1. Introduction

1.1. Background 

Rubella is an acute, contagious viral infection. Although the illness is generally mild in children, it has serious consequences in pregnant women causing fetal death or congenital defects known as Congenital Rubella Syndrome (CRS). Airborne droplets transmit rubella virus when infected people sneeze or cough. Humans are the only known host [1]. The disease affects mostly children, young adults, women of child bearing age and pregnant women. It is common in many resource-constrained countries where vaccination has not yet been introduced. Rubella infection may present as an acute, mild or asymptomatic illness; therefore, the outbreaks may occur without clinical recognition or may be misdiagnosed as measles cases [2]. Congenital Rubella Syndrome (CRS) is an illness in infants that results from maternal infection with rubella virus during pregnancy. When rubella infection occurs during early pregnancy, serious consequences-such as miscarriages, stillbirths, and a constellation of severe birth defects in infants can result. The risk of congenital infection and defects is highest during the first 12 weeks of gestation and decreases after the 12^th week of gestation; defects are rare after infection in the 20^th week (or later) of gestation. 1-3 Common congenital defects of CRS include cataracts, congenital heart disease, hearing impairment, and developmental delay. Infants with CRS often present with more than one sign or symptom consistent with congenital rubella infection. However, infants may present with a single defect, with hearing impairment being the most common single defect [3]. The World Health Organization (WHO) estimates that worldwide more than 110,000 cases of CRS are from developing countries annually [4]. The serum immune response in CRS differs from that seen in rubella (and from many other viral diseases). At birth, the serum of an infant with CRS contains maternally derived rubella-specific IgG antibodies as well as IgG and IgM antibodies synthesized by the fetus. Maternal rubella-specific IgG is also found in normal infants born to women who are immune to rubella. Therefore, rubella-specific IgM is used to diagnose congenital rubella infection in infants. In infants with CRS, rubella-specific IgM can be detected in nearly 100% at age 0-5 months; about 60% at age 6-12 months; and 40% at age 12-18 months; IgM is rarely detected after age 18 months [5]. 

2. Methods 

2.1. Study Setting 

Based on world meters’ report, Ethiopia is the second most populous country in Africa and ranks 14^th in the world with a population of 107.53 million. Children under 5 years of age make up 12.5% of the population. 90% of the population has access to formal health care services. Life expectancy at birth was 55 years, and the total fertility rate was 5.3 children per woman of child bearing age. The country has a surface area of 1.1 million square kilometers and is administratively divided into 9 regional states and 2 city administrations. There are more than 80 linguistic groups in Ethiopia. Djibouti, Eritrea, the Republic of Sudan, the Republic of the South Sudan, Kenya, and Somalia border the country [6,7]. The suspected measles samples for the study are collected from all regions such as Amhara, Oromia, SNNPR, Addis Ababa, Benshangul gumez, Gambela, Tigray, Dire Dawa, Harari and Afar. The sample collection was done by surveillance officers of PHEM, WHO and Health facility surveillance focal person based on Measles guide line. The standard case investigation form was filled at Health facility level and when the sample arrives at the National Measles Laboratory, is one of the WHO accredited laboratory under virology research team at EPHI. The laboratory is a member of the global WHO vaccine-preventable disease laboratory network and works according to WHO standards and protocols. Annually receives Proficiency Penal (PT) and scored an excellent result till 2005.The laboratory send samples quarterly to AFRO Regional Reference Laboratory (RRL) for External Quality Assessment (EQA) and the score is above 95%. All variables of the suspected Measles case on the case investigation form entered to the computer by Measles laboratory data manager. The result of the suspected Measles sample shared to PHEM and WHO on time for intervention. 

2.2. Case Definition 

A suspected measles case was defined as any patient with fever, generalized maculopapular rash, and either cough, or coryza, or conjunctivitis regardless of age and sex [8]. 

Blood samples are collected on suspected cases and all sera are tested for the presence of measles IgM antibody.

This surveillance system provided a platform for identifying suspected rubella cases. Suspect measles cases with sera negative for measles IgM antibody are further tested for rubella. Laboratory confirmed rubella cases were patients who had a positive rubella IgM test results by Enzyme Linked Immunosorbent Assay (ELISA) technique. A confirmed rubella outbreak was defined as a cluster of 5 or more IgM confirmed rubella cases occurring within a month period within a district [9]. 

2.3. Sample Collection 

During 01 January 2010-31 December 2017, all suspected measles cases of blood samples were collected from all regions of Ethiopia by trained staffs about Vaccine Preventable disease (VPD) by WHO collaborating with Ethiopian Public Health Institute at Ethiopia. Demographic and clinical information about the patient was captured through the Case Based Reporting Form (CRF). For measles and rubella testing, samples were transported in a cold box to Ethiopian National Measles and Rubella Laboratory located at the Ethiopian Public Health Institute (EPHI), Addis Ababa, Ethiopia. 

