Low-density asymptomatic parasitemia in southern Zambia does not lead to clinical malaria and is not associated with household transmission: results from a two-year cohort study

Low-density asymptomatic parasitemia in southern Zambia does not lead to clinical malaria and is not associated with household transmission: results from a two-year cohort study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Low-density asymptomatic parasitemia in southern Zambia does not lead to clinical malaria and is not associated with household transmission: results from a two-year cohort study Jessica L. Schue, Anne C. Martin, Japhet Matoba, Caison Sing’anga, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8641961/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 9 You are reading this latest preprint version Abstract Background In low malaria transmission settings targeting elimination, the World Health Organization recommends a combination of mass (e.g., mass test-and-treat), targeted (e.g., chemoprophylaxis or treatment for travelers), and reactive (e.g., reactive drug administration) strategies. Most of these strategies would not identify and treat individuals with asymptomatic parasitemia. This study was conducted in a pre-elimination setting in Southern Province, Zambia to examine risk factors for asymptomatic parasitemia, its epidemiologic relationship to incident clinical malaria, and evidence of its contribution to ongoing transmission to inform policy on whether these parasitemic individuals need to be identified and treated to achieve malaria elimination. Methods An intensive longitudinal cohort study of 197 households within the catchment area of a single health center was designed to capture all clinical malaria cases and episodes of asymptomatic parasitemia between 2018 and 2020. During monthly collections, all household members and overnight visitors were administered a questionnaire and a blood sample was collected to identify Plasmodium falciparum parasitemia by qPCR. Passive surveillance was also established at the local health center to identify cases of clinical malaria. The statistical analysis examined risk factors for parasitemia and associations between asymptomatic parasitemia and subsequent episodes of clinical malaria within individuals and parasitemia in household members. Results Of the 1071 individuals enrolled in the cohort, 144 (13%) were positive by qPCR for P. falciparum at least once during the two-year study period. Monthly parasite prevalence by qPCR never exceeded 4% and parasite density was very low with a median of four parasites/µL. Incidence of self-reported clinical malaria was 46.7 cases per 1000 person-years. Low-density asymptomatic parasitemia was identified in all age groups, including young children. There was no association between asymptomatic parasitemia and clinical malaria within individuals, nor was there an association between asymptomatic parasitemia and subsequent parasitemia in household members beyond one month of the index case. Conclusion Low-level parasitemia was prevalent despite few cases of clinical malaria in this low transmission setting. There was no evidence that low-level asymptomatic parasitemia led to clinical cases of malaria or transmission to other household members. malaria asymptomatic subpatent elimination Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background As of 2025, 47 countries achieved malaria elimination through a combination of mass, targeted, and reactive interventions ( 1 ). Understanding the causes of residual transmission in an area approaching elimination is necessary to deliver appropriate interventions and ultimately achieve malaria elimination. Residual transmission may be due to repeated parasite introductions via imported cases or ongoing low-level transmission from symptomatic infections prior to their treatment and/or untreated chronic, asymptomatic infections ( 2 ). These drivers are countered most effectively with different interventions. For example, reactive test-and-treat can curb transmission introduced from imported cases and symptomatic infections, whereas mass drug administration (MDA) can clear infections that may not result in clinical symptoms or be detected by available diagnostics and thus act as reservoirs for transmission ( 3 – 5 ). Asymptomatic infections with moderate levels of parasitemia are major gametocyte reservoirs in high and moderate transmission settings and contribute disproportionately to onward transmission ( 6 – 8 ). In low transmission settings, where individuals have less protective immunity against clinical disease, asymptomatic parasitemias are typically low-density ( 9 ), undetectable by point-of-care testing ( 10 ), and linked to persistent carriage across transmission seasons ( 11 , 12 ), but are of unknown clinical and public health relevance. Asymptomatic parasitemia in low transmission settings could be important if they are a prelude to clinical malaria in the infected individual or a source of transmission to other individuals. However, few cohort studies have been conducted in pre-elimination settings to understand the relationships between asymptomatic parasitemia and subsequent clinical malaria or asymptomatic parasitemia and subsequent parasitemia in other household members. This work describes a prospective longitudinal cohort study designed to capture all cases of clinical malaria and asymptomatic subpatent parasitemia in a geographically defined population in Southern Province, Zambia. Background prevalence of asymptomatic parasitemia with Plasmodium falciparum is 1–3% by quantitative PCR (qPCR) ( 13 ). Specifically, this study seeks to guide elimination strategies in low transmission settings through examination of asymptomatic parasitemia: its risk factors, its epidemiologic relationship to incident clinical malaria, and evidence of its linkage to ongoing transmission. Methods Study Site A 24-month longitudinal cohort study was conducted from October 2018 through September 2020 in Choma District, Southern Province, Zambia, a low-transmission setting typical of southern Zambia. The region has a tropical savannah climate with a rainy season from December to April, followed by a cool dry season from May to August, and a hot dry season from September to November. The primary malaria vector is Anopheles arabiensis , which peaks during the rainy season ( 14 , 15 ). The study site was a contiguous two-square-kilometer area, defined by natural borders of roads and footpaths, located within the catchment of Mapanza Rural Health Centre (RHC) (Fig. 1 ). Household and participant selection All households within the study site were enumerated using satellite imagery and were invited to participate. Geocoordinates of consenting households were captured by global positioning system (GPS) devices. Throughout the study period, newly constructed and newly occupied households in the study area also were invited to participate. Enrolled households were visited monthly for a minimum of one year. If a household agreed, the study visits continued into a second year. All household residents and overnight visitors older than three months of age were invited to participate. Participants were classified as either a permanent resident, temporary resident (household resident for 2 months or more), or visitor (household resident for fewer than 2 months). Individuals younger than three months or with severe illness other than malaria were excluded. Written informed consent was obtained from each participant 16 years of age or older. Parental or guardian permission was obtained for all children younger than 16 years of age, and assent was obtained from children 13 to 15 years old. Cohort data collection Household and individual surveys were administered monthly, except between April and July 2020 when community data collection was paused in compliance with the Government of the Republic of Zambia’s COVID-19 prevention measures. The household survey recorded household size, amenities, net ownership, and history of indoor residual spraying. Each visit captured the number and condition of nets in the household, where household members slept, and any visitors to the household. House construction variables such as roof and wall material were collected for each sleeping structure within a household complex. Individual surveys captured demographic characteristics, socio-economic indicators, use of nets, times spent indoors versus outdoors, travel history, recent illnesses, and health care seeking behavior. Travel history included the location, purpose, and duration of up to four trips during the previous month. Each participant also had their tympanic temperature taken, and those with a temperature at 38° Celsius or higher were administered a malaria rapid diagnostic test (RDT) (SD Bioline Malaria AG P.f. , Abbott, Abbott Park, Illinois, USA). Participants who tested positive were offered treatment with artemether-lumefantrine ( 16 ). Pregnant women and children under 5 kilograms who tested positive were to be offered transportation to the health center treatment. Dried blood spots (DBS) were collected monthly for detection of P. falciparum parasitemia by qPCR. Health center symptomatic surveillance Passive case detection was established at Mapanza RHC, the catchment area of which included the cohort households, and its affiliated health posts. Individuals older than three months of age who tested positive for malaria by RDT were asked to participate in the study by the health center staff. Those who provided written consent were administered a brief questionnaire, including basic demographic information, recent travel, bed net use, recent illnesses, and whether or not they were participants in the cohort study. DBS were collected from all consenting patients for the detection of P. falciparum by qPCR. Laboratory Testing DNA was extracted from the DBS using a standard saponin and Chelex-100 extraction procedure, and qPCR targeting the P. falciparum cytochrome-b gene was done using SYBR Green PCR Master Mix (Applied Biosystems, Thermo Fischer Scientific Inc, Waltham, MA) ( 10 ). All samples were run in duplicate. Parasite density was estimated based on the cycle threshold, and samples were considered positive if at least one of the two wells had a parasite density above one parasite/µL and a melting point within ± 0.5° Celsius of the melting point of the 3D7 g-DNA control. Positive samples were run on gel electrophoresis to confirm the DNA product size. Data Analysis Parasite prevalence by qPCR and the incidence of clinical malaria using health center surveillance were calculated