Abstract

Rubella virus infection (RVI) is, in most cases, a mild disease, but can cause severe defects in developing fetus and new born known as congenital rubella syndrome (CRS) from maternal infection during pregnancy. Rubella remains poorly controlled in many poor-resourced areas (Nigeria inclusive) despite being a vaccine preventable disease. The study aimed to assess the sero-prevalence of anti-rubella IgG antibody (immunity) and level of susceptibility and associated risk factors to rubella virus infection (RVI) among unimmunized pregnant women in a sub-urban setting. It was a hospital-based, cross-sectional descriptive study, conducted in a public general hospital in Rogo, a sub-urban community located in Kano State, Northwest, Nigeria. A total of 174 consented pregnant women [age range, 13-39 years; mean age, 22.3 ± (SD 2.1) years] attending antenatal care (ANC) clinic of a secondary healthcare centre from July to December, 2017 were randomly selected for the study. Information on bio-data, socio-demographic/risk factors and medical history related to previous RVI were obtained via structured questionnaires and hospital records respectively. Blood sample was collected from each study participant and tested for anti-rubella IgG antibody (past exposure) using ELISA technique. Data were analyzed using SPSS statistical software package version 15.0 and Pearson’s Chi-square or Fisher exact test was used for statistical analysis where applicable. A P-value ≤ 0.05 was considered statistically significant. The sero-prevalence of anti-rubella IgG antibody was 93.6% (163 of 174); and rate of susceptibility to RVI among the study population was 6.4% (11 of 174) and were predominant among the study subjects between ages 16 and 25 years in their first trimester of pregnancy. A significant association exists (P<0.05) between parity (P=0.0021) and history of miscarriage or premature birth (P=0.003) and anti-rubella IgG antibody. Similarly, there is an association between sero-negative IgG antibody and parity (P=0.0021) including occupation (subjects in this category were mainly housewives) (P=0.00541) of the study participants. The high rate of rubella exposure by the study participants indicates the presence and high endemicity of rubella infection in the study area, coupled with significant level of susceptibility to RVI by the study subjects. Hence, there is need for adoption of potent rubella vaccine into the routine national immunization programme to limit the spread of RVI and associated complications and clinical sequelae among the populace.

1. Introduction

Rubella, also widely referred to as German or three day measles is a vaccine-preventable infectious disease caused by rubella virus ^{1, 2}. In most cases, rubella virus infection (RVI) of susceptible children and adults is usually mild, self-limiting and often asymptomatic ^{3, 4, 5}. However, RVI can cause severe congenital abnormalities in newborn, known as congenital rubella syndrome (CRS) especially if it occurs just before conception and during early pregnancy or first trimester (generally within 20 weeks of pregnancy) ^{6, 7}. Though RVI may appear benign in over 90% cases, but can cause miscarriage, fetal death or stillborn baby and low birth weight ⁸ while CRS is often characterized by multiple sequelae defects affecting the heart, brain (causing inflammation or encephalitis), eye (producing cataracts and blindness) and ears (or deafness of the affected children ^{1, 9, 10, 11}.

Rubella virus is categorized and placed under the genus, Rubivirus, being the only member of the genus and an important member of Togaviridae family ^{9, 12}. It is spherical in shape, 40-80nm in diameter, enveloped with a single stranded ribonucleic acid (ssRNA) genome, enclosed by an icosahdral nucleocapsid ^{2, 9}.

The infection by rubella virus is mainly transmitted through person-to-person contact via airborne droplets from infected secretions during coughing or sneezing ^{13, 14, 15}. It is also acquired by developing fetus via intrauterine infection during pregnancy ¹. Rubella virus infection has an incubation period of 2-3 weeks and the virus is known to infect only humans while its infectivity in other living entities has not been established ^{1, 16}.

Rubella infection occurs globally ¹⁷ and the World Health Organization (WHO) ¹⁸ estimates that about 110,000 children are affected by CRS each year and most of them are found in developing countries of Africa and Asia ^{17, 19, 20, 21}. Unvaccinated individuals are at greater risk of acquiring RVI ²² while unimmunized and exposed pregnant females can transmit infection to the fetus via intrauterine dissemination ¹⁰. The rate of transmission and severity of infection vary with gestational age at which such maternal infection occurs ^{8, 23}.

