Evaluating Field Performance of Highly Sensitive Malaria RDT: Detection of Infection Among Febrile Patients, Asymptomatic Pregnant Women and Household Contacts in Mpigi, Uganda

World Health Organisation recommends that malaria case management be based on parasite-based diagnosis in all cases, but currently available tools for clinical use have limitations, including the inability to detect low-level infections. Currently, next-generation highly sensitive rapid diagnostic tests (hsRDT) for Plasmodium falciparum (Pf) are commercially available, but require eld-based validation. This study evaluated the performance of the highly sensitive NxTek™ Eliminate Malaria Pf (NxTek) diagnostic test in health facilities and community settings in Mpigi district, Uganda in comparison to the conventional rapid diagnostic tests (cRDTs).

disease, increased investments in vector control and research [1]. Accurate diagnosis is a critical step in initiating effective management of malaria as well as breaking the malaria transmission chain [2]. In research settings, the use of molecular diagnostic tests has uncovered a large reservoir of malaria parasites previously not detected by microscopy and RDTs [3,4], that contribute to malaria transmission [5][6][7]. Children and pregnant women are some of the most vulnerable population. Worldwide, in 2019, of the estimated 9 million deaths in children younger than 5 years of age, 8% were attributable to malaria. Uganda has the 3rd highest global burden of malaria cases (5% of the cases worldwide) and the 8th highest level of deaths (3% of all malaria death worldwide) [2]. Frequent malaria infections and illness in children also increases the risk of stunting, and makes the child vulnerable to other infections that may cause permanent neurological and cognitive damage [8]. Malaria in pregnancy (MIP) is associated with an increased risk of poor foetal outcomes such as low birth weight (LBW), prenatal, neonatal and infant mortality [9][10][11]. Infection with P. falciparum towards the end of gestation increases the likelihood of placental infection [12]. A review by Desai et al. estimated that approximately one in every four pregnant women in malaria-endemic areas have evidence of placental infection at the time of delivery [13]. In high transmission settings including Uganda, the World Health Organisation (WHO) recommends the use of Sulphadoxine-pyrimethamine (SP) for intermittent preventive treatment in pregnancy (IPTp), as an effective method of preventing malaria in pregnancy. Intermittent screening and treatment (IST) of malaria during pregnancy has been proposed as an alternative, or to augment intermittent preventive treatment in pregnancy (IPTp), where IPTp is failing due to drug resistance. IST of malaria during pregnancy has been proposed to better target e cacious antimalarial treatment of asymptomatic women with evidence of malaria infection, and have been found to be bene cial for women to receive targeted therapy [14]. However, the antenatal parasitaemias are frequently very low density, and the most appropriate screening test for IST has not been de ned. For vulnerable children, integrated community case management (ICCM) is a key intervention for malaria control with the potential to test, treat, and track cases, and enhance community-based surveillance systems. Village health team (VHT) have been equipped to provide care at the community in Uganda.
illnesses such as malaria, pneumonia and diarrhoea. When a child presents to the VHT, a malaria RDT is performed and treatment is offered based on test results. They are supervised by trained coordinators linked to public health facilities. For the study, children with positive cRDTs test results were considered index cases and were enrolled in the study for active case detection (ACD).

