Comparison of Fine-Scale Malaria Strata Derived from Population Survey Data Collected Using Mrdts, Microscopy and Qpcr in South-Eastern Tanzania

Introduction: Malaria-endemic countries are increasingly adopting data-driven risk stratification, often at district or higher regional levels, to guide their intervention strategies. The data typically comes from population-level surveys collected by rapid diagnostic tests (RDTs), which unfortunately perform poorly in low transmission settings. Here, we conducted a high-resolution survey of Plasmodium falciparum prevalence rate (PfPR) in two Tanzanian districts and compared the fine-scale strata obtained using data from RDTs, microscopy and quantitative polymerase chain reaction (qPCR) assays. Methods: A cross-sectional survey was conducted in 35 villages in Ulanga and Kilombero districts, south-eastern Tanzania between 2022 and 2023. We screened 7,628 individuals using RDTs (SD-BIOLINE) and microscopy, with two thirds of the samples further analyzed by qPCR. The data was used to categorize each district and village as having very low (PfPR<1%), low (1%≤PfPR<5%), moderate (5%≤PfPR<30%), or high (PfPR≥30%) parasite prevalence. A generalized linear model was used to analyse infection risk factors. Other metrics, including positive predictive value (PPV), sensitivity, specificity, parasite densities, and Kappa statistics were computed for RDTs or microscopy using qPCR as reference. Results: Significant fine-scale variations in malaria risk were observed within and between districts, with village prevalence ranging from 0% to >50%. Prevalence varied by testing method: Kilombero was low risk by RDTs (PfPR=3%) and microscopy (PfPR=2%) but moderate by qPCR (PfPR=9%); Ulanga was high risk by RDTs (PfPR=39%) and qPCR (PfPR=54%) but moderate by microscopy (PfPR=26%). RDTs and microscopy classified majority of the 35 villages as very low to low risk (18 - 21 villages). In contrast, qPCR classified most villages as moderate to high risk (29 villages). Using qPCR as the reference, PPV for RDTs and microscopy ranged from <20% in very low transmission villages to >80% in moderate to high transmission villages. Sensitivity was 62% for RDTs and 41% for microscopy; specificity was 93% and 96%, respectively. Kappa values were 0.58 for RDTs and 0.42 for microscopy. School-age children (5-15years) had higher malaria prevalence and parasite densities than adults (P<0.001). High-prevalence villages also had higher parasite densities (Spearman r=0.77, P<0.001 for qPCR; r=0.55, P=0.003 for microscopy). Conclusion: This study highlights significant fine-scale variability in malaria risk within and between districts and emphasizes the variable performance of the testing methods when stratifying risk. While RDTs and microscopy were effective in high-transmission areas, they performed poorly in low-transmission settings; and classified most villages as very low or low risk. In contrast, qPCR classified most villages as moderate or high risk. While we cannot conclude on which public health decisions would be subject to change because of these differences, the findings suggest the need for improved testing approaches that are operationally feasible and sufficiently sensitive, to enable precise mapping and effective targeting of malaria in such local contexts. Moreover, public health authorities should recognize the strengths and limitations of their available data when planning local stratification or making decisions. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported in part by the Bill & Melinda Gates Foundation (Grant number Grant No. OPP 1217647 and Grant No. INV-002138 to Ifakara Health Institute). Under the grant conditions of the Foundation, a Creative Commons Attribution 4.0 Generic License has already been assigned to the Author Accepted Manuscript version that might arise from this submission. Support was also received from Rudolf Geigy Foundation through Swiss Tropical & Public Health Institute (to Ifakara Health Institute), Howard Hughes Medical Institute (HHMI) and Gates Foundation (Grants: OPP1099295) awarded to FOO and HMF, Royal Society (Grant No ICA/R1/191238 to SAB University of Glasgow and Ifakara Health Institute). Academy Medical Science Springboard Award (ref: SBF007\100094) to FB. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This work was approved under Ifakara Health Institute Review Board (Ref: IHI/IRB/No: 1 2021) and the National Institute for Medical Research-NIMR (NIMR/HQ/R.8a/Vol. 1X/3735). Permission to publish this work has been granted with reference No: BD. 242/437/01C/6 I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes The dataset supporting the findings is available upon a reasonable request, which can be directed to either the corresponding author, the Ifakara Health Institute ethical review board in Tanzania or National Health Research Ethics Committee in Tanzania with reference to ethical clearance certificate of NIMR/HQ/R.8a/Vol. 1X/3735.