0.561 0.975 0.851 0.688

20 (66.7) 149 (80.1) 178 (79.5) 79 (80.6) 51 (82.3)

1 2.0 (0.87–4.67) 1.9 (0.85–4.42) 2.1 (0.84–5.16) 2.3 (0.85–6.30)

Marital status Married Single Divorced

587 8 5

55 (9.4) 1 (12.5) 1 (20.0)

1 1.4 (0.17–11.44) 2.4 (0.27–22.02)

0.764 0.433

468 (79.7) 6 (75.0) 3 (60.0)

2.6 (0.43–15.87) 2.0 (0.18–22.06) 1

0.294 0.571

Residence Urban Rural

386 214

44 (11.4) 13 (6.1)

2.0 (1.05–3.78) 1

0.036

313 (81.1) 164 (76.6)

1.3 (0.87–1.96) 1

0.196

Educational status No formal education Elementary school High school Certificate and above

146 118 166 170

14 11 18 14

(9.6) (9.3) (10.8) (8.2)

1.2 (0.54–2.57) 1.2 (0.50–2.62) 1.4 (0.65–2.82) 1

0.673 0.747 0.417

120 (82.2) 93 (78.8) 125 (75.3) 139 (81.8)

1.0 (0.58–1.83) 0.8 (0.46–1.50) 0.7 (0.40–1.15) 1

0.922 0.534 0.150

Occupation Civil servant Merchant Farmer Student Housewife Daily laborer

135 67 35 19 317 27

13 (9.6) 6 (9.0) 1 (2.9) 1 (5.3) 34 (10.7) 2 (9.1)

1.3 (0.28–6.27) 1.2 (0.23–6.51) 0.4 (0.03–4.28) 0.7 (0.06–8.26) 1.5 (0.34–6.62) 1

0.717 0.808 0.424 0.773 0.591

105 (77.8) 59 (88.1) 31 (88.6) 17 (89.5) 245 (77.3) 20 (74.0)

1.2 (0.47–3.17) 2.6 (0.83–8.02) 2.7 (0.70–10.47) 3.0 (0.54–16.27) 1.2 (0.48–2.93) 1

0.676 0.101 0.148 0.209 0.703

COR, crude odds ratio; CI, confidence interval.

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those without a history of spontaneous abortion (p = 0.005). In contrast, pregnant women without a previous history of spontaneous abortion had 1.7 times (95% CI 1.04–2.87) the IgG positivity rate of those who had a previous history of spontaneous abortion (p = 0.034) (Table 2).

477 (79.5%) [95% CI: 76.3-82.5]

[95% CI: 6.2-10.2]

Rubella IgM and IgG antibodies in relation to the clinical manifestations

66 (11.0%)

49 (8.2%)

[95% CI: 8.7-13.7]

8 (1.3%)

17

[95% CI: 0.5-2.2] A

B

C

D

Immune status of the pregnant women

Figure 1. The overall prevalence of rubella specific IgM and IgG antibodies among pregnant women in Amhara Regional Sate Referral Hospitals, Ethiopia, December 2015-February 2017. The letter “A”: indicates the number of pregnant women who were positive for both rubella specific IgM and IgG antibodies at a time, “B”: Positive only for rubella IgM antibody, “C”: Positive only for rubella IgG antibody, “D”: Negative for both rubella IgM and IgG antibodies.

pregnant women were in the third trimester of their current pregnancy (p = 0.136). In addition, six (17.1%) of the IgM-positive study participants had a previous history of more than three live births (p = 0.191), one (33.3%) had a previous history of more than three still births (p = 0.205), and six were grand multigravidae (16.2%) (p = 0.175) (Table 2). One hundred and seventy-four (82.5%) of the IgG-positive women were in the third trimester of their current pregnancy (p = 0.096), 29 (83.0%) had a history of more than three previous live births (p = 0.972), 40 (83.3%) had a history of one to three still births (p = 0.434), and 248 (80.8%) were multigravidae (p = 0.346). None of these reproductive history factors was significantly associated with IgM and IgG positivity. However, the pregnant women with a history of one to three previous spontaneous abortions had 2.5 times (95% CI 1.32–4.63) the IgM positivity of