Study design and testing procedures
The study enrolled participants into three cohorts: (1) out-patient cohort (OPD), (2) antenatal cohort (ANC), and (3) community cohort (ICCM). Only participants who provided consent, were of the required age category and without severe disease that required emergency attentions were enrolled in the study.
OPD Cohort: Participants were screened and enrolled at both OPD clinics within the catchment facilities of the 28 health facilities. All children 6 months and older, and adults presenting at the OPD with fever or history of febrile illness in the previous 24 hours, were enrolled. Participants agreed to be tested/investigated multiple times according to the study protocol and testing algorithm used for the day.
ANC cohort: At ANC clinics, pregnant women seeking care and eligible for IPTs in the clinic were medically evaluated and enrolled in the study by the study team. All ANC participants were given a longlasting insecticidal bed net and additional testing according to the national procedures.
Community Cohort: At the community level, the VHT enrolled febrile children under ve years of age and provided a diagnostic test and subsequent case management based on results. Children positive for malaria infection con rmed with cRDTs were de ned as index cases and selected for subsequent testing of the entire household, described here as active case detection (ACD).
Clinical care and diagnostic testing Participants presented to the clinic for routine care (monthly for ANC cohort) or with fever at OPD and Community VHT's homes. Patients with severe disease and requiring inpatient management or referral were excluded from the study. At each visit, clinical interviews were performed, and blood by nger prick and dried blood spots (DBS) were collected, regardless of symptoms. Samples were selected for PCR if both HS-RDTs and cRDTs had been performed regardless of the test results (negative and positive included). The DNA extracted from DBS was tested for the presence of P.falciarum parasitemia using qPCR at Central Public Health Laboratories (CPHL). Fever was described as a temperature of ≥ 38.0 °C. Routine clinical and antenatal care was provided by health workers at the health center, including clinical o cers, midwives, nurses, and other similar cadres of health care staff. Patients with reported or documented fever and any positive RDT result were treated with age-speci c standard dosing of artemether-lumefamefatrine both at ANC and OPD [23]. Participants with asymptomatic parasitaemia were provided anti-malarial therapy at ANC, but had their IPTp delayed for two weeks in accordance with local standard-of-care.

Rapid Diagnostic testing
The Malaria Antigen P.f tests are a one step, rapid, qualitative tests for the detection of HRP-2 speci c to P. falciparum in human blood samples. The NxTek™ Highly-Sensitive Rapid Diagnostic Test (HS-RDT) complete kits used in the study included the following batch numbers and respective expiration dates: (1) 05LDE001A, 20.02.2020; (2) 05LDE003A, 31.03.2020; and (3) 05LDE004A, 01.04.2020, and was compared to CareStart TM Malaria HRP2 (Access Bio, Inc. (Monmouth Jct, New Jersey, USA). All kits were supplied according to the national system supply chain mechanisms in place without monitoring of temperature and humidity in the storage at testing sites. The malaria RDT testing followed the manufacturer's instructions for each speci c malaria RDT brand. The results were recorded by the health worker or CHW at 15 (cRDTs) or 20 (hsRDTs) minutes. Result reading and interpretation were according to the manufacturer's instructions and product package inserts for each malaria RDT brand. If a control line did not appear or only the test result appeared, the result was deemed to be invalid and was repeated.

Testing and Laboratory methods
Participants who met the eligibility criteria were enrolled in the study to undergo testing. A two-phased approach, serial and concurrent, was undertaken to conduct testing of the participants using the RDTs. With serial testing, the patients were rst tested with cRDTs at OPD, and only participants who tested negative were offered an additional HS-RDTs test. With the concurrent testing approach, the participants were tested with cRDT and HS-RDT at the same time, which gave two results for each patient (Figure 1). The DBS were prepared by spotting a drop of blood onto lter paper, air drying it completely, and storing it at room temperature in an individually sealed plastic bag with silica gel (desiccant). P. falciparum DNA was extracted and analysed using standardised methods as previously described [24]. PCR technologists were blinded to the RDT results. As a reference standard, PCR testing targeted a minimum of 500 samples from OPD and ANC cohorts used to determine the diagnostic performance (Sensitivity, Speci city, Positive Predictive Value (PPV) and Negative Predictive Value (NPV)) of the both cRDTs and HS-RDTs

Data recording and transmission
Clinical notes including demographic information, presenting malaria symptoms (febrile illness, fever, joint pains, headache, nausea, vomiting), cRDT and HS-RDT results, and treatment administered were captured in paper format and stored in a controlled location. The results of both cRDTs and HS-RDTs were recorded and transmitted to a central server using the Data Logger SMAPP for further processing and analysis. Real time data were transmitted and enabled the generation of geospatial maps to track malaria prevalence across villages within all sub-counties of the Mpigi district.

