Study setting and population
The study was conducted in Kelwani primary hospital and Derayitu health center of Awra and Gulina district of Afar Region which is found in the north eastern part of Ethiopia. The majority of the communities are pastoralists whose livelihoods depend on livestock, specifically camels, cattle and small ruminants while few are practicing agro-pastoralist and growing crops by irrigation of Awash river.
Study design and sample size determination
A health institution based cross-sectional study design was used to determine sero-prevalence and risk factors of brucellosis among febrile patients visiting health institutes of Awra and Gulina district of Afar region, Ethiopia from February to May 2019. The finding of previous community based sero-prevalence of brucellosis (4.4%) in other pastoral area of the community of the region was used to estimate the sample size . Based on this information, the calculated sample size, at 95% confidence level, 5% degree of accuracy and with 10% compensation for refusal, was 444 respondents.
Study participants, sample and data collection
All patients older than two years who had fever and measured axial body temperature ≥ 37.5 °C during data collection period, willing to provide written consent/assent for participation, was recruited to the study. A total 444 respondents were interviewed in their local language (Afar language) using a structured questionnaire to collect socio-demographic characteristics, sex, age, educational status, marital status, occupational status, residential address (urban/rural), potential risk factors: milk source (large ruminants, small ruminates or camels), ways of milk consumption either raw or boiled, experience of milk consumption from aborted animals, exposure to aborted fetus/ materials of animal without protective equipment, and the clinical features they felt along the onset of days of the features. A 3–5 ml of venous blood was collected from each febrile patients using plain vacutainer tube. Thin and thick blood smears were prepared immediately from each blood samples for the diagnosis of malaria. The remaining sample was kept at room temperature for 30 minutes to facilitate clotting and centrifuged at 3000 rpm for 5 minutes to get clear serum. All sera were separated in a labeled 1.8 ml Cryotubes, transported to Addis Ababa Federal police laboratory in a cold box and stored at 4 °C until testing.
Blood examination for malaria
Malaria was detected from Giemsa stained blood films following the guideline of Ethiopian Ministry of Health for the diagnosis of malaria and identification of Plasmodium species at the health institute .
Blood examination for brucellosis:
Two types of serological tests were used to determine sero-prevalence of brucellosis.
The sera were screened using Rose Bengal Plate Test (RBPT) and positive reactors were further subjected to ELISA. All sera and RBPT reagent and controls were taken out from refrigerator and kept at room temperature for 30 minutes to screen for anti-Brucella antibodies in Addis Ababa Federal police laboratory. As previously described , the smooth, attenuated stained Brucella antigen suspension was mixed with positive and negative controls and serum on circular test card. If specific antibody to Brucella antigen is present in the serum, it reacts with the antigen suspension to produce visible agglutination after shaking on a low speed shaker for four minutes. No agglutination indicates absence of specific antibodies to Brucella antigens. All sera positive for Brucella antibody using RBPT were transported to Armeaur Hansen Research Institute (AHRI) to confirm the anti-Brucella antibodies by IgG ELISA. According to manufacturer’s guideline (Demeditec Brucella abortus IgG ELISA DEBRU01, Germany), qualitative anti-Brucella IgG ELISA was determined based on the principle of the spectrophotometric enzyme immunoassay at the wave length of 450 nm. The calculated absorption for the patient sera were compared with the value of the cut-off standard. If the value of the sample was higher than the cut-off standard, it was considered as positive whereas below the cut-off standard, the result was considered negative.
Descriptive analysis was used to summarize the data in the forms of frequencies and percentages. Pearson Chi2 test was used for testing relationships between brucellosis and malaria infection with each demographic characteristic of study participants. Univariate logistic regression analyses were conducted to establish the association of the putative risk factors with brucellosis and odds ratio at 95% confidence intervals (CI) was considered. All risk factors significant at univariate analysis were considered for multivariate logistic regression analysis to determine the independent association between risk factors and brucellosis at 95% CI. P-value below 0.05 was considered statistical significance.
This study received ethical clearance from the Ethical Review Board of Department of Medical Laboratory Science, College of Health Sciences, Addis Ababa University (DRERC/410/19/MLS). Permission was obtained from Derayitu Health center and Kelwani Primary Hospital. Participants’ information sheet, which contains the objective of the study, inclusion/exclusion criteria, the required data and methods of data collection as well as informed consent/assent document, were prepared in Amharic the national language of the country. Then, the elements of participants’ information sheet initially were orally translated to the local language (Afar Language) and described to each of the study participants or parents in case of children under 18 years by trained local health personnel. Informed consent was obtained from the participants and/or assent in children aged between 12 and 18 years. Blood sample was collected under aseptic condition by experienced laboratory technicians. Study participants who were found positive for malaria were treated according to malaria treatment guideline and the rest were treated with different antibiotics accordingly as per clinician presumptive diagnosis.