across the study period. Monthly parasite prevalence (the proportion of participants parasitemic in a given month) and period prevalence (the proportion of participants ever-parasitemic) were calculated. Annual incidence of clinical malaria was calculated using the cohort cases captured in health facility surveillance and by self-reported confirmed malaria diagnoses at locations other than Mapanza RHC. These were summed and divided by the person-months of the study period (excluding the months the study was paused) and multiplied by twelve to estimate the annual incidence. Descriptive and statistical analysis evaluated risk factors for asymptomatic parasitemia and evidence of the clinical and public health relevance of asymptomatic parasitemia as a prelude to clinical malaria or a source of transmission to other household members. To explore risk factors for asymptomatic parasitemia, multiple analyses were performed. First, summary statistics were stratified by participants who were ever versus never positive by qPCR to examine univariate risk factors associated with being ever positive. Differences were compared using Fisher’s exact test for proportions and the Kruskal-Wallis test for continuous data. Second, univariate regression was used to test for associations between qPCR positivity and age group, travel, and net usage. Finally, to assess if parasitemia occurred randomly across the population, a random forest model was constructed, maximizing predictive power for qPCR using available covariates, and the model fit was compared between the true study population (covariates and associated qPCR outcomes) and an alternate population where the covariates and associated qPCR outcomes were unlinked and qPCR outcomes were randomly assigned. The possible clinical relevance of asymptomatic parasitemia as an indicator of subsequent or prior clinical malaria in the parasitemic individual was explored by examining the association between qPCR positivity and two individual-level outcomes in the six months surrounding the positive event: symptoms reported and self-reported malaria diagnosis. Logistic regression was used to examine the odds ratio of each outcome in those parasitemic by qPCR compared to those who were qPCR negative in each of the three months preceding and following the month in which the outcome was measured. All analyses adjusted p-values for multiple comparisons using a Bonferroni approach. To explore the possible public health importance of asymptomatic parasitemia as a source of transmission to other individuals, associations were examined between individual qPCR positivity and three outcomes in other household members during the three months following the positive event, including symptoms reported, self-reported malaria diagnosis, and qPCR positivity, using the same statistical approach as described above for associations with clinical malaria. To account for potential false positive qPCR results, a sensitivity analysis was conducted for which qPCR positivity was defined as any qPCR positive test with a parasite density of at least 10 parasites/µL. All analyses were done using R version 4.2.3. Results A total of 201 households, some comprised of multiple structures, were identified during the two years of the study, 197 were enrolled, and complete monthly data were obtained for 167 (83%) households. Over the 24 months of the study, 1198 persons were screened and 1071 (89%) were enrolled. Median participant age was 16 years (IQR: 8–28), and 58% of participants were female (Table 1 ). Median household size was 5 people (IQR: 3–7, range: 1–18). Table 1 Longitudinal cohort member demographic characteristics by qPCR positivity for Plasmodium falciparum . Overall Never positive* Ever positive* p-value Household n = 167 n = 80 n = 87 Household size (median [IQR]) 5.0 [3. 0, 7.0] 3.0 [2.0, 6.0] 6.0 [4.0, 8.0] < 0.001 Household has at least one ITN (n, %) 131 (76.6) 54 (74.0) 68 (81.9) 0.314 IRS conducted in household during study period (n, %) 4 ( 2.0) 3 ( 3.8) 1 ( 1.1) 0.554 Household has more than one sleeping structure (n, %) 49 (28.7) 17 (23.3) 31 (37.3) 0.085 Individuals n = 1071 n = 927 n = 144 Number of follow-up visits (median [IQR]) 9.0 [2.0, 16.0] 8.0 [2.0, 15.0] 15.5 [10.0, 19.0] < 0.001 Age (years) (median [IQR]) 16.0 [8.0, 28.0] 16.0 [9.0, 27.0] 13.0 [7.0, 28.3] 0.154 Female sex (n, % of column total) 133 (57.6) 122 (59.2) 11 (44.0) 0.215 Visitors (n, %) 149 (14.9) 142 (16.4) 7 ( 5.2) 0.001 Years of education (median [IQR]) b 7.0 [1.0, 10.0] 7.0 [2.0, 10.0] 7.0 [0.0, 9.0] 0.185 Ever employed (n, %) a,b 369 (34.5) 311 (33.5) 58 (40.3) 0.137 Ever travelled (n, %) c 572 (53.4) 484 (52.2) 88 (61.1) 0.057 % nights using bed net (mean (SD)) 34% (37%) 34% (38%) 38% (35%) 0.246 *Samples were considered qPCR positive if at least one of the two wells had a parasite density above one parasite/µL and a melting point within ± 0.5° Celsius of the control melting point. Never positive individuals were qPCR negative at every study visit. Ever positive individuals were qPCR positive at one or more study visits. Abbreviations: IQR (inter quartile range); a p-values for variables reported as % (n) are from Fisher’s exact test and variables reported as mean or median, (IQR) are from the Kruskal-Wallis test; b Among adults,16 years of age and older; c Among residents and temporary residents Incidence of clinical malaria was low The incidence of clinical malaria in the cohort was 46.7 cases per 1000 person-years, with an incidence of 24.9, 27.1, and 50.7 cases per 1000 person-years in individuals younger than 5 years old, 6 to 15 years, and 15 years and older, respectively. Mapanza RHC recorded 206 confirmed cases of malaria by RDT, of which only four were participants in the cohort study. These four cases occurred during the COVID-19 pandemic when data collection was paused (Table S1). An additional forty cohort members reported a malaria diagnosis at a different health care facility, most commonly, nearby Macha Hospital. None of these forty were qPCR positive in the month of, the month preceding, or the month following their self-reported diagnosis of malaria, although most (n = 34) reported taking Coartem® (artemisinin-lumefantrine), which rapidly clears parasitemia. Three reported finishing their course of medication the day prior to the study visit, ten reported having finished their treatment course within the prior week, and 21 completed their treatment more than one week prior to the study visit. An additional four adult cohort participants reported taking antimalarial medications from a traditional healer, friend, family member, or local chemist and were not included in the case count. Parasite prevalence and density were low in those with asymptomatic parasitemia There were 164 episodes of parasitemia identified by qPCR in 144 individuals ( Table 1 , 13.3% of individuals). No RDT positive participants were identified in the cohort despite testing those who were febrile at the time of the study visit (n = 43). Monthly mean parasite prevalence was 1.5% and highest in May 2019 (4.2%), July 2019 (3.5%), and August 2020 (4.4%) ( Figure S1 ). Most (89%) individuals who were ever parasitemic were only positive once ( Figure 2 ). Fifteen individuals were parasitemic on more than one study visit, for an average of 2.4 monthly visits (Range: 2 - 7) ( Figure 3 ), although none of these individuals self-reported having clinical malaria during the study period. The median parasite density was only four parasites/µL (IQR: 2 - 23) ( Figure 4, Panel B ), much lower than the parasite density for clinical cases at Mapanza RHC (393 parasites/µL, IQR: 41 - 3340). Most (96%) qPCR positive individuals had parasitemia levels below 100 parasites/µL. There was a decreasing trend but no statistical correlation between age and level of parasitemia ( Figure 4, Panel A ) (Pearson correlation coefficient, r = -0.13). While travel, household visitors, and bed net use varied, none were risk factors for parasitemia The odds of parasitemia by qPCR were not different among those who reported travel (odds ratio: 0.82, p-value= 0.45), nor among individuals living in households with others who had travelled (odds ratio: 1.0, p-value= 0.77). The odds of parasitemia by qPCR were not higher in visitors (odds ratio: 1.3, p-value= 0.59) nor in individuals living in households that had a visitor (odds ratio: 1.2, p-value= 0.46). Using a net was not associated with parasitemia by qPCR (odds ratio: 1.0, p-value= 0.34). Common symptoms of malaria were prevalent but not associated with parasitemia There was no statistical association with current or lagged parasitemia and fever, cough or headache ( Figure 5, Panel A ). The prevalence of fever was 7.5% in children younger than 5 years, 3.2% in children 5-15 years, and 5.1% in those older than 15 years. There were 43 instances of fever at the time of the study visit among 40 household residents, but only one febrile individual was parasitemic by qPCR. Univariate analysis examined associations between parasitemia by qPCR and clinical outcomes to assess evidence of the clinical relevance of qPCR positivity. The alpha for the significance threshold was adjusted for multiple comparisons. A) qPCR positivity and symptoms of malaria B) qPCR positivity and self-reported malaria. Participants were asked to self-report malaria diagnoses received at a health facility. There was no evidence that those with parasitemia progressed to clinical malaria Most (97%) of the 147 individuals ever parasitemic by qPCR did not report any episodes of clinical malaria during the 24-month study period. Specifically, no qPCR infected individuals reported clinical malaria in the three months prior or two months following their episode of parasitemia. Three months after individuals self-reported a diagnosis of clinical malaria, their odds of having parasitemia by qPCR were higher than the odds of parasitemia by qPCR in individuals with no history of clinical malaria (univariate odds 7.6, CI [1.8, 33.5] p-value = 0.0066) ( Figure 5, Panel B ), but there was no association in the months preceding the diagnosis of clinical malaria nor during the first or second month following diagnosis. There was no evidence that parasitemia was associated with subsequent clinical malaria in other household members There was no statistical association between parasitemia by qPCR and symptoms of malaria or self-reported malaria in other household members in the three months following the qPCR positive test result (Figure 6, Panels A, B). Individuals with parasitemia by qPCR clustered at the household level, where in a households with