Study area
The study was conducted in South Western Uganda in Bwindi-Mgahinga Conservation Areas (BMCA) which is composed of Bwindi Impenetrable National Park (BINP) and Mgahinga Gorilla National Park; particularly in Kanungu, Rubanda and Kisoro districts which host the two-mountain gorilla national parks. The area is bordering Democratic Republic of Congo to the west and Rwanda to the south (Figure 1).
BINP is part of Bwindi Impenetrable Forest which is situated along the Democratic Republic of the Congo (DRC) border next to the Virunga National Park and on the edge of Albertine Rift; a biodiversity hotspot (8,9) . It’s composed of 321 square kilometres (124 square miles) of both Montane and lowland forest; it is accessible only on foot. BINP is a United Nations Educational, Scientific and Cultural Organization-designated World Heritage Site. It covers an area of 331 square kilometres and is located in the highest parts of Kigezi highlands with an altitude of 1,190 to 2,607 meters above sea level and 60% of this magical park has an elevation of over 2,000 meters above sea level. The highest elevation in the park is Rwamunyonyi hill at the eastern edge and the lowest part of the park is located at its most northern tip (8) .
Mgahinga Gorilla National Park is 33.7 square kilometres and its Uganda’s smallest national park covering the northern slopes of the three northern most Virunga volcanoes, Mt Gahinga, Mt Muhavura and Mt Sabinyo. It’s about 10kilometres south of Kisoro; bordered to the south by the Volcanoes National Park of Rwanda and to the west by the Virunga National Park of the Democratic Republic of Congo. In 1951 the ecosytem was reduced in size from 3370 hectares to 2330 hectares and re-gazetted, to meet growing demand by the local people for more land for cultivation; hence communities cultivate to the edge of the park with no buffer zone (7) . Consequently there is intensification of Human-wildlife interaction and Human-wildlife conflicts. It is the part of the larger Virunga Massif that spreads over the three countries (Uganda, DRC and Rwanda) (7) .
According to the 2020 census report, BMCA landscape has 3,730 indigenous Batwa populations with 1787 males and 1,943 females who regularly interact with the ecosystem as part of their way of life (10) . Individuals from other tribes in the landscape also access the park either illegally, or as park staff from the communities or as part of authorized entry to access resources in multiple-use zones (4) .
Study design
A cross sectional study was conducted on different stratified groups which included boda-boda cyclists, tour guides, people in market places, community members and patients attending health care services to determine the prevalence of COVID-19 in Bwindi-Mgahinga Conservation Area landscape and the predisposing factors associated with it. The health facilities were purposively selected following guidance from the District Health Officer with consideration that they were located in the study sub counties that are closest to the conservation area and they served people in these areas neighboring the BMCA. Tour guides and boda boda cyclists were also selected given that they could interact closely with the trekkers. People in market places and community members in the study sub-counties closest to the conservation area were also selected
Sample size determination
A sample size was determined using Kish Leslie formula below
Where X= sample size, Z= z-score, ME= margin of error, p=population proportion
A prevalence estimate of 50% was assumed at 95% confidence interval considering that no study had been done before, resulting in a minimum sample size of 384 participants. To increase the precision of the study, the sample size was increased to 576 participants.
Data collection
The Kobo collect data collection tool (https://kf.kobotoolbox.org) which was installed on phones was used to collect data using semi structured questionnaires with strict compliance to COVID-19 prevention guidelines. Nasal samples were collected from the different individuals after consenting and the swabs were put in viral transport media and kept at 2 to 8 degrees Celsius.
Sample processing and analysis
Samples were analyzed at UVRI using real-time PCR method for RNA extraction using qiagen kit and amplification by ABI7500 real-time system as described by Thermofisher scientific. Briefly, the process of RNA extraction involved majorly four steps; lysis, binding, wash and elution. The isolated RNA obtained after lysis bound to the silica membrane, washed using wash solutions to remove all unbound proteins. RNA was then eluted by adding elution buffer. Positive and negative controls were also included in the extraction process. This extraction process was then followed by reverse transcription to DNA and amplification of transcribed DNA.
Amplification was done using ABI 7500 Real time PCR system. Briefly, the Nucleic Acid Technology (NAT) kit that was used is designed for detection of ORFlab and N genes of SARS-CoV-2 RNA using fluorescence probing technology in real time PCR. RNA isolated and purified from upper and lower respiratory specimens is reverse transcribed to cDNA and subsequently amplified in the real-time PCR instrument. In the process, the probe anneals to a specific target sequence located between the forward and reverse primers. During the extension phase of the PCR cycle, the 5’ nuclease activity of Taq polymerase degrades the probe, causing the reporter dye to separate from the quencher dye, generating a fluorescent signal. With each cycle, additional reporter dye molecules are cleaved from their respective probes, increasing the fluorescence intensity. Fluorescence intensity is monitored at each PCR cycle by real-time PCR system.
The primers and probes added onto the sample master mix as described by Sansure biotech Inc are designed to detect SARS-CoV-2 RNA qualitatively. The internal control which was also added to the master mix targets the Rnase P gene and it monitors the sample collection, sample handling and the whole real-time PCR process to avoid false negatives. An additional primer/probe set to detect the internal control gene processed with the clinical specimens is also included in the kit (11).
Data analysis
Data in kobo collect software was downloaded as an excel file which was later imported into STATA for statistical analysis. Descriptive statistics were generated and logistic regression performed to determine predisposing factors of COVID-19 prevalence among people living in the BMCA landscape.