Prevalence of and challenges in diagnosing subclinical Plasmodium falciparum infections in Southern Ghana

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However, these efforts are constrained by the high prevalence of subclinical infections which may sustain local disease transmission. The detection and treatment of these subclinical and low-density infection is therefore crucial in monitoring progress towards malaria control and elimination. This study sought to determine the prevalence of subclinical infections in three districts in Ghana, the proportion that could be detected by rapid diagnostic test (RDT), and the occurrence of hrp2 / hrp3 deletions which may impede diagnosis by HRP2-based RDTs. Methods A community-based, cross-sectional study was conducted in the Nkwanta South, Sekyere South, and Ga South districts in Ghana. A total of 1134 whole blood samples were screened by HRP2-based rapid diagnostic test (RDT), expert microscopy, and var ATS qPCR. 304 P. falciparum positive samples were typed for hrp2 / hrp3 deletions by digital PCR (dPCR). Results Parasite prevalence was 57.1% by qPCR, 40.9% by RDT, and 8.4% by microscopy. 33.8% (219/647) of infections were sub-patent. Compared to qPCR, the sensitivity of RDT was 65.7%, and the specificity of 91.9% and thus substantially higher than microscopy (sensitivity 14.4%, specificity 99.4%). Parasite prevalence was highest in children aged 5–15 years (68.2%), followed by adults > 15 years (51.2%) and children < 5 years (45.3%). Prevalence also differed across the three districts, ranging from 44.0% (183/416) in Sekyere South, 55.8% (143/253) in Ga South, to 68.8% (321/466) in Nkwanta South. No hrp2 deletions were observed, and one sample (1/304) carried hrp3 deletion. Conclusion The high prevalence of subclinical malaria infections is likely to be a potential reservoir in sustaining malaria transmission. HRP2-based RDTs detected two-thirds of the subclinical infections. Thus, community test and treatment programs using highly sensitive RDTs could be a valuable strategy to reduce the reservoir. Subclinical low-density Plasmodium falciparum Malaria Figures Figure 1 Figure 2 Figure 3 Introduction Malaria is one of the most devastating parasitic diseases globally. Despite the great strides made towards controlling malaria, the disease was responsible for over 249 million clinical cases and 608,000 associated deaths in 2022, with 95% of the cases reported in sub-Saharan Africa (WHO, 2023). In Ghana, the National Malaria Elimination Programme (NMEP) has increased efforts to disrupt malaria transmission and reduce morbidity. The distribution of insecticide-treated bed nets (ITN), indoor residual spraying (IRS), larviciding, intermittent preventive treatment with sulfadoxine pyrimethamine (IPTp-SP) in pregnancy, and the use of artemisinin-based combination therapies have been effective in reducing the malaria morbidity and mortality rate by 48.7% from 2011 to 2019 (Ghana Health Service, 2020 ). This notwithstanding, malaria remains the leading cause of hospitalization and is responsible for over 50% of out-patient attendances (Shretta et al., 2020 ). Plasmodium falciparum is the predominant malaria parasite accounting for more than 90% of malaria cases in Ghana with P. ovale and P. malariae sharing the remaining 10% (NMCP 2024 ; Agbana et al., 2022 ; Amoah et al., 2019 ; Owusu et al., 2017 ). Subclinical and low-density infections are common across all age groups, and play a critical role in sustaining a large proportion of malaria transmission (Lindblade et al., 2013 ; Waltmann et al., 2015 ; Chen et al., 2016 ). Sustained malaria transmission in endemic areas could be attributed to a significant proportion of subclinical infections. Several studies found that 95% of transmission originates from reservoirs that do not exhibit fever or any acute clinical symptoms (Rek et al., 2022 ; Andolina et al., 2021 ; Sumner et al., 2021 ; Bousema et al., 2014 ). The detection and treatment of these subclinical infections is therefore crucial for malaria control (Assefa et al., 2020 ; Bousema et al., 2014 ). A large number of subclinical infections are characterized by low parasite density. Community and household-based surveillance of subclinical infections as well as point-of-care (POC) detection of malaria, to a great extent, rely on rapid diagnostic tests (RDT) and/ light microscopy. Due to their limited sensitivity, microscopy and RDTs underestimate the true burden of subclinical infections (Mooney et al., 2022 ; Berzosa et al. 2018 ). Unlike RDT, the accuracy of light microscopy is dependent on the expertise of the microscopist examining the slides (WHO, 2016). As a result, light microscopy is reported to have a varying limit of detection of about 50 to 500 parasites/µL (Awosolu et al., 2022 ; Bell et al., 2002; Wongsrichanalai, et al., 2007 ). The latest generation of RDTs achieves a limit of detection of < 50 parasites/µL (Niyukuri et al., 2022 ). The most sensitive RDTs for P. falciparum detection rely on the detection of the HRP2 and HRP3 proteins. Deletions of the hrp2 and/or hrp3 genes have been reported in several countries, particularly in the Horn of Africa (Feleke et al., 2021 ; Golassa et al., 2020 ) and South America (Gamboa et al., 2010 ; Dorado et al., 2016 ), where they pose a significant obstacle to malaria control. In Ghana, deletion frequency appears to be low (Amoah et al, 2016; Bredu et al., 2022 ). To date, studies about the prevalence of subclinical infections and the challenge associated with their detection in Ghana are sparse. In this study, we assessed the prevalence of subclinical malaria infections by qPCR and highly sensitive RDTs, and the occurrence of hrp2/hrp3 gene deletions in three regions of Ghana. Methods 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 var ATS 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 6 o 50’N and, longitude 1 o 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, var ATS qPCR, and hrp2 / 3 deletion typing DNA extraction, var ATS 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 var ATS 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 (χ 2 )-test. Five percent (5%) level of significance was used for all statistical tests. Results Sociodemographic characteristics of the study population A total of 1,134 participants were recruited for the study. These included 466 (41.1%) from Nkwanta South, 416 (36.7%) from Sekyere South, and 252 (22.2%) from Ga South districts (Table 1). 63.6% ( n =720) of the participants were female. The median age was 19 ( IQR =10-43). 56.8% ( n =644) of the participants were adults >15 years, followed by 5-15 years (36.6%, n =415). 68.7% ( n =781) of the participants reported to own an insecticide treated bed net (ITN) but only 27.6% ( n =313) of them reported usage of the ITN 24 hours prior to the study. The use of the ITN was significantly lower in school-aged children 5-15 years (23.8%; n= 99/415) compared to young children ˂5 years 30.9%(n=23/75) and adults 29.6% (n=191/644) ( X 2 =6.2; P =0.04). Table 1: Baseline characteristics of study participants Characteristics N (%) Parasite Prevalence by qPCR n(%) P value Sex Female 720 (63.6%) 392 (54.4%) 0.02 Male 414 (36.5%) 255 (61.6%) Age ˂5 75 (6.6%) 34 (45.3%) 5-15 415 (36.6%) 283 (68.2%) ˂0.0001 >15 644 (56.8%) 330 (51.2%) ITN Ownership Yes No 781 (68.7%) 353 (31.1%) 453 (58.0%) 194 (54.9%) 0.22 ITN usage Yes No Study site 313 (27.6%) 821 (72.4%) 145 (46.3%) 502 (61.1%) ˂0.0001 Nkwanta South 466 (41.1%) 321 (68.8%) Sekyere South 416 (36.7%) 183 (44.0%) ˂0.0001 Ga South 253 (22.2%) 143 (55.8%) P. falciparum prevalence by qPCR, microscopy, and RDT Across all districts, the prevalence of P. falciparum was 57.1% (647/1134) by qPCR., 40.9% (464/1134) by RDT, and 8.4% (96/1134) by microscopy. Of the 464 RDT-positive cases, PfHRP2 was detected in all of them, in 48.5% (225/464) of cases both PfHRP2 and pLDH were detected, and no case was only positive for pLDH. Prevalence by qPCR was highest in Nkwanta South at 68.8% ( n =321/466), followed by Ga South 55.8% ( n =143/252) and Sekyere South 44.0% ( n =183/416) ( X 2 =55.6, P ˂0.0001). The prevalence was highest in children aged 5-15 years (68.2%, 283/415) compared to young children 15years (51.2%, 330/644) ( X 2 =34.1, P 15 years 46.7% ( n =154/330), followed by 23.5% ( n =8/34) in young children < 5 years and 20.1% ( n =57/283) in children aged 5-15 years. Compared to qPCR, the sensitivity of the HRP2-based RDT was 65.7% (95% CI 62.92 - 68.45), and sensitivity of microscopy was 14.37% (95% CI 12.33 – 16.42). Figure 2 shows parasite densities of RDT and microscopy positive and negative samples. 