a qPCR positive individual, the odds of having other positive members in the same month were four times the odds of having no positive household members that month (Odds ratio: 4.0, 95% CI: 2.7, 6.0). However, there were no higher odds of household members being parasitemic by qPCR in the three months following parasitemia in the index case, suggesting no association with household transmission. Univariate analyses examined associations between qPCR positivity and outcomes in other household members to examine whether parasitemia led to onward transmission: A) individual qPCR positivity and symptoms of malaria in household members; B) individual qPCR positivity and self-reported malaria; and C) individual qPCR positivity and qPCR positivity in household members. The results of the sensitivity analysis were consistent with the main results, although, as expected, there were fewer episodes of parasitemia. When restricting the definition of qPCR positivity to those with parasite levels of at least 10 parasites/µL, only 66 episodes of parasitemia were identified in 57 individuals. Full results of the sensitivity analyses are shown in the supplementary materials. Discussion In a two-year longitudinal cohort study in a low transmission setting in southern Zambia, the prevalence of parasitemia was low but persistent throughout the study period and across a wide age range. Parasite density was quite low and most episodes of parasitemia were transient, subsequently either cleared by the immune system or below the limit of detection. There was no evidence to support the hypothesis that low-density parasitemia led to symptomatic malaria. Despite the fact that asymptomatic parasitemia clustered in households within the same month, consistent with the idea of shared household risk factors and consistent with prior research demonstrating spatial clustering of asymptomatic parisitemia. there was no association with an increased risk of parasitemia or reported clinical malaria among household members in subsequent months that would be consistent with onward transmission (13,17). These findings suggest that, in this low transmission setting in southern Zambia, programmatic efforts to identify and treat low level parasitemia of this magnitude are not needed to prevent clinical malaria or achieve malaria elimination. Asymptomatic parasitemia was not associated with typical malaria risk factors of sex, travel, or net usage. In low transmission settings, older individuals are expected to have premunition and thus higher odds of asymptomatic parasitemia of lower density, whereas younger children are assumed to be relatively immunologically naive and to have symptomatic disease when parasitemia (18). Instead, there were no significant decreases in parasite density by age and asymptomatic parasitemia was prevalent in children younger than five years. Other studies have shown that any level of parasitemia carries a risk of onward transmission (19,20). Currently, Southern Province, Zambia deploys a reactive test-and-treat strategy following focal investigations, in which a malaria case at a health center triggers a reactive response around the case’s residence. RDTs are used for screening, which would have missed nearly all the parasitemic individuals identifiedin this cohort. To detect these low-level parasitemias, more sensitive tools are needed (21), but even newer ultra-sensitive RDTs would not have detected the level of parasitemia identified in this study (22). Inherent in interview-based surveys are biases that may come with self-reporting. For example, net usage may be somewhat overestimated due to social desirability bias. However, this is an unavoidable limitation in this context, and the seasonal trends observed in net usage are unlikely to be differentially impacted. A more significant limitation is that most clinical cases were self-reported by individuals who sought care at a health facility not included in the study. However, because these individuals were followed monthly in this cohort, their recall for malaria diagnosis is expected to be relatively accurate. Under the assumption that these self-reported cases are wholly misclassified, the null finding that there is no relationship between clinical and asymptomatic infection may be incorrect, although the absence of clinical malaria in cohort members during the study period supports this conclusion – that these low level parasitemias have no clinical significance. Overall, the repeated longitudinal measurements in this cohort study minimize recall bias and the design is an overall strength. Low-level parasitemia was prevalent in this low transmission setting in southern Zambia. There was no evidence that low-level asymptomatic parasitemia led to clinical malaria or transmission to other household members. Thus, programmatic efforts to identify and treat individuals with low level parasitemia of this magnitude is not warranted to minimize the disease burden or achieve malaria elimination. Declarations Ethics approval and consent to participate The study design and questionnaires were approved by the Johns Hopkins Bloomberg School of Public Health Institutional Review Board and Tropical Diseases Research Center Ethical Review Committee. All adult participants signed informed consents and parents, or guardians, provided consent for children. Consent for publication All authors consent to the publication of this work. Availability of data and materials The data used to support the findings of this study are not publicly available. However, investigators interested in the data can submit a written request to the corresponding author Dr. William Moss ( [email protected] ) and the Macha Research Trust IRB Chairperson ( [email protected] , +260979402560) to assess the request. Competing interests. None. Funding This work was supported by funds from the National Institute of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health (U19AI089680). Author contributions JLS conceived of the study, lead data collection and cleaning, and carried out the initial analytic plan and produced an early draft. ACM conducted additional analyses and wrote the final manuscript. JM, CS, HH, ML, BK, MM, TK, and ES lead field work, data collection, and laboratory processing. AW, DEN, and WJM conceived of the parent study and oversaw data collection. All authors contributed to and reviewed the final manuscript. Acknowledgements The authors gratefully acknowledge all participants and the Zambian communities of Choma District. The authors thank the National Malaria Elimination Centre for their support and all ANTOOMWE study team members and study participants. References Countries and territories certified malaria-free by WHO [Internet]. [cited 2025 July 9]. Available from: https://www.who.int/teams/global-malaria-programme/elimination/countries-and-territories-certified-malaria-free-by-who Ahmed S, Reithinger R, Kaptoge SK, Ngondi JM. Travel Is a Key Risk Factor for Malaria Transmission in Pre-Elimination Settings in Sub-Saharan Africa: A Review of the Literature and Meta-Analysis. Am J Trop Med Hyg. 2020 Oct;103(4):1380–7. Deutsch-Feldman M, Hamapumbu H, Lubinda J, Musonda M, Katowa B, Searle KM, et al. Efficiency of a Malaria Reactive Test-and-Treat Program in Southern Zambia: A Prospective, Observational Study. Am J Trop Med Hyg. 2018 May;98(5):1382–8. Nguyen TD, Tran TNA, Parker DM, White NJ, Boni MF. Antimalarial mass drug administration in large populations and the evolution of drug resistance. PLOS Glob Public Health. 2023 July 26;3(7):e0002200. Pinchoff J, Hamapumbu H, Kobayashi T, Simubali L, Stevenson JC, Norris DE, et al. Factors Associated with Sustained Use of Long-Lasting Insecticide-Treated Nets Following a Reduction in Malaria Transmission in Southern Zambia. Am J Trop Med Hyg. 2015 Nov 4;93(5):954–60. Rek J, Blanken SL, Okoth J, Ayo D, Onyige I, Musasizi E, et al. Asymptomatic School-Aged Children Are Important Drivers of Malaria Transmission in a High Endemicity Setting in Uganda. The Journal of Infectious Diseases. 2022 Aug 15;226(4):708–13. Sumari D, Mwingira F, Selemani M, Mugasa J, Mugittu K, Gwakisa P. Malaria prevalence in asymptomatic and symptomatic children in Kiwangwa, Bagamoyo district, Tanzania. Malar J. 2017 Dec;16(1):1–7. Sumner KM, Freedman E, Abel L, Obala A, Pence BW, Wesolowski A, et al. Genotyping cognate Plasmodium falciparum in humans and mosquitoes to estimate onward transmission of asymptomatic infections. Nat Commun. 2021 Feb 10;12(1):909. Niang M, Thiam LG, Sane R, Diagne N, Talla C, Doucoure S, et al. Substantial asymptomatic submicroscopic Plasmodium carriage during dry season in low transmission areas in Senegal: Implications for malaria control and elimination. PLoS One [Internet]. 2017 Aug 3 [cited 2021 Apr 5];12(8). Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5542561/ Laban NM, Kobayashi T, Hamapumbu H, Sullivan D, Mharakurwa S, Thuma PE, et al. Comparison of a PfHRP2-based rapid diagnostic test and PCR for malaria in a low prevalence setting in rural southern Zambia: implications for elimination. Malar J. 2015 Jan 28;14:25. Ahmad A, Mohammed NI, Joof F, Affara M, Jawara M, Abubakar I, et al. Asymptomatic Plasmodium falciparum carriage and clinical disease: a 5-year community-based longitudinal study in The Gambia. Malaria Journal. 2023 Mar 7;22(1):82. Fola AA, Moser KA, Aydemir O, Hennelly C, Kobayashi T, Shields T, et al. Temporal and spatial analysis of Plasmodium falciparum genomics reveals patterns of parasite connectivity in a low-transmission district in Southern Province, Zambia. Malaria Journal. 2023 July 7;22(1):208. Searle KM, Katowa B, Musonda M, Pringle JC, Hamapumbu H, Matoba J, et al. Sustained Malaria Transmission despite Reactive Screen-and-Treat in a Low-Transmission Area of Southern Zambia. Am J Trop Med Hyg. 2021 Feb;104(2):671–9. Fornadel CM, Norris LC, Glass GE, Norris DE. Analysis of Anopheles arabiensis blood feeding behavior in southern Zambia during the two years after introduction of insecticide-treated bed nets. Am J Trop Med Hyg. 2010 Oct 1;83(4):848–53. Martin, Anne C., Kamilar, Victoria, Simubali, Limonty, Mudenda, Twig, Hamapumbu, Harry, Schue, Jessica L., et al. Seasonal stratification of anopheline abundance and species composition in southern Zambia suggests year-round, season-specific vector control approaches are needed. Malaria Journal. (Accepted October 2025). National Malaria Elimination Centre. Guidelines for the Diagnosis and Treatment of Malaria in Zambia. Ministry of Health, Lusaka, Zambia; 2017. Report No.: 5th Edition. Bhondoekhan FRP, Searle KM, Hamapumbu H, Lubinda M, Matoba J, Musonda M, et al. Improving the efficiency of reactive case detection for malaria elimination in southern Zambia: a cross-sectional study. Malar J. 2020 May 7;19(1):175. Katowa B, Hamapumbu H, Thuma PE, Bérubé S, Wesolowski A, Moss WJ, et al. Declining Age-Specific Seroprevalence and Seroconversion Rates in Plasmodium falciparum from 2009 to 2018 Documents Progress toward Malaria Elimination in Southern Zambia. 