Adults volunteers were recruited during the second rainy season in September 2018 in five villages in the Asante Akim North district in the Ashanti region, Ghana. The district lies within the moist semi-deciduous forest belt of Ghana. The forest areas are characterized by a tropical climate with two rainy seasons . The villagers live in simple houses constructed from local materials and their main occupation is farming. One to three days before recruitment, community health workers visited the representative villages and invited all adults who lived in the catchment area to participate. Exclusion criteria were clinical signs of infection (axillary temperature ≥37,5°C or history of fever in the past 48 hours, headache, chills, myalgia, dizziness, nausea and diarrhoea), pregnancy and puerperium.
On the day of blood collection, field workers informed the villagers again about the exclusion criteria and the planned procedure of blood collection. Participants were only included after providing written informed consent. Age, gender and absence of exclusion criteria were assessed with a questionnaire for each participant. An RDT targeting the histidine-rich protein II antigen specific to P. falciparum and a pan-malarial antigen common to Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae was performed on venous blood immediately to inform all participants about their test result (BinaxNOW Malaria Test; Binax, Inc., Scarborough, ME, USA). According to the WHO guideline, faint test bands were interpreted as malaria positive . Similar to a recent study, faint test bands were defined as being only visible in a good light in agreement of two members of the study team observing the test . If the band was difficult to see in good light but both members of the study team still agreed to see it, the test was interpreted as very faint and reported separately.
For blood counts and PCR, venous blood was collected in EDTA blood tubes (Sarstedt). Blood counts were performed the same day using the Sysmex XP-300 automated hematology analyzer (Sysmex, Kobe, Japan). For PCR analyses EDTA-anticoagulated blood was centrifuged and the pellet was collected and stored at -80°C. Frozen samples were shipped to the National Reference Centre for Tropical Pathogens, Bernhard Nocht Institute of Tropical Medicine, Hamburg, Germany. Nucleic acid was extracted manually from 200 μL of frozen red blood pellet using the QIAamp DNA Blood Mini-Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Screening was performed by genus-specific real-time PCR for Plasmodium species (RealStar Malaria PCR kit 1.0, Altona Diagnostics, Hamburg, Germany) as described before [10, 11].
Samples tested positive were additionally subtyped using species-specific real-time PCR targeting P. falciparum, P. vivax, P. ovale, P. malariae, and Plasmodium knowlesi (Altona Diagnostics, Hamburg, Germany) as described by the manufacturer and others [10, 11]. If the commercial species-specific PCR was negative, a real-time in-house one-tube SybrGreen malaria PCR was performed as described previously. The species-specific in-house PCR targets P. falciparum, P. knowlesi, P. vivax, P. ovale, and P. malariae and was earlier shown to be more inhibition-resistant compared to the commercial PCR . Furthermore, sub-species identification was performed for all P. ovale infections using an in-house PCR according to the protocol of Bauffe et al., as previously described . Data analyses were conducted using R (R Foundation for Statistical Computing, version 3.4.3).
The study was approved by the Committee on Human Research, Publication and Ethics at the Kwame Nkrumah University of Science and Technology, Kumasi, Ghana (CHRPE/AP/455/18).