91.6% ( n =425/464) of samples identified as positive by RDT and 96.8% ( n =93/96) by microscopy were also positive by qPCR. Infections that were detected by all three tests had significantly higher geometric mean parasite density (n=90, 1619 parasite/µL; 95% CI 1007 - 2603) as compared to samples that were detected by qPCR only (Geometric mean = 1.21 parasite/ µL; 95%CI 0.78-1.87) (Figure 2). The geometric mean parasite density of the RDT positive samples (1.31 parasites/µL; 95% CI 50.81 – 100.1) was almost 70-fold higher than RDT negative samples (0.89 parasites/ µL; 95% CI 0.61-1.30)(Figure 2). Table 2. Diagnosis accuracy of RDT and microscopy using qPCR as reference standard RDT Microscopy Sensitivity % (95% C.I) 65.7% (62.9 - 68.5) 14.4% (12.3 – 16.4) Specificity% (95% C.I) 92.% (90.4 – 93,6) 99.4% (98.9 – 99.8) Positive Predictive value% (95% C.I) 91.4% (90.0 – 93.2) 96.9% (95.8 – 97.9) Negative Predictive value%(95% C.I) 66.9% (64.9 – 69.6) 46.6% (43.7 – 49.5) RDT: rapid diagnostic test, C.I : confidence interval, qPCR: quantitative polymerase chain reaction Risk factors associated with subclinical infection In logistic regression analysis, there was no significant association between gender and malaria infection . The temperature ( OR 1.84 95%CI 1.07 – 3.19) and the district from which a participant was sampled were strong predictors of infection. The >15years age group ( OR 2.13 95%CI 1.60 – 2.83) was significantly associated with malaria infection when compared to children < 5years old (Table 3). Individuals who reported usage of insecticide treated nets 24 hours prior to the study had lower odds of malaria infection ( OR 0.54 95% CI 0.421 – 0.714)(Table 3). Table 3. Logistic regression for potential risk factors associated with qPCR detectable malaria OR P value 95% CI Lower Upper Age 15 2.127 ˂0.0001 1.600 2.829 Gender Male Ref Female 1.217 0.154 0.929 1.595 District Sekyere South Ref Ga South 0.492 ˂0.0001 0.350 0.692 Nkwanta South 0.272 ˂0.0001 0.202 0.367 Temperature 1.846 0.028 1.069 3.188 ITN usage No Ref Yes 0.548 ˂0.0001 0.421 0.714 P value >0.05 significant, OR Odds ratio, District various sampling areas Plasmodium falciparum hrp2 / hrp3 deletion typing by dPCR Deletion typing was conducted on 304 samples (166 from Nkwanta South, 86 from Ga South, and 52 from Sekyere South). For both hrp2/hrp3 typing, 98.7% ( n =300) of the samples met the inclusion criteria of ≥ 5 partitions positive for tRNA. No hrp2 deletion were observed. One of the isolates from Nkwanta South district in Oti region examined carried a deletion of the hrp3 gene. Figure 4 shows the plot of a wild-type samples and the sample with deletion of hrp3 gene. Discussion In this study, the prevalence of subclinical P. falciparum infection was determined using microscopy, RDTs of the latest generation, and qPCR. By qPCR, 57% of the population tested positive. Two-thirds of infections were were also detected by RDT. Light microscopy showed very poor sensitivity (14%) . This finding is consistent with earlier studies that reported considerable prevalence of the parasite reservoir in the form of subclinical infections across sub-Saharan Africa (Agbana et al., 2022; Slater et al., 2019; Okell et al., 2012, Steenkeste et al., 2010). The ultrasensitive RDT used in the present study detected about two-thirds of infections and may therefore offer possibilities for shrinking the reservoir of subclinical infections e.g. through mass testing and treatment (MTAT), reactive case detection (RCD), or focal test and treat (FTAT) programs in Ghana. Subclinical infections were shown to be the major source of transmission in several countries (Andolina et al., 2021; Sumner et al., 2021). Their ability to infect mosquitos greatly depends on their gametocyte density, which depends on parasite density (Tadesse et al., 2018). In mosquito feeding experiments, very low parasite density samples did not infect mosquitos (Churcher et al., 2017). A study in Uganda found that subclinical infections were the source of >99% of infected mosquitos (Andolina et al., 2021), yet, submicroscopic infections were the source of only approximately 15% of transmission. The sensitivity of the RDT used in the current study exceeded the sensitivity of microscopy. Thus, using this RDT in a well-designed program to screen for submicroscopic infections and administer treatment to those testing positive, presumably a large proportion of the subclinical infectious reservoir could be cleared. The sensitivity of HRP2-based RDTs could be affected by deletions of hrp2/3 genes. In this study, no hrp2 deletions were observed, and one sample carried a hrp3 deletion, corroborating previous reports of low deletion frequency in Ghana ((Amoah et al, 2016; Bredu et al., 2022)). Hence, HRP2-based RDTs remain an appropriate tool to detect P. falciparum infections in Ghana. Consistent with previous studies, the prevalence of infection was higher in school age children (5-15 years) compared to young children 15years, and they may serve as an important reservoir for onward parasite transmission (Coalson et al. 2018; Afriyie et al., 2023). These patterns can be explained by the frequent exposure of adolescents to the parasite and the gradual acquisition of immunity in older individuals (O’Flaherty et al., 2022; Stresman et al., 2014). The high prevalence of infection is likely a result of the limited utilization of preventive interventions such as insecticide treated nets (ITN) as observed in this study (Walldorf et al., 2015). In Ghana, ITNs are distributed to all regions across the country especially in endemic communities as well as antenatal and Child welfare clinics (Jaeger et al., 2016). This study showed that over two-thirds of the participants possessed an ITN but only around one quarter reported usage prior to the study. Individuals who reported usage of ITN had reduced likelihood of being infected, corroborating their vital role it plays in preventing malaria infections. Previous studies indicated that the use of ITNs has been crucial in reducing malaria cases in Ghana by approximately 68% since the year 2000 (Ngonghala, 2022; McCreesh et al., 2018). In conclusion, the current study found a high prevalence of subclinical infections by qPCR. Periodic reassessment of the burden and distribution of subclinical infections may be of crucial importance to strengthening malaria surveillance and understanding the impact of control. No hrp2 deletions were detected, thus HRP2-based RDTs remain efficient for P. falciparum detection in Ghana. The highly sensitive RDT used in this present study detected two thirds of infections. Screen-and-treat campaigns using these RDTs could be a vital tool in Ghana’s l malaria surveillance campaign and to accelerate progress towards malaria control and elimination. Abbreviations PCR: Polymerase chain reaction; dPCR: Digital polymerase chain reaction; qPCR: Quantitative polymerase chain reaction; hrp2/3 : Plasmodium falciparum histidine rich protein 2/3; ITN: Insecticide treated bed net; RDT: Rapid diagnostic test; var ATS: var acidic terminal sequence; IQR: interquartile range Declarations Author contributions Study concept and design: KB, CK. Data generation: AHM, CK, KB, SOA, TKA, KBA, EVT and CAVA. Data analysis and statistical review: AHM, CK, KBA, CAVA and SOA. Drafting: AHM. Revision of manuscript: KB, CK, AHM, MGA, ABT. Critical appraisal and approval for submission: all authors. All authors read and approved the final manuscript Funding This project (TMA2016CDF1605) is part of the EDCTP2 programme supported by the European Union. CK was supported by BMGF Investment #005898. 