2023 July 5 [cited 2025 July 14]; Available from: https://www.ajtmh.org/view/journals/tpmd/109/1/article-p134.xml Bousema T, Drakeley C. Epidemiology and Infectivity of Plasmodium falciparum and Plasmodium vivax Gametocytes in Relation to Malaria Control and Elimination. Clin Microbiol Rev. 2011 Apr;24(2):377–410. Gonçalves BP, Kapulu MC, Sawa P, Guelbéogo WM, Tiono AB, Grignard L, et al. Examining the human infectious reservoir for Plasmodium falciparum malaria in areas of differing transmission intensity. Nat Commun. 2017 Dec;8(1):1133. Bousema T, Okell L, Felger I, Drakeley C. Asymptomatic malaria infections: detectability, transmissibility and public health relevance. Nat Rev Microbiol. 2014 Dec;12(12):833–40. Acquah FK, Donu D, Obboh EK, Bredu D, Mawuli B, Amponsah JA, et al. Diagnostic performance of an ultrasensitive HRP2-based malaria rapid diagnostic test kit used in surveys of afebrile people living in Southern Ghana. Malar J. 2021 Dec;20(1):125. Additional Declarations No competing interests reported. Supplementary Files Supplement.LowdensityparasitemiaandclinicalrelevancecohortJan142026.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 16 Mar, 2026 Reviews received at journal 02 Mar, 2026 Reviews received at journal 23 Feb, 2026 Reviewers agreed at journal 13 Feb, 2026 Reviewers agreed at journal 01 Feb, 2026 Reviewers invited by journal 27 Jan, 2026 Editor assigned by journal 21 Jan, 2026 Submission checks completed at journal 20 Jan, 2026 First submitted to journal 19 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Schue","email":"","orcid":"","institution":"Johns Hopkins Bloomberg School of Public Health","correspondingAuthor":false,"prefix":"","firstName":"Jessica","middleName":"L.","lastName":"Schue","suffix":""},{"id":581472476,"identity":"a8961f85-be83-45b1-a282-df41deb6bfbc","order_by":1,"name":"Anne C. Martin","email":"","orcid":"","institution":"Johns Hopkins Bloomberg School of Public Health","correspondingAuthor":false,"prefix":"","firstName":"Anne","middleName":"C.","lastName":"Martin","suffix":""},{"id":581472479,"identity":"25d6db1d-8654-4b50-881f-a6c9b85178b2","order_by":2,"name":"Japhet Matoba","email":"","orcid":"","institution":"Macha Research Trust","correspondingAuthor":false,"prefix":"","firstName":"Japhet","middleName":"","lastName":"Matoba","suffix":""},{"id":581472480,"identity":"b0716f40-6adb-4235-8512-07f9e5f89d15","order_by":3,"name":"Caison Sing’anga","email":"","orcid":"","institution":"Macha Research Trust","correspondingAuthor":false,"prefix":"","firstName":"Caison","middleName":"","lastName":"Sing’anga","suffix":""},{"id":581472483,"identity":"4bbaaccd-951d-48cb-8fe9-2b7044134cff","order_by":4,"name":"Mukuma Lubinda","email":"","orcid":"","institution":"Macha Research Trust","correspondingAuthor":false,"prefix":"","firstName":"Mukuma","middleName":"","lastName":"Lubinda","suffix":""},{"id":581472485,"identity":"a480e84a-98eb-44b4-9c4a-b659f2984f48","order_by":5,"name":"Ben Katowa","email":"","orcid":"","institution":"Macha Research Trust","correspondingAuthor":false,"prefix":"","firstName":"Ben","middleName":"","lastName":"Katowa","suffix":""},{"id":581472487,"identity":"529877be-850d-499d-8f1b-fa29b48ae6c7","order_by":6,"name":"Michael Musonda","email":"","orcid":"","institution":"Macha Research Trust","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Musonda","suffix":""},{"id":581472488,"identity":"9d90d9de-0b93-4187-b2f3-01e4cec19166","order_by":7,"name":"Sophie Berube","email":"","orcid":"","institution":"University of Florida","correspondingAuthor":false,"prefix":"","firstName":"Sophie","middleName":"","lastName":"Berube","suffix":""},{"id":581472489,"identity":"434030ba-8935-4061-b730-a803afa4970b","order_by":8,"name":"Timothy Shields","email":"","orcid":"","institution":"Johns Hopkins Bloomberg School of Public Health","correspondingAuthor":false,"prefix":"","firstName":"Timothy","middleName":"","lastName":"Shields","suffix":""},{"id":581472490,"identity":"7fcd8e9e-4d1a-4efe-be09-b92afcc01c87","order_by":9,"name":"Tamaki Kobayashi","email":"","orcid":"","institution":"Johns Hopkins Bloomberg School of Public Health","correspondingAuthor":false,"prefix":"","firstName":"Tamaki","middleName":"","lastName":"Kobayashi","suffix":""},{"id":581472492,"identity":"7380e8ad-6a67-42c2-8b43-1cb36ab2e328","order_by":10,"name":"Harry Hamapumbu","email":"","orcid":"","institution":"Macha Research Trust","correspondingAuthor":false,"prefix":"","firstName":"Harry","middleName":"","lastName":"Hamapumbu","suffix":""},{"id":581472494,"identity":"3a29019e-440e-4e45-bde5-cfdab5598abe","order_by":11,"name":"Edgar Simulundu","email":"","orcid":"","institution":"Zambia National Public Health Institute","correspondingAuthor":false,"prefix":"","firstName":"Edgar","middleName":"","lastName":"Simulundu","suffix":""},{"id":581472495,"identity":"64d1fe86-fc37-47b8-81f5-b3d21591300f","order_by":12,"name":"Douglas E. Norris","email":"","orcid":"","institution":"Johns Hopkins Bloomberg School of Public Health","correspondingAuthor":false,"prefix":"","firstName":"Douglas","middleName":"E.","lastName":"Norris","suffix":""},{"id":581472496,"identity":"79b7e772-b118-4faa-9733-c07c7924ab02","order_by":13,"name":"Amy Wesolowksi","email":"","orcid":"","institution":"Johns Hopkins Bloomberg School of Public Health","correspondingAuthor":false,"prefix":"","firstName":"Amy","middleName":"","lastName":"Wesolowksi","suffix":""},{"id":581472498,"identity":"6ac6aad6-bbb3-496d-aabd-70b5190a0c4f","order_by":14,"name":"William J. Moss","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAqElEQVRIiWNgGAWjYBACgwNg0obZAETzEK+lII1kLR8OM5Cg5drhwx9/GJxnN5dIYHzwto0ILWa309KkeQxuM1vOSGA2nEuclhwzZgagFoMbCWzSvERqMQY67BxIC/tvorTY384xkOAxOAC2hZkoLZYQvyQzG5x52Cw55xwRWgxuJwND7I9dssHx5IMf3pQRoQUGkhkYGBtIUA8EdqQpHwWjYBSMghEFALp+N6ZD2Rv6AAAAAElFTkSuQmCC","orcid":"","institution":"Johns Hopkins Bloomberg School of Public Health","correspondingAuthor":true,"prefix":"","firstName":"William","middleName":"J.","lastName":"Moss","suffix":""}],"badges":[],"createdAt":"2026-01-19 17:41:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8641961/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8641961/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101459849,"identity":"bcbd3934-6f89-4086-82e4-1fc14712c9b7","added_by":"auto","created_at":"2026-01-30 01:33:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":656142,"visible":true,"origin":"","legend":"\u003cp\u003eMap of enrolled households in the catchment area of Mapanza Rural Health Center.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8641961/v1/6fd36835c5275a7c7a5f434a.png"},{"id":101459850,"identity":"37bf94d7-370e-4e85-bcfd-de1ced0b7a6b","added_by":"auto","created_at":"2026-01-30 01:33:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":204710,"visible":true,"origin":"","legend":"\u003cp\u003eIndividual qPCR results of cohort members over time for those who were ever positive. Each line represents one individual cohort participant ordered by age. Cohort data collection was paused from April to July in 2020 in compliance with the Government of Zambia’s COVID-19 prevention measures.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8641961/v1/6e08184d4859fbb116a151e5.png"},{"id":101459853,"identity":"2550969b-0f67-4d2e-b0e5-47dc2bb3ce41","added_by":"auto","created_at":"2026-01-30 01:33:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":70271,"visible":true,"origin":"","legend":"\u003cp\u003eFrequency and timing of parasitemia by qPCR among individuals who tested positive more than once. None self-reported or had confirmed clinical malaria.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8641961/v1/0edd75d9217cc8698655b8b6.png"},{"id":101459854,"identity":"daac2336-5c3f-4fe4-b9dd-c5571285d047","added_by":"auto","created_at":"2026-01-30 01:33:40","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":124492,"visible":true,"origin":"","legend":"\u003cp\u003eParasitemia levels by age among qPCR positive cohort members\u003c/p\u003e\n\u003cp\u003eA) Parasitemia by qPCR in log base 10 by age with a Loess smoothing line for trend. B) Raw parasite density frequency plots stratified by cohort participants and health center cases. The blue line represents cohort participants, and the red line represents health facility cases. The dotted line is 100 parasites/µL, the accepted limit of detection of RDTs.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8641961/v1/1e16d7fd25bd779b747bff05.png"},{"id":101459852,"identity":"499d3c59-5fb1-4697-b71c-f975fda8a26c","added_by":"auto","created_at":"2026-01-30 01:33:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":129426,"visible":true,"origin":"","legend":"\u003cp\u003eNo associations between individual qPCR positivity and clinical disease\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8641961/v1/0e0233a61a3a64a5eea8c08c.png"},{"id":101459848,"identity":"d9ed4328-5d47-4fab-9206-444a5953a1bb","added_by":"auto","created_at":"2026-01-30 01:33:39","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":91581,"visible":true,"origin":"","legend":"\u003cp\u003eNo associations between individual parasitemia by qPCR and clinical malaria or subsequent parasitemia in household members.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8641961/v1/324f28d73b175030e2da6782.png"},{"id":101459892,"identity":"03fa04aa-f412-4436-adf2-ad2bfbaac540","added_by":"auto","created_at":"2026-01-30 01:33:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1787322,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8641961/v1/4adcc6d3-fd6e-48e3-98b0-3fbe5b17bf12.pdf"},{"id":101459851,"identity":"c35127ed-9a6a-4596-a35a-dabc5bafe093","added_by":"auto","created_at":"2026-01-30 01:33:39","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2683899,"visible":true,"origin":"","legend":"","description":"","filename":"Supplement.LowdensityparasitemiaandclinicalrelevancecohortJan142026.docx","url":"https://assets-eu.researchsquare.com/files/rs-8641961/v1/926f9e1555b3a32256eafe9e.