The introduction of universal immunization at national levels of many industrialized regions including Europe, North America and parts of Asia such as Japan and Australia has greatly reduced rubella outbreak and thus limits the risk of acquisition of RVI especially among children and females of childbearing age ^{24, 25, 26, 27}. However, rubella remains a significant public health concern in many developing countries where rubella immunization programme is yet to be implemented ^{5, 12, 28, 29}.

In Nigeria, rubella immunization has not been included in the national immunization schedule especially for children and pregnant women attending antenatal care ³⁰. Despite concerted efforts to promote rubella vaccination for the susceptible groups and general populace, persistent knowledge gap exists in most developing nations of the world coupled with limited epidemiological data regarding the burden of the disease and the risk or vulnerability to RVI among females of childbearing age in those areas (Nigeria inclusive) ^{29, 31}.

The study sought to assess the level of vulnerability of unvaccinated pregnant women to RVI as there were no records of serological evaluation of protective IgG antibody among this category of women at the study area to determine their immunity or otherwise. It was also hoped that the data obtained from the study would be useful in the sensitization of the stakeholders and authorities at all levels of government in poor-resourced areas including Nigeria, about the critical need to adopt effective deterrent measures against the continuous spread of rubella virus among the populace to forestall associated morbidity and complications.

2. Materials and Methods

This study was carried out in Rogo, a rural development town in Kano State, North west Nigeria. Rogo is a Local Government Area (RGLA) covering a total area of 802km² with population of 227,742 ³². Rogo town lies between Latitude 11°34N and Longitude 7°50’E with an altitude range of 400m to 800m above the seal level ³². The area is populated mainly by Hausa-Fulani tribes while significant number of other tribes in Nigeria including Yoruba, Igbo, Ebira and other smaller ethnic groups are also found in the community. The main occupations of the inhabitants are agriculture, animal husbandry, trading while others are artisans or civil servants.

Healthcare delivery available in the area is mainly through primary and secondary healthcare centers as well as a few private clinics. Rogo General Hospital (RGH) plays a significant role in the healthcare delivery being the major secondary healthcare center serving the residents of Rogo town and other surrounding towns and villages. The hospital also provides immunization services for those who require them, including antenatal attendees.

The hospital (RGH) was chosen for the study because of its semi-urban location while most of the previous similar researches in Nigeria were mainly carried out in large tertiary hospitals located in urban centers.

It was a cross-sectional descriptive and hospital-based study carried out between July and December, 2017 following approval by Kano State Hospitals Management Board’s Ethical Committee.

The minimum acceptable sample (including attrition rate) of 174 used in this study was determined using the procedures as proposed earlier ^{33, 34} for the estimation of sample size. It was based on previous prevalence of anti-rubella IgG antibody of 97.9 % ⁴⁶ The participants were of different age groups, educational and socio-economic backgrounds at different stages (trimesters) of pregnancy.

All eligible pregnant women attending ANC at RGH were verbally informed in English language translated into local Hausa language about the study usually after routine ANC health education. After giving their consents, a total of one hundred and seventy four (174) of the pregnant women were selected and enrolled for the study. Systematic random sampling technique was used to recruit the participants as follows:

The first served pregnant women and every second woman thereafter who consented to participate in the study and without history of vaccination to rubella were recruited for the study until the required sample size was obtained ³⁰. The purpose and objectives of the study were once again explained to all participants prior to the administration of structured questionnaires and collection of samples.

Information on participants obtained includes that of bio-data, socio-demography consisting age, educational status, occupation as well as trimester of pregnancy and number of pregnancy (parity). Others were history of miscarriage, stillbirth or having babies with congenital defects and present or past experience of skin rash (exanthema) ^{23, 35}. The specific information on other medical history regarding measles-mumps-rubella (MMR) vaccination was also obtained from the hospital records and was corroborated by the study participants.