In this study, four (26.7%) of the IgM-positive pregnant women had lymphadenopathy (p = 0.082), four (14.3%) had a runny nose (p = 0.381), and six (17.1%) had a sore throat (p = 0.119) at the time of data collection. In addition, 19 (12.2%) had a headache (p = 0.187), five (20.8%) had an inflamed eye (p = 0.062), and three (17.6%) had jaundice (p = 0.255). In contrast, 471 (79.8%) of the IgG-positive pregnant women had no arthralgia/arthritis (p = 0.138) and 467 (79.8%) had no lymphadenopathy (p = 0.124). In addition, 458 (80.1%) of the IgG-positive pregnant women had no runny/stuffy nose (p = 0.124) and 403 (80.9%) had no malaise (p = 0.058). Furthermore, 361 (81.3%) of the IgG-positive pregnant women had no headache (p = 0.066) and 465 (79.8%) had no jaundice (p = 0.360). However, none of them had significant association (Table 3). There was a significant association between the presence of a maculopapular rash and rubella-specific IgM positivity in the multivariate logistic regression analysis. The pregnant women with a maculopapular rash had 3.5 times (95% CI 1.464–8.649) the IgM positivity of those without a maculopapular rash (p = 0.005). There was also a significant association between the presence or absence of a maculopapular rash and rubella-specific IgG positivity in the multivariate logistic regression analysis. The pregnant women without a maculopapular rash had 2.5 times (95% CI 1.120– 5.691) the protective antibody of those who had a maculopapular rash at the time of data collection (p = 0.026) (Table 3).

Table 2 Rubella IgM and IgG antibodies in relation to the reproductive history of the pregnant women in Amhara Regional State referral hospitals, Ethiopia, December 2015 to February 2017. Reproductive characteristics

Number tested

IgM-positive

Only IgG-positive

Positive, n (%)

COR (95% CI)

p-Value

Positive, n (%)

COR (95% CI)

p-Value

Trimester at the time of data collection First trimester 211 Second trimester 191 Third trimester 198

18 (8.5) 13 (6.8) 26 (13.1)

1 0.8 (0.37–1.64) 1.6 (0.86–3.10)

0.518 0.136

174 (82.5) 153 (80.1) 150 (75.8)

1.5 (0.93–2.44) 1.3 (0.80–2.10) 1

0.096 0.302

History of live births None 1–3 >3

256 309 35

25 (9.8) 26 (8.4) 6 (17.1)

1 0.9 (0.48–1.51) 1.9 (0.72–5.05)

0.577 0.191

197 (77.0) 251 (81.2) 29 (83.0)

1 1.3 (0.86–1.95) 1.5 (0.57–3.65)

History of spontaneous abortion None 1–3

510 90

41 (8.0) 16 (17.8)

1 2.5 (1.32–4.63)

413 (81.0) 64 (71.1)

1.7 (1.04–2.87) 1

0.034

0.005

History of stillbirth None 1–3 >3

549 48 3

52 (9.5) 4 (8.3) 1 (33.3)

1 0.9 (0.30–2.52) 4. 8 (0.43–53.6)

0.795 0.205

435 (79.2) 40 (83.3) 2 (66.7)

1.9 (0.17–21.23) 2.5 (0.20–31.0) 1

0.599 0.434

Having malformed children None 1–3

576 24

55 (9.5) 2 (8.3)

1.2 (0.27–5.07) 1

459 (79.7) 18 (75.0)

1.3 (0.51–3.37) 1

0.578

Gravidity Primigravida Multigravida Grand multigravida

256 307 37

23 (9.0) 28 (9.1) 6 (16.2)

1 1.0 (0.57–1.83) 1.96 (0.74–5.19)

200 (78.1) 248 (80.8) 29 (78.4)

1 1.18 (0.78–1.77) 1.0 (0.44–2.34)

COR, crude odds ratio; CI, confidence interval.