Data analysis
Data were analysed using STATA (version 13; STATA Corp., College Station, TX, USA). The proportion of participants with no detectable infection by both cRDTs, HS-RDTs and PCR were made for different cohorts and age strata. The analysis data set included a hard paper questionnaire, double entered into the EPIDATA database and digital data was captured by the Data loggers displayed on dashboards. The analysis compared test results between HS-RDTs and cRDTs to estimate the magnitude of undetected low parasite densities, with qPCR as quality control. Qualitative methods were used to understand suitability of the tools. Kappa statistics with 95% CI were used to assess the agreement between the cRDT and the HS-RDT. Using qPCR as the reference standard, clinical performance of the cRDT and HS-RDTs were calculated (sensitivity, speci city, PPV and NPV) for the presence of P. falciparum. Focus Group Discussions (FGDs) and individual structured key interviews were conducted to collect qualitative information on the health workers' experience and perception on the use of the HS-RDTs. Participants were purposely selected to obtain a representation of both the facility and village health workers with differences in the range of abilities. A total of ve interviews were conducted of which three were focus group discussions (FGDs) conducted among the facility-based health workers, one FGD for VHTs and one structured interview for the key informant interview (KII) with the District malaria focal point-person. The discussions were conducted by a team of three study personnel (including a moderator and a note taker), following topic guides that had been speci cally developed for this study. The discussions were conducted either in English and/or the local language (Luganda), depending on which was convenient for the health workers. The audio les were transcribed into texts, which were then thematically analysed.