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Low-density asymptomatic parasitemia in southern Zambia does not lead to clinical malaria and is not associated with household transmission: results from a two-year cohort study","fulltext":[{"header":"Background","content":"\u003cp\u003eAs of 2025, 47 countries achieved malaria elimination through a combination of mass, targeted, and reactive interventions (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Understanding the causes of residual transmission in an area approaching elimination is necessary to deliver appropriate interventions and ultimately achieve malaria elimination. Residual transmission may be due to repeated parasite introductions via imported cases or ongoing low-level transmission from symptomatic infections prior to their treatment and/or untreated chronic, asymptomatic infections (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). These drivers are countered most effectively with different interventions. For example, reactive test-and-treat can curb transmission introduced from imported cases and symptomatic infections, whereas mass drug administration (MDA) can clear infections that may not result in clinical symptoms or be detected by available diagnostics and thus act as reservoirs for transmission (\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAsymptomatic infections with moderate levels of parasitemia are major gametocyte reservoirs in high and moderate transmission settings and contribute disproportionately to onward transmission (\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). In low transmission settings, where individuals have less protective immunity against clinical disease, asymptomatic parasitemias are typically low-density (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e), undetectable by point-of-care testing (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e), and linked to persistent carriage across transmission seasons (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), but are of unknown clinical and public health relevance. Asymptomatic parasitemia in low transmission settings could be important if they are a prelude to clinical malaria in the infected individual or a source of transmission to other individuals. However, few cohort studies have been conducted in pre-elimination settings to understand the relationships between asymptomatic parasitemia and subsequent clinical malaria or asymptomatic parasitemia and subsequent parasitemia in other household members.\u003c/p\u003e \u003cp\u003eThis work describes a prospective longitudinal cohort study designed to capture all cases of clinical malaria and asymptomatic subpatent parasitemia in a geographically defined population in Southern Province, Zambia. Background prevalence of asymptomatic parasitemia with \u003cem\u003ePlasmodium falciparum\u003c/em\u003e is 1\u0026ndash;3% by quantitative PCR (qPCR) (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Specifically, this study seeks to guide elimination strategies in low transmission settings through examination of asymptomatic parasitemia: its risk factors, its epidemiologic relationship to incident clinical malaria, and evidence of its linkage to ongoing transmission.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy Site\u003c/h2\u003e \u003cp\u003eA 24-month longitudinal cohort study was conducted from October 2018 through September 2020 in Choma District, Southern Province, Zambia, a low-transmission setting typical of southern Zambia. The region has a tropical savannah climate with a rainy season from December to April, followed by a cool dry season from May to August, and a hot dry season from September to November. The primary malaria vector is \u003cem\u003eAnopheles arabiensis\u003c/em\u003e, which peaks during the rainy season (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). The study site was a contiguous two-square-kilometer area, defined by natural borders of roads and footpaths, located within the catchment of Mapanza Rural Health Centre (RHC) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHousehold and participant selection\u003c/h3\u003e\n\u003cp\u003eAll households within the study site were enumerated using satellite imagery and were invited to participate. Geocoordinates of consenting households were captured by global positioning system (GPS) devices. Throughout the study period, newly constructed and newly occupied households in the study area also were invited to participate. Enrolled households were visited monthly for a minimum of one year. If a household agreed, the study visits continued into a second year.\u003c/p\u003e \u003cp\u003eAll household residents and overnight visitors older than three months of age were invited to participate. Participants were classified as either a permanent resident, temporary resident (household resident for 2 months or more), or visitor (household resident for fewer than 2 months). Individuals younger than three months or with severe illness other than malaria were excluded. Written informed consent was obtained from each participant 16 years of age or older. Parental or guardian permission was obtained for all children younger than 16 years of age, and assent was obtained from children 13 to 15 years old.\u003c/p\u003e\n\u003ch3\u003eCohort data collection\u003c/h3\u003e\n\u003cp\u003eHousehold and individual surveys were administered monthly, except between April and July 2020 when community data collection was paused in compliance with the Government of the Republic of Zambia\u0026rsquo;s COVID-19 prevention measures. The household survey recorded household size, amenities, net ownership, and history of indoor residual spraying. Each visit captured the number and condition of nets in the household, where household members slept, and any visitors to the household. House construction variables such as roof and wall material were collected for each sleeping structure within a household complex. Individual surveys captured demographic characteristics, socio-economic indicators, use of nets, times spent indoors versus outdoors, travel history, recent illnesses, and health care seeking behavior. Travel history included the location, purpose, and duration of up to four trips during the previous month. Each participant also had their tympanic temperature taken, and those with a temperature at 38\u0026deg; Celsius or higher were administered a malaria rapid diagnostic test (RDT) (SD Bioline Malaria AG \u003cem\u003eP.f.\u003c/em\u003e, Abbott, Abbott Park, Illinois, USA). Participants who tested positive were offered treatment with artemether-lumefantrine (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Pregnant women and children under 5 kilograms who tested positive were to be offered transportation to the health center treatment. Dried blood spots (DBS) were collected monthly for detection of \u003cem\u003eP. falciparum\u003c/em\u003e parasitemia by qPCR.\u003c/p\u003e\n\u003ch3\u003eHealth center symptomatic surveillance\u003c/h3\u003e\n\u003cp\u003ePassive case detection was established at Mapanza RHC, the catchment area of which included the cohort households, and its affiliated health posts. Individuals older than three months of age who tested positive for malaria by RDT were asked to participate in the study by the health center staff. Those who provided written consent were administered a brief questionnaire, including basic demographic information, recent travel, bed net use, recent illnesses, and whether or not they were participants in the cohort study. DBS were collected from all consenting patients for the detection of \u003cem\u003eP. falciparum\u003c/em\u003e by qPCR.\u003c/p\u003e\n\u003ch3\u003eLaboratory Testing\u003c/h3\u003e\n\u003cp\u003eDNA was extracted from the DBS using a standard saponin and Chelex-100 extraction procedure, and qPCR targeting the \u003cem\u003eP. falciparum cytochrome-b\u003c/em\u003e gene was done using SYBR Green PCR Master Mix (Applied Biosystems, Thermo Fischer Scientific Inc, Waltham, MA) (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). All samples were run in duplicate. Parasite density was estimated based on the cycle threshold, and samples were considered positive if at least one of the two wells had a parasite density above one parasite/\u0026micro;L and a melting point within \u0026plusmn;\u0026thinsp;0.5\u0026deg; Celsius of the melting point of the 3D7 g-DNA control. Positive samples were run on gel electrophoresis to confirm the DNA product size.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eData Analysis\u003c/h2\u003e \u003cp\u003eParasite prevalence by qPCR and the incidence of clinical malaria using health center surveillance were calculated across the study period. Monthly parasite prevalence (the proportion of participants parasitemic in a given month) and period prevalence (the proportion of participants ever-parasitemic) were calculated. Annual incidence of clinical malaria was calculated using the cohort cases captured in health facility surveillance and by self-reported confirmed malaria diagnoses at locations other than Mapanza RHC. These were summed and divided by the person-months of the study period (excluding the months the study was paused) and multiplied by twelve to estimate the annual incidence.\u003c/p\u003e \u003cp\u003eDescriptive and statistical analysis evaluated risk factors for asymptomatic parasitemia and evidence of the clinical and public health relevance of asymptomatic parasitemia as a prelude to clinical malaria or a source of transmission to other household members. To explore risk factors for asymptomatic parasitemia, multiple analyses were performed. First, summary statistics were stratified by participants who were ever versus never positive by qPCR to examine univariate risk factors associated with being ever positive. Differences were compared using Fisher\u0026rsquo;s exact test for proportions and the Kruskal-Wallis test for continuous data. Second, univariate regression was used to test for associations between qPCR positivity and age group, travel, and net usage. Finally, to assess if parasitemia occurred randomly across the population, a random forest model was constructed, maximizing predictive power for qPCR using available covariates, and the model fit was compared between the true study population (covariates and associated qPCR outcomes) and an alternate population where the covariates and associated qPCR outcomes were unlinked and qPCR outcomes were randomly assigned.