0.842

0.955 0.175

0.212 0.434

0.346 0.972

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Table 3 Rubella IgM and IgG antibodies in relation to the clinical information of the pregnant women in Amhara Regional State referral hospitals, Ethiopia, December 2015 to February 2017. Clinical information

Number tested

IgM-positive

Only IgG-positive

Positive, n (%)

COR (95% CI)

Positive, n (%)

COR (95% CI)

130 (80.2) 347 (79.2)

1.1 (0.68–1.67) 1

17 (56.7) 460 (80.7)

1 3.2 (1.51–6.78)a

6 (60.0) 471 (79.8)

1 2.6 (0.73–9.50)

10 (66.7) 467 (79.8)

1 2.0 (0.66–5.90)

1.6 (0.55–4.88) 1

19 (67.9) 458 (80.1)

1 1.9 (0.84–4.32)

6 (17.1) 51 (9.0)

2.1 (0.83–5.26) 1

23 (65.7) 454 (80.4)

1 2.1 (1.03–4.42)a

102 498

10 (9.8) 47 (9.4)

1.0 (0.51–2.14) 1

74 (72.5) 403 (80.9)

1 1.6 (0.98–2.62)

Headache Yes No

156 444

19 (12.2) 38 (8.6)

1.5 (0.83–2.67) 1

116 (74.4) 361 (81.3)

1 1.5 (0.97–2.31)

Inflamed/red eyes Yes No

24 576

5 (20.8) 52 (9.0)

2.7 (0.95–7.40) 1

15 (62.5) 462 (80.2)

1 2.4 (1.04–5.70)a

Jaundice Yes No

17 583

3 (17.6) 54 (9.3)

2.1 (0.59–7.54) 1

12 (70.6) 465 (79.8)

1 1.6 (0.57–4.75)

Mild fever Yes No

162 438

11 (6.8) 46 (10.5)

1 1.6 (0.81–3.19)

Maculopapular rash Yes No

30 570

8 (26.7) 49 (8.6)

3.9 (1.64–9.14)a 1

Arthralgia/arthritis Yes No

10 590

1 (10.0) 56 (9.5)

1.1 (0.13–8.52) 1

Lymphadenopathy Yes No

15 585

4 (26.7) 53 (9.1)

3.7 (1.12–11.86)a 1

Runny or stuffy nose Yes 28 No 572

4 (14.3) 53 (9.3)

Sore throat Yes No

35 565

General malaise Yes No

AOR (95% CI)

3.5 (1.46–8.65)

3.0 (0.87–10.1)

p-Value

0.005

0.082

AOR (95% CI)

p-Value

0.026 2.5 (1.12–5.69)

0.315 1.5 (0.68–3.33)

0.170 1.9 (0.77–4.58)

COR, crude odds ratio; AOR, adjusted odds ratio; CI, confidence interval. a Significant association (p < 0.05).

Rubella IgM and IgG positivity in relation to the possible factors The majority of the IgM-positive pregnant women, nine (17.0%), had more than three children living in the given house (p = 0.240). Similarly, 249 (81.9%) of the IgG-positive pregnant women had one to three children living in the house (p = 0.727) and 18 (81.8%) had a history of blood transfusion (p = 0.639). None of these possible risk factors showed a statistically significant association in relation to either IgM or IgG positivity (Table 4). However, there were statistically significant differences in IgM positivity in relation to frequent exposure to children and study site on both univariate and multivariate analysis. The pregnant women who had frequent exposure to children in their daily activities had 2.8 times (95% CI 1.6–5.1) the IgM positivity of those who had no daily exposure to children (p = 0.001). In addition, the pregnant women from Dessie Referral Hospital had 2.8 times (95% CI 1.546–5.160) the IgM positivity of those pregnant women from University of Gondar Referral Hospital (Table 4) (p = 0.001).

Discussion Rubella IgM and IgG antibodies are important immunoglobulins to study when investigating the prevalence of rubella in a given

area (Olajide et al., 2015). The presence of only IgM or both IgM and IgG antibodies at the same time indicates an acute/recent rubella virus infection. However, the presence of IgG antibody in the absence of IgM is a seromarker of immunity against rubella virus (Taneja and Sharma, 2012; Peter, 2015). The absence of both IgM and IgG antibodies indicates susceptibility to acquiring rubella infection. In this study, both rubella-specific IgM and IgG antibodies were analyzed among pregnant women to determine acute/recent infections and the levels of immunity against rubella virus infection in the pre-vaccine era in Ethiopia. The overall seroprevalence of rubella among pregnant women was found to be 89% (95% CI 86.3–91.3%). A similar finding has been reported from other African countries such as Senegal (90.1%) (Dromigny et al., 2003) and Namibia (85.0%) (Jonas et al., 2016). However, the overall seroprevalence in this study is higher than reports from other African countries like the Democratic Republic of Congo (58.97%) (Zanga et al., 2017), Sudan (65%) (Hamdan et al., 2011), and Nigeria (68%) (Bamgboye et al., 2004), and lower than reports from Burkina Faso (95%) (Tahita et al., 2013a,b) and Zimbabwe (92%) (Mamvura et al., 2015). This variation in different studies might be due to the difference in the endemicity of the virus, the variation in the sample size of the studies, the laboratory methods used, and differences in the cut-off points of the assays used.