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Identification of the asymptomatic Plasmodium falciparum and Plasmodium vivax gametocyte reservoir under different transmission intensities. PLoS neglected tropical diseases , 15 (8), e0009672. Laishram, D. D., Sutton, P. L., Nanda, N., Sharma, V. L., Sobti, R. C., Carlton, J. M., & Joshi, H. (2012). The complexities of malaria disease manifestations with a focus on asymptomatic malaria. Malaria Journal, 11 (1), 1-15. Lee, N., Gatton, M. L., Pelecanos, A., Bubb, M., Gonzalez, I., Bell, D., ... & McCarthy, J. S. (2012). Identification of optimal epitopes for Plasmodium falciparum rapid diagnostic tests that target histidine-rich proteins 2 and 3. Journal of clinical microbiology , 50 (4), 1397-1405. Lindblade, K. A., Steinhardt, L., Samuels, A., Kachur, S. P., & Slutsker, L. (2013). The silent threat: asymptomatic parasitemia and malaria transmission. Expert review of anti-infective therapy, 11(6), 623-639. Mbanefo, Afoma, and Nirbhay Kumar. "Evaluation of malaria diagnostic methods as a key for successful control and elimination programs." Tropical Medicine and Infectious Disease 5, no. 2 (2020): 102. McCreesh, P., Mumbengegwi, D., Roberts, K., Tambo, M., Smith, J., Whittemore, B., ... & Hsiang, M. S. (2018). Subpatent malaria in a low transmission African setting: a cross-sectional study using rapid diagnostic testing (RDT) and loop-mediated isothermal amplification (LAMP) from Zambezi region, Namibia. Malaria journal , 17 , 1-11. Mooney, J. P., DonVito, S. M., Jahateh, M., Bittaye, H., Bottomley, C., D’Alessandro, U., & Riley, E. M. (2022). Dry season prevalence of Plasmodium falciparum in asymptomatic gambian children, with a comparative evaluation of diagnostic methods. Malaria journal , 21 (1), 171. Mutala, A. H., Badu, K., Owusu, C., Agordzo, S. K., Tweneboah, A., Abbas, D. A., & Addo, M. G. (2019). Impact of malaria on haematological parameters of urban, peri-urban and rural residents in the Ashanti region of Ghana: a cross-sectional study. AAS Open Research , 2 . NMCP 2024. National Malaria Control Program 2024 – 208 Malaria Strategic Plan Ngonghala, C. N. (2022). Assessing the impact of insecticide-treated nets in the face of insecticide resistance on malaria control. Journal of Theoretical Biology , 555 , 111281. Niyukuri, D., Sinzinkayo, D., Troth, E. V., Oduma, C. O., Barengayabo, M., Ndereyimana, M., ... & Koepfli, C. (2022). Performance of highly sensitive and conventional rapid diagnostic tests for clinical and subclinical Plasmodium falciparum infections, and hrp2/3 deletion status in Burundi. PLOS Global Public Health , 2 (7), e0000828. O'Flaherty, K., Roe, M., & Fowkes, F. J. (2022). The role of naturally acquired antimalarial antibodies in subclinical Plasmodium spp. infection. Journal of Leukocyte Biology , 111 (5), 1097-1105. Okell, L. C., Bousema, T., Griffin, J. T., Ouédraogo, A. L., Ghani, A. C., & Drakeley, C. J. (2012). Factors determining the occurrence of submicroscopic malaria infections and their relevance for control. Nature communications , 3 (1), 1237. Owusu, E. D., Brown, C. A., Grobusch, M. P., & Mens, P. (2017). Prevalence of Plasmodium falciparum and non-P. falciparum infections in a highland district in Ghana, and the influence of HIV and sickle cell disease. Malaria journal , 16 , 1-8. Park, S. H., Jegal, S., Ahn, S. K., Jung, H., Lee, J., Na, B. K., ... & Kim, T. S. (2020). Diagnostic performance of three rapid diagnostic test kits for malaria parasite Plasmodium falciparum. The Korean Journal of Parasitology , 58 (2), 147. Rek, J., Blanken, S. L., Okoth, J., Ayo, D., Onyige, I., Musasizi, E., ... & Conrad, M. D. (2022). Asymptomatic school-aged children are important drivers of malaria transmission in a high endemicity setting in Uganda. The Journal of infectious diseases , 226 (4), 708-713. Sumner, K. M., Freedman, E., Abel, L., Obala, A., Pence, B. W., Wesolowski, A., ... & Taylor, S. M. (2021). Genotyping cognate Plasmodium falciparum in humans and mosquitoes to estimate onward transmission of asymptomatic infections. Nature communications , 12 (1), 909. Slater, H. C., Ross, A., Felger, I., Hofmann, N. E., Robinson, L., Cook, J., ... & Okell, L. C. (2019). The temporal dynamics and infectiousness of subpatent Plasmodium falciparum infections in relation to parasite density. Nature communications , 10 (1), 1433. Shretta, Rima, Sheetal P. Silal, Keziah Malm, Wahjib Mohammed, Joel Narh, Danielle Piccinini, Kathryn Bertram, Jessica Rockwood, and Matt Lynch. "Estimating the risk of declining funding for malaria in Ghana: the case for continued investment in the malaria response." Malaria journal 19, no. 1 (2020): 1-15. Steenkeste, N., Rogers, W. O., Okell, L., Jeanne, I., Incardona, S., Duval, L., ... & Rogier, C. (2010). Sub-microscopic malaria cases and mixed malaria infection in a remote area of high malaria endemicity in Rattanakiri province, Cambodia: implication for malaria elimination. Malaria journal , 9 , 1-11. Stresman, G. H., Stevenson, J. C., Ngwu, N., Marube, E., Owaga, C., Drakeley, C., ... & Cox, J. (2014). High levels of asymptomatic and subpatent Plasmodium falciparum parasite carriage at health facilities in an area of heterogeneous malaria transmission intensity in the Kenyan highlands. The American journal of tropical medicine and hygiene , 91 (6), 1101. Sumner, K. M., Freedman, E., Abel, L., Obala, A., Pence, B. W., Wesolowski, A., ... & Taylor, S. M. (2021). Genotyping cognate Plasmodium falciparum in humans and mosquitoes to estimate onward transmission of asymptomatic infections. Nature communications , 12 (1), 909. Tadesse, F. G., Slater, H. C., Chali, W., Teelen, K., Lanke, K., Belachew, M., ... & Bousema, T. (2018). The relative contribution of symptomatic and asymptomatic Plasmodium vivax and Plasmodium falciparum infections to the infectious reservoir in a low-endemic setting in Ethiopia. Clinical infectious diseases , 66 (12), 1883-1891. Vera-Arias, C. A., Holzschuh, A., Oduma, C. O., Badu, K., Abdul-Hakim, M., Yukich, J., ... & Koepfli, C. (2022). High-throughput Plasmodium falciparum hrp2 and hrp3 gene deletion typing by digital PCR to monitor malaria rapid diagnostic test efficacy. Elife , 11 , e72083. Walldorf, J. A., Cohee, L. M., Coalson, J. E., Bauleni, A., Nkanaunena, K., Kapito-Tembo, A., ... & Laufer, M. K. (2015). School-age children are a reservoir of malaria infection in Malawi. PloS one , 10 (7), e0134061. Waltmann, A., Darcy, A. W., Harris, I., Koepfli, C., Lodo, J., Vahi, V., ... & Mueller, I. (2015). High rates of asymptomatic, sub-microscopic Plasmodium vivax infection and disappearing Plasmodium falciparum malaria in an area of low transmission in Solomon Islands. PLoS neglected tropical diseases , 9 (5), e0003758. Wongsrichanalai, C., Barcus, M. J., Muth, S., Sutamihardja, A., & Wernsdorfer, W. H. (2007). A review of malaria diagnostic tools: microscopy and rapid diagnostic test (RDT). Defining and Defeating the Intolerable Burden of Malaria III: Progress and Perspectives: Supplement to Volume 77 (6) of American Journal of Tropical Medicine and Hygiene . World Health Organisaion. Plasmodium falciparum hrp2/3gene deletions. Malaria Policy Advisory Committee Meeting. Geneva: World Health Organization; 2016. World Health Organisation (2016) False-negative RDT results and implications of new reports of P. falciparum histidine-rich protein 2/3 gene deletions World Health Organisation World Health Organization. (2016). Malaria microscopy standard operating procedures. World Health Organization. (2022). World malaria report 2022 . World Health Organization. Additional Declarations No competing interests reported. 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Bottom left: Negative partitions for both target and tRNA are shown in gray. Top left: Partitions positive for the target gene) are shown in yellow. Top right: Partitions positive for both target and tRNA are shown in light blue. \u003cstrong\u003e(A)\u003c/strong\u003e Wild type sample with no deletion. \u003cstrong\u003e(B)\u003c/strong\u003e \u003cem\u003ehrp3\u003c/em\u003e deletion sample: Droplets positive for tRNA (control), but no droplets are positive for the target gene (\u003cem\u003ehrp3\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4462230/v1/06e163b1ce224122947602fc.png"},{"id":58422920,"identity":"74a69721-8334-4971-8e8d-39e7e7386c9f","added_by":"auto","created_at":"2024-06-15 16:16:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1159201,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4462230/v1/3e3c720e-b603-45dc-b498-53460d347f90.