\u003c/p\u003e \u003cp\u003eThe possible clinical relevance of asymptomatic parasitemia as an indicator of subsequent or prior clinical malaria in the parasitemic individual was explored by examining the association between qPCR positivity and two individual-level outcomes in the six months surrounding the positive event: symptoms reported and self-reported malaria diagnosis. Logistic regression was used to examine the odds ratio of each outcome in those parasitemic by qPCR compared to those who were qPCR negative in each of the three months preceding and following the month in which the outcome was measured. All analyses adjusted p-values for multiple comparisons using a Bonferroni approach.\u003c/p\u003e \u003cp\u003eTo explore the possible public health importance of asymptomatic parasitemia as a source of transmission to other individuals, associations were examined between individual qPCR positivity and three outcomes in other household members during the three months \u003cem\u003efollowing\u003c/em\u003e the positive event, including symptoms reported, self-reported malaria diagnosis, and qPCR positivity, using the same statistical approach as described above for associations with clinical malaria.\u003c/p\u003e \u003cp\u003eTo account for potential false positive qPCR results, a sensitivity analysis was conducted for which qPCR positivity was defined as any qPCR positive test with a parasite density of at least 10 parasites/\u0026micro;L. All analyses were done using R version 4.2.3.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eA total of 201 households, some comprised of multiple structures, were identified during the two years of the study, 197 were enrolled, and complete monthly data were obtained for 167 (83%) households. Over the 24 months of the study, 1198 persons were screened and 1071 (89%) were enrolled. Median participant age was 16 years (IQR: 8\u0026ndash;28), and 58% of participants were female (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Median household size was 5 people (IQR: 3\u0026ndash;7, range: 1\u0026ndash;18).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLongitudinal cohort member demographic characteristics by qPCR positivity for \u003cem\u003ePlasmodium falciparum\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOverall\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNever positive*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEver positive*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHousehold\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;167\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHousehold size (median [IQR])\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.0 [3. 0, 7.0]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.0 [2.0, 6.0]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.0 [4.0, 8.0]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHousehold has at least one ITN (n, %)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e131 (76.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e54 (74.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e68 (81.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.314\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIRS conducted in household during study period (n, %)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 ( 2.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3 ( 3.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 ( 1.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.554\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHousehold has more than one sleeping structure (n, %)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e49 (28.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17 (23.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e31 (37.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.085\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIndividuals\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;1071\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;927\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;144\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber of follow-up visits (median [IQR])\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.0 [2.0, 16.0]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.0 [2.0, 15.0]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.5 [10.0, 19.0]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (years) (median [IQR])\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16.0 [8.0, 28.0]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.0 [9.0, 27.0]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.0 [7.0, 28.3]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.154\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemale sex (n, % of column total)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e133 (57.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e122 (59.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11 (44.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.215\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVisitors (n, %)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e149 (14.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e142 (16.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7 ( 5.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYears of education (median [IQR])\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.0 [1.0, 10.0]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.0 [2.0, 10.0]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.0 [0.0, 9.0]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.185\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEver employed (n, %)\u003csup\u003ea,b\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e369 (34.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e311 (33.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e58 (40.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.137\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEver travelled (n, %)\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e572 (53.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e484 (52.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e88 (61.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.057\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e% nights using bed net (mean (SD))\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e34% (37%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34% (38%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e38% (35%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.246\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e*Samples were considered qPCR positive if at least one of the two wells had a parasite density above one parasite/\u0026micro;L and a melting point within \u0026plusmn;\u0026thinsp;0.5\u0026deg; Celsius of the control melting point. Never positive individuals were qPCR negative at every study visit. Ever positive individuals were qPCR positive at one or more study visits.\u003c/p\u003e\u003cp\u003eAbbreviations: IQR (inter quartile range); \u003csup\u003ea\u003c/sup\u003e p-values for variables reported as % (n) are from Fisher\u0026rsquo;s exact test and variables reported as mean or median, (IQR) are from the Kruskal-Wallis test; \u003csup\u003eb\u003c/sup\u003e Among adults,16 years of age and older; \u003csup\u003ec\u003c/sup\u003e Among residents and temporary residents\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIncidence of clinical malaria was low\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe incidence of clinical malaria in the cohort was 46.7 cases per 1000 person-years, with an incidence of 24.9, 27.1, and 50.7 cases per 1000 person-years in individuals younger than 5 years old, 6 to 15 years, and 15 years and older, respectively. Mapanza RHC recorded 206 confirmed cases of malaria by RDT, of which only four were participants in the cohort study. These four cases occurred during the COVID-19 pandemic when data collection was paused (Table S1). An additional forty cohort members reported a malaria diagnosis at a different health care facility, most commonly, nearby Macha Hospital. None of these forty were qPCR positive in the month of, the month preceding, or the month following their self-reported diagnosis of malaria, although most (n = 34) reported taking Coartem\u0026reg; (artemisinin-lumefantrine), which rapidly clears parasitemia. Three reported finishing their course of medication the day prior to the study visit, ten reported having finished their treatment course within the prior week, and 21 completed their treatment more than one week prior to the study visit. An additional four adult cohort participants reported taking antimalarial medications from a traditional healer, friend, family member, or local chemist and were not included in the case count.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eParasite prevalence and density were low in those with asymptomatic parasitemia\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThere were 164 episodes of parasitemia identified by qPCR in 144 individuals (\u003cstrong\u003eTable 1\u003c/strong\u003e, 13.3% of individuals). No RDT positive participants were identified in the cohort despite testing those who were febrile at the time of the study visit (n = 43). Monthly mean parasite prevalence was 1.5% and highest in May 2019 (4.2%), July 2019 (3.5%), and August 2020 (4.4%) (\u003cstrong\u003eFigure S1\u003c/strong\u003e). Most (89%) individuals who were ever parasitemic were only positive once (\u003cstrong\u003eFigure 2\u003c/strong\u003e). Fifteen individuals were parasitemic on more than one study visit, for an average of 2.4 monthly visits (Range: 2 - 7) (\u003cstrong\u003eFigure 3\u003c/strong\u003e), although none of these individuals self-reported having clinical malaria during the study period.\u0026nbsp;\u003c/p\u003e\u003cp\u003eThe median parasite density was only four parasites/\u0026micro;L (IQR: 2 - 23) (\u003cstrong\u003eFigure 4, Panel B\u003c/strong\u003e), much lower than the parasite density for clinical cases at Mapanza RHC (393 parasites/\u0026micro;L, IQR: 41 - 3340). Most (96%) qPCR positive individuals had parasitemia levels below 100 parasites/\u0026micro;L. There was a decreasing trend but no statistical correlation between age and level of parasitemia (\u003cstrong\u003eFigure 4, Panel A\u003c/strong\u003e) (Pearson correlation coefficient, r = -0.13). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eWhile travel, household visitors, and bed net use varied, none were risk factors for parasitemia\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe odds of parasitemia by qPCR were not different among those who reported travel (odds ratio: 0.82, p-value= 0.45), nor among individuals living in households with others who had travelled (odds ratio: 1.0, p-value= 0.77). The odds of parasitemia by qPCR were not higher in visitors (odds ratio: 1.3, p-value= 0.59) nor in individuals living in households that had a visitor (odds ratio: 1.2, p-value= 0.46). Using a net was not associated with parasitemia by qPCR (odds ratio: 1.0, p-value= 0.34).