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Table 4 Rubella IgM and IgG antibodies in relation to possible factors for rubella virus infection among pregnant women in Amhara Regional State referral hospitals, Ethiopia, December 2015 to February 2017 Possible risk factors

Number tested

IgM-positive

Only IgG-positive

Positive, n (%)

COR (95% CI)

AOR (95% CI)

p-Value

Positive, n (%)

COR (95% CI)

p-Value

2.8 (1.6–5.1)

0.001

216 (79.4) 261 (79.6)

1 1.0 (0.68–1.50)

0.961

0.727 0.456

Frequent exposure to children Yes No

272 328

38 (14.0) 19 (5.8)

2.6 (1.45–4.70)a 1

Number of children in a house None 1–3 >3

243 304 53

27 (11.1) 21 (6.9) 9 (17.0)

1 0.6 (0.33–1.08) 1.6 (0.72–3.72)

189 (77.8) 249 (81.9) 39 (73.6)

1 1.1 (0.66–1.82) 1.5 (0.54–3.93)

History of blood transfusion Yes No

22 578

2 (9.1) 55 (9.5)

1 1.1 (0.24–4.62)

18 (81.8) 459 (79.4)

1.4 (0.33–6.22) 1

Study site University of Gondar Referral Hospital Felege-Hiwot Referral Hospital Dessie Referral Hospital

358 115 127

26 (7.3) 8 (7.0) 23 (18.1)

1 0.9 (0.42–2.17) 2.8 (1.55–5.16)a

288 (80.4) 96 (83.5) 93 (73.2)

1 1.2 (0.60–2.23) 1.1 (0.59–2.05)

1 0.9 (0.4–2.0) 2.9 (1.6–5.4)

0.001

0.639

0.663 0.762

COR, crude odds ratio; AOR, adjusted odds ratio; CI, confidence interval. a Significant association (p < 0.05).

Although there is some variation among countries in terms of the concentration of IgG antibodies considered to be protective (WHO, 2011b), based on the previous recommendations of the US National Committee for Clinical Laboratory Standards (NCCLS) (Skendzel, 1996), international agreements and guidelines (Cutts and Vynnycky, 1999; Dimech et al., 2008), in the absence of IgM, pregnant women who had rubella IgG levels 10 IU/ml were classified as immune and those with IgG levels <10 IU/ml were classified as susceptible. In the present study, 79.5% (95% CI 76.3–82.5%) of the pregnant women had IgG levels of >10 IU/ml. None of these pregnant women had a previous history of rubella vaccination and they were immune from wild-type rubella infections. This might be due to the endemicity of the virus in the study area and sustained previous infections of the study participants before conception or during their childhood, as rubella infection is common among children and teenagers in the country (Shiferaw et al., 2016). The prevalence of rubella IgG in this study was also comparable to that reported in Burkina Faso (77%) (Tahita et al., 2013a), but it was higher than the prevalence reported in Niger (53%) (Onakewhor and Chiwuzie, 2011) and southern India (65%) (Padmaja et al., 2010). However, the IgG positivity rate in this study was lower than that found in studies conducted in other countries like Nigeria (97.9%) (Mohammed et al., 2010), Cameroon (88.6%) (Fokunang et al., 2010), Turkey (96.1%) (Tamer et al., 2008), Italy (85.8%) (Calimeri et al., 2012), and Mexico (97.1%) (Alvarado-Esquivel et al., 2016). These variations in rubella IgG positivity in different countries might be due to the difference in the endemicity of the rubella virus and the presence or absence of rubella vaccination in their immunization programs. According to the WHO, the incidence of rubella in Ethiopia was 7.27 per million inhabitants in 2017 and 5.39 per million inhabitants in 2018 (WHO, 2018). However, due to the benign nature of the virus and lack of independent rubella surveillance system in the country, most of the rubella reports might be from measles-suspected cases, as discussed earlier. The existing burden of the virus among women of child-bearing age might therefore be underestimated. In the present study, 9.5% of the pregnant women were positive for rubella IgM. As rubella IgM mostly declines quickly and is usually undetectable at 2–3 months after the infection (Vardas, 2011), the present result indicates the presence of acute rubella virus infection at 0–3 months before the blood