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Prevalence of and challenges in diagnosing subclinical Plasmodium falciparum infections in Southern Ghana","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMalaria is one of the most devastating parasitic diseases globally. Despite the great strides made towards controlling malaria, the disease was responsible for over 249\u0026nbsp;million clinical cases and 608,000 associated deaths in 2022, with 95% of the cases reported in sub-Saharan Africa (WHO, 2023). In Ghana, the National Malaria Elimination Programme (NMEP) has increased efforts to disrupt malaria transmission and reduce morbidity. The distribution of insecticide-treated bed nets (ITN), indoor residual spraying (IRS), larviciding, intermittent preventive treatment with sulfadoxine pyrimethamine (IPTp-SP) in pregnancy, and the use of artemisinin-based combination therapies have been effective in reducing the malaria morbidity and mortality rate by 48.7% from 2011 to 2019 (Ghana Health Service, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This notwithstanding, malaria remains the leading cause of hospitalization and is responsible for over 50% of out-patient attendances (Shretta et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). \u003cem\u003ePlasmodium falciparum\u003c/em\u003e is the predominant malaria parasite accounting for more than 90% of malaria cases in Ghana with \u003cem\u003eP. ovale\u003c/em\u003e and \u003cem\u003eP. malariae\u003c/em\u003e sharing the remaining 10% (NMCP \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Agbana et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Amoah et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Owusu et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSubclinical and low-density infections are common across all age groups, and play a critical role in sustaining a large proportion of malaria transmission (Lindblade et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Waltmann et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Chen et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Sustained malaria transmission in endemic areas could be attributed to a significant proportion of subclinical infections. Several studies found that 95% of transmission originates from reservoirs that do not exhibit fever or any acute clinical symptoms (Rek et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Andolina et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Sumner et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Bousema et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The detection and treatment of these subclinical infections is therefore crucial for malaria control (Assefa et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Bousema et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA large number of subclinical infections are characterized by low parasite density. Community and household-based surveillance of subclinical infections as well as point-of-care (POC) detection of malaria, to a great extent, rely on rapid diagnostic tests (RDT) and/ light microscopy. Due to their limited sensitivity, microscopy and RDTs underestimate the true burden of subclinical infections (Mooney et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Berzosa et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Unlike RDT, the accuracy of light microscopy is dependent on the expertise of the microscopist examining the slides (WHO, 2016). As a result, light microscopy is reported to have a varying limit of detection of about 50 to 500 parasites/\u0026micro;L (Awosolu et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Bell et al., 2002; Wongsrichanalai, et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The latest generation of RDTs achieves a limit of detection of \u0026lt;\u0026thinsp;50 parasites/\u0026micro;L (Niyukuri et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe most sensitive RDTs for \u003cem\u003eP. falciparum\u003c/em\u003e detection rely on the detection of the HRP2 and HRP3 proteins. Deletions of the \u003cem\u003ehrp2\u003c/em\u003e and/or \u003cem\u003ehrp3\u003c/em\u003e genes have been reported in several countries, particularly in the Horn of Africa (Feleke et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Golassa et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and South America (Gamboa et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Dorado et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), where they pose a significant obstacle to malaria control. In Ghana, deletion frequency appears to be low (Amoah et al, 2016; Bredu et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo date, studies about the prevalence of subclinical infections and the challenge associated with their detection in Ghana are sparse. In this study, we assessed the prevalence of subclinical malaria infections by qPCR and highly sensitive RDTs, and the occurrence of \u003cem\u003ehrp2/hrp3\u003c/em\u003e gene deletions in three regions of Ghana.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eDefinition of terms\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSubclinical malaria infection, herein, was defined as \u003cem\u003ePlasmodium falciparum\u003c/em\u003e positive individual/case without fever (i.e. temperature \u0026le;37.5\u0026deg;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 \u003cem\u003evar\u003c/em\u003eATS qPCR (Hofmann et al., 2015). \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the\u0026nbsp;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).\u0026nbsp;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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStudy areas and population\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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 6\u003csup\u003eo\u003c/sup\u003e 50\u0026rsquo;N and, longitude 1\u003csup\u003eo\u003c/sup\u003e 40\u0026rsquo;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\u0026deg;35\u0026rsquo;N and longitude 0\u0026deg;10\u0026rsquo; 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). \u0026nbsp;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)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStudy design and sample collection\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCross-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\u0026rsquo;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 \u0026micro;L 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. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSample processing and Laboratory investigations\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRapid Diagnostic Test\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Biocredit Malaria Ag Pf (pLDH/HRPII) RDT (lot no: HOO6C007D) manufactured by Rapigen, Inc. was used to diagnose \u003cem\u003ePlasmodium falciparum\u003c/em\u003e 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 \u003cem\u003ehrp2\u003c/em\u003e/\u003cem\u003ehrp3\u003c/em\u003e 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\u0026rsquo;s protocol and results recorded after 15-20 minutes. In case an RDT showed very faint bands, the test was repeated.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicroscopy\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThick and thin blood films were prepared. The thin blood film (2\u0026mu;L) 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 \u003cem\u003eet al.,\u003c/em\u003e 2017). The parasites quantified were expressed as parasite per microliter of blood.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA extraction, \u003cem\u003evar\u003c/em\u003eATS qPCR, and \u003cem\u003ehrp2\u003c/em\u003e/\u003cem\u003e3\u003c/em\u003e deletion typing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDNA extraction, \u003cem\u003evar\u003c/em\u003eATS qPCR and \u003cem\u003ehrp2/3\u003c/em\u003e deletion typing were performed at the University of Notre Dame, USA. Genomic DNA was extracted from 100\u0026micro;L of blood using the Macherey-Nagel Nucleomag extraction kits (D\u0026uuml;ren, Germany) and eluted in equal volume of elution buffer. qPCR was performed on ThermoFisher QuantStudio 3 instrument in a total reaction volume of 12 \u0026micro;L, including 4 \u0026micro;L of DNA. The multicopy \u003cem\u003evar\u003c/em\u003eATS 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 \u003cem\u003eP.falciparum\u003c/em\u003e parasites quantified by dPCR were run along field samples.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSamples positive for \u003cem\u003eP. falciparum\u003c/em\u003e and with a Ct value of \u0026le; 28 were typed for deletion of the \u003cem\u003ehrp2\u003c/em\u003e and \u003cem\u003ehrp3\u003c/em\u003e genes by digital PCR (Vera-Arias et al., 2022). In the absence of the \u003cem\u003ehrp2\u003c/em\u003e gene, anti-HRP2 antibodies also recognize the HRP3 protein, a structural homolog that shares numerous epitopes with HRP2 (Lee et al., 2012). \u0026nbsp;In the dPCR assay, primers targeting \u003cem\u003ehrp2\u003c/em\u003e or \u003cem\u003ehrp3\u003c/em\u003e genes are multiplexed with an assay targeting serine-tRNA ligase (PF3D7_0717700\u003cstrong\u003e)\u003c/strong\u003e as control. The ratio of positive partitions of \u003cem\u003ehrp2\u003c/em\u003e/\u003cem\u003ehrp3\u003c/em\u003e to tRNA indicates the presence or absence of \u003cem\u003ehrp2\u003c/em\u003e or \u003cem\u003ehrp3.\u0026nbsp;\u003c/em\u003eFor 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 \u0026le; 5 positive droplets were observed for the reference gene.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData 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 (\u0026chi;\u003csup\u003e2\u003c/sup\u003e)-test. \u0026nbsp;Five percent (5%) level of significance was used for all statistical tests.