Five milliliter (5ml) of venous blood was aseptically collected from each subject by venipuncture. This was dispensed into an appropriately labeled anticoagulant-free sample tube, screw-capped and serum was obtained by centrifugation at 3000rpm for 5 minutes, ^{29, 36} and transferred into a correspondingly labeled cryo tube.

The sera were transported in cold chain to Microbiology Department Laboratory Unit of Aminu Kano Teaching Hospital (AKTH), Kano, Nigeria and kept frozen at -20°C until analyzed.

The enzyme-linked immuno-sorbent assay (ELISA) (Acon Laboratories Incorp. San Diego, USA) technique was used for the analysis.

The test kit is a solid phase enzyme immunoassay based on indirect principle for qualitative and quantitative detection or measurement of antibodies to rubella in human serum or plasma. It employs micro-well plates pre-coated with purified rubella antigens. When patient’s serum containing homologous antibodies, and appropriately prepared diluents are added, and incubated at required time (such as room temperature) for a specific period of time, will lead to the formation of immobilized rubella antigen-antibody complexes. Thus the rubella specific antibody (IgM or IgG) binds to the coated antigen in the wells while all unbound materials are washed away following subsequent washing.

Enzyme conjugate is added and binds to the antigen-antibody complex. A chromogenic substrate is then added after excess enzyme conjugate is washed off with washing buffer. The enzyme conjugate catalytic reaction is then stopped with an acid (such as Sulfuric acid) at a specific time and temperature. The intensity of colour generated is proportional to the quantity of specific immunoglobulin antibody in the test sample. The results are read using a micro-plate or ELISA reader at a specific wave length (eg. 450nm) and compared in a parallel way with calibrator and controls (eg. positive and negative controls) ^{4, 50}.

Sera and reagents for the serological testing were brought at room temperature (28⁰C) prior to analysis. Each serum sample was tested for the presence of anti-rubella IgG antibody using an Enzyme-Linked Immuno-sorbent Assay (ELISA) kit (Acon Laboratories Incorp., San Diego, USA) according to manufacturer’s instructions.

Briefly, 100µl of diluents serum (1:100) was added to wells of a micro-titer plate coated with rubella virus antigen and incubated at room temperature for 30 minutes. The wells were then washed three times with buffer and followed by addition of 100µl of peroxidase-labeled rabbit anti-human IgG to each well. The wells were incubated at room temperature for 30 minutes and protected from light. The wells were washed once again with buffer while 100µl TMB/H₂O₂ chromogen substrate was added to each well. The wells were again incubated at room temperature for 15 minutes. The reaction was then stopped by adding 100µl 0.5M sulfuric acid to each well and gently mixed. The optical density at 450nm was read using an ELISA reader (Tecan infinite F50; Tecan Trading AG, Mannedorf, Switzerland).

The experiment was conducted in duplicates; positive and negative control sera were also included. The results were recorded as rubella IgG concentration in international unit per milliliter (IU/ml). Rubella immunity was defined as anti-rubella IgG level ≥ 10 IU/ml; an equivocal result was considered as anti-rubella IgG level 8-9.9 IU/ml while no rubella immunity was defined as anti-rubella IgG level <8 IU/ml ^{4, 50}.

The results of the study were analyzed using the statistical package for social Sciences (SPSS) version 15.0 windows (SPSS Incorp., Chicago IL., USA) to generate frequency distribution and percentage prevalence scores of the various parameters or variables. Data were thus summarized as proportions or percentages. The association between past exposure to RVI or presence of IgG protective antibody (Immunity) and socio-demographic factors were measured by Pearson’s Chi-square or Fisher’s exact test as applicable. A P-value of ≤ 0.05 was considered as significant.

3. Results

A total of 174 pregnant women who gave informed consent were randomly selected and enrolled in the study. The mean age of the subjects was 23.2± (SD 2.1) years, ranging from 13 to 39 years. All the participants presented with married status at the time of investigation. Seventy (70, 40.2%) were in their first trimester while 98 or 56.3% in second and 6 (3.5%) in third trimester of pregnancy. Parity ranges from 0-7 while 39 or 22.4% were nulliparous, 23 (13.2%) primiparous and 112 (64.4%) multiparous with different educational backgrounds (Table 1). None of the women had received rubella vaccine in the past or prior to this study.