samples were obtained. Of the total pregnant women, 8.2% had both rubella IgM and IgG antibodies. As rubella virus re-infection following natural immunity is very rare (Mendelson et al., 2006), the pregnant women who had both IgM and IgG antibodies might have been in the resolving stages of primary rubella infections. Since the majority of these pregnant women were in the third trimester of pregnancy, they might have acquired the infection during the first or second trimester of pregnancy and subsequently developed IgG antibodies within 30 days of infection (Navigator, 2013). This indicates that these groups of pregnant women might not be immune before becoming pregnant and their fetuses may not be excluded from rubella-associated risks. Although there is a scarcity of data about CRS in the country, as indicated earlier (Mekonnen, 2017), the newborns from women infected with rubella during early pregnancy might acquire a congenital rubella infection and be born with rubella-associated congenital anomalies or CRS. Therefore, the screening of women of child-bearing age before conception or during pregnancy might be crucial to reduce the consequences of acute rubella infection during pregnancy. A similar IgM seroprevalence was also reported in Nigeria (9.2%) (Onakewhor and Chiwuzie, 2011). However, the IgM positivity rate in the present study was higher than those reported recently from Southern Ethiopia (Tamirat et al., 2017) and Turkey (2%) (Tamer et al., 2009). In contrast, the present study result was lower than that in another report from Nigeria (38.8%) (Olajide et al., 2015). These variations in rubella-specific IgM positivity might be due to the difference in endemicity of the rubella virus and sustained transmission in susceptible groups, differences in population density, variations in temperature/humidity, and the presence or absence of rubella vaccination, as discussed earlier. No statistically significant difference in rubella IgM and IgG positivity was found in relation to most socio-demographic characteristics of the pregnant women in this study. A similar finding was also reported in a recent study in Southern Ethiopia (Tamirat et al., 2017), and in other studies in Nigeria (Pennap and Egwa, 2016) and Namibia (Jonas et al., 2016). However, a statistically significant association between IgM positivity and area of residence was found in the present study; pregnant women from urban settings had two times the IgM positivity of those from rural settings. Although further study of rubella virus transmission dynamics in rural and urban settings is needed, this difference in IgM positivity between the two settings might be due to

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differences in population density. The high population density in urban areas might increase the contact rate and, as discussed earlier, pregnant women without protective levels of rubella immunity might acquire the infections. A similar finding was also reported in the pre-vaccine era in other countries (Goodson et al., 2011; Assaad and Ljungars-Esteves, 1985; Hinman et al., 1998). In this study, there was no statistically significant difference in rubella antibody positivity according to the trimester of pregnancy of the women, as has been reported in other studies (Agbede, 2011; Olajide et al., 2015). However, emphasis should be placed on those pregnant women who have had recent or acute infections due to the teratogenic nature of the virus (Lee and Bowden, 2000). In the present study, there was a statistically significant association between a previous history of spontaneous abortion and IgM positivity. Pregnant women with a history of one to three previous spontaneous abortions had 2.5 times the IgM positivity rate of those without a history of spontaneous abortion. An explanation for this is that those pregnant women who have a previous bad obstetric history (BOH) may be more vulnerable to acquiring acute rubella infections (Priyanka et al., 2017). Although the mechanism is not clear and further studies are needed, a similar finding has also been reported in other studies (Noor et al., 2015; Abdolreza et al., 2011). With regard to the relationship between IgG positivity and previous reproductive history, there was a statistically significant difference in the levels of IgG according to the absence of a previous history of spontaneous abortion. Pregnant women without a previous history of spontaneous abortion had 1.7 times the IgG positivity of those who had a previous history of spontaneous abortion. An explanation for this is that these groups of pregnant women might have acquired the rubella infection during their childhood and developed protective immunity against rubella virus at their reproductive age. Even though the clinical manifestations of rubella are nonspecific and it is difficult to diagnose clinically (WHO, 2011a), the present study found statistically significant differences in rubella IgM or IgG positivity according to the presence or absence of certain clinical manifestations in the pregnant women at the time of data collection. Although it was not statistically significant in the multivariate analysis, pregnant women with lymphadenopathy had three times the IgM positivity rate of those without lymphadenopathy. The pregnant women with a maculopapular rash had 3.5 times the IgM positivity of those without a maculopapular rash. In contrast, pregnant women without a maculopapular rash had 2.5 times the protective IgG antibody compared to those who had a maculopapular rash. There was, however, no statistically significant difference in the presence or absence of other clinical manifestations and IgM/IgG positivity. The lack of association between IgM/IgG positivity and most clinical manifestations may be due to the mild nature of rubella infections (Edlich et al., 2005). Furthermore, most patients with rubella may recover without any complications or sequelae and pass unnoticed, as rubella virus mostly causes a self-limiting disease in postnatal infections (Mwambe et al., 2014; Edlich et al., 2005; Forrest and Mense, 2008). When a comparison was made of the possible risk factors and rubella IgM positivity, the pregnant women who had frequent exposure to children in their daily activities had 2.8 times the IgM positivity of those who had no daily exposure. This can be explained by the fact that rubella infection is more common in childhood (Junaid et al., 2011) and children might harbor and spread the infection to susceptible pregnant women. Similarly, the pregnant women from Dessie Referral Hospital had 2.8 times the IgM positivity rate of pregnant women from University of Gondar Referral Hospital. This indicates that there may be epidemiological