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eSociodemographic characteristics of the study population\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 1,134 participants were recruited for the study. These included 466 (41.1%) from Nkwanta South, 416 (36.7%) from Sekyere South, and 252 (22.2%) from Ga South districts (Table 1). 63.6% (\u003cem\u003en\u003c/em\u003e=720) of the participants were female. \u0026nbsp;The median age was 19 (\u003cem\u003eIQR\u003c/em\u003e=10-43). 56.8% (\u003cem\u003en\u003c/em\u003e=644) of the participants were adults \u0026gt;15 years, followed by 5-15 years (36.6%, \u003cem\u003en\u003c/em\u003e=415). 68.7% (\u003cem\u003en\u003c/em\u003e=781) of the participants reported to own an insecticide treated bed net (ITN) but only 27.6% (\u003cem\u003en\u003c/em\u003e=313) of them reported usage of the ITN 24 hours prior to the study. The use of the ITN was significantly lower in school-aged children 5-15 years (23.8%; \u003cem\u003en=\u003c/em\u003e99/415) compared to young children ˂5 years 30.9%(n=23/75) and adults 29.6% (n=191/644) (\u003cem\u003eX\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e=6.2; \u003cem\u003eP\u003c/em\u003e=0.04).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1:\u0026nbsp;\u003c/strong\u003eBaseline characteristics of study participants\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"597\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.633165829145728%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCharacteristics\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.262981574539364%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eN (%)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.64824120603015%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eParasite Prevalence \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eby qPCR n(%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.45561139028476%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eP value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.633165829145728%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSex\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.262981574539364%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.64824120603015%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.45561139028476%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.633165829145728%\" valign=\"top\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.262981574539364%\" valign=\"top\"\u003e\n \u003cp\u003e720 (63.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.64824120603015%\" valign=\"top\"\u003e\n \u003cp\u003e392 (54.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.45561139028476%\" valign=\"top\"\u003e\n \u003cp\u003e0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.633165829145728%\" valign=\"top\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.262981574539364%\" valign=\"top\"\u003e\n \u003cp\u003e414 (36.5%)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.64824120603015%\" valign=\"top\"\u003e\n \u003cp\u003e255 (61.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.45561139028476%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.633165829145728%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.262981574539364%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.64824120603015%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.45561139028476%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.633165829145728%\" valign=\"top\"\u003e\n \u003cp\u003e˂5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.262981574539364%\" valign=\"top\"\u003e\n \u003cp\u003e75 (6.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.64824120603015%\" valign=\"top\"\u003e\n \u003cp\u003e34 (45.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.45561139028476%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.633165829145728%\" valign=\"top\"\u003e\n \u003cp\u003e5-15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.262981574539364%\" valign=\"top\"\u003e\n \u003cp\u003e415 (36.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.64824120603015%\" valign=\"top\"\u003e\n \u003cp\u003e283 (68.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.45561139028476%\" valign=\"top\"\u003e\n \u003cp\u003e˂0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.633165829145728%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026gt;15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.262981574539364%\" valign=\"top\"\u003e\n \u003cp\u003e644 (56.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.64824120603015%\" valign=\"top\"\u003e\n \u003cp\u003e330 (51.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.45561139028476%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.633165829145728%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eITN Ownership\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eYes\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.262981574539364%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e781 (68.7%)\u003c/p\u003e\n \u003cp\u003e353 (31.1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.64824120603015%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e453 (58.0%)\u003c/p\u003e\n \u003cp\u003e194 (54.9%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.45561139028476%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.633165829145728%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eITN usage\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eStudy site\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.262981574539364%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e313 (27.6%)\u003c/p\u003e\n \u003cp\u003e821 (72.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.64824120603015%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e145 (46.3%)\u003c/p\u003e\n \u003cp\u003e502 (61.1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.45561139028476%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e˂0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.633165829145728%\" valign=\"top\"\u003e\n \u003cp\u003eNkwanta South\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.262981574539364%\" valign=\"top\"\u003e\n \u003cp\u003e466 (41.1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.64824120603015%\" valign=\"top\"\u003e\n \u003cp\u003e321 (68.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.45561139028476%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.633165829145728%\" valign=\"top\"\u003e\n \u003cp\u003eSekyere South\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.262981574539364%\" valign=\"top\"\u003e\n \u003cp\u003e416 (36.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.64824120603015%\" valign=\"top\"\u003e\n \u003cp\u003e183 (44.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.45561139028476%\" valign=\"top\"\u003e\n \u003cp\u003e˂0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.633165829145728%\" valign=\"top\"\u003e\n \u003cp\u003eGa South\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.262981574539364%\" valign=\"top\"\u003e\n \u003cp\u003e253 (22.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.64824120603015%\" valign=\"top\"\u003e\n \u003cp\u003e143 (55.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.45561139028476%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eP. falciparum\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;prevalence by qPCR, microscopy, and RDT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAcross all districts, the prevalence of \u003cem\u003eP. falciparum\u003c/em\u003e was 57.1% (647/1134) by qPCR., 40.9% (464/1134) by RDT, and 8.4% (96/1134) by microscopy. Of the 464 RDT-positive cases, PfHRP2 was detected in all of them, in 48.5% (225/464) of cases both PfHRP2 and pLDH were detected, and no case was only positive for pLDH. \u0026nbsp;Prevalence by qPCR was highest in Nkwanta South at 68.8% (\u003cem\u003en\u003c/em\u003e=321/466), followed by Ga South 55.8% (\u003cem\u003en\u003c/em\u003e=143/252) and Sekyere South 44.0% (\u003cem\u003en\u003c/em\u003e=183/416) (\u003cem\u003eX\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e=55.6, \u003cem\u003eP\u0026nbsp;\u003c/em\u003e˂0.0001). The prevalence was highest in children aged 5-15 years (68.2%, 283/415) compared to young children \u0026lt;5 years (45.3%, 34/75) and adults \u0026gt;15years (51.2%, 330/644) (\u003cem\u003eX\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e=34.1, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001). 