2.4. Laboratory Method 

First, all samples were tested for measles specific IgM antibody and those samples having negative or two sets of indeterminate (equivocal) measles results were tested for rubella specific IgM by indirect ELISA technique, with sensitivity of 98% and specificity of 97.3%, using a commercially available standard kit (Siemens Diagnostics, Marburg, Germany). A serum/plasma sample of 5 μl volume was diluted in a 1:21 ratio using diluting plate (two wells for one sample). 150 μl of diluted sample was then transferred to a rubella antigen coated test plate and incubated at 37^°C for an hour. Then the plate was washed with an ELISA plate washer to remove unattached antibodies and debris, and 100 μl enzyme labeled anti-human IgM working solution was added to the wells and incubated at 37^°C for an hour. After washing, a substrate-chromogen working solution was added and incubated at room temperature for 30 min to allow the labeled enzyme (if any) break the substrate and give color through the chromogen. Finally, a stop solution was added to stop the substrate-enzyme reaction and the Optical Densities (OD) of the wells were read with an ELISA reader. Based on the protocol, the read out was recorded in two programs of the machine. One, the OD value of each well was given (antigen and control OD). Second, the calculated change in OD of each sample (antigen well OD minus control well OD) was recorded. Those samples having a change in OD value of >0.2 were registered as positive and those <0.1 were negative for rubella virus IgM. Samples with a change in OD between 0.1 and 0.2 were recorded as indeterminate (equivocal). All samples were tested once for rubella Ig M by national measles laboratory staffs who had trained by WHO with collaborating Ethiopian Public Health Institute at Ethiopia. 

2.5. Data Analysis 

The laboratory results and patient information from the case report form were entered into an Epi-info based electronic database. The case-based surveillance data were regularly consolidated, cleaned, analyzed and disseminated to stakeholders for action including the Federal Ministry of Health (FMOH), Public Health Emergency Management (PHEM), World Health Organization (WHO) Ethiopia Country office and WHO Regional Office for Africa. Data for the purpose of this study were extracted and analyzed by Epi-Info software version 3.5.4. 

2.6. Quality Assurance 

The Ethiopian National Measles and Rubella laboratory is member of the global WHO vaccine-preventable diseases laboratory network, as such, it is subjected to periodic quality control checks and accredited annually in order to deliver credible results for the program. All the equipment and materials of the laboratory were supplied by WHO. Standard Operating Procedures (SOPs) and job aids were available for lab activities. The lab receives External Quality Assessment (EQA) samples once a year and sends 10% Quality Control (QC) samples quarterly. To check the validity of each run, kit and in house control materials (negative and positive) were used and patient results were reported only for a valid run. The lab performance of ≥95% accuracy for both EQA and QC in the study period is a witness for credible result delivery. 

3. Result 

From January 2010 through December 2017, a total of 32,959 suspected measles cases were investigated with blood specimen collection for confirmatory laboratory testing. Of these, 20,214(61.3%) were tested for rubella-specific IgM antibody according to laboratory procedures. Of the 20,214 cases tested, 333were samples of age ranges from 0 month to 6 months. From this 333 samples 14 were laboratory-confirmed as rubella, 247 had test results that were negative for rubella IgM, 13 had test results that were indeterminate (Table 1). 

From 2010-2017, a total of 537 suspected measles cases were investigated with blood specimen collection for confirmatory laboratory testing for age below 6 months’ age. Of these, 333 (62.0%) were tested for rubella-specific IgM antibody according to laboratory procedures; however, in 2010 and 2014, because of a shortage of measles and rubella IgM test kits, 45and 14 serum samples were not tested respectively. Of the 333-specimen tested for rubella IgM, the highest number of specimens,109 cases were tested on 2010 and followed 61 cases by year 2012 (Table 2). 

During 2010-2017, rubella cases were reported from all 11 regions of Ethiopia, and cases were distributed widely throughout the country (Table 3) but cases reported for age below 6 months were reported from 5 regions of Ethiopia. Seasonal distribution of cases occurred each year and peaked on May 4(0.29%) (Figure 1).

The majority of the lab-confirmed rubella cases were the Oromia Region (35.7%) followed by SNNPR (28.6%), Amhara (21.4%) and Addis Ababa Region (14.3%) (Table 3). These regions are the four most populous regions in the country. 

4. Discussion

Currently, rubella vaccination is not part of child routine immunization services in Ethiopia and a standalone surveillance system for rubella and CRS does not exist. In the major urban centers, some private practitioners provide Rubella Containing Vaccine (RCV) to infants at 9 months of age or older in the form of measles-rubella vaccine, but the coverage is unknown among the general population as the services is not monitored yet. Little attention has been given to rubella as it is not considered a killer disease. The major impetus behind rubella vaccination and rubella related studies is to reduce the risk of CRS. Unfortunately, there is no recent data on the incidence of CRS in Ethiopia to guide evidence-based decision making for rubella vaccine introduction [10]. 