differences in the circulation of rubella virus within the country. In addition, there may also have been differences in temperature and humidity between the study sites at the time of data collection. These differences might have contributed to the differences in active transmission of rubella virus in the study areas. Although maternal immunity is protective against intrauterine rubella infection (Aboudy et al., 1997), around 11% (95% CI 8.7–13.7%) of the study participants had IgG levels 10 IU/ml; these women were classified as seronegative and represent the susceptible group. A similar finding has also been reported in previous studies (Yadav et al., 1995; Pooja and Piyush, 2012; Oyinloye et al., 2013). In developing countries, about 10–25% of women have been reported to be seronegative (Cutts et al., 2000b; Gavin et al., 2015), and countries with high rates of susceptibility to rubella virus among women of child-bearing age might be at risk of CRS (Lambert et al., 2015). The susceptibility rate of 10% among adult women could result in outbreaks of CRS (WHO, 2011b). Therefore, attention must be paid to the susceptible group of women in this study in order to reduce the risk of CRS in their future pregnancies. Limitations of the study Due to the lack facilities, it was not possible to use advanced laboratory techniques like RT-PCR for the diagnosis of rubella. Furthermore, due to the nature of the study (cross-sectional study) and reagent constraints, it was not possible to obtain convalescent sera from each rubella IgM-positive/IgG-negative study participant. Since the study was conducted only in the selected referral hospitals of Amhara Regional State, Ethiopia, a large-scale community-based study might be important. However, as there is scarcity of data about rubella among pregnant women in the country, the information provided by this study might serve as a baseline for the study area and increase awareness for health decision-makers and collaborators in the country so that the longterm health consequences can be reconsidered. Conclusions The seroprevalence of rubella virus was found to be high, and many (9.5%) of the pregnant women had acute rubella virus infections at the time of data collection. This implies that the virus is endemic in the study areas. Despite 79.5% of pregnant women having IgG levels >10 IU/ml and being immune to natural/wildtype rubella virus infections, about 11% of the pregnant women were found to be non-immune and represent the susceptible group. These pregnant women may be at risk of developing rubella-associated congenital anomalies in their future pregnancies. Hence, the screening of women of child-bearing age before conception, introduction of rubella vaccination, and a strong surveillance system might be important to reduce rubellaassociated health complications in the country. Acknowledgements We would like to thank the University of Gondar for funding the project. Our special thanks also go to all of the study participants, data collectors, and other staff at the respective referral hospitals for their cooperation during the data collection process. Ethical approval The study was conducted after obtaining institutional ethical clearance from the Ethics Committee of the University of Gondar. A letter of agreement and the cooperation of the clinical director/ chief executive officer of each referral hospital were obtained.