Anti-rubella IgG antibody was detected in 93.6% (163/174) of the study participants and negative in the rest 11 or 6.4 percent (Table 2). Although there was a preponderance of the previous RVI (presence of anti-rubella IgG antibody) among 16-20 years age stratification however, there was no statistical difference (P>0.05; P=0.8112) between age groups and prevalence of anti-rubella IgG antibody. Meanwhile, immunoglobulin G antibody (IgG) to rubella was detected in subjects from deferent parity groups, trimester stages and educational backgrounds but there were no significant relationships (P>0.05) between the variables of age (P=0.8112), education (P=0.09312) and stage of pregnancy (trimester) (P=3.4211) and previous exposure to this IgG antibody (Table 1). However, significant association exists between presence of anti-rubella IgG antibody and parity (P=0.0021) (Table 1), and medical history of miscarriage or premature deliveries (P=0.0032) (Table 3).

Furthermore, the anti-rubella IgG antibody was detected in 27 (15.5%) of subjects who have had either stillbirths (P=0.0121), congenital/neonatal defects (4, 2.3%; P=0.0891) or maculo-papular rash (Exanthem) (14, 8%; P = 1.0822) with no significant relationships (P>0.05) found between the prevalence of anti-rubella IgG antibody and those characteristic sequelae of previous RVI among the study participants.

However, the anti-rubella IgG antibody sero-negative cases (11, 6.4%) were found mainly among our subjects who were full-time housewives (100%), primiparous, with primary education (45.5% in each case) and were between ages of 16 and 25 years (54.6%) in their first trimester of pregnancy (54.5%) (Table 1). Statistical analysis indicates an association between sero-negative IgG antibody and occupation (this category of our subjects were mainly housewives) (P=0.00541) and parity (P=0.0021) of the study population.

4. Discussion

Rubella or German measles remains an important public health problem especially in low income and developing countries of the world due to its teratogenic effects on pregnant women and CRS sequelae arising from poor control measures ^{4, 37}. Vaccination and recovery from previous infection by rubella virus resulting in the production of protective IgG antibodies confer long lasting immunity to the disease or may reduce the severity of re-infection ^{38, 39}.

In this study, we detected a prevalence of 93.6% anti-rubella IgG antibodies in pregnant women in Rogo, a sub-urban community of North western region of Nigeria. This result (which shows a significant level of past or natural exposure to RVI as our subjects were not vaccinated against rubella prior to this study) was significantly higher than other results reported at different locations in Nigeria. For instance, rubella IgG prevalence of 54.1% was recorded in Maiduguri, North east Nigeria ³⁵, 68.5% in Ibadan, south west Nigeria ⁴⁰, 76% in Lagos, Nigeria ^31, and elsewhere outside Nigeria including Sudan (65.3%) ⁴¹ and Cameroon (88.6%) ⁴². Our result was however, similar to other published data in Mwanza, Tanzania (92.6%) ⁴³ and Kerala in India (94.3%) ⁴⁴.

In contrast however, the rates of RVI reported in Ilesha and Ibadan, South west Nigeria (96.6%) by the same investigators ⁴⁵ and Zaria North west Nigeria (97.9%) ⁴⁶ were relatively higher than the present finding. The high level of rubella immunity observed in pregnant women surveyed may be due to persistent endemicity of rubella virus in the study area resulting in the high risk of infection and thus high level of natural immunity ^{47, 48}. This is more or less expected as many areas in sub-Saharan Africa are characterized by poor-socio-economic factors where virus infections such as RVI thrives ³⁰. Thus large proportion of the populace are often exposed to RVI at an early stage of life ³⁵.

The difference in rate of past exposure among different sub-populations obviously reflects a varying epidemiologic characteristics of RVI in different socio-economic environment ^{49, 50}. Also, the high rates of previous exposure to rubella in the present study and other studies in Nigeria is an indication that RVI is widespread and persists in a continuous circulation in Nigeria ^{35, 40, 45, 46}.