33.8% (219/647) of infections were exclusively detected by qPCR. The proportion of sub-patent infections was highest in adults \u0026gt;15 years 46.7% (\u003cem\u003en\u003c/em\u003e=154/330), followed by 23.5% (\u003cem\u003en\u003c/em\u003e=8/34) in young children \u0026lt; 5 years and 20.1% (\u003cem\u003en\u003c/em\u003e=57/283) in children aged 5-15 years.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCompared to qPCR, the sensitivity of the HRP2-based RDT was 65.7% (95% CI 62.92 - 68.45), and sensitivity of microscopy was 14.37% (95% CI 12.33 \u0026ndash; 16.42). Figure 2 shows parasite densities of RDT and microscopy positive and negative samples. 91.6% (\u003cem\u003en\u003c/em\u003e=425/464) of samples identified as positive by RDT and 96.8% (\u003cem\u003en\u003c/em\u003e=93/96) by microscopy were also positive by qPCR.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Infections that were detected by all three tests had significantly higher geometric mean parasite density (n=90, 1619 parasite/\u0026micro;L; 95% CI 1007 - 2603) as compared to samples that were detected by qPCR only (Geometric mean = 1.21 parasite/ \u0026micro;L; 95%CI 0.78-1.87) (Figure 2). The geometric mean parasite density of the RDT positive samples (1.31 parasites/\u0026micro;L; 95% CI 50.81 \u0026ndash; 100.1) was almost 70-fold higher than RDT negative samples (0.89 parasites/ \u0026micro;L; 95% CI 0.61-1.30)(Figure 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u0026nbsp;\u003c/strong\u003eDiagnosis accuracy of RDT and microscopy using qPCR as reference standard\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"634\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.321766561514195%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.126182965299684%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eRDT\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.55205047318612%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMicroscopy\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.321766561514195%\" valign=\"top\"\u003e\n \u003cp\u003eSensitivity % (95% C.I)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.126182965299684%\" valign=\"top\"\u003e\n \u003cp\u003e65.7% (62.9 - 68.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.55205047318612%\" valign=\"top\"\u003e\n \u003cp\u003e14.4% (12.3 \u0026ndash; 16.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.321766561514195%\" valign=\"top\"\u003e\n \u003cp\u003eSpecificity% (95% C.I)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.126182965299684%\" valign=\"top\"\u003e\n \u003cp\u003e92.% (90.4 \u0026ndash; 93,6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.55205047318612%\" valign=\"top\"\u003e\n \u003cp\u003e99.4% (98.9 \u0026ndash; 99.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.321766561514195%\" valign=\"top\"\u003e\n \u003cp\u003ePositive Predictive value% (95% C.I)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.126182965299684%\" valign=\"top\"\u003e\n \u003cp\u003e91.4% (90.0 \u0026ndash; 93.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.55205047318612%\" valign=\"top\"\u003e\n \u003cp\u003e96.9% (95.8 \u0026ndash; 97.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.321766561514195%\" valign=\"top\"\u003e\n \u003cp\u003eNegative Predictive value%(95% C.I)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.126182965299684%\" valign=\"top\"\u003e\n \u003cp\u003e66.9% (64.9 \u0026ndash; 69.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.55205047318612%\" valign=\"top\"\u003e\n \u003cp\u003e46.6% (43.7 \u0026ndash; 49.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cem\u003eRDT:\u0026nbsp;\u003c/em\u003erapid diagnostic test, \u003cem\u003eC.I\u003c/em\u003e: confidence interval, \u003cem\u003eqPCR:\u0026nbsp;\u003c/em\u003equantitative polymerase chain reaction\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRisk factors associated with subclinical infection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn logistic regression analysis, there was no significant association between gender and malaria infection . The temperature (\u003cem\u003eOR\u003c/em\u003e 1.84 95%CI 1.07 \u0026ndash; 3.19) and the district from which a participant was sampled were strong predictors of infection. \u0026nbsp;The \u0026gt;15years age group (\u003cem\u003eOR\u003c/em\u003e 2.13 95%CI 1.60 \u0026ndash; 2.83) was significantly associated with malaria infection when compared to children \u0026lt; 5years old (Table 3). \u0026nbsp;Individuals who reported usage of insecticide treated nets 24 hours prior to the study had lower odds of malaria infection (\u003cem\u003eOR\u003c/em\u003e 0.54 95% CI 0.421 \u0026ndash; 0.714)(Table 3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3.\u0026nbsp;\u003c/strong\u003eLogistic regression for potential risk factors associated with qPCR detectable malaria\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"29.37062937062937%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.755244755244757%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eOR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.503496503496503%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eP value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.37062937062937%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e95% CI\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"29.37062937062937%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.755244755244757%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.503496503496503%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.909090909090908%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eLower\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.461538461538462%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eUpper\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"29.37062937062937%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.755244755244757%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.503496503496503%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.909090909090908%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.461538461538462%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"29.37062937062937%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026lt; 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.755244755244757%\" valign=\"top\"\u003e\n \u003cp\u003eRef\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.503496503496503%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.909090909090908%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.461538461538462%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"29.37062937062937%\" valign=\"top\"\u003e\n \u003cp\u003e5-15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.755244755244757%\" valign=\"top\"\u003e\n \u003cp\u003e0.763\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.503496503496503%\" valign=\"top\"\u003e\n \u003cp\u003e0.232\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.909090909090908%\" valign=\"top\"\u003e\n \u003cp\u003e0.490\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.461538461538462%\" valign=\"top\"\u003e\n \u003cp\u003e1.189\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"29.37062937062937%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026gt;15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.755244755244757%\" valign=\"top\"\u003e\n \u003cp\u003e2.127\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.503496503496503%\" valign=\"top\"\u003e\n \u003cp\u003e˂0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.909090909090908%\" valign=\"top\"\u003e\n \u003cp\u003e1.600\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.461538461538462%\" valign=\"top\"\u003e\n \u003cp\u003e2.829\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"29.37062937062937%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eGender\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.755244755244757%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.503496503496503%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.909090909090908%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.461538461538462%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"29.37062937062937%\" valign=\"top\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.755244755244757%\" valign=\"top\"\u003e\n \u003cp\u003eRef\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.503496503496503%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.909090909090908%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.461538461538462%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"29.37062937062937%\" valign=\"top\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.755244755244757%\" valign=\"top\"\u003e\n \u003cp\u003e1.217\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.503496503496503%\" valign=\"top\"\u003e\n \u003cp\u003e0.154\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.909090909090908%\" valign=\"top\"\u003e\n \u003cp\u003e0.929\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.461538461538462%\" valign=\"top\"\u003e\n \u003cp\u003e1.595\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"29.37062937062937%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eDistrict\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.755244755244757%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.503496503496503%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.909090909090908%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.461538461538462%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"29.37062937062937%\" valign=\"top\"\u003e\n \u003cp\u003eSekyere South\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.755244755244757%\" valign=\"top\"\u003e\n \u003cp\u003eRef\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.503496503496503%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.909090909090908%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.461538461538462%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"29.37062937062937%\" valign=\"top\"\u003e\n \u003cp\u003eGa South\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.755244755244757%\" valign=\"top\"\u003e\n \u003cp\u003e0.492\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.503496503496503%\" valign=\"top\"\u003e\n \u003cp\u003e˂0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.909090909090908%\" valign=\"top\"\u003e\n \u003cp\u003e0.350\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.461538461538462%\" valign=\"top\"\u003e\n \u003cp\u003e0.692\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"29.37062937062937%\" valign=\"top\"\u003e\n \u003cp\u003eNkwanta South\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.755244755244757%\" valign=\"top\"\u003e\n \u003cp\u003e0.272\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.503496503496503%\" valign=\"top\"\u003e\n \u003cp\u003e˂0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.909090909090908%\" valign=\"top\"\u003e\n \u003cp\u003e0.202\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.461538461538462%\" valign=\"top\"\u003e\n \u003cp\u003e0.367\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"29.37062937062937%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTemperature\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.755244755244757%\" valign=\"top\"\u003e\n \u003cp\u003e1.846\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.503496503496503%\" valign=\"top\"\u003e\n \u003cp\u003e0.028\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.909090909090908%\" valign=\"top\"\u003e\n \u003cp\u003e1.069\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.461538461538462%\" valign=\"top\"\u003e\n \u003cp\u003e3.188\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"29.37062937062937%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eITN usage\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.755244755244757%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.503496503496503%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.909090909090908%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.461538461538462%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"29.37062937062937%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.755244755244757%\" valign=\"top\"\u003e\n \u003cp\u003eRef\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.503496503496503%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.909090909090908%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.461538461538462%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"29.37062937062937%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eYes\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.755244755244757%\" valign=\"top\"\u003e\n \u003cp\u003e0.548\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.503496503496503%\" valign=\"top\"\u003e\n \u003cp\u003e˂0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.909090909090908%\" valign=\"top\"\u003e\n \u003cp\u003e0.421\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.461538461538462%\" valign=\"top\"\u003e\n \u003cp\u003e0.714\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cem\u003eP value\u003c/em\u003e \u0026gt;0.05 significant, \u003cem\u003eOR\u003c/em\u003e Odds ratio, \u003cem\u003eDistrict\u003c/em\u003e various sampling areas \u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePlasmodium falciparum hrp2\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e/\u003cem\u003ehrp3\u003c/em\u003e deletion typing by dPCR\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDeletion typing was conducted on 304 samples (166 from Nkwanta South, 86 from Ga South, and 52 from Sekyere South). For both \u003cem\u003ehrp2/hrp3\u003c/em\u003e typing, 98.7% (\u003cem\u003en\u003c/em\u003e=300) of the samples met the inclusion criteria of \u0026ge; 5 partitions positive for tRNA. No \u003cem\u003ehrp2\u003c/em\u003e deletion were observed. One of the isolates from Nkwanta South district in Oti region examined carried a deletion of the \u003cem\u003ehrp3\u003c/em\u003e gene. Figure 4 shows the plot of a wild-type samples and the sample with deletion of \u003cem\u003ehrp3\u0026nbsp;\u003c/em\u003egene.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, the prevalence of subclinical \u003cem\u003eP. falciparum\u003c/em\u003e infection was determined using microscopy, RDTs of the latest generation, and qPCR. By qPCR, 57% of the population tested positive. Two-thirds of infections were were also detected by RDT. Light microscopy showed very poor sensitivity (14%) . This finding is consistent with earlier studies that reported considerable prevalence of the parasite reservoir in the form of subclinical infections across sub-Saharan Africa (Agbana et al., 2022; Slater et al., 2019; Okell et al., 2012, Steenkeste et al., 2010).\u003c/p\u003e\n\u003cp\u003eThe ultrasensitive RDT used in the present study detected about two-thirds of infections and may therefore offer possibilities for shrinking the reservoir of subclinical infections e.g. through mass testing and treatment (MTAT), reactive case detection (RCD), or focal test and treat (FTAT) programs in Ghana. Subclinical infections were shown to be the major source of transmission in several countries (Andolina et al., 2021; Sumner et al., 2021). Their ability to infect mosquitos greatly depends on their gametocyte density, which \u0026nbsp;depends on parasite density (Tadesse et al., 2018). In mosquito feeding experiments, very low parasite density samples did not infect mosquitos (Churcher et al., 2017). A study in Uganda found that subclinical infections were the source of \u0026gt;99% of infected mosquitos (Andolina et al., 2021), yet, submicroscopic infections were the source of only approximately 15% of transmission. The sensitivity of the RDT used in the current study exceeded the sensitivity of microscopy. Thus, using this RDT in a well-designed program to screen for submicroscopic infections and administer treatment to those testing positive, presumably a large proportion of the subclinical infectious reservoir could be cleared.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe sensitivity of HRP2-based RDTs could be affected by deletions of \u003cem\u003ehrp2/3\u003c/em\u003e genes. In this study, no \u003cem\u003ehrp2\u003c/em\u003e deletions were observed, and one sample carried a \u003cem\u003ehrp3\u003c/em\u003e deletion, corroborating previous reports of \u0026nbsp;low deletion frequency in Ghana ((Amoah et al, 2016; Bredu et al., 2022)). Hence, HRP2-based RDTs remain an appropriate tool to detect \u003cem\u003eP. falciparum\u0026nbsp;\u003c/em\u003einfections in Ghana.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConsistent with previous studies, the prevalence of infection was higher in school age children (5-15 years) compared to young children \u0026lt;5 years and adults \u0026gt;15years, and they may serve as an important reservoir for onward parasite transmission (Coalson et al. 2018; Afriyie et al., 2023). These patterns can be explained by the frequent exposure of adolescents to the parasite and the gradual acquisition of immunity in older individuals (O\u0026rsquo;Flaherty et al., 2022; Stresman et al., 2014).