In this study, 277 (61%) of the 537 specimens from individuals children below 6-month age with suspected measles cases had test results that were negative for measles IgM antibodies and later had negative or indeterminate rubella test results. One of the reasons for this may be the timing of the collection of the specimens. It is known that specimens collected within the first 3 days of the onset of measles rash or after 28 days may not have antibody levels high enough to be detected using the standard serological tests. However, according to the VPD surveillance manual of CRS, infants can shed the virus for prolonged periods, (up to 1 year of age or longer) infants with CRS should be considered infectious until they are at least 1-year-old. 

The majority of infants will shed virus for 3 months after birth, so screening will typically start at 3 months after a decline would reasonably be expected the collection of specimens is encouraged any time after the three months after the onset of rash, so that opportunities for laboratory testing are not missed. Another possible reason is the presence of other febrile rash illnesses, such as chickenpox, erythema infectiosum, roseola infantum, meningococcal infections, scarlet fever, entero viral infections, and drug rashes. The integration of rubella testing alongside measles case-based surveillance provides an opportunity to gain understanding about the epidemiology of rubella infection. Since 1999, countries in the American Region of the WHO have conducted integrated rubella and measles surveillance [11]. For purposes of integrated surveillance, a patient who presents with generalized rash and fever or is suspected by a health care worker of having measles or rubella infection is considered to have a suspected measles or rubella case [12]. The adoption of a case definition that is more sensitive to rubella, similar to the one used in Pan American Health Organization, would likely lead to an increase in case detection and a better understanding of the rubella epidemiology without losing sensitivity for detecting measles. To better understand the burden and epidemiology of rubella and CRS, Ethiopia may consider adopting a comprehensive approach to surveillance, including a more inclusive case definition for measles and rubella, establishing sentinel surveillance for CRS, and conducting appropriate studies to assist in defining the rubella susceptibility profile in school-aged girls and women of child bearing age. Information from such studies will be useful in the consideration of appropriate rubella control strategies in Ethiopia. 

The findings of this study are subjected to several limitations. First, the case definition to detect rubella cases was designed for measles cases and as a result may under-estimate the true burden of rubella in the country. Second, since up to 50% of rubella cases are asymptomatic, the case definition used would not be able to identify all rubella cases. Cases without symptoms, mild symptoms or without a rash would not have been identified. Thirdly, our analysis was not able to determine neither the prevalence of current rubella infection nor the immune status of reproductive age females in order to predict the risk of CRS in the population. Finally, we were unable to identify any epidemiologically-linked or clinically confirmed rubella cases during the study period as there is no ongoing rubella- specific surveillance. This would result in a decrease in the number of reported rubella cases and outbreaks in the country.

Figure 1: Rubella -positive cases by month, 2010 -2017, Ethiopia.

Table 1: Number of Specimens Tested for Rubella Immunoglobulin (Ig)M Antibodies and Results of age ranged from 0 to 6 months, Ethiopia, 2010-2017.

Table 2: Number of Specimens Tested for Rubella Immunoglobulin (Ig)M Antibodies and Results of age below 6 months’ age by year, Ethiopia, 2010-2017.

Table 3: Distribution of Laboratory-Confirmed Rubella Cases by Region for age below 6 months, Ethiopia, 2010-2017.

1. CareHealth, Infection, Rubella, August 2015 Archived:http:chealth canoe.com/chnnel condition info details asp?disease id=252&channel id =1020&relation id=71085
2. Mariambo MM, Aboud S, Mushi MF, Groß U, Mshana SE, et al. (2016) Serological evidence of acute rubella infection among under-fives in Mwanza: a threat to increasing rates of congenital rubella syndrome in Tanzania. Ital J Pediatr 42: 54.
3. Tatiana L, Susan R, Emily A, Joseph I. VPD Chapter 15: Congenital Rubella Syndrome Surveillance Manual.
4. Esete Y (2017) Rubella virus among pregnant women, Lambert Acadamic Publisher.
5. Vaccine Assessment and Monitoring Team (1999) Guidelines for surveillance of congenital rubella syndrome and rubella, Field test version.
6. Population of Ethiopia (2015): Ethiopia Population clock, United Nation Department of Economics and Social Affairs: Population Division.
7. Ethiopia National Expanded Program On Immunization. Comprehensive Multi Year Plan 2016-2020 Federal Ministry of Health, Addis Ababa.
8. Manual for the laboratory diagnosis of measles and rubella virus infection, Second edition, August 2007.
9. African Regional Guidelines for Measles and Rubella Surveillance, WHO Regional Office for Africa, 2015.
10. Mekonen G, Berhane B, Kathleen G, Ayesheshem A, Mesfin T, et al. (2016) Epidemiology of rubella virus cases in the pre-vaccination era of Ethiopia, 2009-2015.
11. Kassahun M, Tesfaye B, Balcha M, Wendemagegn K (2011) The Journal of Infectious Diseases, The Epidemiology of Rubella Disease in Ethiopia: Data From the Measles Case-Based Surveillance System, 204: S239-S242.
12. Castillo-Solorzano C, Carrasco P, Tambini G, Reef S, Brana M, et al. (2003) New horizons in the control of rubella and prevention of congenital rubella syndrome in the Americas. J Infect Dis 187: 146-152.