The result of the present study showed that 6.4% of the pregnant women were negative for IgG antibodies and hence were not immune to RVI. This is not surprising as different sub-populations in Nigeria (including women of childbearing age) are not immune against rubella because rubella vaccination has not been incorporated into the routine immunization programme in the country ^{40, 45}.

A study from Bangladesh ⁵¹ also demonstrated high susceptibility rate of 15.7% among the populace studied. Inaccessibility to rubella vaccination on a large scale in different poor-resourced areas of the world was reaffirmed during the present study as none of the pregnant women surveyed was immunized using a well-established standard protocol for immunization against rubella prior to our study. The implication of such scenario is that significant proportion of pregnant women and indeed all women of childbearing age, in that community are potentially susceptible to rubella with attendant risk of having children with congenital defects or other complications during pregnancy ^{3, 8, 10}.

In the present study, the rate of past exposure to rubella was not influenced by stages of pregnancy (trimester, P>0.05) in contrast to previous reports in Nigeria ^{27, 28}.

However, it has been suggested that there is a greater risk (up to 90% of vertical transmission by those who suffer from RVI during the first trimester of pregnancy, especially when the pregnancy is less than 20 weeks) ²⁹. This is further compounded by absence of effective and safe treatment for mothers and developing fetuses at this stage and may result in fetal wastage or CRS ^{15, 24}. In any of these scenarios, a routine prenatal screening and general vaccination for females and children would help to monitor and prevent RVI complications and sequelae in these sub-population groups.

Regarding the distributions of rubella exposure and susceptibility, the women who were in age brackets 16-20 years and 21-30 years had greater rates of protective (IgG) antibody or immunity to RVI (though not statistically significant, P>0.05) as compared to those in age stratification ≤15 years and ≥ 30 years. This result corroborates the reports from Burkina Faso ⁵² which stated that rate of exposure and natural acquisition of immunity to rubella was influenced by age. These results however, contrast the published data in Togo ¹⁶ which concluded otherwise.

However, all the pregnant women found to be susceptible to rubella (IgG sero-negative) in this study were full housewives (who did not engage in any outdoor occupation or economic activities) aged between 16 to 25 years. This finding however, contrasts the suggestion that susceptibility to RVI may be influenced by regular exposure or contact with larger outdoor environmental conditions or risk factors where rubella virus may be circulating in the locality ⁵. It was however, in line with the published data by various investigators ^{9, 39} suggesting that occupation was not significantly associated with RVIs except in hospital settings and daycare centers where occupation is considered as high risk to un-immunized healthcare workers.

The results of this study showed that there was a relationship between previous exposure to RVI and parity including history of miscarriage or premature birth. This is in consonant with widespread opinion that certain clinical sequelae of RVI such as medical history of low birth weight and premature birth may serve as indications of previous exposure to rubella infection by un-immunized females in rubella endemic areas ^{4, 53}.

Our study revealed that there was no significant link between past exposure to rubella infection and some socio-demographic characteristics of our subjects such as education and age of pregnancy (trimester). The present results contradict the reports from various studies elsewhere especially in Africa ^{30, 32} in which educational attainment by the study population and age of pregnancy were associated with the rates of previous exposure to rubella disease.

In conclusion, the study reveals a high rate of past exposure to rubella virus by the pregnant women surveyed with significant proportion of susceptible individuals to RVI among them. These women can therefore be infected and also transmit RVIs to others in the community. There is urgent need for the integration of rubella vaccination into national routine immunization program in Nigeria to checkmate the continuous spread of RVI in various communities.

It will also mitigate rubella-associated complications among un-immunized females of childbearing age/pregnant women and children born of infected mothers.

Acknowledgements

The authors would like to thank the Kano State Hospital Management Board for granting the permission to use the Rogo General Hospital (RGH), and the management of Aminu Kano Teaching Hospital (AKTH), for allowing the use of their laboratory facility. The authors also extend their appreciations to the staff of Antenatal Care (ANC) Clinic of RGH for their cooperation and support, and the participants who took part in the study.

Declaration of Conflict of Interest

No conflict of interest declared.

References