Results
A total of 28 private and government-sponsored health facilities providing routine antenatal and curative out-patient care were selected as study sites. Patient enrolment and testing was conducted between April and December 2019 in Mpigi district. A total of 312 health workers were trained during the whole course of the study, among whom 217 were VHTs and 95 were facility health workers during eight training sessions. The trained health workers were issued with HS-RDTs and android phones as data loggers based on the patient load estimates for 3 months. Of these, 217 (70%) were VHW, and two-thirds were female. The study team screened and enrolled 29,333 clients within the three cohorts. The demographic details of the participants enrolled are summarised in Table 1.
Symptomatic participants at OPD: Out of the 11,511 patients screened, 8,585 (74.6%) were tested, 1,553 in a serial procedure (cRDT rst, then HS-RDT for the initial negative). Out of the 1,553, only 165 (11%) were positive with cRDT. The subset of negative cRDT patients were tested again with HS-RDTs. Out of 1,386 (1,553-165) patients, 167 (12.1%) were positive on HS-RDTs, documented as the undetected magnitude of low-density infections with two missing results. Of the 7,032 patients tested with both cRDTs and HS-RDTs performed by the concurrent testing approach, a 7% difference was observed, with 447 (6.4%) cRDT and 933 (13.3%) HS-RDT positive for malaria infection. The calculated level of agreement between the two test kit results was 91.2% (6,410/7,032), kappa statistics = 0.51 (p<0.001). The details are shown in Table 2.
Asymptomatic pregnant participants at ANC: A total of 3,446 women tested at ANC were tested with HS-RDT, of whom 13% (67/514) were positive. When cRDTs and HS-RDTs were performed using the concurrent testing approach, 10.4% (310/2,976) (95% CI 9.3-11.6) were HS-RDTs positive compared to 7.0% (209/2,976) (95% CI 6.2-8.0) for cRDTs. The percentage increment detected due to low-density infections was 48% (310-209/209) (95% CI: 41.6-55.1). The level of agreement between the two tests was 96.1%, kappa statistics = 0.758 (p<0.001. Febrile children among Community cohort: Seventy-three percent (3,009/ 4,131) of the febrile children tested for malaria using cRDTs in the VHT provider cohort were children under ve years of age. The number of cRDTs that tested positive among the children were 2,397 (79.7%), who were treated and denoted as index cases. When asymptomatic households of these children were tested with HS-RDTs, 21.1% (1,877/8,888) were found to be positive. The proportions of the asymptomatic infections were: (1) 26.3% (672/2,555) under-ve years of age, (2) 25% (546/2,192) 6-12 years of age (school going age group), and (3) 16.3% (653/4,010) 13 years of age and above. One hundred and one (101) participants had missing age information and were removed from further strati ed analysis (Table 3A and B). Invalid results at all testing points were repeated so that a decision for treatment was made. At the community level, invalid results were ve out of the 4,131 (0.12%) for cRDTs and fourteen out of 8,888 (0.16%) for the HS-RDT test kits.
Polymerase Chain Reaction (PCR): Out of the 10,008 parallel tests performed at OPD (7,032) and ANC (2,976) a the subset of 497 paired samples analysed for P. falciparum infection using qPCR. The samples at OPD were from febrile symptomatic patients and xx from OPD were asymptomatic women seeking routine care, For the total PCR tests done, 52.2% (95% CI 46.3-58.0) had detectable P. falciparum infection, with wide variability as shown by the CT values(Appendix 1). The sensitivity was 75.5% (95% CI: 69.7-80.7) for HS-RDTs compared to 45.8% (95% CI: 39.6-52.2) for the cRDTs for any detected parasite for the symptomatic cases at OPD. The speci city, positive and negative predictive values are shown in Table 4.
Acceptability and ease of use of new technology: All 250 health workers were able to provide data throughout the study period. Each user of the smartphone device required an operator account. The data logger was used to transmit test results for both cRDT and HS-RDTs and associated patient demographic information, as well as a tracked sample number (ticket). Thirty-seven (37) of 217 (17%) community health workers did not enter the test results in the data logger, even when they were testing and keeping the hard-copy records. The majority, (75%) of the data transfers were transmitted and received during the normal working time (8:00am-5:00pm) and the remaining data was sent at the end of the day or at night. (Figure 3).
Health workers scanned the digital Near Field Communication (NFC) feature that provided the Lot and Batch numbers as well as expiration dates for 4,753 tests. Such details were useful for logistics management, quality control, and tracking activities. Analysis of the data showed that all the kits were utilised within the shelf life-time before April 2020 ( Figure 4).
In Mpigi, 20 participants, 8 VHTs and 14 clinic staff participated in the FGDs and KII. Overall, the users of HS-RDTs found them easy to manipulate, given their routine use of malaria RDTs, and experienced minimal challenges transmitting test results on the android mobile phones. The skill improved with time as they used the phones and additional training for those who were struggling.
Acceptability of new technology was observed and demonstrated by the users. In general, health workers reported that using the new technology gave them an opportunity to have peer discussions on the quality of the results generated. The summary of responses and views are summarized in Table 5. Participation in the study restored con dence among health workers (VHTs), and raised their motivation to serve their communities (quote 2a, 2b). The health workers made personal adjustments to accommodate the work requirement by transmitting data at a convenient time during the study (Quote 2c) The tools were a useful demonstration of quality of the diagnostic process at all levels. When both cRDTs and HS-RDTs were used in the community, VHTs used kits as a way to con rm RDT quality and restore con dence in RDT results in situations where doubt existed. For example, when health workers encountered a discrepancy between their clinical impression (that a patient had malaria) and a negative RDT result, HS-RDT use was reported to help resolve the uncertainty (Quotes 3 and 4). Some health workers mentioned that HS-RDTs were useful in detecting asymptomatic malaria infection among ANC women. (Quote 4) Health workers had previously been trained to check RDT expiration dates and desiccant packets as a means of quality control. When HS-RDTs and Data Loggers (SMAPP) were introduced, the same information was obtained by scanning the near eld communications (NFC) tag, and imbedded method similar to reading a bar code. This introduced a new quality-control option which was integrated into routine practice, reported as useful and fully embraced. (Quote 5). Use of SMAPP phones to transmit data was time-saving, improved the quality of the data collection, reporting, analysis, and tracking of commodities (Quotes 6, 6b and 7).