\u003c/p\u003e\n\u003cp\u003eThe high prevalence of infection is likely a result of the limited utilization of preventive interventions such as insecticide treated nets (ITN) as observed in this study (Walldorf et al., 2015). In Ghana, ITNs are distributed to all regions across the country especially in endemic communities as well as antenatal and Child welfare clinics (Jaeger et al., 2016). This study showed that over two-thirds of the participants possessed an ITN but only around one quarter reported usage prior to the study. Individuals who reported usage of ITN had reduced likelihood of being infected, corroborating their vital role it plays in preventing malaria infections. Previous studies indicated that the use of ITNs has been crucial in reducing malaria cases in Ghana by approximately 68% since the year 2000 (Ngonghala, 2022; McCreesh et al., 2018).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn conclusion, the current study found a high prevalence of subclinical infections by qPCR. Periodic reassessment of the burden and distribution of subclinical infections may be of crucial importance to strengthening malaria surveillance and understanding the impact of control. No \u003cem\u003ehrp2\u003c/em\u003e deletions were detected, thus HRP2-based RDTs remain efficient for \u003cem\u003eP. falciparum\u0026nbsp;\u003c/em\u003edetection in Ghana. The highly sensitive RDT used in this present study detected two thirds of infections. Screen-and-treat campaigns using these RDTs could be a vital tool in Ghana\u0026rsquo;s l malaria surveillance campaign and to accelerate progress towards malaria control and elimination.\u0026nbsp;\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003ePCR: Polymerase chain reaction; dPCR: Digital polymerase chain reaction; qPCR: Quantitative polymerase chain reaction; \u003cem\u003ehrp2/3\u003c/em\u003e: Plasmodium falciparum histidine rich protein 2/3; ITN: Insecticide treated bed net; RDT: Rapid diagnostic test; \u003cem\u003evar\u003c/em\u003eATS: var acidic terminal sequence; IQR: interquartile range\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStudy concept and design: KB, CK. Data generation: AHM, CK, KB, SOA, TKA, KBA, EVT and CAVA. Data analysis and statistical review: AHM, CK, KBA, CAVA and SOA. Drafting: AHM. Revision of manuscript: KB, CK, AHM, MGA, ABT. \u0026nbsp;Critical appraisal and approval for submission: all authors. All authors read and approved the final manuscript\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project (TMA2016CDF1605) is part of the EDCTP2 programme supported by the European Union. CK was supported by BMGF Investment #005898. The research was also supported by the U.S. National Institute of Allergy and Infectious Disease (award number R01-AI- 143809, subaward to KNUST SPC-1000004635 / GR123009). \u0026nbsp;.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe will like to thank Emmanuel Agbodogli, the municipal disease control officer in Nkwanta South and the participants from Nkwanta south, Sekyere South and Ga South distrcits. We also acknowledge the contributions of the research assistants in Vector-Borne Infectious Disease Research Group-KNUST.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors declare they are no competing interest\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAfriyie, S. O., Addison, T. K., Gebre, Y., Mutala, A. H., Antwi, K. B., Abbas, D. A., ... \u0026amp; Badu, K. (2023). Accuracy of diagnosis among clinical malaria patients: comparing microscopy, RDT and a highly sensitive quantitative PCR looking at the implications for submicroscopic infections. \u003cem\u003eMalaria Journal\u003c/em\u003e, \u003cem\u003e22\u003c/em\u003e, 1.\u003c/li\u003e\n \u003cli\u003eAgbana, H. B., Rogier, E., Lo, A., Abukari, Z., Jones, S., Gyan, B., ... \u0026amp; Amoah, L. E. (2022). 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Sub-microscopic malaria cases and mixed malaria infection in a remote area of high malaria endemicity in Rattanakiri province, Cambodia: implication for malaria elimination. \u003cem\u003eMalaria journal\u003c/em\u003e, \u003cem\u003e9\u003c/em\u003e, 1-11.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eStresman, G. H., Stevenson, J. C., Ngwu, N., Marube, E., Owaga, C., Drakeley, C., ... \u0026amp; Cox, J. (2014). High levels of asymptomatic and subpatent Plasmodium falciparum parasite carriage at health facilities in an area of heterogeneous malaria transmission intensity in the Kenyan highlands. \u003cem\u003eThe American journal of tropical medicine and hygiene\u003c/em\u003e, \u003cem\u003e91\u003c/em\u003e(6), 1101.\u003c/li\u003e\n \u003cli\u003eSumner, K. M., Freedman, E., Abel, L., Obala, A., Pence, B. W., Wesolowski, A., ... \u0026amp; Taylor, S. M. (2021). 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Geneva: World Health Organization; 2016.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eWorld Health Organisation (2016) False-negative RDT results and implications of new reports of P. falciparum histidine-rich protein 2/3 gene deletions World Health Organisation\u003c/li\u003e\n \u003cli\u003eWorld Health Organization. (2016). Malaria microscopy standard operating procedures.\u003c/li\u003e\n \u003cli\u003eWorld Health Organization. (2022). \u003cem\u003eWorld malaria report 2022\u003c/em\u003e. World Health Organization.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Subclinical, low-density, Plasmodium falciparum, Malaria","lastPublishedDoi":"10.21203/rs.3.rs-4462230/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4462230/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eMany national malaria elimination programmes (NMEP) are intensifying campaigns for malaria control and elimination. However, these efforts are constrained by the high prevalence of subclinical infections which may sustain local disease transmission. The detection and treatment of these subclinical and low-density infection is therefore crucial in monitoring progress towards malaria control and elimination. This study sought to determine the prevalence of subclinical infections in three districts in Ghana, the proportion that could be detected by rapid diagnostic test (RDT), and the occurrence of \u003cem\u003ehrp2\u003c/em\u003e/\u003cem\u003ehrp3\u003c/em\u003e deletions which may impede diagnosis by HRP2-based RDTs.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA community-based, cross-sectional study was conducted in the Nkwanta South, Sekyere South, and Ga South districts in Ghana. A total of 1134 whole blood samples were screened by HRP2-based rapid diagnostic test (RDT), expert microscopy, and \u003cem\u003evar\u003c/em\u003eATS qPCR. 304 \u003cem\u003eP. falciparum\u003c/em\u003e positive samples were typed for \u003cem\u003ehrp2\u003c/em\u003e/\u003cem\u003ehrp3\u003c/em\u003e deletions by digital PCR (dPCR).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eParasite prevalence was 57.1% by qPCR, 40.9% by RDT, and 8.4% by microscopy. 33.8% (219/647) of infections were sub-patent. Compared to qPCR, the sensitivity of RDT was 65.7%, and the specificity of 91.9% and thus substantially higher than microscopy (sensitivity 14.4%, specificity 99.4%). Parasite prevalence was highest in children aged 5\u0026ndash;15 years (68.2%), followed by adults\u0026thinsp;\u0026gt;\u0026thinsp;15 years (51.2%) and children\u0026thinsp;\u0026lt;\u0026thinsp;5 years (45.3%). Prevalence also differed across the three districts, ranging from 44.0% (183/416) in Sekyere South, 55.8% (143/253) in Ga South, to 68.8% (321/466) in Nkwanta South. No \u003cem\u003ehrp2\u003c/em\u003e deletions were observed, and one sample (1/304) carried \u003cem\u003ehrp3\u003c/em\u003e deletion.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe high prevalence of subclinical malaria infections is likely to be a potential reservoir in sustaining malaria transmission. HRP2-based RDTs detected two-thirds of the subclinical infections. Thus, community test and treatment programs using highly sensitive RDTs could be a valuable strategy to reduce the reservoir.\u003c/p\u003e","manuscriptTitle":"Prevalence of and challenges in diagnosing subclinical Plasmodium falciparum infections in Southern Ghana","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-07 23:00:19","doi":"10.21203/rs.3.rs-4462230/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0a91af7f-7f5e-4749-919a-9fbf9777e5f5","owner":[],"postedDate":"June 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-06-15T16:08:10+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-07 23:00:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4462230","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4462230","identity":"rs-4462230","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}