Definition of terms
Subclinical malaria infection, herein, was defined as Plasmodium falciparum positive individual/case without fever (i.e. temperature ≤37.5°C) at the time of sampling as well as the absence of any malaria related symptoms (Laishram et al., 2012). Subpatent malaria infections were defined as infections exclusively detected (positive) by only varATS qPCR (Hofmann et al., 2015).
Ethical Approval
The study was approved by the Committee on Human Research, Publication and Ethics of the Kwame Nkrumah University of Science and Technology, School of Medical Sciences and Komfo Anokye Teaching Hospital (CHRPE/AP/030/20), and the ethics review board of University of Notre Dame, USA (approval no. 19-04-5321). Before sample collection, written informed consent was obtained from participants aged 18 years and above. For children between the ages of 8 to 17 years, consent of a parent or legal guardian was obtained, followed by written assent of the child.
Study areas and population
The study was conducted at three districts/regions in Ghana; Afamananso in Sekyere South District (Ashanti Region), Obom in Ga South (Greater Accra Region) and Gekrong, Pawa, Nsuogya, and Keri (Nkwanta South Municipal) in the Oti Region of Ghana (Figure 1). Sekyere South District (latitude 6o 50’N and, longitude 1o 40’W) is one of the 43 districts in the Ashanti region of Ghana and located about 40 kilometers away from Kumasi on the Kumasi-Mampong road. Sekyere South has a population of 120,076 distributed into 29,892 households (GSS, 2021). The Ga South district lies within latitude 5°35’N and longitude 0°10’ W and occupies a land area of 284.08 square kilometers with about 412 communities. Ga South has a population of 350,121 while Nkwanta South Municipal in Oti region occupies a land area of 2,473 square kilometers with a population of 135,936 (GSS, 2021). The rural population together with the urban dwellers in the Nkwanta district are distributed in about 22,429 households with an average household size of 4.2 persons (GSS, 2021)
Study design and sample collection
Cross-sectional community-based surveys were conducted at different time points in the aforementioned areas. Samples were obtained in Sekyere South from December 2020 to January 2021 while data were collected from Nkwanta South and Ga South in October and July, 2021 respectively. Prior to the commencement of the study, opinion leaders in each community were engaged where aims and procedures of the study were explained clearly in layman’s term. Upon visiting the communities, persons of all age groups, both male and females, were invited to take part in the study. The temperature of each participant was recorded using infrared thermometers. Two milliliter of venous blood was obtained from consenting participants into EDTA tubes by trained phlebotomists. Thick and thin blood smears were prepared for microscopy and RDT diagnosis performed. An aliquot of 200 µL of whole blood was pipetted into 1.5ml Eppendorf tubes and transported on ice to the Vector-Borne Infectious Disease laboratory at Kwame Nkrumah University of Science and Technology(KNUST), Kumasi, Ghana for storage and for further laboratory analysis.
Sample processing and Laboratory investigations
Rapid Diagnostic Test
The Biocredit Malaria Ag Pf (pLDH/HRPII) RDT (lot no: HOO6C007D) manufactured by Rapigen, Inc. was used to diagnose Plasmodium falciparum in the study. In addition to the control band, the test kit has two bands (HRP2 and pLDH), allowing for the phenotypic detection of potential hrp2/hrp3 deletions. The performance of this RDT has been evaluated before, and it was found to be more sensitive than AccessBio Carestart Malaria Pf RDT, the test routinely used by the Ghana NMCP (Niyukuri et al., 2022; Park et al., 2020). The test kit was used according to the manufacturer’s protocol and results recorded after 15-20 minutes. In case an RDT showed very faint bands, the test was repeated.
Microscopy
Thick and thin blood films were prepared. The thin blood film (2μL) was fixed with absolute methanol. Blood smears were stained with 10% Giemsa solution and examined under light microscope by two expert microscopists. Parasites were quantified after counting 200 or 500 White Blood Cells (WBC) (Mutala et al., 2019; Afriyie et al., 2023). A slide was declared negative when no malaria parasite was seen after scanning 100 high power fields (HPFs) (Gatton et al., 2017). The parasites quantified were expressed as parasite per microliter of blood.
DNA extraction, varATS qPCR, and hrp2/3 deletion typing
DNA extraction, varATS qPCR and hrp2/3 deletion typing were performed at the University of Notre Dame, USA. Genomic DNA was extracted from 100µL of blood using the Macherey-Nagel Nucleomag extraction kits (Düren, Germany) and eluted in equal volume of elution buffer. qPCR was performed on ThermoFisher QuantStudio 3 instrument in a total reaction volume of 12 µL, including 4 µL of DNA. The multicopy varATS gene was amplified. This gene is present in approximately 60 copies per parasite genome, of which approximately 20 copies are amplified (Hofmann et al., 2015). To obtain absolute density estimates, dilution series of cultured NF54 P.falciparum parasites quantified by dPCR were run along field samples.
Samples positive for P. falciparum and with a Ct value of ≤ 28 were typed for deletion of the hrp2 and hrp3 genes by digital PCR (Vera-Arias et al., 2022). In the absence of the hrp2 gene, anti-HRP2 antibodies also recognize the HRP3 protein, a structural homolog that shares numerous epitopes with HRP2 (Lee et al., 2012). In the dPCR assay, primers targeting hrp2 or hrp3 genes are multiplexed with an assay targeting serine-tRNA ligase (PF3D7_0717700) as control. The ratio of positive partitions of hrp2/hrp3 to tRNA indicates the presence or absence of hrp2 or hrp3. For the analysis of dPCR assay, a minimum of five droplets positive for the reference (tRNA) gene were considered. Samples were re-run if deletion were recorded but ≤ 5 positive droplets were observed for the reference gene.
Data analysis
Data was analyzed using GraphPad Prism 8.0 (San Diago, California), Stata 17 (Stata Corp. LLC, College Station, Texas, USA) and IBM SPSS Version 27 (Armonk, New York). Binary logistic regression was used to assess the association of potential risk factors (age, sex temperature and community) with qPCR results. Prevalence between age groups was compared using the chi-square (χ2)-test. Five percent (5%) level of significance was used for all statistical tests.