Discussion
Local magnitude of undetected malaria infections: Rapid diagnostic tests (RDTs) are the only practical method to provide parasite-based early diagnosis in remote and poorly resourced areas, where most malaria cases and malaria mortality occur [2]. Evidence from the study demonstrated that the use of HS-RDTs yielded a two-fold increase in the number of malaria detections among patients seeking care for febrile illness at OPD in health facilities compared to conventional tests. This was consistent for the two testing approaches. The reported positive cases were twice as high with HS-RDTs across the different study populations of clinical symptomatic patients and asymptomatic pregnant women (6.4% with cRDT, compared to 13.3% for HS-RDT). When tests were done on the same patients, the HS-RDT detected twice the number of positives as cRDT. Similarly, HS-RDTs testing in asymptomatic pregnant women attending ANC showed a 13% prevalence. The magnitude of undetected malaria infections in symptomatic patients at OPD and non-febrile patients giving negative results using the conventional test kit was higher. The positive cases that had been missed by the cRDT but detected by the HS-RDT may represent undetected malaria infections with low parasite densities. This evidence correlates with results from earlier studies in Uganda [25] and Colombia [26] that demonstrated that HS-RDTs may offer an improved parasitological diagnosis of malaria with a signi cantly lower LOD. The rst of these is the NxTek™ Eliminate Malaria Pf. RDT hsRDT) for Pf HRP2. This test has a ten-fold improvement in Limit of Detection (LOD) from 800 pg/mL to 80 pg/mL Pf HRP2, as demonstrated with recombinant Pf HRP2 and several native Pf HRP2 types from axenic parasite cultures [25]. Further validation of the HS-RDTs with a panel of clinical specimens from asymptomatic study participants in Myanmar and Uganda showed that the order of magnitude of improvement in LOD indicates: (1) the ability of the test to reliably detect infections with as low as 1 pg/µL as compared to the 50-200 pg/µL detected by microscopy or current RDTs, and (2) the detection of Pf HRP2 as low as 25 pg/mL as compared to 800 pg/mL by current RDTs [25]. The results indicate a signi cant increase in sensitivity for P. falciparum detection in low-density sub-clinical infections with the HS-RDTs compared to the current commercial RDTs. Additionally, asymptomatic cases were detected in Uganda and Myanmar [25].
The school-going age group had a disproportionally higher malaria positivity rate among asymptomatic individuals.
The positive cases that had been missed by the conventional RDT (cRDT) but detected by the HS-RDT may represent undetected malaria infections with low parasite densities.
The majority of the services offered at OPD were utilised by women and school going children. Prevention emphasis should now focus on the new vulnerable groups observed in the study population of school going children. The disease burden seems to be shifting to the school age group, and therefore, routine testing and prevention approaches to cover this group needs to be introduced under school health care programmes.
Improved diagnosis is the rst step in providing appropriate treatment. The use of a more robust test kit will ensure that all malaria cases receive the appropriate treatment, and the negative cases are provided alternative care. Effective diagnosis and treatment result in reduced disease burden.
ANC Cohort: In Uganda, pregnant women are provided IPTp with the assumption that they will not have any parasites. In this study, the introduction of testing in ANC identi ed asymptomatic pregnant women who bene tted from the appropriate timely treatment. For the asymptomatic pregnant women visiting ANC, the magnitude of undetected malaria infections was 13%. Antenatal parasitaemias are frequently very low and a testing tool for this population is yet to be de ned. A nested analysis of 198 women, showed that close to half of these women in the low to medium transmission setting had detectable malaria infection. HS-RDTs showed a sensitivity of 75% (13% positive) against PCR. The higher sensitivity of the HRP2-detecting RDT may re ect the detection of circulating antigen from placenta sequestered malaria parasites, or recent spikes in parasite density remaining as persistent antigenemia.
While it is reasonable to postulate that higher parasite densities in circulation may cause greater harm to mother and child, a greater understanding of the clinical signi cance of the third of PCR-positive women, who were undetectable by the most sensitive RDT and so would not receive treatment under a RDT-based testing approach, is needed. These ndings are not similar to earlier studies performed in Eastern Uganda and Burkina Faso [14].
Currently, achieving greater sensitivity is possible with the next-generation highly sensitive RDTs, supplementing the use of a nucleic acid ampli cation test (NAAT) assay. . However, development of panspecies malaria are yet to be developed. As IPTp is reduced, programmes will have to make this costbene t trade-off of introducing sensitive tests like HS-RDTs. A previous study showed that improving the diagnostic sensitivity to 20 parasites per microlitre increased the proportion of detection of infection by 49% in Burkina Faso [27].
The updated WHO recommendation is that all pregnant women in malaria endemic countries must receive at least three doses of IPTp. However, the coverage of IPTp in most of sub-Saharan Africa remains below international targets. Given the low coverage and other factors that limit the effectiveness of IPTp, it is not unusual to nd pregnant women with malaria infection during ANC testing surveys in endemic areas [14,28]. These ndings showed a malaria prevalence of up to 13% among ANC clients, justifying the need to test and treat. This difference between the two testing tools is expected among the asymptomatic pregnant women in this setting because of low parasite densities.
ICCM treatment: CHWs, their supervisors at the district, and study facilitators commended the reactive case detection (RCD) system for improving access to malaria services, and signi cantly detecting a number of cases in their areas. The main implementation barriers included lack of supplies such as rain gear, transportation such as bicycles, communication (e.g. di culties in maintaining cell phone charge to transmit data by phone), and inconsistent supply chain (e.g., inadequate numbers of RDT kits and antimalarial drugs to test and treat uncomplicated cases).
Future efforts to reduce the spread of malaria further will require moving beyond the treatment of clinical infections to targeting malaria transmission more broadly in the community. As such, the accurate identi cation of asymptomatic human infections, which can sustain a large proportion of transmission, is becoming a vital component of control and elimination programmes [29].
The relative importance of these very low parasite infections has been debated in relation to their contribution to malaria transmission [29][30][31][32][33][34][35]. In areas working towards malaria elimination, the low undetected infections act as a reservoir of infection, even when they do not contribute to the burden of malaria cases [32].
To our knowledge, this is the rst study to conduct a RCD, using the existing structures of the ICCM, where the index cases of a positive under ve years of age child was the basis for tracking household members (contacts). RCD seeks to enhance malaria surveillance and control, by identifying and treating parasitaemic individuals residing near index cases.
Many VHTs found the RDC feasible and effective, similar to other places in Zambia [36] One in every ve household members tested using the HS-RDT was found to have malaria parasites antigens. The majority of these were in the school going age group. This may call for targeted intervention at schools like mass drug administration (MDA).