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCommon symptoms of malaria were prevalent but not associated with parasitemia\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThere was no statistical association with current or lagged parasitemia and fever, cough or headache (\u003cstrong\u003eFigure 5, Panel A\u003c/strong\u003e). The prevalence of fever was 7.5% in children younger than 5 years, 3.2% in children 5-15 years, and 5.1% in those older than 15 years. There were 43 instances of fever at the time of the study visit among 40 household residents, but only one febrile individual was parasitemic by qPCR.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eUnivariate analysis examined associations between parasitemia by qPCR and clinical outcomes to assess evidence of the clinical relevance of qPCR positivity. The alpha for the significance threshold was adjusted for multiple comparisons. \u0026nbsp;A) qPCR positivity and symptoms of malaria B) qPCR positivity and self-reported malaria. Participants were asked to self-report malaria diagnoses received at a health facility.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThere was no evidence that those with parasitemia progressed to clinical malaria\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMost (97%) of the 147 individuals ever parasitemic by qPCR did not report any episodes of clinical malaria during the 24-month study period. Specifically, no qPCR infected individuals reported clinical malaria in the three months prior or two months following their episode of parasitemia. Three months after individuals self-reported a diagnosis of clinical malaria, their odds of having parasitemia by qPCR were higher than the odds of parasitemia by qPCR in individuals with no history of clinical malaria (univariate odds 7.6, CI [1.8, 33.5] p-value = 0.0066) (\u003cstrong\u003eFigure 5, Panel B\u003c/strong\u003e), but there was no association in the months preceding the diagnosis of clinical malaria nor during the first or second month following diagnosis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThere was no evidence that parasitemia was associated with subsequent clinical malaria in other household members\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThere was no statistical association between parasitemia by qPCR and symptoms of malaria or self-reported malaria in other household members in the three months following the qPCR positive test result (Figure 6, Panels A, B). Individuals with parasitemia by qPCR clustered at the household level, where in a households with a qPCR positive individual, the odds of having other positive members in the same month were four times the odds of having no positive household members that month (Odds ratio: 4.0, 95% CI: 2.7, 6.0). However, there were no higher odds of household members being parasitemic by qPCR in the three months following parasitemia in the index case, suggesting no association with household transmission.\u003c/p\u003e\n\u003cp\u003eUnivariate analyses examined associations between qPCR positivity and outcomes in other household members to examine whether parasitemia led to onward transmission: A) individual qPCR positivity and symptoms of malaria in household members; B) individual qPCR positivity and self-reported malaria; and C) individual qPCR positivity and qPCR positivity in household members.\u003c/p\u003e\n\u003cp\u003eThe results of the sensitivity analysis were consistent with the main results, although, as expected, there were fewer episodes of parasitemia. When restricting the definition of qPCR positivity to those with parasite levels of at least 10 parasites/\u0026micro;L, only 66 episodes of parasitemia were identified in 57 individuals. Full results of the sensitivity analyses are shown in the supplementary materials.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn a two-year longitudinal cohort study in a low transmission setting in southern Zambia, the prevalence of parasitemia was low but persistent throughout the study period and across a wide age range. Parasite density was quite low and most episodes of parasitemia were transient, subsequently either cleared by the immune system or below the limit of detection. There was no evidence to support the hypothesis that low-density parasitemia led to symptomatic malaria. Despite the fact that asymptomatic parasitemia clustered in households within the same month, consistent with the idea of shared household risk factors and consistent with prior research demonstrating spatial clustering of asymptomatic parisitemia. there was no association with an increased risk of parasitemia or reported clinical malaria among household members in subsequent months that would be consistent with onward transmission (13,17). These findings suggest that, in this low transmission setting in southern Zambia, programmatic efforts to identify and treat low level parasitemia of this magnitude are not needed to prevent clinical malaria or achieve malaria elimination.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAsymptomatic parasitemia was not associated with typical malaria risk factors of sex, travel, or net usage. In low transmission settings, older individuals are expected to have premunition and thus higher odds of asymptomatic parasitemia of lower density, whereas younger children are assumed to be relatively immunologically naive and to have symptomatic disease when parasitemia (18). Instead, there were no significant decreases in parasite density by age and asymptomatic parasitemia was prevalent in children younger than five years.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOther studies have shown that any level of parasitemia carries a risk of onward transmission (19,20). Currently, Southern Province, Zambia deploys a reactive test-and-treat strategy following focal investigations, in which a malaria case at a health center triggers a reactive response around the case\u0026rsquo;s residence. RDTs are used for screening, which would have missed nearly all the parasitemic individuals identifiedin this cohort. To detect these low-level parasitemias, more sensitive tools are needed (21), but even newer ultra-sensitive RDTs would not have detected the level of parasitemia identified in this study (22).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInherent in interview-based surveys are biases that may come with self-reporting. For example, net usage may be somewhat overestimated due to social desirability bias. However, this is an unavoidable limitation in this context, and the seasonal trends observed in net usage are unlikely to be differentially impacted. A more significant limitation is that most clinical cases were self-reported by individuals who sought care at a health facility not included in the study. However, because these individuals were followed monthly in this cohort, their recall for malaria diagnosis is expected to be relatively accurate. Under the assumption that these self-reported cases are wholly misclassified, the null finding that there is no relationship between clinical and asymptomatic infection may be incorrect, although the absence of clinical malaria in cohort members during the study period supports this conclusion \u0026ndash; that these low level parasitemias have no clinical significance. Overall, the repeated longitudinal measurements in this cohort study minimize recall bias and the design is an overall strength.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLow-level parasitemia was prevalent in this low transmission setting in southern Zambia. There was no evidence that low-level asymptomatic parasitemia led to clinical malaria or transmission to other household members. Thus, programmatic efforts to identify and treat individuals with low level parasitemia of this magnitude is not warranted to minimize the disease burden or achieve malaria elimination.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study design and questionnaires were approved by the Johns Hopkins Bloomberg School of Public Health Institutional Review Board and Tropical Diseases Research Center Ethical Review Committee. All adult participants signed informed consents and parents, or guardians, provided consent for children.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors consent to the publication of this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data used to support the findings of this study are not publicly available. However, investigators interested in the data can submit a written request to the corresponding author Dr. William Moss ([email protected]) and the Macha Research Trust IRB Chairperson ([email protected], +260979402560) to assess the request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by funds from the National Institute of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health (U19AI089680).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJLS conceived of the study, lead data collection and cleaning, and carried out the initial analytic plan and produced an early draft. ACM conducted additional analyses and wrote the final manuscript. JM, CS, HH, ML, BK, MM, TK, and ES lead field work, data collection, and laboratory processing. AW, DEN, and WJM conceived of the parent study and oversaw data collection. All authors contributed to and reviewed the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors gratefully acknowledge all participants and the Zambian communities of Choma District. The authors thank the National Malaria Elimination Centre for their support and all ANTOOMWE study team members and study participants.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCountries and territories certified malaria-free by WHO [Internet]. [cited 2025 July 9]. Available from: https://www.who.int/teams/global-malaria-programme/elimination/countries-and-territories-certified-malaria-free-by-who\u003c/li\u003e\n\u003cli\u003eAhmed S, Reithinger R, Kaptoge SK, Ngondi JM. Travel Is a Key Risk Factor for Malaria Transmission in Pre-Elimination Settings in Sub-Saharan Africa: A Review of the Literature and Meta-Analysis. Am J Trop Med Hyg. 2020 Oct;103(4):1380\u0026ndash;7. \u003c/li\u003e\n\u003cli\u003eDeutsch-Feldman M, Hamapumbu H, Lubinda J, Musonda M, Katowa B, Searle KM, et al. Efficiency of a Malaria Reactive Test-and-Treat Program in Southern Zambia: A Prospective, Observational Study. Am J Trop Med Hyg. 2018 May;98(5):1382\u0026ndash;8. \u003c/li\u003e\n\u003cli\u003eNguyen TD, Tran TNA, Parker DM, White NJ, Boni MF. Antimalarial mass drug administration in large populations and the evolution of drug resistance. PLOS Glob Public Health. 