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Informed consent was also obtained from each study participant, as per the National Research Ethics Review Guidelines (FDRE-MST, 2014). We also obtained written consent from each study participant to publish the findings in a peer-reviewed journal for the scientific community. Funding For data collection and laboratory reagents/materials, funding was obtained from the University of Gondar. Conflict of interest The authors declare that no competing interest exists with respect to the authorship and/or publication of this research paper. Author contributions YW: Participated in the conception, design and proposed the research idea, data collection, data clearance, entry, analysis and interpretation of the findings and drafting the manuscript and write-up. MT: Participated in the conception, design and proposed the research idea, supervision/consultations during data collection and interpretations of the findings. BA: Participated in consultation during data collection and interpretations of the findings. GF, MW, and MB: Participated in data collection and interpretations of the findings. BT: Participated in the conception, design and proposed the research idea, supervision/consultations during data collection and interpretations of the findings. All authors reviewed and approved the final manuscript. References Abdolreza Sotoodeh Jahromi, Akbar Kazemi, Gita Manshoori, Abdolhossien Madani, Seyed-Hamid Moosavy, Bita Seddigh. Seroprevalence of rubella virus in women with spontaneous abortion. Am J Infect Dis 2011;7(1):16–9. Aboudy Y, Fogel A, Barnea B, Mendelson E, Yosef L, Frank T. Subclinical rubella reinfection during pregnancy followed by transmission of virus to the fetus. J Infect 1997;34:273–6. Adam O, Makkawi T, Kannan A, Osman ME. Seroprevalence of rubella among pregnant women in Khartoum state, Sudan. East Mediterr Health J 2013;9 (9):812–5. Adewumi Olubusuyi M, Olayinka Adebowale O, Olusola Babatunde A, Faleye Temitope OC, Sule Waidi F, Adesina Olubukola. Epidemiological evaluation of rubella virus infection among pregnant women in Ibadan, Nigeria. Peer J 2014;1 (2):613. Agbede OO. Sero-prevalence of antenatal rubella in UITH. Open Public Health J 2011;5:10–1. Al-Rubai B, Aboud M, Hamza W. Evaluation of anti- rubella antibodies among childbearing age women in Babylon Governorate. Med J Babylon 2010;7:2. Alleman Mary M, Wannemuehler Kathleen A, Hao Lijuan, Perelyginab Ludmila, Icenogle Joseph P, Vynnycky Emilia, et al. Estimating the burden of rubella virus infection and congenital rubella syndrome through a rubella immunity assessment among pregnant women in the Democratic Republic of the Congo: potential impact on vaccination policy. Vaccine 2016;34:6502–11. Alvarado-Esquivel Cosme, Hernandez-Tinoco Jesus, Sanchez-Anguiano Luis Francisco, Ramos-Nevarez Agar, Cerrillo-Soto Sandra Margarita, Salas-Pacheco Jose Manuel. Rubella immune status in pregnant women in a Northern Mexican City. J Clin Med Res 2016;8(9). Assaad F, Ljungars-Esteves K. Rubella-world impact. Rev Infect Dis 1985;7:29–36. Bamgboye AE, Afolabi KA, Esumeh FI, Enweani IB. Prevalence of rubella antibody in pregnant women in Ibadan, Nigeria. West Afr Med J 2004;23(3):245–8. CDC. Control and prevention of rubella: evaluation and management of suspected outbreaks, rubella in pregnant women, and surveillance for congenital rubella syndrome. MMWR Recomm Rep 2001;50(RR12):1–23. CDC. Progress toward control of rubella and prevention of congenital rubella syndrome worldwide, 2009. Morbid Mortal Weekly Rep 2010;59(40):1307–10. Calimeri CA, Fauci VLA, Squeri R, Grillo OC, Lo Giudice D. Prevalence of serum antirubella virus antibodies among pregnant women in southern Italy. Int J Gynecol Obstet 2012;116(3):211–3. Cradock-Watson JE, Ridehalg MKS, Anderson MJ, Pattison JR. Outcome of asymptomatic infection with rubella virus during pregnancy. J Hyg 1981;87 (2):147–54. Cutts FT, Vynnycky E. Modelling the incidence of congenital rubella syndrome in developing countries. Indian J Epidemiol 1999;28(6):1176–84.

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