Training and capacity development:
Our study showed that health workers across different cadres and with different levels of pre-service training were able to learn how to effectively perform the HS-RDT, from a one-day training session.
Because the HS-RDTs have the same design and usability features as the current cRDTs, they were easily adopted in health facilities and community settings and detected a high proportion of people with lowdensity P. falciparum infections. The only additional training needed beyond the one-day training was speci cally for the data logger usage and this was done through continuous mentorship where need was identi ed.
The HS-RDTs exhibited excellent speci city. The advantage of this study is that it was performed with clinical specimens from an endemic region, in real-life resource-limited settings. The results depict performance under eld conditions. This validates the ndings from laboratory studies of high accuracy of the new tools [37] Data logger: To our knowledge, the smartphone loaded with the data logger mobile app was used by POC clinicians to log patient results in real-time, and for the rst time, piloted in Uganda during the HS-RDT malaria study. There was no damage due to a fall or mishandling for all 250 devices deployed for 6 months of the study, which can be attributed to the sturdy protection casing. Both the hardware and software are secure in that and it allows only the app and associated phone system settings to run.
Routine review of data submitted using the data logger, guided identi cation of individuals whose phones were not visible on the server and/or had data errors, received additional mentorship and support. The data logger allowed for the capture of GPS coordinates, as the results were transmitted to the central server. The accurate GPS coordinate elements captured along with each transmitted result allowed the application to create geospatial outputs for analysis of the disease. When this was utilised, interventions could be targeted to the identi ed locations and populations.
The feature of data cost savings enables only the data logger to access internet data for sending results, while all the other background apps are disabled. Charging of mobile devices has been one of the main challenges of implementing real-time electronic datasolutions in rural areas.
Limitations: The three tests were not compared against microscopy. In different transmission settings, where parasitemia and HRP-2 distribution vary, the improvement in the detection of HS-RDTs versus cRDTs is not consistent. Future studies should be conducted in varied settings to validate the importance of improvement in the detection of asymptomatic infections. However, many other studies have shown that the performance of RDTs is superior to routine microscopy, both in symptomatic and asymptomatic populations [27,29].
This study did not consider the whole spectrum of case management beyond diagnostic results, and therefore recommends further research on how to handle cases of low parasitemia among asymptomatic patients in the community.
The recent development and distribution of P. falciparum pfhrp2 and/or pfhrp3 gene deletion is not fully characterised in this part of the country. The deletions, if present, may have reduced the speci city of both HS-RDTs and cRDTs. In other studies, HS-RDTs were found to have the same threshold of crossreactivity with HRP3 similar to cRDTs [38]. HS-RDTs was found to have a 10 fold lower parasitaemia limit of detection (LOD) than SD-RDTs [37] .