2023 July 26;3(7):e0002200. \u003c/li\u003e\n\u003cli\u003ePinchoff J, Hamapumbu H, Kobayashi T, Simubali L, Stevenson JC, Norris DE, et al. Factors Associated with Sustained Use of Long-Lasting Insecticide-Treated Nets Following a Reduction in Malaria Transmission in Southern Zambia. Am J Trop Med Hyg. 2015 Nov 4;93(5):954\u0026ndash;60. \u003c/li\u003e\n\u003cli\u003eRek J, Blanken SL, Okoth J, Ayo D, Onyige I, Musasizi E, et al. Asymptomatic School-Aged Children Are Important Drivers of Malaria Transmission in a High Endemicity Setting in Uganda. The Journal of Infectious Diseases. 2022 Aug 15;226(4):708\u0026ndash;13. \u003c/li\u003e\n\u003cli\u003eSumari D, Mwingira F, Selemani M, Mugasa J, Mugittu K, Gwakisa P. Malaria prevalence in asymptomatic and symptomatic children in Kiwangwa, Bagamoyo district, Tanzania. Malar J. 2017 Dec;16(1):1\u0026ndash;7. \u003c/li\u003e\n\u003cli\u003eSumner KM, Freedman E, Abel L, Obala A, Pence BW, Wesolowski A, et al. Genotyping cognate Plasmodium falciparum in humans and mosquitoes to estimate onward transmission of asymptomatic infections. Nat Commun. 2021 Feb 10;12(1):909. \u003c/li\u003e\n\u003cli\u003eNiang M, Thiam LG, Sane R, Diagne N, Talla C, Doucoure S, et al. Substantial asymptomatic submicroscopic Plasmodium carriage during dry season in low transmission areas in Senegal: Implications for malaria control and elimination. PLoS One [Internet]. 2017 Aug 3 [cited 2021 Apr 5];12(8). Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5542561/\u003c/li\u003e\n\u003cli\u003eLaban NM, Kobayashi T, Hamapumbu H, Sullivan D, Mharakurwa S, Thuma PE, et al. Comparison of a PfHRP2-based rapid diagnostic test and PCR for malaria in a low prevalence setting in rural southern Zambia: implications for elimination. Malar J. 2015 Jan 28;14:25. \u003c/li\u003e\n\u003cli\u003eAhmad A, Mohammed NI, Joof F, Affara M, Jawara M, Abubakar I, et al. Asymptomatic Plasmodium falciparum carriage and clinical disease: a 5-year community-based longitudinal study in The Gambia. Malaria Journal. 2023 Mar 7;22(1):82. \u003c/li\u003e\n\u003cli\u003eFola AA, Moser KA, Aydemir O, Hennelly C, Kobayashi T, Shields T, et al. Temporal and spatial analysis of Plasmodium falciparum genomics reveals patterns of parasite connectivity in a low-transmission district in Southern Province, Zambia. Malaria Journal. 2023 July 7;22(1):208. \u003c/li\u003e\n\u003cli\u003eSearle KM, Katowa B, Musonda M, Pringle JC, Hamapumbu H, Matoba J, et al. Sustained Malaria Transmission despite Reactive Screen-and-Treat in a Low-Transmission Area of Southern Zambia. Am J Trop Med Hyg. 2021 Feb;104(2):671\u0026ndash;9. \u003c/li\u003e\n\u003cli\u003eFornadel CM, Norris LC, Glass GE, Norris DE. Analysis of Anopheles arabiensis blood feeding behavior in southern Zambia during the two years after introduction of insecticide-treated bed nets. Am J Trop Med Hyg. 2010 Oct 1;83(4):848\u0026ndash;53. \u003c/li\u003e\n\u003cli\u003eMartin, Anne C., Kamilar, Victoria, Simubali, Limonty, Mudenda, Twig, Hamapumbu, Harry, Schue, Jessica L., et al. Seasonal stratification of anopheline abundance and species composition in southern Zambia suggests year-round, season-specific vector control approaches are needed. Malaria Journal. (Accepted October 2025). \u003c/li\u003e\n\u003cli\u003eNational Malaria Elimination Centre. Guidelines for the Diagnosis and Treatment of Malaria in Zambia. Ministry of Health, Lusaka, Zambia; 2017. Report No.: 5th Edition. \u003c/li\u003e\n\u003cli\u003eBhondoekhan FRP, Searle KM, Hamapumbu H, Lubinda M, Matoba J, Musonda M, et al. Improving the efficiency of reactive case detection for malaria elimination in southern Zambia: a cross-sectional study. Malar J. 2020 May 7;19(1):175. \u003c/li\u003e\n\u003cli\u003eKatowa B, Hamapumbu H, Thuma PE, B\u0026eacute;rub\u0026eacute; S, Wesolowski A, Moss WJ, et al. Declining Age-Specific Seroprevalence and Seroconversion Rates in Plasmodium falciparum from 2009 to 2018 Documents Progress toward Malaria Elimination in Southern Zambia. 2023 July 5 [cited 2025 July 14]; Available from: https://www.ajtmh.org/view/journals/tpmd/109/1/article-p134.xml\u003c/li\u003e\n\u003cli\u003eBousema T, Drakeley C. Epidemiology and Infectivity of \u003cem\u003ePlasmodium falciparum\u003c/em\u003e and \u003cem\u003ePlasmodium vivax\u003c/em\u003e Gametocytes in Relation to Malaria Control and Elimination. Clin Microbiol Rev. 2011 Apr;24(2):377\u0026ndash;410. \u003c/li\u003e\n\u003cli\u003eGon\u0026ccedil;alves BP, Kapulu MC, Sawa P, Guelb\u0026eacute;ogo WM, Tiono AB, Grignard L, et al. Examining the human infectious reservoir for \u003cem\u003ePlasmodium falciparum\u003c/em\u003e malaria in areas of differing transmission intensity. Nat Commun. 2017 Dec;8(1):1133. \u003c/li\u003e\n\u003cli\u003eBousema T, Okell L, Felger I, Drakeley C. Asymptomatic malaria infections: detectability, transmissibility and public health relevance. Nat Rev Microbiol. 2014 Dec;12(12):833\u0026ndash;40. \u003c/li\u003e\n\u003cli\u003eAcquah FK, Donu D, Obboh EK, Bredu D, Mawuli B, Amponsah JA, et al. Diagnostic performance of an ultrasensitive HRP2-based malaria rapid diagnostic test kit used in surveys of afebrile people living in Southern Ghana. Malar J. 2021 Dec;20(1):125. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"malaria-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"malj","sideBox":"Learn more about [Malaria Journal](http://malariajournal.biomedcentral.com/)","snPcode":"12936","submissionUrl":"https://submission.nature.com/new-submission/12936/3","title":"Malaria Journal","twitterHandle":"@malariajournal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"malaria, asymptomatic, subpatent, elimination","lastPublishedDoi":"10.21203/rs.3.rs-8641961/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8641961/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eIn low malaria transmission settings targeting elimination, the World Health Organization recommends a combination of mass (e.g., mass test-and-treat), targeted (e.g., chemoprophylaxis or treatment for travelers), and reactive (e.g., reactive drug administration) strategies. Most of these strategies would not identify and treat individuals with asymptomatic parasitemia. This study was conducted in a pre-elimination setting in Southern Province, Zambia to examine risk factors for asymptomatic parasitemia, its epidemiologic relationship to incident clinical malaria, and evidence of its contribution to ongoing transmission to inform policy on whether these parasitemic individuals need to be identified and treated to achieve malaria elimination.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eAn intensive longitudinal cohort study of 197 households within the catchment area of a single health center was designed to capture all clinical malaria cases and episodes of asymptomatic parasitemia between 2018 and 2020. During monthly collections, all household members and overnight visitors were administered a questionnaire and a blood sample was collected to identify \u003cem\u003ePlasmodium falciparum\u003c/em\u003e parasitemia by qPCR. Passive surveillance was also established at the local health center to identify cases of clinical malaria. The statistical analysis examined risk factors for parasitemia and associations between asymptomatic parasitemia and subsequent episodes of clinical malaria within individuals and parasitemia in household members.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eOf the 1071 individuals enrolled in the cohort, 144 (13%) were positive by qPCR for \u003cem\u003eP. falciparum\u003c/em\u003e at least once during the two-year study period. Monthly parasite prevalence by qPCR never exceeded 4% and parasite density was very low with a median of four parasites/\u0026micro;L. Incidence of self-reported clinical malaria was 46.7 cases per 1000 person-years. Low-density asymptomatic parasitemia was identified in all age groups, including young children. There was no association between asymptomatic parasitemia and clinical malaria within individuals, nor was there an association between asymptomatic parasitemia and subsequent parasitemia in household members beyond one month of the index case.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eLow-level parasitemia was prevalent despite few cases of clinical malaria in this low transmission setting. There was no evidence that low-level asymptomatic parasitemia led to clinical cases of malaria or transmission to other household members.\u003c/p\u003e","manuscriptTitle":"Low-density asymptomatic parasitemia in southern Zambia does not lead to clinical malaria and is not associated with household transmission: results from a two-year cohort study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-30 01:33:32","doi":"10.21203/rs.3.rs-8641961/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-16T15:34:44+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-02T23:36:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-24T01:09:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"58481042654635924768725449809819614394","date":"2026-02-13T19:09:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"114475661966220584307177706656033707831","date":"2026-02-01T22:44:11+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-27T17:13:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-21T15:49:08+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-20T18:01:36+00:00","index":"","fulltext":""},{"type":"submitted","content":"Malaria Journal","date":"2026-01-19T16:45:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"malaria-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"malj","sideBox":"Learn more about [Malaria Journal](http://malariajournal.biomedcentral.com/)","snPcode":"12936","submissionUrl":"https://submission.nature.com/new-submission/12936/3","title":"Malaria Journal","twitterHandle":"@malariajournal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1c7352e6-e6ec-42cf-bc66-6f4f9a6525fc","owner":[],"postedDate":"January 30th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-03-16T15:39:47+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-30 01:33:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8641961","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8641961","identity":"rs-8641961","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}