Conclusion
This study was conducted in an operational manner, putting the tools in the hands of many and diverse health caregivers in their routine settings, to demonstrate feasibility and acceptability of HS-RDTs both at health facilities and community care providers (VHTs). This can be translated into routine practice, using new technology of data collection and reporting among all health workers, including ICCMs and reactive case detection for malaria surveillance.
The health workers did not require additional training to perform the HS-RDTs except for a single demonstration and provision of a chart on how to conduct the test. The HS-RDTs have the same design and usability features as the current RDTs, and hence was easily adopted. In comparison to conventional RDTs, the HS-RDT was able to detect low parasite densities among both symptomatic and asymptomatic patients.
This study demonstrated the ability to detect asymptomatic cases, increased case ndings using HS-RDT, leading to the elimination of a malaria reservoir through treatment of asymptomatic patients, active case detection and linking the community households to care, which is the actual goal of malaria elimination. This may help identify active case detection strategies that will successfully reduce the parasite reservoirs.
Previous concerns about overtreatment, incorrect diagnosis of febrile patients, increased burden to malaria control programmes due to identi cation of more malaria cases may be valid and need to be better understood in large scale out implementation pilots. Further, research is needed to better understand how a more sensitive test can be implemented appropriately in a manner that is costeffective. This evidence supports the earlier propositions by Das et. al. that the HS-RDT is an effective diagnostic tool for malaria control and elimination programmes [37].

Declarations
Ethical Approval and consent to participate The study protocol was reviewed and approved by the Uganda National Council for Science and Technology and institutional review board of Mildmay Uganda Research Ethics Committee (REF#:0402-2019). Informed consent was obtained from all participants and parents or guardians of the participating children. Additional consent was obtained from household members from the community, in which an index case is identi ed by village health teams (VHT).

Consent for publication
Not applicable Availability of data and materials The datasets analysed during the current study are available from the corresponding author on reasonable request.

Funding
The main sponsor of the study was Abbott International with additional funding from Ministry of health. Abbott did not have any role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. The staff and investigators were ministry of health and local government employees. Abbott through a public-private corporation with the Ministry of health provided kits and Mobile phones used to send the test results. DJK was WHO supported.

Competing interests
There authors declare on competing interest. The study sponsors did not have any role in the study design, conduct or interpretation of the results.     Table 4: Performance evaluation of HS-RDT and cRDT for the detection of of P. falciparum in peripheral blood during antenatal and Out-patient department visits with PCR as reference method.
Quote 6b "It helps in monitoring of malaria data without forging it. This will help to monitor the use of testing kits and the anti-malarial which are supposed to be administered to clients that have tested positive. This will help to minimize on the misuse." Buwama HCIII -Health Worker: Quote 7 "It's quick and new technology. Normally we have to go to the district with a compilation of the data to be sent, but with this you just send data without going to the district." Mitala Maria HCIII-Health worker  ANC cohort (please also add the description) Figure 3 The majority, (75%) of the data transfers were transmitted and received during the normal working time (8:00am-5:00pm) and the remaining data was sent at the end of the day or at night.